il 21  (PeproTech)

 
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    Name:
    Anti Human IL 21
    Description:
    Rabbit Anti Human IL 21 Source Polyclonal Rabbit Formulation Lyophilized Produced from sera of rabbits pre immunized with highly pure 98 recombinant hIL 21 Anti Human IL 21 specific antibody was purified by affinity chromatography employing immobilized hIL 21 matrix
    Catalog Number:
    500-P191-100UG
    Price:
    250.00
    Category:
    Antibodies
    Source:
    Polyclonal Rabbit
    Quantity:
    100UG
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    Structured Review

    PeproTech il 21
    Ex vivo expansion and characterization of <t>IL-15+IL-21</t> stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Rabbit Anti Human IL 21 Source Polyclonal Rabbit Formulation Lyophilized Produced from sera of rabbits pre immunized with highly pure 98 recombinant hIL 21 Anti Human IL 21 specific antibody was purified by affinity chromatography employing immobilized hIL 21 matrix
    https://www.bioz.com/result/il 21/product/PeproTech
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    il 21 - by Bioz Stars, 2021-06
    95/100 stars

    Images

    1) Product Images from "The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation"

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02816

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.
    Figure Legend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Techniques Used: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Techniques Used: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.
    Figure Legend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Techniques Used: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Techniques Used: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay

    2) Product Images from "The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses"

    Article Title: The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses

    Journal: Nature Communications

    doi: 10.1038/s41467-021-23220-x

    Leptin promotes IL-21 production in a STAT3-dependent manner. a B220 - CD4 + CD25 - CD44 + CD62L - CXCR5 + PD-1 + T FH cells (1 × 10 5 ) and B220 + Fas + GL-7 + GC B cells (1 × 10 5 ) from 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin (NP-OVA) immunized WT and db/db mice (9 dpi) were co-cultured with 5 µg/mL NP-OVA for 9 days. In the db/db T FH :WT B-cell culture, IL-21 (10 ng/mL) was added. Anti-NP IgG1 titers in the supernatant were measured by ELISA (IL-21_10 ng/mL: * P = 0.0403, WT: ** P = 0.0033). b ELISA measurement of IL-21 in cultured naive CD4 + T cells from WT mice with anti-CD3/CD28 and leptin (0-200 ng/mL) stimulation for 3 days (100 ng/mL: ** P = 0.0100, 200 ng/mL: ** P = 0.0441). c Western blot showing Stat3 phosphorylation (p-Stat3) in WT naive CD4 + T cells stimulated with 200 ng/mL leptin for 2 h. Values showing the fold changes relative to the non-treated control. d Binding of Stat3 to the Il21 promoter. Stat3-ChIP assays were performed on WT naive CD4 + T cells treated with 200 ng/mL leptin for 3 h. Results showing PCR products (left) and values for the fold changes in ChIP enrichment relative to non-treated control (right) (100 ng/mL: ** P = 0.0096, 200 ng/mL: ** P = 0.0043). e Schematic of Il21 promoter construction and luciferase assay. f The transcriptional activity of the Il21 promoter. Dual-Luciferase reporter assay for the Il21 promoter in 293 T cells with 200 ng/mL leptin treatment for 12 h (100 ng/mL: ** P = 0.0082, 200 ng/mL: ** P = 0.0016). g Cd4-Cre : Stat3 +/+ or Cd4-Cre : Stat3 fl/fl naive CD4 + T cells were stimulated under IL-21-inducing conditions with 200 ng/mL leptin for 3 days. Results for IL-21 secretion showing representative FACS plots (left) and statistics (right) (nil: * P = 0.0111, Leptin: * P = 0.0144). Data are shown for individuals (dots) and mean (bars) values, and analysed by two-way ANOVA ( a ) one-way ANOVA ( b , d , f ) or Mann–Whitney U-test ( g ). * P
    Figure Legend Snippet: Leptin promotes IL-21 production in a STAT3-dependent manner. a B220 - CD4 + CD25 - CD44 + CD62L - CXCR5 + PD-1 + T FH cells (1 × 10 5 ) and B220 + Fas + GL-7 + GC B cells (1 × 10 5 ) from 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin (NP-OVA) immunized WT and db/db mice (9 dpi) were co-cultured with 5 µg/mL NP-OVA for 9 days. In the db/db T FH :WT B-cell culture, IL-21 (10 ng/mL) was added. Anti-NP IgG1 titers in the supernatant were measured by ELISA (IL-21_10 ng/mL: * P = 0.0403, WT: ** P = 0.0033). b ELISA measurement of IL-21 in cultured naive CD4 + T cells from WT mice with anti-CD3/CD28 and leptin (0-200 ng/mL) stimulation for 3 days (100 ng/mL: ** P = 0.0100, 200 ng/mL: ** P = 0.0441). c Western blot showing Stat3 phosphorylation (p-Stat3) in WT naive CD4 + T cells stimulated with 200 ng/mL leptin for 2 h. Values showing the fold changes relative to the non-treated control. d Binding of Stat3 to the Il21 promoter. Stat3-ChIP assays were performed on WT naive CD4 + T cells treated with 200 ng/mL leptin for 3 h. Results showing PCR products (left) and values for the fold changes in ChIP enrichment relative to non-treated control (right) (100 ng/mL: ** P = 0.0096, 200 ng/mL: ** P = 0.0043). e Schematic of Il21 promoter construction and luciferase assay. f The transcriptional activity of the Il21 promoter. Dual-Luciferase reporter assay for the Il21 promoter in 293 T cells with 200 ng/mL leptin treatment for 12 h (100 ng/mL: ** P = 0.0082, 200 ng/mL: ** P = 0.0016). g Cd4-Cre : Stat3 +/+ or Cd4-Cre : Stat3 fl/fl naive CD4 + T cells were stimulated under IL-21-inducing conditions with 200 ng/mL leptin for 3 days. Results for IL-21 secretion showing representative FACS plots (left) and statistics (right) (nil: * P = 0.0111, Leptin: * P = 0.0144). Data are shown for individuals (dots) and mean (bars) values, and analysed by two-way ANOVA ( a ) one-way ANOVA ( b , d , f ) or Mann–Whitney U-test ( g ). * P

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase, Activity Assay, Reporter Assay, FACS, MANN-WHITNEY

    Defective T FH cells responses in Cd4-Cre:LepR fl/fl mice after influenza virus infection. a – e Cd4-Cre:LepR +/+ mice (grey, n = 11) and Cd4-Cre:LepR fl/fl mice (red, n = 8) were infected with influenza virus A/X-31 (H3N2). Mediastinal lymph nodes were analysed at day 9 post-infection. Representative FACS plots and statistics showing the CD4 + Foxp3 - CD44 high effector cells, CD4 + Foxp3 + T REG cells ( a ), CD44 + CXCR5 + Bcl6 + T FH ( b ), (* P = 0.0499, * P = 0.0347), IL-21 production ( c ), (* P = 0.0313, * P = 0.0142), B220 + GL-7 + Fas + GC B cells ( d ) (* P = 0.0151, * P = 0.0249) and B220 - CD138 + TACI + ASCs ( e ) (* P = 0.0250, * P = 0.0409) 9 days post influenza viral infection. f Cd4-Cre:LepR +/+ and Cd4-Cre:LepR fl/fl mice were intranasally challenged with H1N1 influenza virus and virus-specific IgG1, IgG2b and IgG2c in sera were measured by ELISA 9 days post-infection ( n = 7 per genotype) (IgG1: * P = 0.0183, IgG2b: * P
    Figure Legend Snippet: Defective T FH cells responses in Cd4-Cre:LepR fl/fl mice after influenza virus infection. a – e Cd4-Cre:LepR +/+ mice (grey, n = 11) and Cd4-Cre:LepR fl/fl mice (red, n = 8) were infected with influenza virus A/X-31 (H3N2). Mediastinal lymph nodes were analysed at day 9 post-infection. Representative FACS plots and statistics showing the CD4 + Foxp3 - CD44 high effector cells, CD4 + Foxp3 + T REG cells ( a ), CD44 + CXCR5 + Bcl6 + T FH ( b ), (* P = 0.0499, * P = 0.0347), IL-21 production ( c ), (* P = 0.0313, * P = 0.0142), B220 + GL-7 + Fas + GC B cells ( d ) (* P = 0.0151, * P = 0.0249) and B220 - CD138 + TACI + ASCs ( e ) (* P = 0.0250, * P = 0.0409) 9 days post influenza viral infection. f Cd4-Cre:LepR +/+ and Cd4-Cre:LepR fl/fl mice were intranasally challenged with H1N1 influenza virus and virus-specific IgG1, IgG2b and IgG2c in sera were measured by ELISA 9 days post-infection ( n = 7 per genotype) (IgG1: * P = 0.0183, IgG2b: * P

    Techniques Used: Mouse Assay, Infection, FACS, Enzyme-linked Immunosorbent Assay

    3) Product Images from "PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases"

    Article Title: PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases

    Journal: JCI Insight

    doi: 10.1172/jci.insight.126457

    TNF-α induces PPP2R2B methylation, abolishes B55β expression, and impairs CWID in healthy T cells. ( A ) T cells from HDs were activated and expanded in the presence of IL-2 for 10 days. In addition to IL-2, at days 0, 2, 4, 6, and 8, the indicated cytokines (TNF-α, IFN-α, IL-6, IL-21, or IL-17) were added to the culture. At day 10, cells were counted, washed, and replated in the absence of IL-2 and proinflammatory cytokines. ( B ) Apoptosis (annexin V + SYTOX Orange − ) was quantified before (0 hours) and after IL-2 withdrawal ( n = 3–6). * P
    Figure Legend Snippet: TNF-α induces PPP2R2B methylation, abolishes B55β expression, and impairs CWID in healthy T cells. ( A ) T cells from HDs were activated and expanded in the presence of IL-2 for 10 days. In addition to IL-2, at days 0, 2, 4, 6, and 8, the indicated cytokines (TNF-α, IFN-α, IL-6, IL-21, or IL-17) were added to the culture. At day 10, cells were counted, washed, and replated in the absence of IL-2 and proinflammatory cytokines. ( B ) Apoptosis (annexin V + SYTOX Orange − ) was quantified before (0 hours) and after IL-2 withdrawal ( n = 3–6). * P

    Techniques Used: Methylation, Expressing

    4) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    5) Product Images from "CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD"

    Article Title: CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD

    Journal: JCI Insight

    doi: 10.1172/jci.insight.136185

    TGF-β from CAR-Tregs play a major role in the suppression of B cell proliferation and IgG production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of various neutralizing antibodies (10 μg/mL) ( n = 4–5). ( B ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells after coculture with CD19-CAR Tregs in the presence of TGF-β type 1 receptor inhibitor RepSox (0.1 and 1 μM). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panel ( n = 3). NC, negative control; PC, positive control; Cont IgG control IgG. ( C ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of RepSox (1 μM) ( n = 3). ( A ) Data are representative of independent experiments using samples from 2 healthy donors. ( B and C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P
    Figure Legend Snippet: TGF-β from CAR-Tregs play a major role in the suppression of B cell proliferation and IgG production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of various neutralizing antibodies (10 μg/mL) ( n = 4–5). ( B ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells after coculture with CD19-CAR Tregs in the presence of TGF-β type 1 receptor inhibitor RepSox (0.1 and 1 μM). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panel ( n = 3). NC, negative control; PC, positive control; Cont IgG control IgG. ( C ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of RepSox (1 μM) ( n = 3). ( A ) Data are representative of independent experiments using samples from 2 healthy donors. ( B and C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P

    Techniques Used: Produced, Labeling, Negative Control, Positive Control

    CD19-targeted CAR Tregs efficiently suppress B cells and antibody production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells 3 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1 and 1:1 (B cells/Tregs) ( n = 3). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panels. ( B ) Total IgG antibody levels produced by B cells and flow cytometric analysis of differentiated B cells (CD4 – FVD – IgD – CD38 + ) 7 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1, 1:0.3, and 1:1 (B cells/Tregs) ( n = 3–4). HD, healthy donor. ( C ) Total IgA antibody levels after coculture ( n = 3). ( A and B ) Data are representative of independent experiments using samples from 2 healthy donors. ( C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P
    Figure Legend Snippet: CD19-targeted CAR Tregs efficiently suppress B cells and antibody production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells 3 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1 and 1:1 (B cells/Tregs) ( n = 3). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panels. ( B ) Total IgG antibody levels produced by B cells and flow cytometric analysis of differentiated B cells (CD4 – FVD – IgD – CD38 + ) 7 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1, 1:0.3, and 1:1 (B cells/Tregs) ( n = 3–4). HD, healthy donor. ( C ) Total IgA antibody levels after coculture ( n = 3). ( A and B ) Data are representative of independent experiments using samples from 2 healthy donors. ( C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P

    Techniques Used: Labeling, Produced

    6) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    7) Product Images from "Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure"

    Article Title: Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.641362

    High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p
    Figure Legend Snippet: High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p

    Techniques Used: Flow Cytometry, MANN-WHITNEY

    Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p
    Figure Legend Snippet: Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, AST Assay

    CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Flow Cytometry

    Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Cell Culture, Dilution Assay, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure"

    Article Title: Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.641362

    High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p
    Figure Legend Snippet: High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p

    Techniques Used: Flow Cytometry, MANN-WHITNEY

    Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p
    Figure Legend Snippet: Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, AST Assay

    CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Flow Cytometry

    Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Cell Culture, Dilution Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    10) Product Images from "Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets"

    Article Title: Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

    Journal: Cells

    doi: 10.3390/cells9122633

    pSTAT and NF-κB signaling in naïve and memory B cells upon B cell activation via BCR, CD40 or IL4/Il-21. A total of 10,000 human naïve (CD19 + CD27 − IgD + ) or memory (CD19 + CD27 + ) B cells ( n = 3) were stimulated after sorting with an anti-Ig F(ab) 2 mix (5 μg/mL) either together or not together with CD40L-expressing 3T3 cells and recombinant IL-4 (25 ng/mL) and/or IL-21 (50 ng/mL) cytokines. Multiple signaling proteins were analyzed by phosphoflow analysis up to 72 h of stimulation. ( A ) Representative FACS plots of the sorting strategy for purification of CD19 + CD27 + memory and CD19 + CD27 − IgD + naïve B cells. ( B ) Quantification of GeoMFI of signaling proteins in stimulated sorted naïve or memory B cells after 30 min, 24 h or 72 h stimulation with varying IL-21 stimulations. Fold change was calculated normalizing expression to unstimulated condition. p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test. * p
    Figure Legend Snippet: pSTAT and NF-κB signaling in naïve and memory B cells upon B cell activation via BCR, CD40 or IL4/Il-21. A total of 10,000 human naïve (CD19 + CD27 − IgD + ) or memory (CD19 + CD27 + ) B cells ( n = 3) were stimulated after sorting with an anti-Ig F(ab) 2 mix (5 μg/mL) either together or not together with CD40L-expressing 3T3 cells and recombinant IL-4 (25 ng/mL) and/or IL-21 (50 ng/mL) cytokines. Multiple signaling proteins were analyzed by phosphoflow analysis up to 72 h of stimulation. ( A ) Representative FACS plots of the sorting strategy for purification of CD19 + CD27 + memory and CD19 + CD27 − IgD + naïve B cells. ( B ) Quantification of GeoMFI of signaling proteins in stimulated sorted naïve or memory B cells after 30 min, 24 h or 72 h stimulation with varying IL-21 stimulations. Fold change was calculated normalizing expression to unstimulated condition. p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test. * p

    Techniques Used: Activation Assay, Expressing, Recombinant, FACS, Purification

    Transcription factor analysis in stimulated human B cells by flow cytometry. Human B cells ( n = 3) were stimulated with an anti-Ig F(ab)2 mix (5 μg/mL) and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines for 6 days and analyzed for the mRNA and protein expression of multiple transcription factors. ( A ) Schematic representation of the expression of B cell-, GC cell- and ASC-defining transcription factors after T cell-dependent B cell stimulation. ( B ) Representative FACS plot of the sorting strategy for the CD27/CD38 subpopulations for analysis by semiquantitative RT-PCR. A more stringent gating strategy was used here to prevent the contamination of subpopulations during sorting. ( C ) Quantification of relative gene expression of PAX5 , c-MYC , BCL6 , BLIMP1 , XBP-1 and AICDA in different CD27/CD38 subpopulations as measured by semiquantitative RT-PCR. All results were normalized to the internal control 18S rRNA. Expression was calculated relative to the CD27 − CD38 − subpopulation (set at value of 1). Each dot represents an independent donor, and mean values are represented as bars ( n = 3). ( D ) Representative FACS plot of the CD27/CD38 subpopulations that were stained for transcription factors and analyzed by FACS. ( E ) Quantification of the GeoMFI of PAX5, c-MYC, BCL6, BLIMP1, XBP-1s (active spliced isoform) and AID stained for in different CD27/CD38 subpopulations (left) and corresponding histogram overlays (right). Values depicted next to histograms represent the corresponding GeoMFI. Each dot represents an independent donor, and mean values are represented as bars ( n = 3). p values were calculated using Tukey’s multiple-comparison test, * p
    Figure Legend Snippet: Transcription factor analysis in stimulated human B cells by flow cytometry. Human B cells ( n = 3) were stimulated with an anti-Ig F(ab)2 mix (5 μg/mL) and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines for 6 days and analyzed for the mRNA and protein expression of multiple transcription factors. ( A ) Schematic representation of the expression of B cell-, GC cell- and ASC-defining transcription factors after T cell-dependent B cell stimulation. ( B ) Representative FACS plot of the sorting strategy for the CD27/CD38 subpopulations for analysis by semiquantitative RT-PCR. A more stringent gating strategy was used here to prevent the contamination of subpopulations during sorting. ( C ) Quantification of relative gene expression of PAX5 , c-MYC , BCL6 , BLIMP1 , XBP-1 and AICDA in different CD27/CD38 subpopulations as measured by semiquantitative RT-PCR. All results were normalized to the internal control 18S rRNA. Expression was calculated relative to the CD27 − CD38 − subpopulation (set at value of 1). Each dot represents an independent donor, and mean values are represented as bars ( n = 3). ( D ) Representative FACS plot of the CD27/CD38 subpopulations that were stained for transcription factors and analyzed by FACS. ( E ) Quantification of the GeoMFI of PAX5, c-MYC, BCL6, BLIMP1, XBP-1s (active spliced isoform) and AID stained for in different CD27/CD38 subpopulations (left) and corresponding histogram overlays (right). Values depicted next to histograms represent the corresponding GeoMFI. Each dot represents an independent donor, and mean values are represented as bars ( n = 3). p values were calculated using Tukey’s multiple-comparison test, * p

    Techniques Used: Flow Cytometry, Recombinant, Expressing, Cell Stimulation, FACS, Reverse Transcription Polymerase Chain Reaction, Staining

    Uniform Manifold Approximation and Projection (UMAP) analysis of combined membrane marker and TF marker analyses upon B cell differentiation. UMAP clustering analysis on B cells stained for CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 after 6-day culture with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( A ) UMAP 2D scatter plot of 60,000 living, single CD19 + B cells from 3 donors (left), and the different CD27/CD38 subpopulations overlaid (right). UMAP settings were as follows: distance function, Euclidean; number of neighbors, 30; minimal distance, 0.5; and number of components, 2. ( B ) Heatmap of the relative protein expression of CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 included in the UMAP analysis overlaid on the UMAP 2D scatter plot. ( C ) Heatmap of the relative protein expression of PAX5 and BLIMP1 within the isolated CD27 + CD38 − population (top) and CD27 − CD38 + population (bottom) overlaid on the UMAP 2D scatter plot.
    Figure Legend Snippet: Uniform Manifold Approximation and Projection (UMAP) analysis of combined membrane marker and TF marker analyses upon B cell differentiation. UMAP clustering analysis on B cells stained for CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 after 6-day culture with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( A ) UMAP 2D scatter plot of 60,000 living, single CD19 + B cells from 3 donors (left), and the different CD27/CD38 subpopulations overlaid (right). UMAP settings were as follows: distance function, Euclidean; number of neighbors, 30; minimal distance, 0.5; and number of components, 2. ( B ) Heatmap of the relative protein expression of CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 included in the UMAP analysis overlaid on the UMAP 2D scatter plot. ( C ) Heatmap of the relative protein expression of PAX5 and BLIMP1 within the isolated CD27 + CD38 − population (top) and CD27 − CD38 + population (bottom) overlaid on the UMAP 2D scatter plot.

    Techniques Used: Marker, Cell Differentiation, Staining, Expressing, Recombinant, Isolation

    Phosphoflow analysis of STAT5 phosphorylation unveils heterogeneity over time in CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative histogram overlays of p-STAT5 staining in CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the GeoMFI of p-STAT5 within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p
    Figure Legend Snippet: Phosphoflow analysis of STAT5 phosphorylation unveils heterogeneity over time in CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative histogram overlays of p-STAT5 staining in CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the GeoMFI of p-STAT5 within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p

    Techniques Used: Expressing, Recombinant, Staining

    Phosphoflow analysis of stimulated human B cells show differences between CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative FACS plots show the gating strategy for CD19 + and CD27/CD38 subpopulations by phosphoflow analysis after 96 h stimulation. ( B – E ) Representative histogram overlays of p-STAT1 ( B ), p-STAT3 ( C ), p-STAT6 ( D ) and NF-κB p65 ( E ) staining in unstimulated and stimulated CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the geometric MFI (GeoMFI) within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. For NF-κB p65 (E), fold change was calculated by normalizing to the expression in unstimulated CD19 + cells (set at value of 1). n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p
    Figure Legend Snippet: Phosphoflow analysis of stimulated human B cells show differences between CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative FACS plots show the gating strategy for CD19 + and CD27/CD38 subpopulations by phosphoflow analysis after 96 h stimulation. ( B – E ) Representative histogram overlays of p-STAT1 ( B ), p-STAT3 ( C ), p-STAT6 ( D ) and NF-κB p65 ( E ) staining in unstimulated and stimulated CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the geometric MFI (GeoMFI) within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. For NF-κB p65 (E), fold change was calculated by normalizing to the expression in unstimulated CD19 + cells (set at value of 1). n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p

    Techniques Used: Expressing, Recombinant, FACS, Staining

    Efficient in vitro generation of human B cell and antibody-secreting cell subpopulations. Total human B cells ( n = 3) were stimulated in vitro to generate multiple B cell and antibody-secreting cell (ASC) subpopulations. ( A ) Schematic overview of the culture system used to induce B cell differentiation. A total of 5000 CD19 + human B cells were stimulated with a human-CD40L-expressing 3T3 feeder layer; an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM, IgG and IgA; and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( B ) Representative FACS plot after 6 days of culture based on expression of CD27 and CD38. ( C ) Quantification of the relative percentages of CD27 and CD38 subpopulations in the total CD19 + B cell population between 3 and 6 days of culture. n = 4; p values were calculated by 2-way ANOVA with Tukey’s multiple-comparison test; * p
    Figure Legend Snippet: Efficient in vitro generation of human B cell and antibody-secreting cell subpopulations. Total human B cells ( n = 3) were stimulated in vitro to generate multiple B cell and antibody-secreting cell (ASC) subpopulations. ( A ) Schematic overview of the culture system used to induce B cell differentiation. A total of 5000 CD19 + human B cells were stimulated with a human-CD40L-expressing 3T3 feeder layer; an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM, IgG and IgA; and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( B ) Representative FACS plot after 6 days of culture based on expression of CD27 and CD38. ( C ) Quantification of the relative percentages of CD27 and CD38 subpopulations in the total CD19 + B cell population between 3 and 6 days of culture. n = 4; p values were calculated by 2-way ANOVA with Tukey’s multiple-comparison test; * p

    Techniques Used: In Vitro, Cell Differentiation, Expressing, Recombinant, FACS

    11) Product Images from "A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation"

    Article Title: A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01851

    Natural killer (NK) cytotoxicity measured by peripheral blood mononuclear cells (PBMC) NK cytotoxicity (40:1) and whole blood (WB)-based NK cytotoxicity (200 μL) according to the absolute number of NK cells (low, three donors; medium, 11 donors; high, 14 donors). NK cytotoxicity results using (A) PBMCs and WB activated overnight with (B) IL-2, (C) IL-2/IL-18, (D) IL-2/IL-21, and (E) IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (* p
    Figure Legend Snippet: Natural killer (NK) cytotoxicity measured by peripheral blood mononuclear cells (PBMC) NK cytotoxicity (40:1) and whole blood (WB)-based NK cytotoxicity (200 μL) according to the absolute number of NK cells (low, three donors; medium, 11 donors; high, 14 donors). NK cytotoxicity results using (A) PBMCs and WB activated overnight with (B) IL-2, (C) IL-2/IL-18, (D) IL-2/IL-21, and (E) IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (* p

    Techniques Used: Western Blot

    CD107a expression of peripheral blood mononuclear cells (PBMCs) and whole blood (WB) under various cytokine combinations and incubation times. PBMCs or WB samples were mixed with media containing various cytokine combinations (no cytokines (media), IL-2, IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21) for the indicated durations of time, and CD107a assays were performed either immediately or following overnight incubation. Samples were incubated for 3 h with or without K562 cells. (A) Gating strategy of CD107a measurement. CD107a surface expression was determined as CD3 − CD56 + CD107a + by flow cytometry. (B,C) Bar graphs indicate the percentage of CD107a + NK cells in PBMCs following cytokine stimulation for 3 h or overnight. (D,E) Bar graphs indicate percentages of CD107a + NK cells in WB following cytokine stimulation for 3 h and overnight. Data are represented as the mean ± SEM of results from five donors (* p
    Figure Legend Snippet: CD107a expression of peripheral blood mononuclear cells (PBMCs) and whole blood (WB) under various cytokine combinations and incubation times. PBMCs or WB samples were mixed with media containing various cytokine combinations (no cytokines (media), IL-2, IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21) for the indicated durations of time, and CD107a assays were performed either immediately or following overnight incubation. Samples were incubated for 3 h with or without K562 cells. (A) Gating strategy of CD107a measurement. CD107a surface expression was determined as CD3 − CD56 + CD107a + by flow cytometry. (B,C) Bar graphs indicate the percentage of CD107a + NK cells in PBMCs following cytokine stimulation for 3 h or overnight. (D,E) Bar graphs indicate percentages of CD107a + NK cells in WB following cytokine stimulation for 3 h and overnight. Data are represented as the mean ± SEM of results from five donors (* p

    Techniques Used: Expressing, Western Blot, Incubation, Flow Cytometry

    NK cytotoxicity correlations between peripheral blood mononuclear cells (PBMCs) and overnight cytokine-activated whole blood (WB) assays. Natural killer (NK) cytotoxicity assays were performed using PBMCs at an E:T ratio of (A) 40:1, (B) 20:1, and (C) 10:1 and 200 μL WB ( n = 28 donors). Correlation between PBMCs and IL-2-, IL-2/IL-18-, IL-2/IL-21-, and IL-2/IL-18/IL-21-treated WB.
    Figure Legend Snippet: NK cytotoxicity correlations between peripheral blood mononuclear cells (PBMCs) and overnight cytokine-activated whole blood (WB) assays. Natural killer (NK) cytotoxicity assays were performed using PBMCs at an E:T ratio of (A) 40:1, (B) 20:1, and (C) 10:1 and 200 μL WB ( n = 28 donors). Correlation between PBMCs and IL-2-, IL-2/IL-18-, IL-2/IL-21-, and IL-2/IL-18/IL-21-treated WB.

    Techniques Used: Western Blot

    Comparison of natural killer (NK) cytotoxicity in healthy donors ( n = 28) and patients with liver diseases ( n = 26) using the whole blood (WB)-based NK cytotoxicity assay (200 μL). WB was activated overnight with IL-2, IL-2/IL-18, and IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (*** p
    Figure Legend Snippet: Comparison of natural killer (NK) cytotoxicity in healthy donors ( n = 28) and patients with liver diseases ( n = 26) using the whole blood (WB)-based NK cytotoxicity assay (200 μL). WB was activated overnight with IL-2, IL-2/IL-18, and IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (*** p

    Techniques Used: Western Blot, Cytotoxicity Assay

    12) Product Images from "Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets"

    Article Title: Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets

    Journal: Cells

    doi: 10.3390/cells9122633

    pSTAT and NF-κB signaling in naïve and memory B cells upon B cell activation via BCR, CD40 or IL4/Il-21. A total of 10,000 human naïve (CD19 + CD27 − IgD + ) or memory (CD19 + CD27 + ) B cells ( n = 3) were stimulated after sorting with an anti-Ig F(ab) 2 mix (5 μg/mL) either together or not together with CD40L-expressing 3T3 cells and recombinant IL-4 (25 ng/mL) and/or IL-21 (50 ng/mL) cytokines. Multiple signaling proteins were analyzed by phosphoflow analysis up to 72 h of stimulation. ( A ) Representative FACS plots of the sorting strategy for purification of CD19 + CD27 + memory and CD19 + CD27 − IgD + naïve B cells. ( B ) Quantification of GeoMFI of signaling proteins in stimulated sorted naïve or memory B cells after 30 min, 24 h or 72 h stimulation with varying IL-21 stimulations. Fold change was calculated normalizing expression to unstimulated condition. p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test. * p
    Figure Legend Snippet: pSTAT and NF-κB signaling in naïve and memory B cells upon B cell activation via BCR, CD40 or IL4/Il-21. A total of 10,000 human naïve (CD19 + CD27 − IgD + ) or memory (CD19 + CD27 + ) B cells ( n = 3) were stimulated after sorting with an anti-Ig F(ab) 2 mix (5 μg/mL) either together or not together with CD40L-expressing 3T3 cells and recombinant IL-4 (25 ng/mL) and/or IL-21 (50 ng/mL) cytokines. Multiple signaling proteins were analyzed by phosphoflow analysis up to 72 h of stimulation. ( A ) Representative FACS plots of the sorting strategy for purification of CD19 + CD27 + memory and CD19 + CD27 − IgD + naïve B cells. ( B ) Quantification of GeoMFI of signaling proteins in stimulated sorted naïve or memory B cells after 30 min, 24 h or 72 h stimulation with varying IL-21 stimulations. Fold change was calculated normalizing expression to unstimulated condition. p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test. * p

    Techniques Used: Activation Assay, Expressing, Recombinant, FACS, Purification

    Transcription factor analysis in stimulated human B cells by flow cytometry. Human B cells ( n = 3) were stimulated with an anti-Ig F(ab)2 mix (5 μg/mL) and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines for 6 days and analyzed for the mRNA and protein expression of multiple transcription factors. ( A ) Schematic representation of the expression of B cell-, GC cell- and ASC-defining transcription factors after T cell-dependent B cell stimulation. ( B ) Representative FACS plot of the sorting strategy for the CD27/CD38 subpopulations for analysis by semiquantitative RT-PCR. A more stringent gating strategy was used here to prevent the contamination of subpopulations during sorting. ( C ) Quantification of relative gene expression of PAX5 , c-MYC , BCL6 , BLIMP1 , XBP-1 and AICDA in different CD27/CD38 subpopulations as measured by semiquantitative RT-PCR. All results were normalized to the internal control 18S rRNA. Expression was calculated relative to the CD27 − CD38 − subpopulation (set at value of 1). Each dot represents an independent donor, and mean values are represented as bars ( n = 3). ( D ) Representative FACS plot of the CD27/CD38 subpopulations that were stained for transcription factors and analyzed by FACS. ( E ) Quantification of the GeoMFI of PAX5, c-MYC, BCL6, BLIMP1, XBP-1s (active spliced isoform) and AID stained for in different CD27/CD38 subpopulations (left) and corresponding histogram overlays (right). Values depicted next to histograms represent the corresponding GeoMFI. Each dot represents an independent donor, and mean values are represented as bars ( n = 3). p values were calculated using Tukey’s multiple-comparison test, * p
    Figure Legend Snippet: Transcription factor analysis in stimulated human B cells by flow cytometry. Human B cells ( n = 3) were stimulated with an anti-Ig F(ab)2 mix (5 μg/mL) and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines for 6 days and analyzed for the mRNA and protein expression of multiple transcription factors. ( A ) Schematic representation of the expression of B cell-, GC cell- and ASC-defining transcription factors after T cell-dependent B cell stimulation. ( B ) Representative FACS plot of the sorting strategy for the CD27/CD38 subpopulations for analysis by semiquantitative RT-PCR. A more stringent gating strategy was used here to prevent the contamination of subpopulations during sorting. ( C ) Quantification of relative gene expression of PAX5 , c-MYC , BCL6 , BLIMP1 , XBP-1 and AICDA in different CD27/CD38 subpopulations as measured by semiquantitative RT-PCR. All results were normalized to the internal control 18S rRNA. Expression was calculated relative to the CD27 − CD38 − subpopulation (set at value of 1). Each dot represents an independent donor, and mean values are represented as bars ( n = 3). ( D ) Representative FACS plot of the CD27/CD38 subpopulations that were stained for transcription factors and analyzed by FACS. ( E ) Quantification of the GeoMFI of PAX5, c-MYC, BCL6, BLIMP1, XBP-1s (active spliced isoform) and AID stained for in different CD27/CD38 subpopulations (left) and corresponding histogram overlays (right). Values depicted next to histograms represent the corresponding GeoMFI. Each dot represents an independent donor, and mean values are represented as bars ( n = 3). p values were calculated using Tukey’s multiple-comparison test, * p

    Techniques Used: Flow Cytometry, Recombinant, Expressing, Cell Stimulation, FACS, Reverse Transcription Polymerase Chain Reaction, Staining

    Uniform Manifold Approximation and Projection (UMAP) analysis of combined membrane marker and TF marker analyses upon B cell differentiation. UMAP clustering analysis on B cells stained for CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 after 6-day culture with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( A ) UMAP 2D scatter plot of 60,000 living, single CD19 + B cells from 3 donors (left), and the different CD27/CD38 subpopulations overlaid (right). UMAP settings were as follows: distance function, Euclidean; number of neighbors, 30; minimal distance, 0.5; and number of components, 2. ( B ) Heatmap of the relative protein expression of CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 included in the UMAP analysis overlaid on the UMAP 2D scatter plot. ( C ) Heatmap of the relative protein expression of PAX5 and BLIMP1 within the isolated CD27 + CD38 − population (top) and CD27 − CD38 + population (bottom) overlaid on the UMAP 2D scatter plot.
    Figure Legend Snippet: Uniform Manifold Approximation and Projection (UMAP) analysis of combined membrane marker and TF marker analyses upon B cell differentiation. UMAP clustering analysis on B cells stained for CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 after 6-day culture with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( A ) UMAP 2D scatter plot of 60,000 living, single CD19 + B cells from 3 donors (left), and the different CD27/CD38 subpopulations overlaid (right). UMAP settings were as follows: distance function, Euclidean; number of neighbors, 30; minimal distance, 0.5; and number of components, 2. ( B ) Heatmap of the relative protein expression of CD19, CD27, CD38, PAX5, BLIMP1 and BCL6 included in the UMAP analysis overlaid on the UMAP 2D scatter plot. ( C ) Heatmap of the relative protein expression of PAX5 and BLIMP1 within the isolated CD27 + CD38 − population (top) and CD27 − CD38 + population (bottom) overlaid on the UMAP 2D scatter plot.

    Techniques Used: Marker, Cell Differentiation, Staining, Expressing, Recombinant, Isolation

    Phosphoflow analysis of STAT5 phosphorylation unveils heterogeneity over time in CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative histogram overlays of p-STAT5 staining in CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the GeoMFI of p-STAT5 within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p
    Figure Legend Snippet: Phosphoflow analysis of STAT5 phosphorylation unveils heterogeneity over time in CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative histogram overlays of p-STAT5 staining in CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the GeoMFI of p-STAT5 within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p

    Techniques Used: Expressing, Recombinant, Staining

    Phosphoflow analysis of stimulated human B cells show differences between CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative FACS plots show the gating strategy for CD19 + and CD27/CD38 subpopulations by phosphoflow analysis after 96 h stimulation. ( B – E ) Representative histogram overlays of p-STAT1 ( B ), p-STAT3 ( C ), p-STAT6 ( D ) and NF-κB p65 ( E ) staining in unstimulated and stimulated CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the geometric MFI (GeoMFI) within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. For NF-κB p65 (E), fold change was calculated by normalizing to the expression in unstimulated CD19 + cells (set at value of 1). n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p
    Figure Legend Snippet: Phosphoflow analysis of stimulated human B cells show differences between CD27/CD38 subpopulations. Human B cells ( n = 3–4) were stimulated with a human-CD40L-expressing 3T3 feeder layer, an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM/IgG/IgA and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines, and multiple signaling proteins were analyzed by phosphoflow analysis over the course of a 6-day culture. ( A ) Representative FACS plots show the gating strategy for CD19 + and CD27/CD38 subpopulations by phosphoflow analysis after 96 h stimulation. ( B – E ) Representative histogram overlays of p-STAT1 ( B ), p-STAT3 ( C ), p-STAT6 ( D ) and NF-κB p65 ( E ) staining in unstimulated and stimulated CD19 + and CD27/CD38 subpopulations after 6 and 72 h stimulation (left), and the quantification of the geometric MFI (GeoMFI) within the different subpopulations over the course of 6 days of culture (right). Values depicted next to histograms represent the corresponding GeoMFI. For NF-κB p65 (E), fold change was calculated by normalizing to the expression in unstimulated CD19 + cells (set at value of 1). n = 3–4; p values were calculated using a mixed-effect analysis with Tukey’s multiple-comparison test; * p

    Techniques Used: Expressing, Recombinant, FACS, Staining

    Efficient in vitro generation of human B cell and antibody-secreting cell subpopulations. Total human B cells ( n = 3) were stimulated in vitro to generate multiple B cell and antibody-secreting cell (ASC) subpopulations. ( A ) Schematic overview of the culture system used to induce B cell differentiation. A total of 5000 CD19 + human B cells were stimulated with a human-CD40L-expressing 3T3 feeder layer; an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM, IgG and IgA; and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( B ) Representative FACS plot after 6 days of culture based on expression of CD27 and CD38. ( C ) Quantification of the relative percentages of CD27 and CD38 subpopulations in the total CD19 + B cell population between 3 and 6 days of culture. n = 4; p values were calculated by 2-way ANOVA with Tukey’s multiple-comparison test; * p
    Figure Legend Snippet: Efficient in vitro generation of human B cell and antibody-secreting cell subpopulations. Total human B cells ( n = 3) were stimulated in vitro to generate multiple B cell and antibody-secreting cell (ASC) subpopulations. ( A ) Schematic overview of the culture system used to induce B cell differentiation. A total of 5000 CD19 + human B cells were stimulated with a human-CD40L-expressing 3T3 feeder layer; an anti-Ig F(ab) 2 mix (5 μg/mL) targeting IgM, IgG and IgA; and recombinant IL-4 (25 ng/mL) and IL-21 (50 ng/mL) cytokines. ( B ) Representative FACS plot after 6 days of culture based on expression of CD27 and CD38. ( C ) Quantification of the relative percentages of CD27 and CD38 subpopulations in the total CD19 + B cell population between 3 and 6 days of culture. n = 4; p values were calculated by 2-way ANOVA with Tukey’s multiple-comparison test; * p

    Techniques Used: In Vitro, Cell Differentiation, Expressing, Recombinant, FACS

    13) Product Images from "Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota"

    Article Title: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota

    Journal: bioRxiv

    doi: 10.1101/2021.04.29.439534

    IgA Increases Systemic BCAAs Capable of Enhancing IgG Production (A) Heat map of significantly differentially abundant plasma metabolites from 9 HCs, 4 IGAD-R patients and 3 IGAD-NR patients. Data represent standardized peak areas and have been row mean-centered and row-normalized. Metabolites highlighted in bold are discussed in the text. SPA, standardized peak area. The positioning of the methyl group on methyl-2-oxvaleric acid was unable to be differentiated between attachment to either the third or fourth carbon of the molecule. (B) Plasma concentration of leucine, isoleucine and valine in donors described in A. Standardized peak area compared to internal standard and quantitative estimation (μM) was performed by normalizing relative peak areas and comparing to standard curve. (C) ELISA of IgG secreted by B cells from 7-10 HCs upon exposure of peripheral blood mononuclear cells to medium alone (ctrl) or T cell-associated stimuli CD40L and IL-21 for 6 days in the presence of decreasing amounts of BCAAs. From left to right: complete BCAA-sufficient media, (120 mg/L BCAAs at a 2:5:5 ratio of L-valine, L-leucine, and L-isoleucine), BCAA-depleted media with one tenth as much BCAAs (12 mg/L BCAAs at the same ratio as above), and BCAA-deficient media with no BCAAs. (D) ELISA of IgM secreted by B cells from 10 HCs upon exposure of peripheral blood mononuclear cells for 6 days to medium alone (ctrl) or the TI ligand CpG-DNA in the presence of decreasing amounts of BCAAs as in (C). Metabolomics (A, B) were from one experiment. In vitro BCAA experiments (C, D) summarize two independent experiments involving peripheral blood mononuclear cells from 5 HCs each. Data are presented with mean and significance was determined through Kruskal-Wallis test with Dunn’s correction for multiple comparisons. *p
    Figure Legend Snippet: IgA Increases Systemic BCAAs Capable of Enhancing IgG Production (A) Heat map of significantly differentially abundant plasma metabolites from 9 HCs, 4 IGAD-R patients and 3 IGAD-NR patients. Data represent standardized peak areas and have been row mean-centered and row-normalized. Metabolites highlighted in bold are discussed in the text. SPA, standardized peak area. The positioning of the methyl group on methyl-2-oxvaleric acid was unable to be differentiated between attachment to either the third or fourth carbon of the molecule. (B) Plasma concentration of leucine, isoleucine and valine in donors described in A. Standardized peak area compared to internal standard and quantitative estimation (μM) was performed by normalizing relative peak areas and comparing to standard curve. (C) ELISA of IgG secreted by B cells from 7-10 HCs upon exposure of peripheral blood mononuclear cells to medium alone (ctrl) or T cell-associated stimuli CD40L and IL-21 for 6 days in the presence of decreasing amounts of BCAAs. From left to right: complete BCAA-sufficient media, (120 mg/L BCAAs at a 2:5:5 ratio of L-valine, L-leucine, and L-isoleucine), BCAA-depleted media with one tenth as much BCAAs (12 mg/L BCAAs at the same ratio as above), and BCAA-deficient media with no BCAAs. (D) ELISA of IgM secreted by B cells from 10 HCs upon exposure of peripheral blood mononuclear cells for 6 days to medium alone (ctrl) or the TI ligand CpG-DNA in the presence of decreasing amounts of BCAAs as in (C). Metabolomics (A, B) were from one experiment. In vitro BCAA experiments (C, D) summarize two independent experiments involving peripheral blood mononuclear cells from 5 HCs each. Data are presented with mean and significance was determined through Kruskal-Wallis test with Dunn’s correction for multiple comparisons. *p

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, In Vitro

    14) Product Images from "The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation"

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02816

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.
    Figure Legend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Techniques Used: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Techniques Used: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.
    Figure Legend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Techniques Used: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Techniques Used: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay

    15) Product Images from "Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure"

    Article Title: Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.641362

    High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p
    Figure Legend Snippet: High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p

    Techniques Used: Flow Cytometry, MANN-WHITNEY

    Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p
    Figure Legend Snippet: Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, AST Assay

    CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Flow Cytometry

    Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Cell Culture, Dilution Assay, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Sustained antibody response to ZIKV infection induced by NS1 protein is accompanied by the progressive appearance of autoreactive antibodies and cross-reactive B cell clones"

    Article Title: Sustained antibody response to ZIKV infection induced by NS1 protein is accompanied by the progressive appearance of autoreactive antibodies and cross-reactive B cell clones

    Journal: bioRxiv

    doi: 10.1101/2020.12.24.423863

    GC B cells produce both virus specific and autoreactive antibodies. (A) GC B cells (CD38 lo/− GL-7 + gated on B220 + CD138 − ) in the spleen of ZIKV infected mice at 14 days post infection (left). Kinetics of frequency of follicular (FO) and germinal center (GC) B cells after infection is represented in right panel. ( B ) Subcutaneous infection experimental design indicating the time points of serum samples and lymphoid tissue collections from control mice (MOCK), mice immunized UV-inactivated virus (iZIKV) and infected mice (ZIKV). ( C ) Kinetics of serum IgG specific to ZIKV NS1 and VLPs. OD Sum is the summation of ODs of four serum dilutions (1:40, 1:120, 1:360 and 1:1080). ( D ) Representative plots of GC B cells (CD38 lo/− GL-7 + gated on B220 + CD138 − ) at day 14 post infection. Mice were injected in the left footpad. ( E ) Kinetics of frequency of germinal center (GC) B cells in left popliteal lymph nodes after infection (ZIKV) or immunization (iZIKV). ( F-H ) GC B cells from popliteal lymph nodes of infected mice were sorted and cultured in decreasing cells numbers/well (300, 100, 30, 10 cells/well). Supernatants were collected on day 7 and screened for IgG secretion by ELISA. Supernatants that revealed the presence of IgG were tested for antigen specificity by ELISA (F and G) or immunoblot against mouse brain tissue as source of self-antigens (H). Frequencies of IgG + GC B cells that bound NS1 (F), VLP (G) or self-antigens (H) were calculated utilizing Poisson distribution. Self-antigens used for frequency determination are indicated by arrows. Cell culture was performed on a monolayer of gamma-irradiated (20 Gy) NB40L feeder cells (3 × 10 3 cells/well), LPS (30 μg/mL) and IL-21 (2 ng/mL).
    Figure Legend Snippet: GC B cells produce both virus specific and autoreactive antibodies. (A) GC B cells (CD38 lo/− GL-7 + gated on B220 + CD138 − ) in the spleen of ZIKV infected mice at 14 days post infection (left). Kinetics of frequency of follicular (FO) and germinal center (GC) B cells after infection is represented in right panel. ( B ) Subcutaneous infection experimental design indicating the time points of serum samples and lymphoid tissue collections from control mice (MOCK), mice immunized UV-inactivated virus (iZIKV) and infected mice (ZIKV). ( C ) Kinetics of serum IgG specific to ZIKV NS1 and VLPs. OD Sum is the summation of ODs of four serum dilutions (1:40, 1:120, 1:360 and 1:1080). ( D ) Representative plots of GC B cells (CD38 lo/− GL-7 + gated on B220 + CD138 − ) at day 14 post infection. Mice were injected in the left footpad. ( E ) Kinetics of frequency of germinal center (GC) B cells in left popliteal lymph nodes after infection (ZIKV) or immunization (iZIKV). ( F-H ) GC B cells from popliteal lymph nodes of infected mice were sorted and cultured in decreasing cells numbers/well (300, 100, 30, 10 cells/well). Supernatants were collected on day 7 and screened for IgG secretion by ELISA. Supernatants that revealed the presence of IgG were tested for antigen specificity by ELISA (F and G) or immunoblot against mouse brain tissue as source of self-antigens (H). Frequencies of IgG + GC B cells that bound NS1 (F), VLP (G) or self-antigens (H) were calculated utilizing Poisson distribution. Self-antigens used for frequency determination are indicated by arrows. Cell culture was performed on a monolayer of gamma-irradiated (20 Gy) NB40L feeder cells (3 × 10 3 cells/well), LPS (30 μg/mL) and IL-21 (2 ng/mL).

    Techniques Used: Infection, Mouse Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation

    Antigen-specificity of B cells in germinal centers after immunization with ZIKV VLP and NS1. (A) Experimental design indicating the time points of foot pad subcutaneous immunization, serum samples and popliteal lymph nodes collections. (B) Kinetics of serum levels of IgG binding to ZIKV NS1 recombinant protein or ZIKV VLP, measured by ELISA. OD Sum is the sum of ODs of four serum dilutions (1:40, 1:120, 1:360 and 1:1080). ( C ) Representative plots of GC B cells (CD38 lo/− GL-7 + gated on B220 + CD138 − ) at day 14 post immunization. Mice were immunized in the left footpad. ( D ) Kinetics of frequency of germinal center (GC) B cells in left popliteal lymph nodes after immunization with NS1 (2 ug/mouse), VLP (2 ug/mouse) or both (4 ug of NS1 and 2 ug of VLP/mouse). Immunizations were adjuvanted with R848 (1ug/mouse). ( E ) GC B cells from popliteal lymph nodes of immunized mice were sorted and cultured in decreasing cells numbers/well (300, 100, 30, 10 cells/well). Supernatants were collected on day 7 and screened for IgG secretion by ELISA. Supernatants that revealed the presence of IgG were tested for antigen specificity by ELISA. Frequencies of IgG + GC B cells that bound VLP (upper panel) or NS1 (lower panel) were calculated utilizing Poisson distribution and are summarized on the right graph. Cell culture was performed on a monolayer of gamma-irradiated (20 Gy) NB40L feeder cells (3 × 10 3 cells/well), LPS (30 μg/mL) and IL-21 (2 ng/mL). ( F ) OD of IgG + supernatants of different cell numbers/well binding to ZIKV NS1, measured by ELISA.
    Figure Legend Snippet: Antigen-specificity of B cells in germinal centers after immunization with ZIKV VLP and NS1. (A) Experimental design indicating the time points of foot pad subcutaneous immunization, serum samples and popliteal lymph nodes collections. (B) Kinetics of serum levels of IgG binding to ZIKV NS1 recombinant protein or ZIKV VLP, measured by ELISA. OD Sum is the sum of ODs of four serum dilutions (1:40, 1:120, 1:360 and 1:1080). ( C ) Representative plots of GC B cells (CD38 lo/− GL-7 + gated on B220 + CD138 − ) at day 14 post immunization. Mice were immunized in the left footpad. ( D ) Kinetics of frequency of germinal center (GC) B cells in left popliteal lymph nodes after immunization with NS1 (2 ug/mouse), VLP (2 ug/mouse) or both (4 ug of NS1 and 2 ug of VLP/mouse). Immunizations were adjuvanted with R848 (1ug/mouse). ( E ) GC B cells from popliteal lymph nodes of immunized mice were sorted and cultured in decreasing cells numbers/well (300, 100, 30, 10 cells/well). Supernatants were collected on day 7 and screened for IgG secretion by ELISA. Supernatants that revealed the presence of IgG were tested for antigen specificity by ELISA. Frequencies of IgG + GC B cells that bound VLP (upper panel) or NS1 (lower panel) were calculated utilizing Poisson distribution and are summarized on the right graph. Cell culture was performed on a monolayer of gamma-irradiated (20 Gy) NB40L feeder cells (3 × 10 3 cells/well), LPS (30 μg/mL) and IL-21 (2 ng/mL). ( F ) OD of IgG + supernatants of different cell numbers/well binding to ZIKV NS1, measured by ELISA.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Mouse Assay, Cell Culture, Irradiation

    Self-reactivity of GC B after ZIKV NS1 immunization. Single GC B cells were sorted and cultured on a monolayer of gamma-irradiated feeder cells (1×10 3 cells/well) expressing CD40L, BAFF and IL-21 (Kuraoka et al., 2016). After 7 days, supernatants were collected for binding assays and cells were harvested for Igh sequencing. (A) Frequency of IgG + single GC B cell culture supernatants that bound to ZIKV NS1. Supernatants were screened for IgG production and IgG + wells were tested for binding to ZIKV NS1 protein by ELISA. (B) Clonal distribution of GC B cells found to bind to NS1 (upper panel) or that did not bind to NS1 (lower panel). Size of the slice is proportional to the clone frequency. Colored slices represent variants of clones that were f ound both as binders and non-binders. Right panel represents the frequency of expanded clones among binders and non-binders at specific time points after immunization. (C) Number of somatic mutations found in VH segments from each GC B cell sequenced grouped based on binding to NS1. (D) CDR-H3 average hydrophobicity index variation at different time points grouped based on binding to NS1. (E) Distribution of OD in NS1 ELISA with single cell culture supernatants related to the presence of charged amino acids. Red dots indicate the presence of 3 or more charged amino acids in CDR-H3 at different time points. (F-H) Single GC B cell culture supernatants were tested for binding to NS1 by ELISA and to self-antigens by immunoblot. (F) Clone counts of NS1 binders (black bars) and NS1 non binders (white bars) separated by self-reactivity. (G) Number of somatic mutations found in VH segments from each GC B cell sequenced grouped based on binding to NS1 and self-reactivity assessed by immunoblot. (H) Clonality, somatic hypermutation, binding to NS1, self-reactivity, hydrophobicity and charged amino acid usage ploted by time after immunization. Clonality corresponds to the number of variants of each clone found in the dataset. SHM is represented by the number of VH mutations found in each sequence. Anti-NS1 indicates the OD obtained by ELISA. Self-reactivity corresponds to the number of bands found for each supernatant in mouse tissue extracts (brain and/or muscle). Hydrophobicity corresponds to the average hydrophobicity of the CDR-H3 loop, hydrophobic and charged CDR-H3 sequences are shown in blue and red, respectively. Charged AA indicates the number of charged amino acids found in CDR-H3 loop.
    Figure Legend Snippet: Self-reactivity of GC B after ZIKV NS1 immunization. Single GC B cells were sorted and cultured on a monolayer of gamma-irradiated feeder cells (1×10 3 cells/well) expressing CD40L, BAFF and IL-21 (Kuraoka et al., 2016). After 7 days, supernatants were collected for binding assays and cells were harvested for Igh sequencing. (A) Frequency of IgG + single GC B cell culture supernatants that bound to ZIKV NS1. Supernatants were screened for IgG production and IgG + wells were tested for binding to ZIKV NS1 protein by ELISA. (B) Clonal distribution of GC B cells found to bind to NS1 (upper panel) or that did not bind to NS1 (lower panel). Size of the slice is proportional to the clone frequency. Colored slices represent variants of clones that were f ound both as binders and non-binders. Right panel represents the frequency of expanded clones among binders and non-binders at specific time points after immunization. (C) Number of somatic mutations found in VH segments from each GC B cell sequenced grouped based on binding to NS1. (D) CDR-H3 average hydrophobicity index variation at different time points grouped based on binding to NS1. (E) Distribution of OD in NS1 ELISA with single cell culture supernatants related to the presence of charged amino acids. Red dots indicate the presence of 3 or more charged amino acids in CDR-H3 at different time points. (F-H) Single GC B cell culture supernatants were tested for binding to NS1 by ELISA and to self-antigens by immunoblot. (F) Clone counts of NS1 binders (black bars) and NS1 non binders (white bars) separated by self-reactivity. (G) Number of somatic mutations found in VH segments from each GC B cell sequenced grouped based on binding to NS1 and self-reactivity assessed by immunoblot. (H) Clonality, somatic hypermutation, binding to NS1, self-reactivity, hydrophobicity and charged amino acid usage ploted by time after immunization. Clonality corresponds to the number of variants of each clone found in the dataset. SHM is represented by the number of VH mutations found in each sequence. Anti-NS1 indicates the OD obtained by ELISA. Self-reactivity corresponds to the number of bands found for each supernatant in mouse tissue extracts (brain and/or muscle). Hydrophobicity corresponds to the average hydrophobicity of the CDR-H3 loop, hydrophobic and charged CDR-H3 sequences are shown in blue and red, respectively. Charged AA indicates the number of charged amino acids found in CDR-H3 loop.

    Techniques Used: Cell Culture, Irradiation, Expressing, Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Clone Assay

    17) Product Images from "Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma"

    Article Title: Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma

    Journal: eLife

    doi: 10.7554/eLife.53244

    Identification of low-frequency antigen-specific T cells following peptide-specific expansion. CD8 + T cells isolated from M40 TILs were stimulated with B*18:01-artificial APCs pulsed with 10 μg/ml MAGE-A3 167-176 peptide and cultured with 10 IU/ml IL-2, 10 ng/ml IL-15, and 30 ng/ml IL-21 for 14 days. ( A ) Data from staining with the indicated multimers before stimulation (day 0) and 14 days after stimulation (day 14) are shown. The B*18:01/HIV gag 161-170 multimer was used as a control. The percentage of multimer + cells in CD8 + T cells is shown. ( B ) IFN-γ production by the M40 TILs in a B*18:01/MAGE-A3 167-176 -specific manner following peptide-specific stimulation. CD8 + T cells stimulated with B*18:01-artificial APCs pulsed with the MAGE-A3 167-176 peptide were employed as responder cells in IFN-γ ELISPOT analysis. T2 cells or T2 cells transduced with HLA-B*18:01 (T2-B*18:01) pulsed with MAGE-A3 167-176 or the HIV gag 161-170 control peptide were used as stimulator cells. The data shown represent the mean ± SD of experiments performed in triplicate. All the results are representative of at least two independent experiments. **p
    Figure Legend Snippet: Identification of low-frequency antigen-specific T cells following peptide-specific expansion. CD8 + T cells isolated from M40 TILs were stimulated with B*18:01-artificial APCs pulsed with 10 μg/ml MAGE-A3 167-176 peptide and cultured with 10 IU/ml IL-2, 10 ng/ml IL-15, and 30 ng/ml IL-21 for 14 days. ( A ) Data from staining with the indicated multimers before stimulation (day 0) and 14 days after stimulation (day 14) are shown. The B*18:01/HIV gag 161-170 multimer was used as a control. The percentage of multimer + cells in CD8 + T cells is shown. ( B ) IFN-γ production by the M40 TILs in a B*18:01/MAGE-A3 167-176 -specific manner following peptide-specific stimulation. CD8 + T cells stimulated with B*18:01-artificial APCs pulsed with the MAGE-A3 167-176 peptide were employed as responder cells in IFN-γ ELISPOT analysis. T2 cells or T2 cells transduced with HLA-B*18:01 (T2-B*18:01) pulsed with MAGE-A3 167-176 or the HIV gag 161-170 control peptide were used as stimulator cells. The data shown represent the mean ± SD of experiments performed in triplicate. All the results are representative of at least two independent experiments. **p

    Techniques Used: Isolation, Cell Culture, Staining, Enzyme-linked Immunospot, Transduction

    18) Product Images from "PD-L1 up-regulation restrains Th17 cell differentiation in STAT3 loss- and STAT1 gain-of-function patients"

    Article Title: PD-L1 up-regulation restrains Th17 cell differentiation in STAT3 loss- and STAT1 gain-of-function patients

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161427

    STAT3 LOF and STAT1 GOF cells have enhanced pSTAT1 that is partially dependent on impaired induction of SOCS3. (A) pSTAT1 (left) and pSTAT3 (right) in PBMCs after stimulation with IL-6, IL-21, or IL-27 for 15 min. (B) Total STAT1 in PBMCS before stimulation (left) and after cytokine stimulation (right) for 15 min. (C and D) SOCS3 and SOCS1 mRNA (C) and SOCS3 protein (D) levels in naive CD4 T cells after stimulation with IL-6, IL-21, or IL-27 for 1 h. (E and F) Naive (CD45RO − ) CD4 + T cells were transfected with SOCS3–GFP or GFP–control or SOCS3 –RNAi or scramble RNAi–control. IL-27 was added 5 h after transfection, and pSTAT1 levels were analyzed 18–24 h later. One-way ANOVA (A–D) and paired and Student’s t tests (E and F) were performed. *, P
    Figure Legend Snippet: STAT3 LOF and STAT1 GOF cells have enhanced pSTAT1 that is partially dependent on impaired induction of SOCS3. (A) pSTAT1 (left) and pSTAT3 (right) in PBMCs after stimulation with IL-6, IL-21, or IL-27 for 15 min. (B) Total STAT1 in PBMCS before stimulation (left) and after cytokine stimulation (right) for 15 min. (C and D) SOCS3 and SOCS1 mRNA (C) and SOCS3 protein (D) levels in naive CD4 T cells after stimulation with IL-6, IL-21, or IL-27 for 1 h. (E and F) Naive (CD45RO − ) CD4 + T cells were transfected with SOCS3–GFP or GFP–control or SOCS3 –RNAi or scramble RNAi–control. IL-27 was added 5 h after transfection, and pSTAT1 levels were analyzed 18–24 h later. One-way ANOVA (A–D) and paired and Student’s t tests (E and F) were performed. *, P

    Techniques Used: Transfection

    19) Product Images from "Targeting IL-21 to tumor-reactive T cells enhances memory T cell responses and anti-PD-1 antibody therapy"

    Article Title: Targeting IL-21 to tumor-reactive T cells enhances memory T cell responses and anti-PD-1 antibody therapy

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21241-0

    Superior antitumor effect of PD-1Ab21 treatment. a Balb/c mice transplanted s.c. with CT26 cells (left) or C57BL/6 mice transplanted s.c. with MC38 cells (right) were treated with anti-PD-1, PD-1Ab + IL-21, or PD-1Ab21 ( n = 5 mice /group). b Balb/c mice ( n = 5 mice /group) transplanted s.c. with TUBO cells were treated with anti-Her2/neu antibody alone or in combination with PD-1Ab + IL-21 or PD-1Ab21. c C57BL/6 mice ( n = 5 mice /group) transferred with 1 × 10 6 naïve CD90.1 + OT-1 cells 1 d before inoculation of B16-OVA cells were immunized with poly I:C and OVA 257-264 peptide, followed by treatment with anti-PD-1, PD-1Ab + IL-21, or PD-1Ab21. Tumor length (a) and width (b) were measured and tumor volume was calculated as (ab 2 /2). Tumor growth is shown as mean tumor size ±SEM over time. Results were compared using two-way ANOVA followed by Tukey’s multiple comparison test. a CT26: * p = 0.0167, ** p = 0.0024, **** p
    Figure Legend Snippet: Superior antitumor effect of PD-1Ab21 treatment. a Balb/c mice transplanted s.c. with CT26 cells (left) or C57BL/6 mice transplanted s.c. with MC38 cells (right) were treated with anti-PD-1, PD-1Ab + IL-21, or PD-1Ab21 ( n = 5 mice /group). b Balb/c mice ( n = 5 mice /group) transplanted s.c. with TUBO cells were treated with anti-Her2/neu antibody alone or in combination with PD-1Ab + IL-21 or PD-1Ab21. c C57BL/6 mice ( n = 5 mice /group) transferred with 1 × 10 6 naïve CD90.1 + OT-1 cells 1 d before inoculation of B16-OVA cells were immunized with poly I:C and OVA 257-264 peptide, followed by treatment with anti-PD-1, PD-1Ab + IL-21, or PD-1Ab21. Tumor length (a) and width (b) were measured and tumor volume was calculated as (ab 2 /2). Tumor growth is shown as mean tumor size ±SEM over time. Results were compared using two-way ANOVA followed by Tukey’s multiple comparison test. a CT26: * p = 0.0167, ** p = 0.0024, **** p

    Techniques Used: Mouse Assay

    The mechanisms underlying the antitumor effect of PD-1Ab21 treatment. a PD-1-receptor occupancy by anti-PD-1 antibody and PD-1Ab21 in blood and tumor. B16-OVA tumor-bearing mice with transferred OT-1 cells were immunized with poly I:C and OVA 257-264 peptide on day 6, followed by i.p. injections of anti-PD-1 or PD-1Ab21 on day 9 after tumor inoculation. Blood and tumors were harvested and the binding of a fluorescent-conjugated anti-PD-1 antibody to OT-1 cells was analyzed by flow cytometry at the indicated time post injections of antibody or PD-1Ab21. One representative experiment out of three independent experiments is shown. b Percentage of CD8 + T cells with naïve phenotype and T SCM in DLNs and spleens of CT26 tumor-bearing mice in different treatment groups as described in Fig. 5a ( n = 5 mice /group). Mice were sacrificed 1 d after last injections of antibody or proteins, and lymphocytes were prepared from DLNs and spleens for flow cytometry analysis. c – e B16-OVA-bearing mice with transferred OT-1 cells were immunized with OVA 257-264 peptide/ poly I:C, followed by treatment with anti-PD-1, PD-1Ab21 or combination of IL-21 and PD-1Ab, and then sacrificed 1 d after last injections of antibody or protein ( n = 5 mice /group). DLNs, spleens, and tumors were harvested for flow cytometry analysis. c Frequencies of OT-1 cells in DLN, spleen and tumor. d Phenotypes of OT-1 cells in DLNs. e Phenotypes of OT-1 cells in TILs. Data are presented as mean ± SEM and are representative of at least three independent experiments. Results were compared using one-way ANOVA followed by Tukey’s multiple comparison test. b * p = 0.0208 (DLN CD44 lo CD62L hi ), * p = 0.0431 (DLN CD44 lo CD62L hi CD122 + ScaI + ), ** p = 0.0013. c DLN: ** p = 0.007, *** p = 0.0004; Tumor: *** p = 0.0002. e * p = 0.0214 (Vac vs. Vac+α-PD-1), * p = 0.0234 (Vac+α-PD-1 vs. Vac+PD-1Ab21), ** p = 0.0035 (Vac vs. Vac+PD-1Ab21), ** p = 0.0012 (Vac+α-PD-1 vs. Vac+PD-1Ab21). **** p
    Figure Legend Snippet: The mechanisms underlying the antitumor effect of PD-1Ab21 treatment. a PD-1-receptor occupancy by anti-PD-1 antibody and PD-1Ab21 in blood and tumor. B16-OVA tumor-bearing mice with transferred OT-1 cells were immunized with poly I:C and OVA 257-264 peptide on day 6, followed by i.p. injections of anti-PD-1 or PD-1Ab21 on day 9 after tumor inoculation. Blood and tumors were harvested and the binding of a fluorescent-conjugated anti-PD-1 antibody to OT-1 cells was analyzed by flow cytometry at the indicated time post injections of antibody or PD-1Ab21. One representative experiment out of three independent experiments is shown. b Percentage of CD8 + T cells with naïve phenotype and T SCM in DLNs and spleens of CT26 tumor-bearing mice in different treatment groups as described in Fig. 5a ( n = 5 mice /group). Mice were sacrificed 1 d after last injections of antibody or proteins, and lymphocytes were prepared from DLNs and spleens for flow cytometry analysis. c – e B16-OVA-bearing mice with transferred OT-1 cells were immunized with OVA 257-264 peptide/ poly I:C, followed by treatment with anti-PD-1, PD-1Ab21 or combination of IL-21 and PD-1Ab, and then sacrificed 1 d after last injections of antibody or protein ( n = 5 mice /group). DLNs, spleens, and tumors were harvested for flow cytometry analysis. c Frequencies of OT-1 cells in DLN, spleen and tumor. d Phenotypes of OT-1 cells in DLNs. e Phenotypes of OT-1 cells in TILs. Data are presented as mean ± SEM and are representative of at least three independent experiments. Results were compared using one-way ANOVA followed by Tukey’s multiple comparison test. b * p = 0.0208 (DLN CD44 lo CD62L hi ), * p = 0.0431 (DLN CD44 lo CD62L hi CD122 + ScaI + ), ** p = 0.0013. c DLN: ** p = 0.007, *** p = 0.0004; Tumor: *** p = 0.0002. e * p = 0.0214 (Vac vs. Vac+α-PD-1), * p = 0.0234 (Vac+α-PD-1 vs. Vac+PD-1Ab21), ** p = 0.0035 (Vac vs. Vac+PD-1Ab21), ** p = 0.0012 (Vac+α-PD-1 vs. Vac+PD-1Ab21). **** p

    Techniques Used: Mouse Assay, Binding Assay, Flow Cytometry

    Cloning, expression, and characterization of PD-1Ab and PD-1Ab21. a Schematic of the cloning strategy (left) and domain assembly (right) of PD-1Ab21. b SDS-PAGE analysis of the purified proteins PD-1Ab (30 kDa) and PD-1Ab21 (48 kDa). MW, molecular weight. c Analytical size-exclusion chromatography of PD-1Ab21. Expected elution volumes were as follows: monomer, 15 ml; dimer, 13 ml; polymer, 12 ml. Protein elution was monitored by measuring absorbance at 280 nm. d Binding of PD-1Ab21 to PD-1 + cells. EG7 (left) or activated OT-1 cells (right) were stained without (as control, filled grey histogram) or with PD-1Ab21 in the presence (dashed line histogram) or absence (black line histogram) of anti-PD-1 antibody, and then followed by staining with anti-flag antibody (top). Or the cells were stained without (as control, filled grey histogram) or with PD-L1IgFc in the presence (dashed line histogram) or absence (black line histogram) of PD-1Ab21, and then followed by staining with anti-hIg antibody (bottom). e Detection of IL-21 bioactivities. Baf3 cells were cultured with medium only (as control), IL-21 or PD-1Ab21 for 3 d and counted using a cell counter ( n = 3 replicates /group). Results are presented as mean ± SEM and are representative of more than three independent experiments.
    Figure Legend Snippet: Cloning, expression, and characterization of PD-1Ab and PD-1Ab21. a Schematic of the cloning strategy (left) and domain assembly (right) of PD-1Ab21. b SDS-PAGE analysis of the purified proteins PD-1Ab (30 kDa) and PD-1Ab21 (48 kDa). MW, molecular weight. c Analytical size-exclusion chromatography of PD-1Ab21. Expected elution volumes were as follows: monomer, 15 ml; dimer, 13 ml; polymer, 12 ml. Protein elution was monitored by measuring absorbance at 280 nm. d Binding of PD-1Ab21 to PD-1 + cells. EG7 (left) or activated OT-1 cells (right) were stained without (as control, filled grey histogram) or with PD-1Ab21 in the presence (dashed line histogram) or absence (black line histogram) of anti-PD-1 antibody, and then followed by staining with anti-flag antibody (top). Or the cells were stained without (as control, filled grey histogram) or with PD-L1IgFc in the presence (dashed line histogram) or absence (black line histogram) of PD-1Ab21, and then followed by staining with anti-hIg antibody (bottom). e Detection of IL-21 bioactivities. Baf3 cells were cultured with medium only (as control), IL-21 or PD-1Ab21 for 3 d and counted using a cell counter ( n = 3 replicates /group). Results are presented as mean ± SEM and are representative of more than three independent experiments.

    Techniques Used: Clone Assay, Expressing, SDS Page, Purification, Molecular Weight, Size-exclusion Chromatography, Binding Assay, Staining, Cell Culture

    20) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    21) Product Images from "CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD"

    Article Title: CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD

    Journal: JCI Insight

    doi: 10.1172/jci.insight.136185

    TGF-β from CAR-Tregs play a major role in the suppression of B cell proliferation and IgG production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of various neutralizing antibodies (10 μg/mL) ( n = 4–5). ( B ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells after coculture with CD19-CAR Tregs in the presence of TGF-β type 1 receptor inhibitor RepSox (0.1 and 1 μM). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panel ( n = 3). NC, negative control; PC, positive control; Cont IgG control IgG. ( C ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of RepSox (1 μM) ( n = 3). ( A ) Data are representative of independent experiments using samples from 2 healthy donors. ( B and C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P
    Figure Legend Snippet: TGF-β from CAR-Tregs play a major role in the suppression of B cell proliferation and IgG production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of various neutralizing antibodies (10 μg/mL) ( n = 4–5). ( B ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells after coculture with CD19-CAR Tregs in the presence of TGF-β type 1 receptor inhibitor RepSox (0.1 and 1 μM). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panel ( n = 3). NC, negative control; PC, positive control; Cont IgG control IgG. ( C ) Total IgG antibody levels produced by primary human B cells 7 days after coculture with CD19-CAR Tregs in the presence of RepSox (1 μM) ( n = 3). ( A ) Data are representative of independent experiments using samples from 2 healthy donors. ( B and C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P

    Techniques Used: Produced, Labeling, Negative Control, Positive Control

    CD19-targeted CAR Tregs efficiently suppress B cells and antibody production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells 3 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1 and 1:1 (B cells/Tregs) ( n = 3). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panels. ( B ) Total IgG antibody levels produced by B cells and flow cytometric analysis of differentiated B cells (CD4 – FVD – IgD – CD38 + ) 7 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1, 1:0.3, and 1:1 (B cells/Tregs) ( n = 3–4). HD, healthy donor. ( C ) Total IgA antibody levels after coculture ( n = 3). ( A and B ) Data are representative of independent experiments using samples from 2 healthy donors. ( C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P
    Figure Legend Snippet: CD19-targeted CAR Tregs efficiently suppress B cells and antibody production. Primary human B cells were stimulated with anti-IgM and anti-CD40 antibodies in the presence of IL-21. ( A ) Flow cytometric analysis of CellTrace Violet dilution of CellTrace Violet–labeled primary human B cells 3 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1 and 1:1 (B cells/Tregs) ( n = 3). The fraction of CellTrace Violet lo B cells in the absence of drug and CD19-CAR Tregs is shown as 100% in the right panels. ( B ) Total IgG antibody levels produced by B cells and flow cytometric analysis of differentiated B cells (CD4 – FVD – IgD – CD38 + ) 7 days after coculture with CD19-CAR Tregs or polyclonal Tregs at ratios of 1:0.1, 1:0.3, and 1:1 (B cells/Tregs) ( n = 3–4). HD, healthy donor. ( C ) Total IgA antibody levels after coculture ( n = 3). ( A and B ) Data are representative of independent experiments using samples from 2 healthy donors. ( C ) Data were collected using human samples provided by 1 healthy donor. ( A – C ) P values were determined using 1-way ANOVA (* P

    Techniques Used: Labeling, Produced

    22) Product Images from "Generation of functional murine CD11c+ age-associated B cells in the absence of B cell T-bet expression"

    Article Title: Generation of functional murine CD11c+ age-associated B cells in the absence of B cell T-bet expression

    Journal: European journal of immunology

    doi: 10.1002/eji.201847641

    In vitro generation of CD11b + CD11c + ABCs independently of T-bet. (A) Percentage of CD11b + CD11c + B cells after in vitro stimulation of WT and Tbx21 −/− CD43-microbead purified splenic B cells with indicated combinations of anti-IgM, R848, IFN-γ, IL-21 and anti-CD40. Error bars indicate mean plus standard error of the mean (S.E.M.) ** p
    Figure Legend Snippet: In vitro generation of CD11b + CD11c + ABCs independently of T-bet. (A) Percentage of CD11b + CD11c + B cells after in vitro stimulation of WT and Tbx21 −/− CD43-microbead purified splenic B cells with indicated combinations of anti-IgM, R848, IFN-γ, IL-21 and anti-CD40. Error bars indicate mean plus standard error of the mean (S.E.M.) ** p

    Techniques Used: In Vitro, Purification

    23) Product Images from "T cells expressing the lupus susceptibility allele Pbx1d enhance autoimmunity and atherosclerosis in dyslipidemic mice"

    Article Title: T cells expressing the lupus susceptibility allele Pbx1d enhance autoimmunity and atherosclerosis in dyslipidemic mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.138274

    Cholesterol reduces Tfh cell expansion in vitro. ( A ) Representative CD4 + -gated FACS plots showing the percentage of CD4 + CXCR5 + PD-1 + Tfh cells differentiated in vitro from CD4 + T cells purified from B6-IL-21 VFP and Pbx1d-Tg-IL-21 VFP mice in the presence of vehicle, CD-CHO, or CS. ( B and C ) Frequency of Tfh cells corresponding to the assay shown in A . ( D ) Representative CD4 + -gated FACS plots showing the percentage of IL-21 + CD4 + T cells differentiated in vitro from B6-IL-21 VFP and Pbx1d-Tg-IL-21 VFP CD4 + T cells in the presence of vehicle, CD-CHO, or CS. Frequency of IL-21 + in CD4 + T cells ( E and F ) and IL-21 MFI ( G and H ). ( I and J ) Percentage of IL-21 + cells remaining in CD4 + CD44 + IL-21 + cells isolated from B6-IL-21 VFP and Pbx1d-Tg-IL-21 VFP mice after 24 hours’ stimulation with anti-CD3 and anti-CD28 antibodies in the presence of vehicle, CD-CHO, or CS. Paired t tests; * P
    Figure Legend Snippet: Cholesterol reduces Tfh cell expansion in vitro. ( A ) Representative CD4 + -gated FACS plots showing the percentage of CD4 + CXCR5 + PD-1 + Tfh cells differentiated in vitro from CD4 + T cells purified from B6-IL-21 VFP and Pbx1d-Tg-IL-21 VFP mice in the presence of vehicle, CD-CHO, or CS. ( B and C ) Frequency of Tfh cells corresponding to the assay shown in A . ( D ) Representative CD4 + -gated FACS plots showing the percentage of IL-21 + CD4 + T cells differentiated in vitro from B6-IL-21 VFP and Pbx1d-Tg-IL-21 VFP CD4 + T cells in the presence of vehicle, CD-CHO, or CS. Frequency of IL-21 + in CD4 + T cells ( E and F ) and IL-21 MFI ( G and H ). ( I and J ) Percentage of IL-21 + cells remaining in CD4 + CD44 + IL-21 + cells isolated from B6-IL-21 VFP and Pbx1d-Tg-IL-21 VFP mice after 24 hours’ stimulation with anti-CD3 and anti-CD28 antibodies in the presence of vehicle, CD-CHO, or CS. Paired t tests; * P

    Techniques Used: In Vitro, FACS, Purification, Mouse Assay, Isolation

    24) Product Images from "Five patterns of cell signaling pathways associated with cell behavior"

    Article Title: Five patterns of cell signaling pathways associated with cell behavior

    Journal: bioRxiv

    doi: 10.1101/2020.08.04.235986

    Analysis of homeostatic ERK pathway using FCM in T cells and neutrophils. Human peripheral blood cells were stimulated with IFN-α (100 U/mL), IL-21 (1 nM), G-CSF (1 nM), or a combination of these cytokines for 30 min. After the stimulations, the cells were fixed/permeabilized and stained with Pacific Blue-conjugated anti-human CD3 mAb, PE-conjugated anti-GPI-80 mAb, and Alexa647-conjugated anti-phosphorylated ERK1/2 mAb. The cells were measured by FCM (FACSCanto II). CD3 + cells were gated for T cell analysis (A and B), and GPI-80 + cells were gated for neutrophil analysis (D and E). MFI and CV of phosphorylated ERK1/2 (pERK1/2) in each gated cell population were analyzed. The correlations between MFI and CV on T cells and neutrophils are shown in (C) and (F), respectively. The data consisted of more than four independent experiments, and the statistical significances of correlation were calculated with Pearson’s correlation coefficient ( p value is indicated in each figure).
    Figure Legend Snippet: Analysis of homeostatic ERK pathway using FCM in T cells and neutrophils. Human peripheral blood cells were stimulated with IFN-α (100 U/mL), IL-21 (1 nM), G-CSF (1 nM), or a combination of these cytokines for 30 min. After the stimulations, the cells were fixed/permeabilized and stained with Pacific Blue-conjugated anti-human CD3 mAb, PE-conjugated anti-GPI-80 mAb, and Alexa647-conjugated anti-phosphorylated ERK1/2 mAb. The cells were measured by FCM (FACSCanto II). CD3 + cells were gated for T cell analysis (A and B), and GPI-80 + cells were gated for neutrophil analysis (D and E). MFI and CV of phosphorylated ERK1/2 (pERK1/2) in each gated cell population were analyzed. The correlations between MFI and CV on T cells and neutrophils are shown in (C) and (F), respectively. The data consisted of more than four independent experiments, and the statistical significances of correlation were calculated with Pearson’s correlation coefficient ( p value is indicated in each figure).

    Techniques Used: Staining

    25) Product Images from "IL-21 is a broad negative regulator of IgE class switch recombination in mouse and human B cells"

    Article Title: IL-21 is a broad negative regulator of IgE class switch recombination in mouse and human B cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20190472

    Models of the regulation of IgE CSR. (A) Our data indicate that while IL-4 promotes both IgE and IgG1 CSR, IL-21 inhibits IgE CSR but promotes IgG1 CSR. IL-21 appears to be the key negative regulator of IgE under a broad range of conditions in both mouse and human B cells. We observed that IFN-γ was required for IgG2a/c CSR but in vivo had no physiological impact on IgE and IgG1 CSR under the conditions tested. (B) ). The model does not exclude the possibility that other signaling pathways downstream of these receptors may also contribute to IgE regulation. The hatched lines show an expanded view of the regulation of IgE germline transcription by these receptors in C. (C) Model of the regulation of IgE germline transcription by the relative strength of IL-4R, CD40, and IL-21R signaling in B cells. Quantitative differences in signals from these receptors may depend on the extent and/or duration of receptor ligation (such as differences in the relative amounts of IL-4, IL-21, and CD40L expressed by T cells, versus the duration of T cell–B cell contacts). Three cases are provided for consideration: (1) IgE germline transcription is promoted in the context of strong IL-4R and CD40 signals. The CD40 signals attenuate the inhibitory signals downstream of the IL-21R. (2) When CD40 signals are weaker, strong IL-21R signals can inhibit IgE germline transcription, even in the presence of strong IL-4R signals. (3) When IL-4R signals are too weak, only minimal IgE germline transcription would occur regardless of the relative strength of CD40 and IL-21R signals. Note that in all three cases, IgG1 germline transcription would be promoted by these signals, and thus cases 2 or 3 would lead to a bias toward IgG1 CSR rather than IgE CSR.
    Figure Legend Snippet: Models of the regulation of IgE CSR. (A) Our data indicate that while IL-4 promotes both IgE and IgG1 CSR, IL-21 inhibits IgE CSR but promotes IgG1 CSR. IL-21 appears to be the key negative regulator of IgE under a broad range of conditions in both mouse and human B cells. We observed that IFN-γ was required for IgG2a/c CSR but in vivo had no physiological impact on IgE and IgG1 CSR under the conditions tested. (B) ). The model does not exclude the possibility that other signaling pathways downstream of these receptors may also contribute to IgE regulation. The hatched lines show an expanded view of the regulation of IgE germline transcription by these receptors in C. (C) Model of the regulation of IgE germline transcription by the relative strength of IL-4R, CD40, and IL-21R signaling in B cells. Quantitative differences in signals from these receptors may depend on the extent and/or duration of receptor ligation (such as differences in the relative amounts of IL-4, IL-21, and CD40L expressed by T cells, versus the duration of T cell–B cell contacts). Three cases are provided for consideration: (1) IgE germline transcription is promoted in the context of strong IL-4R and CD40 signals. The CD40 signals attenuate the inhibitory signals downstream of the IL-21R. (2) When CD40 signals are weaker, strong IL-21R signals can inhibit IgE germline transcription, even in the presence of strong IL-4R signals. (3) When IL-4R signals are too weak, only minimal IgE germline transcription would occur regardless of the relative strength of CD40 and IL-21R signals. Note that in all three cases, IgG1 germline transcription would be promoted by these signals, and thus cases 2 or 3 would lead to a bias toward IgG1 CSR rather than IgE CSR.

    Techniques Used: In Vivo, Ligation

    IL-21 inhibits IgE and promotes IgG1 responses of cultured human B cells depending on the strength of CD40 and IL-4 signals. Total B cells (A–E) or naive B cells (F–H) were purified from human tonsils and cultured for 7 d with anti-CD40 (as indicated) and IL-4 (20 ng/ml, except in H), in the presence or absence of IL-21 (20 ng/ml). (A–C) Representative flow cytometry (A) and quantification of the frequency (B) and total number (C) of IgG + and IgE + cells among IgD − cells from cultures of total B cells with the indicated concentrations of anti-CD40 and a fixed concentration of IL-4 (20 ng/ml) in the presence or absence of IL-21. (D) Representative flow cytometry showing proliferation of total B cells cultured from human tonsils with anti-CD40 (100 ng/ml) and IL-4, in the presence or absence of IL-21, as measured by dilution of CellTrace Violet. (E) Frequency of activated (IgD − ) cells among total tonsil B cells after culture with anti-CD40 (as indicated) and IL-4, in the presence or absence of IL-21, quantified as a percentage of live cells. (F–H) Representative flow cytometry (F) and quantification (G and H) of the frequency of IgG4 + , IgG1 + , and IgE + B cells, quantified as a percentage of IgD − cells, after culturing naive B cells. (F) Naive B cells were cultured with anti-CD40 (20 ng/ml) and IL-4 in the presence or absence of IL-21. Cells were pregated as IgD – (left panels) to identify IgG4 + cells; gates for IgG1 + and IgE + cells were then drawn within the IgG4 − population (right panels). IgG1 + and IgG4 + populations were gated sequentially to account for cross-reactivity of the anti-IgG1 antibody with IgG4. (G) Naive B cells were cultured with a variable concentration of anti-CD40 as indicated with a fixed concentration of IL-4 (20 ng/ml), in the presence or absence of IL-21. (H) Naive B cells were cultured with a fixed concentration of anti-CD40 (100 ng/ml) and a variable concentration of IL-4, as indicated, in the presence or absence of IL-21. Dots represent data points from individual donors. Bars represent arithmetic (B, E, G, and H) or geometric (C) means. n.s., not significant; *, P
    Figure Legend Snippet: IL-21 inhibits IgE and promotes IgG1 responses of cultured human B cells depending on the strength of CD40 and IL-4 signals. Total B cells (A–E) or naive B cells (F–H) were purified from human tonsils and cultured for 7 d with anti-CD40 (as indicated) and IL-4 (20 ng/ml, except in H), in the presence or absence of IL-21 (20 ng/ml). (A–C) Representative flow cytometry (A) and quantification of the frequency (B) and total number (C) of IgG + and IgE + cells among IgD − cells from cultures of total B cells with the indicated concentrations of anti-CD40 and a fixed concentration of IL-4 (20 ng/ml) in the presence or absence of IL-21. (D) Representative flow cytometry showing proliferation of total B cells cultured from human tonsils with anti-CD40 (100 ng/ml) and IL-4, in the presence or absence of IL-21, as measured by dilution of CellTrace Violet. (E) Frequency of activated (IgD − ) cells among total tonsil B cells after culture with anti-CD40 (as indicated) and IL-4, in the presence or absence of IL-21, quantified as a percentage of live cells. (F–H) Representative flow cytometry (F) and quantification (G and H) of the frequency of IgG4 + , IgG1 + , and IgE + B cells, quantified as a percentage of IgD − cells, after culturing naive B cells. (F) Naive B cells were cultured with anti-CD40 (20 ng/ml) and IL-4 in the presence or absence of IL-21. Cells were pregated as IgD – (left panels) to identify IgG4 + cells; gates for IgG1 + and IgE + cells were then drawn within the IgG4 − population (right panels). IgG1 + and IgG4 + populations were gated sequentially to account for cross-reactivity of the anti-IgG1 antibody with IgG4. (G) Naive B cells were cultured with a variable concentration of anti-CD40 as indicated with a fixed concentration of IL-4 (20 ng/ml), in the presence or absence of IL-21. (H) Naive B cells were cultured with a fixed concentration of anti-CD40 (100 ng/ml) and a variable concentration of IL-4, as indicated, in the presence or absence of IL-21. Dots represent data points from individual donors. Bars represent arithmetic (B, E, G, and H) or geometric (C) means. n.s., not significant; *, P

    Techniques Used: Cell Culture, Purification, Flow Cytometry, Concentration Assay

    IL-21 inhibits IgE and promotes IgG1 responses of cultured mouse B cells depending on the strength of CD40 and IL-4 signals. (A and B) Representative flow cytometry of IgD − activated B cells (A) and quantification of the frequency of IgE + and IgG1 + cells (B). Purified B cells were cultured for 4 d with a fixed concentration of IL-4 (12.5 ng/ml) and the indicated concentrations of anti-CD40 (α-CD40) in the presence or absence of IL-21 (50 ng/ml). (C) Quantification of the frequency of IgE + and IgG1 + cells among IgD – activated B cells. Splenocytes were cultured for 4 d with a fixed concentration of anti-CD40 (125 ng/ml) and the indicated concentrations of IL-4 in the presence or absence of IL-21 (25 ng/ml). (D) Representative flow cytometry of B cells (gated as B220 + ) after splenocytes were incubated with the indicated cytokines (IL-21, IFN-γ, or IL-10; 100 ng/ml each) for 20 min at 37°C. (E) Representative flow cytometry of B cells (gated as B220 + ) and CD4 + T cells (gated as CD4 + ) after splenocytes were incubated with the indicated cytokines (IL-21, IL-6, or IL-4; 50 ng/ml each) for 15 min at 37°C. (F) Quantification of the frequency of IgE + and IgG1 + cells among IgD − activated B cells. Purified B cells were cultured for 4 d with anti-CD40 (125 ng/ml) and IL-4 (12.5 ng/ml) alone (Ctrl) or with the addition of the indicated cytokines (IL-21 50 ng/ml, IFN-γ 25 ng/ml, or IL-10 25 ng/ml). (G) Representative flow cytometry of IgE + and IgG1 + B cells within the population of class-switched B cells (B220 + IgD − IgM − ) after splenocytes were cultured for 4 d with anti-CD40 (62.5 ng/ml) and IL-4 (25 ng/ml) in the presence or absence of IL-6 (81 ng/ml). Cells were from WT mice on a B6 background or Boy/J CD45.1 congenic mice bred in our colony. Dots represent data points of cells from individual mice, and bars represent the mean (B, C, and F). n.s., not significant; *, P
    Figure Legend Snippet: IL-21 inhibits IgE and promotes IgG1 responses of cultured mouse B cells depending on the strength of CD40 and IL-4 signals. (A and B) Representative flow cytometry of IgD − activated B cells (A) and quantification of the frequency of IgE + and IgG1 + cells (B). Purified B cells were cultured for 4 d with a fixed concentration of IL-4 (12.5 ng/ml) and the indicated concentrations of anti-CD40 (α-CD40) in the presence or absence of IL-21 (50 ng/ml). (C) Quantification of the frequency of IgE + and IgG1 + cells among IgD – activated B cells. Splenocytes were cultured for 4 d with a fixed concentration of anti-CD40 (125 ng/ml) and the indicated concentrations of IL-4 in the presence or absence of IL-21 (25 ng/ml). (D) Representative flow cytometry of B cells (gated as B220 + ) after splenocytes were incubated with the indicated cytokines (IL-21, IFN-γ, or IL-10; 100 ng/ml each) for 20 min at 37°C. (E) Representative flow cytometry of B cells (gated as B220 + ) and CD4 + T cells (gated as CD4 + ) after splenocytes were incubated with the indicated cytokines (IL-21, IL-6, or IL-4; 50 ng/ml each) for 15 min at 37°C. (F) Quantification of the frequency of IgE + and IgG1 + cells among IgD − activated B cells. Purified B cells were cultured for 4 d with anti-CD40 (125 ng/ml) and IL-4 (12.5 ng/ml) alone (Ctrl) or with the addition of the indicated cytokines (IL-21 50 ng/ml, IFN-γ 25 ng/ml, or IL-10 25 ng/ml). (G) Representative flow cytometry of IgE + and IgG1 + B cells within the population of class-switched B cells (B220 + IgD − IgM − ) after splenocytes were cultured for 4 d with anti-CD40 (62.5 ng/ml) and IL-4 (25 ng/ml) in the presence or absence of IL-6 (81 ng/ml). Cells were from WT mice on a B6 background or Boy/J CD45.1 congenic mice bred in our colony. Dots represent data points of cells from individual mice, and bars represent the mean (B, C, and F). n.s., not significant; *, P

    Techniques Used: Cell Culture, Flow Cytometry, Purification, Concentration Assay, Incubation, Mouse Assay

    26) Product Images from "The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation"

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02816

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.
    Figure Legend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Techniques Used: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Techniques Used: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.
    Figure Legend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Techniques Used: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Techniques Used: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay

    27) Product Images from "The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation"

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02816

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.
    Figure Legend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Techniques Used: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Techniques Used: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.
    Figure Legend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Techniques Used: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Techniques Used: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay

    28) Product Images from "Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure"

    Article Title: Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.641362

    High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p
    Figure Legend Snippet: High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p

    Techniques Used: Flow Cytometry, MANN-WHITNEY

    Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p
    Figure Legend Snippet: Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, AST Assay

    CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Flow Cytometry

    Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Cell Culture, Dilution Assay, Enzyme-linked Immunosorbent Assay

    29) Product Images from "Tumour necrosis factor alpha promotes secretion of 14-3-3η by inducing necroptosis in macrophages"

    Article Title: Tumour necrosis factor alpha promotes secretion of 14-3-3η by inducing necroptosis in macrophages

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-020-2110-9

    14-3-3η is detectable in culture supernatants of macrophages derived from HD and treated with TNF-α. The culture supernatants of macrophages cultured with or without diamide, TNF-α (100 ng/ml; n = 3), IL-1β (10 ng/ml; n = 3), IL-6/sIL-6R (10 ng/ml; n = 3), or IL-21 (10 ng/ml; n = 3) were subjected to WB. Recombinant 14-3-3η was used as a positive control. BSA was used as a loading control and was stained with CBB-R350. Representative images from three independent experiments are shown
    Figure Legend Snippet: 14-3-3η is detectable in culture supernatants of macrophages derived from HD and treated with TNF-α. The culture supernatants of macrophages cultured with or without diamide, TNF-α (100 ng/ml; n = 3), IL-1β (10 ng/ml; n = 3), IL-6/sIL-6R (10 ng/ml; n = 3), or IL-21 (10 ng/ml; n = 3) were subjected to WB. Recombinant 14-3-3η was used as a positive control. BSA was used as a loading control and was stained with CBB-R350. Representative images from three independent experiments are shown

    Techniques Used: Derivative Assay, Cell Culture, Western Blot, Recombinant, Positive Control, Staining

    30) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    31) Product Images from "A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation"

    Article Title: A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.01851

    Natural killer (NK) cytotoxicity measured by peripheral blood mononuclear cells (PBMC) NK cytotoxicity (40:1) and whole blood (WB)-based NK cytotoxicity (200 μL) according to the absolute number of NK cells (low, three donors; medium, 11 donors; high, 14 donors). NK cytotoxicity results using (A) PBMCs and WB activated overnight with (B) IL-2, (C) IL-2/IL-18, (D) IL-2/IL-21, and (E) IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (* p
    Figure Legend Snippet: Natural killer (NK) cytotoxicity measured by peripheral blood mononuclear cells (PBMC) NK cytotoxicity (40:1) and whole blood (WB)-based NK cytotoxicity (200 μL) according to the absolute number of NK cells (low, three donors; medium, 11 donors; high, 14 donors). NK cytotoxicity results using (A) PBMCs and WB activated overnight with (B) IL-2, (C) IL-2/IL-18, (D) IL-2/IL-21, and (E) IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (* p

    Techniques Used: Western Blot

    CD107a expression of peripheral blood mononuclear cells (PBMCs) and whole blood (WB) under various cytokine combinations and incubation times. PBMCs or WB samples were mixed with media containing various cytokine combinations (no cytokines (media), IL-2, IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21) for the indicated durations of time, and CD107a assays were performed either immediately or following overnight incubation. Samples were incubated for 3 h with or without K562 cells. (A) Gating strategy of CD107a measurement. CD107a surface expression was determined as CD3 − CD56 + CD107a + by flow cytometry. (B,C) Bar graphs indicate the percentage of CD107a + NK cells in PBMCs following cytokine stimulation for 3 h or overnight. (D,E) Bar graphs indicate percentages of CD107a + NK cells in WB following cytokine stimulation for 3 h and overnight. Data are represented as the mean ± SEM of results from five donors (* p
    Figure Legend Snippet: CD107a expression of peripheral blood mononuclear cells (PBMCs) and whole blood (WB) under various cytokine combinations and incubation times. PBMCs or WB samples were mixed with media containing various cytokine combinations (no cytokines (media), IL-2, IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21) for the indicated durations of time, and CD107a assays were performed either immediately or following overnight incubation. Samples were incubated for 3 h with or without K562 cells. (A) Gating strategy of CD107a measurement. CD107a surface expression was determined as CD3 − CD56 + CD107a + by flow cytometry. (B,C) Bar graphs indicate the percentage of CD107a + NK cells in PBMCs following cytokine stimulation for 3 h or overnight. (D,E) Bar graphs indicate percentages of CD107a + NK cells in WB following cytokine stimulation for 3 h and overnight. Data are represented as the mean ± SEM of results from five donors (* p

    Techniques Used: Expressing, Western Blot, Incubation, Flow Cytometry

    NK cytotoxicity correlations between peripheral blood mononuclear cells (PBMCs) and overnight cytokine-activated whole blood (WB) assays. Natural killer (NK) cytotoxicity assays were performed using PBMCs at an E:T ratio of (A) 40:1, (B) 20:1, and (C) 10:1 and 200 μL WB ( n = 28 donors). Correlation between PBMCs and IL-2-, IL-2/IL-18-, IL-2/IL-21-, and IL-2/IL-18/IL-21-treated WB.
    Figure Legend Snippet: NK cytotoxicity correlations between peripheral blood mononuclear cells (PBMCs) and overnight cytokine-activated whole blood (WB) assays. Natural killer (NK) cytotoxicity assays were performed using PBMCs at an E:T ratio of (A) 40:1, (B) 20:1, and (C) 10:1 and 200 μL WB ( n = 28 donors). Correlation between PBMCs and IL-2-, IL-2/IL-18-, IL-2/IL-21-, and IL-2/IL-18/IL-21-treated WB.

    Techniques Used: Western Blot

    Comparison of natural killer (NK) cytotoxicity in healthy donors ( n = 28) and patients with liver diseases ( n = 26) using the whole blood (WB)-based NK cytotoxicity assay (200 μL). WB was activated overnight with IL-2, IL-2/IL-18, and IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (*** p
    Figure Legend Snippet: Comparison of natural killer (NK) cytotoxicity in healthy donors ( n = 28) and patients with liver diseases ( n = 26) using the whole blood (WB)-based NK cytotoxicity assay (200 μL). WB was activated overnight with IL-2, IL-2/IL-18, and IL-2/IL-18/IL-21. Data are represented as the mean ± SEM (*** p

    Techniques Used: Western Blot, Cytotoxicity Assay

    32) Product Images from "Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure"

    Article Title: Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.641362

    High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p
    Figure Legend Snippet: High frequency of Tfh cells in HBV-ACLF patients was associated with disease severity. (A) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF (n = 36), M-CHB (n = 21), S-CHB (n = 32) patients and HC (n = 10) subjects were demonstrated by flow cytometry. (B) The frequencies of CD4 + CXCR5 + , CD4 + CXCR5 + ICOS + and CD4 + CXCR5 + IL-21 + Tfh cells in the PBMCs from HBV-ACLF, M-CHB, S-CHB and HC subjects were analyzed using Mann-Whitney U test. (C) The correlation between frequency of CD4 + CXCR5 + ICOS + Tfh cells and MELD score was analyzed using Spearman correlation analysis. (D) The frequency of CD4 + CXCR5 + ICOS + Tfh cells from ameliorated (n = 7) and non-ameliorated patients (n = 29) were analyzed by flow cytometry . (E) The frequencies of Tfh cells from ameliorated patients (n = 7) over 8- and 12-week treatment were analyzed by flow cytometry. Interclass comparison was made using Wilcoxon’s signed-rank test. * p

    Techniques Used: Flow Cytometry, MANN-WHITNEY

    Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p
    Figure Legend Snippet: Concentrations of serum cytokines were compared among groups. (A) The levels of cytokines (IL-12p70, IL-21, IL-17, TNF, IL-8, IL-6, IL-2, IL-4, IL-1β, IL-10, IFN-γ and TGFβ) in serum from HBV-ACLF (n = 36), S-CHB (n = 21), M-CHB (n = 32) patients and HC (n = 10) subjects were detected by ELISA. p values are tested for the significance of comparisons using Man-Whitney U test. (B–E) The correlations of IL-21 level with the frequency of CD4 + CXCR5 + ICOS + Tfh cells (B) , MELD score (C) , levels of ALT (D) and AST (E) in HBV-ACLF patients. r , the spearman rank order correlation coefficient. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, AST Assay

    CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: CD4 + CXCR5 + ICOS + Tfh cells were induced by cytokines or serum of HBV-ACLF patients. (A, D) The naïve CD4 + T cells of HC subjects (n = 6) were stimulated by the RPMI complete medium as control (CTR), dynabeads ® human T-activator CD3/CD28, IL-12 (10 ng/ml), IL-21 (20 ng/ml), IL-12+IL-21 and IL-17 (10 ng/ml) for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (B, E) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HC subjects (n = 6), and serum from HBV-ACLF patients for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. (C, F) The naïve CD4 + T cells of HC subjects were stimulated by CTR, dynabeads ® human T-activator CD3/CD28, serum from HBV-ACLF patients (1:8) with or without IL-12 (1 µg/ml), IL-21 (1 µg/ml), IL-12/21 and IL-17(1 µg/ml) antibody (the antibody experiments were performed in the presence of 1:8 HBV-ACLF serum), for 72 hours in vitro , and the frequencies of CD4 + CXCR5 + ICOS + Tfh cells were demonstrated by flow cytometry, respectively. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Flow Cytometry

    Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p
    Figure Legend Snippet: Tfh cells after stimulation of HBV-ACLF patients’ serum induced the proliferation and IgG production of B cells in vitro . (A, B) Naïve CD4 + T cells stimulated with the RPMI complete medium (CTR), with or without dynabeads ® human T-activator CD3/CD28 and HBV-ACLF patients’ serum, were cultured with naïve B cells in the presence of a surperantigen. The proliferation of CD19 + B cell in the CFSE dilution assay was evaluated quantitatively by comparing the percentages of cells that underwent cell division at least once. (C, D) IgG production (C) and IL-21 release (D) were dosed in the culture supernatants through ELISA. Representative data of independent experiments are shown as median (range). * p

    Techniques Used: In Vitro, Cell Culture, Dilution Assay, Enzyme-linked Immunosorbent Assay

    33) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    34) Product Images from "IRF4 haploinsufficiency in a family with Whipple’s disease"

    Article Title: IRF4 haploinsufficiency in a family with Whipple’s disease

    Journal: eLife

    doi: 10.7554/eLife.32340

    Ex vivo cytokine production by CD4+ memory T cells from patients and controls. Naive CD4 + T cells from healthy unrelated controls and patients ( P2 and P3 ) were stimulated with TAE beads alone or under Th1, Th2, Th17 or Tfh polarizing conditions. The production of IL-10, IL-21, IL-17A, IL-17F and IFN-γ was measured 5 days later, in the corresponding polarizing conditions. No significant differences were observed between healthy unrelated controls and patients.
    Figure Legend Snippet: Ex vivo cytokine production by CD4+ memory T cells from patients and controls. Naive CD4 + T cells from healthy unrelated controls and patients ( P2 and P3 ) were stimulated with TAE beads alone or under Th1, Th2, Th17 or Tfh polarizing conditions. The production of IL-10, IL-21, IL-17A, IL-17F and IFN-γ was measured 5 days later, in the corresponding polarizing conditions. No significant differences were observed between healthy unrelated controls and patients.

    Techniques Used: Ex Vivo

    35) Product Images from "Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception"

    Article Title: Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception

    Journal: eLife

    doi: 10.7554/eLife.53226

    Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.
    Figure Legend Snippet: Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency. ( A ) Absolute numbers of PD-1 subsets in spleens. ( B ) Frequency of IL-21 producers in spleens. ( C, D ) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Data are presented as the mean ± s.e.m. Figure 5A : Frequencies of PD-1 subpopulation. Data represent two independent experiments with n = 4 mice per organ. Figure 5B : Frequencies of IL-21 producers in spleens. Data represent two independent experiments with n = 4 mice per organ. Figure 5C : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ. Figure 5D : Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent two independent experiments with n = 4 mice per organ.

    Techniques Used: Functional Assay, Mouse Assay

    Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.
    Figure Legend Snippet: Functional comparison of CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi cells in B and T cell co-cultures. ( A ) Gating strategy used to sort CXCR3 + PD-1 lo Tsh, CXCR3 - PD-1 lo CD4 + T cells and PD-1 hi CXCR5 +/- . Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of two-years old C57Bl/6 mice. Lower row (black dot plots) shows purity and phenotype of the sorted populations. ( B ) Gating strategy to sort B220 + CD19 + B cells. Pseudo-color plots on the left-hand side show pre-sorted cells prepared from pooled splenocytes of 2-year-old C57Bl/6 mice and enriched for B cells by negative magnetic cell sort on Miltenyi column. Black dot plots on the right-hand side show B220 + CD19 + B cells purity after FACS-sort. ( C ) PMA/ionomycin-stimulated pre-sorted cells from pooled splenocytes of two-years old C57Bl/6 mice were assessed for CD40L, IL-21, and IFN-γ production. Pseudo-color plots show gating strategy for identification of CD4 T cell subsets and contour plots show cytokine production by the assessed populations. ( D ) Analysis of 5 days co-cultures by flow cytometry. Representative plots from co-culture wells with B cells and PD-1 hi cells (upper plots) and B cells and CXCR3 + PD-1 lo Tsh cells (lower plots) are shown. Data are representative of two independent experiments. Legends for figure source data.

    Techniques Used: Functional Assay, Mouse Assay, FACS, Flow Cytometry, Co-Culture Assay

    PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.
    Figure Legend Snippet: PRI results can be confirmed with viSNE and conventional analysis. ( A ) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 ( A, D ). ( B ) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the PD-1hi area and red circles the IFN-γ hi area. ( C ) Bin plots displaying PD-1 (x-axis) and IL-21 (y-axis) with cell density and MFI+ of IFN-γ, Bcl6, CXCR5 and ICOS. Cell frequencies per quadrant are calculated on the number of cells per sample (black), number of Z + cells per sample (green), and number of Z + cells per quadrant to all Z+ cells (blue). Grey bins contain less than 10 Z + cells. ( D ) FlowJo color map with PD-1 (x-axis), IFN-γ (y-axis) and MFI of IL-21 (Z parameter). ( E ) 3D heatmap plots showing MFI+ of Bcl6 (left) and IL-21 (right) as relief on PD-1 (x-axis) and IFN-γ (y-axis). Data are representative for at least two independent experiments with old diseased mice with ( A ) n = 3–11 mice and ( B–E ) n ≥ 3 mice. A, Data are presented as the mean ± s.e.m. Figure 6—figure supplement 1A : Frequencies of protein expressions sub-divided into regions. Data represent three independent experiments with n = 3–11 mice.

    Techniques Used: Mouse Assay

    The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .
    Figure Legend Snippet: The majority of IL-21 is produced by non-Tfh cells. ( A ) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. ( B, C ) PRI-based statistical analysis of marker co-expression in young and old mice. ( D ) Bin plots of PD-1 (x-axis) vs. CXCR5 (y-axis) with heatmap of frequency (top) and expression level (bottom) per bin of Tfh and B cell interaction proteins. Cell frequencies per quadrant are calculated on the number of cells per sample (black) and number of Z + cells per sample (green). Grey bins contain less than 10 Z + cells. ( D ) Data represent two experiments with n = 6 mice in total. ( B, C ) Samples were compared using the unpaired two-tailed t-test. Data are presented as the mean ± s.e.m. Figure 3—figure supplement 1A : Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines. Figure 3—figure supplement 1B, C : Frequencies from IL-21 + subpopulations extracted from PRI bin plots. Data as in Figure 3—source data 1 .

    Techniques Used: Produced, Marker, Expressing, Mouse Assay, Two Tailed Test

    36) Product Images from "The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation"

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02816

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.
    Figure Legend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Techniques Used: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Techniques Used: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.
    Figure Legend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Techniques Used: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Techniques Used: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay

    37) Product Images from "The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation"

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02816

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.
    Figure Legend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Techniques Used: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Techniques Used: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Figure Legend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Techniques Used: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.
    Figure Legend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Techniques Used: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p
    Figure Legend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Techniques Used: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay

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    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation
    Article Snippet: This observation was seen in both NK cell media but was even more pronounced for NK cells cultivated in NK MACS® medium. .. Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis. .. However, specific cell lysis was slightly higher after ex vivo culture in X-VIVOTM 10 medium compared to that in NK MACS® medium.

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    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation
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    Ex Vivo:

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation
    Article Snippet: .. Optimizing ex vivo NK Cell Activation Protocol for CD3/CD19-Depleted Cells by IL-21 As NK cells expanded with IL-15 after CD3/CD19-depletion showed the highest expansion rate and cytotoxic capacity against NB cell lines, we further aimed to optimize this protocol based on previous data indicating a further gain in NK cell cytotoxicity by a boost with IL-21 ( ). .. Therefore, the cytokine stimulation has been prolonged by the addition of IL-21 prior to harvesting.

    Activation Assay:

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation
    Article Snippet: .. Optimizing ex vivo NK Cell Activation Protocol for CD3/CD19-Depleted Cells by IL-21 As NK cells expanded with IL-15 after CD3/CD19-depletion showed the highest expansion rate and cytotoxic capacity against NB cell lines, we further aimed to optimize this protocol based on previous data indicating a further gain in NK cell cytotoxicity by a boost with IL-21 ( ). .. Therefore, the cytokine stimulation has been prolonged by the addition of IL-21 prior to harvesting.

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  • 99
    PeproTech recombinant il 4
    Adoptively transferred T reg cells lose Foxp3 expression and convert to Il4 -expressing cells after secondary infection with H. polygyrus . (A) Experimental model. T reg cells (HpTR; CD4 + TCRβ + Il4 GFP– Foxp3 RFP+ CD25 high ) were sort purified from male Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d. nT cells (CD4 + TCRβ + CD25 – CD44 low Il4 GFP– Foxp3 RFP– ) were sort purified from the spleen and MLN of naive male Il4 GFP Foxp3 RFP mice. HpTR or nT cells were transferred to male Tcra −/− mice further subjected to a secondary infection with H. polygyrus . (B) Representative FACS plots of Foxp3 RFP , Il4 GFP , and CD25 expression in HpTR cells before and after sort and day 42 after transfer. (C and D) Proportion of Foxp3 RFP+ CD25 high cells in HpTR recipients day 42 after transfer and relative to Foxp3 RFP expression before transfer (C) and proportion of CD4 + TCRβ + Il4 GFP+ Foxp3 RFP– cells in the spleen, MLN, and PPs of nT or HpTR cell recipients day 42 after transfer (D). Data are representative of at least five independent experiments with three to five mice per group. (E) qRT-PCR validation of Foxp3 and Il4 expression relative (rel.) to Hprt in sort-purified nT→ Il4 GFP+ , HpTR→ Il4 GFP+ , or HpTR cells. Data are representative of three experiments with cells pooled from three to five recipients. (F) Frequency of CD4 + CD44 high <t>IL-4</t> + cells in the MLN of nT and HpTR cell recipients as measured by intracellular cytokine staining. Data are representative of three independent experiments with four mice per group. CD4 + TCRβ + Foxp3 RFP+ CD25 high or CD25 low cells were sort purified from Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d and transferred to Tcra −/− mice subjected to a secondary infection with H. polygyrus . (G) Representative FACS plots of Foxp3 RFP and CD25 expression in donor HpTR cells presort. (H) Proportion of CD4 + TCRβ + Il4 GFP+ cells in the PPs of Hp 2° recipients day 42 after transfer. Data are representative of two independent experiments with four to five mice per group. Adopt., adoptively. Error bars represent SEM.
    Recombinant Il 4, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant il 4/product/PeproTech
    Average 99 stars, based on 1 article reviews
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    recombinant il 4 - by Bioz Stars, 2021-06
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    95
    PeproTech il 21
    Ex vivo expansion and characterization of <t>IL-15+IL-21</t> stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p
    Il 21, supplied by PeproTech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 21/product/PeproTech
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    il 21 - by Bioz Stars, 2021-06
    95/100 stars
      Buy from Supplier

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    Rabbit Biotinylated Anti Murine IL 21 Source Polyclonal Rabbit Formulation Lyophilized Produced from sera of rabbits pre immunized with highly pure 98 recombinant mIL 21 Murine Interleukin 21 Anti Murine
      Buy from Supplier

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    Rabbit Anti Human IL 21 Source Polyclonal Rabbit Formulation Lyophilized Produced from sera of rabbits pre immunized with highly pure 98 recombinant hIL 21 Anti Human IL 21 specific antibody
      Buy from Supplier

    Image Search Results


    Adoptively transferred T reg cells lose Foxp3 expression and convert to Il4 -expressing cells after secondary infection with H. polygyrus . (A) Experimental model. T reg cells (HpTR; CD4 + TCRβ + Il4 GFP– Foxp3 RFP+ CD25 high ) were sort purified from male Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d. nT cells (CD4 + TCRβ + CD25 – CD44 low Il4 GFP– Foxp3 RFP– ) were sort purified from the spleen and MLN of naive male Il4 GFP Foxp3 RFP mice. HpTR or nT cells were transferred to male Tcra −/− mice further subjected to a secondary infection with H. polygyrus . (B) Representative FACS plots of Foxp3 RFP , Il4 GFP , and CD25 expression in HpTR cells before and after sort and day 42 after transfer. (C and D) Proportion of Foxp3 RFP+ CD25 high cells in HpTR recipients day 42 after transfer and relative to Foxp3 RFP expression before transfer (C) and proportion of CD4 + TCRβ + Il4 GFP+ Foxp3 RFP– cells in the spleen, MLN, and PPs of nT or HpTR cell recipients day 42 after transfer (D). Data are representative of at least five independent experiments with three to five mice per group. (E) qRT-PCR validation of Foxp3 and Il4 expression relative (rel.) to Hprt in sort-purified nT→ Il4 GFP+ , HpTR→ Il4 GFP+ , or HpTR cells. Data are representative of three experiments with cells pooled from three to five recipients. (F) Frequency of CD4 + CD44 high IL-4 + cells in the MLN of nT and HpTR cell recipients as measured by intracellular cytokine staining. Data are representative of three independent experiments with four mice per group. CD4 + TCRβ + Foxp3 RFP+ CD25 high or CD25 low cells were sort purified from Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d and transferred to Tcra −/− mice subjected to a secondary infection with H. polygyrus . (G) Representative FACS plots of Foxp3 RFP and CD25 expression in donor HpTR cells presort. (H) Proportion of CD4 + TCRβ + Il4 GFP+ cells in the PPs of Hp 2° recipients day 42 after transfer. Data are representative of two independent experiments with four to five mice per group. Adopt., adoptively. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: Adoptively transferred T reg cells lose Foxp3 expression and convert to Il4 -expressing cells after secondary infection with H. polygyrus . (A) Experimental model. T reg cells (HpTR; CD4 + TCRβ + Il4 GFP– Foxp3 RFP+ CD25 high ) were sort purified from male Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d. nT cells (CD4 + TCRβ + CD25 – CD44 low Il4 GFP– Foxp3 RFP– ) were sort purified from the spleen and MLN of naive male Il4 GFP Foxp3 RFP mice. HpTR or nT cells were transferred to male Tcra −/− mice further subjected to a secondary infection with H. polygyrus . (B) Representative FACS plots of Foxp3 RFP , Il4 GFP , and CD25 expression in HpTR cells before and after sort and day 42 after transfer. (C and D) Proportion of Foxp3 RFP+ CD25 high cells in HpTR recipients day 42 after transfer and relative to Foxp3 RFP expression before transfer (C) and proportion of CD4 + TCRβ + Il4 GFP+ Foxp3 RFP– cells in the spleen, MLN, and PPs of nT or HpTR cell recipients day 42 after transfer (D). Data are representative of at least five independent experiments with three to five mice per group. (E) qRT-PCR validation of Foxp3 and Il4 expression relative (rel.) to Hprt in sort-purified nT→ Il4 GFP+ , HpTR→ Il4 GFP+ , or HpTR cells. Data are representative of three experiments with cells pooled from three to five recipients. (F) Frequency of CD4 + CD44 high IL-4 + cells in the MLN of nT and HpTR cell recipients as measured by intracellular cytokine staining. Data are representative of three independent experiments with four mice per group. CD4 + TCRβ + Foxp3 RFP+ CD25 high or CD25 low cells were sort purified from Il4 GFP Foxp3 RFP mice infected with H. polygyrus for 14 d and transferred to Tcra −/− mice subjected to a secondary infection with H. polygyrus . (G) Representative FACS plots of Foxp3 RFP and CD25 expression in donor HpTR cells presort. (H) Proportion of CD4 + TCRβ + Il4 GFP+ cells in the PPs of Hp 2° recipients day 42 after transfer. Data are representative of two independent experiments with four to five mice per group. Adopt., adoptively. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: Expressing, Infection, Purification, Mouse Assay, FACS, Quantitative RT-PCR, Staining

    Ex-Foxp3 Th2 cells secrete type-2 cytokines, promote AAMφ in vitro, and are sufficient to drive the expulsion of H. polygyrus . (A–J) T reg, Th2, and ex-Foxp3 Th2 cells were sort purified from Hp 1° and Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice and stimulated with PMA/ionomycin for 24 h. (A) Concentration of IL-4, IL-13, IL-5, and IL-2 in the supernatant of restimulated cells. Three technical replicates were used. (B) BMDMs were cultured with FACS-purified T cells for 24 h or with media and recombinant IL-4 + IL-13. (C) Expression of Arg1 and Retnla in stimulated BMDMs. Data are representative of two to three independent experiments with three technical replicates. Sort-purified T cells were pooled from three to four donor mice. rel., relative. (D) Th2 and ex-Foxp3 Th2 cells were sort purified from Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice at day 14 after infection and transferred to Hp 1°C57BL/6 recipients 2 d after infection. (E) Intestinal worm burden at day 21 after infection. Data represent two pooled experiments with 6–10 mice per group. (F) Experimental model (see model in Fig. 6 D ). Absolute number of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE– and CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleens is shown. Four mice per group were used. (G) Small intestine expression of key type-2 response genes, expressed relative (rel.) to Hprt . Four mice per group were used. (H and I) IgE (H)- and H. polygyrus (I)–specific IgG1 levels in the serum. Data represent two independent experiments with three to four mice per group. a.u., arbitrary units. (J) Intestinal worm count day 14 after infection. Data represent two independent experiments with five to seven mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: Ex-Foxp3 Th2 cells secrete type-2 cytokines, promote AAMφ in vitro, and are sufficient to drive the expulsion of H. polygyrus . (A–J) T reg, Th2, and ex-Foxp3 Th2 cells were sort purified from Hp 1° and Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice and stimulated with PMA/ionomycin for 24 h. (A) Concentration of IL-4, IL-13, IL-5, and IL-2 in the supernatant of restimulated cells. Three technical replicates were used. (B) BMDMs were cultured with FACS-purified T cells for 24 h or with media and recombinant IL-4 + IL-13. (C) Expression of Arg1 and Retnla in stimulated BMDMs. Data are representative of two to three independent experiments with three technical replicates. Sort-purified T cells were pooled from three to four donor mice. rel., relative. (D) Th2 and ex-Foxp3 Th2 cells were sort purified from Hp 2° Il4 GFP Foxp3 YFP/Cre R26R FP635 mice at day 14 after infection and transferred to Hp 1°C57BL/6 recipients 2 d after infection. (E) Intestinal worm burden at day 21 after infection. Data represent two pooled experiments with 6–10 mice per group. (F) Experimental model (see model in Fig. 6 D ). Absolute number of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE– and CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleens is shown. Four mice per group were used. (G) Small intestine expression of key type-2 response genes, expressed relative (rel.) to Hprt . Four mice per group were used. (H and I) IgE (H)- and H. polygyrus (I)–specific IgG1 levels in the serum. Data represent two independent experiments with three to four mice per group. a.u., arbitrary units. (J) Intestinal worm count day 14 after infection. Data represent two independent experiments with five to seven mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: In Vitro, Purification, Mouse Assay, Concentration Assay, Cell Culture, FACS, Recombinant, Expressing, Infection, MANN-WHITNEY

    IL-4 is sufficient to promote T reg to Th2 cell conversion in vitro. (A–G) HpTR cells and nT cells were sort purified from Il4 GFP Foxp3 RFP reporter mice as described in Fig. 2 . Sorted cells were stimulated with recombinant IL-4 at 37°C for 15 min or with media as a control. (A) Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells. Data are representative of three independent experiments. Sorted T cells were pooled from three to four mice. mW, molecular weight. (B and C) nT or HpTR cells were cultured with anti-CD3/CD28 and IL-2, with and without the addition of IL-4. Representative FACS plots (B) and graph (C) showing the frequency of CD4 + TCRβ + Foxp3 RFP+ and CD4 + TCRβ + Il4 GFP+ cells in day 7 cultures are shown. HpTR cells were cultured with anti-CD3/CD28 and IL-2, with increasing concentrations of IL-4, and cells were harvested for FACS or qRT-PCR at day 7. (D and E) Frequency of CD4 + TCRβ + Il4 GFP+ (D) and CD4 + TCRβ + Foxp3 RFP+ (E) cells in day 7 cultures. (F and G) Gene expression of Gata3 (relative [rel] to Hprt ; F) and mir182 (relative to rnu6b ; G) in cultured cells at day 7. Data are representative of two independent experiments. Cells were sort purified from four to six mice. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: IL-4 is sufficient to promote T reg to Th2 cell conversion in vitro. (A–G) HpTR cells and nT cells were sort purified from Il4 GFP Foxp3 RFP reporter mice as described in Fig. 2 . Sorted cells were stimulated with recombinant IL-4 at 37°C for 15 min or with media as a control. (A) Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells. Data are representative of three independent experiments. Sorted T cells were pooled from three to four mice. mW, molecular weight. (B and C) nT or HpTR cells were cultured with anti-CD3/CD28 and IL-2, with and without the addition of IL-4. Representative FACS plots (B) and graph (C) showing the frequency of CD4 + TCRβ + Foxp3 RFP+ and CD4 + TCRβ + Il4 GFP+ cells in day 7 cultures are shown. HpTR cells were cultured with anti-CD3/CD28 and IL-2, with increasing concentrations of IL-4, and cells were harvested for FACS or qRT-PCR at day 7. (D and E) Frequency of CD4 + TCRβ + Il4 GFP+ (D) and CD4 + TCRβ + Foxp3 RFP+ (E) cells in day 7 cultures. (F and G) Gene expression of Gata3 (relative [rel] to Hprt ; F) and mir182 (relative to rnu6b ; G) in cultured cells at day 7. Data are representative of two independent experiments. Cells were sort purified from four to six mice. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: In Vitro, Purification, Mouse Assay, Recombinant, Molecular Weight, Cell Culture, FACS, Quantitative RT-PCR, Expressing, MANN-WHITNEY

    IL-4 signaling in T reg cells is required for the development of ex-Foxp3 Th2 cells in vivo. (A and B) nT, Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt HpTR cells were sort purified from naive (A) or Hp 1° (B) Il4ra wt/wt or Il4ra fl/fl fate-reporter mice day 14 after infection and stimulated with recombinant IL-4 at 37°C for 15 min or media as a control. Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells are shown. Data are representative of two independent experiments. Sorted T cells were pooled from two to three mice. mW, molecular weight. (C) Proportion and absolute number of Foxp3 YFP+ cells in the spleen of naive Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt fate-reporter mice. Data are representative of two independent experiments. (D) Experimental model. Il4 GFP Foxp3 YFP/Cre R26R FP635 Il4ra fl/fl mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on day 14–15 and reinfected on day 28. Mice were harvested at day 7 after infection. (E) Representative FACS plots of Foxp3 FATE expression within CD4 + TCRβ + Il4 GFP+ cells in Il4ra fl/fl or Il4ra wt/wt Hp 2° mice day 7 after infection. (F and G) Proportion (F) and absolute number (G) of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleen. Data represent two independent experiments with three to four mice per group. (H) Experimental model. T reg cells were sort purified from Il4ra wt/wt or Il4ra fl/fl Hp 1° mice day 14 after infection. Il4ra wt/wt or Il4ra fl/fl T reg cells were transferred to Tcra −/− mice. Recipient mice were infected with H. polygyrus , treated with pyrantel embonate at days 14–15, and then infected with H. polygyrus at day 35 and harvested at day 42 after transfer (see experimental model in Fig. 2 A ). (I and J) Representative FACS plots of CD4 + TCRβ + Il4 GFP+ cells (I) and frequency of Il4 GFP+ cells (J) in the spleen and MLN of Il4ra fl/fl or Il4ra wt/wt HpTR-recipient mice day 14 after infection. Data are representative of two experiments with three to five mice per group. Adopt., adoptively. (K) Experimental model. In brief, Il4ra fl/fl or Il4ra wt/wt mice were subjected to a model of HDM/alum sensitization. i.t., intratracheally. (L) Frequency of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in mediastinal LNs (medLN) and lungs 24 h after challenge. (M) Proportion of eosinophils in the BAL fluid. Data represent two independent experiments with four to nine mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths

    doi: 10.1084/jem.20161104

    Figure Lengend Snippet: IL-4 signaling in T reg cells is required for the development of ex-Foxp3 Th2 cells in vivo. (A and B) nT, Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt HpTR cells were sort purified from naive (A) or Hp 1° (B) Il4ra wt/wt or Il4ra fl/fl fate-reporter mice day 14 after infection and stimulated with recombinant IL-4 at 37°C for 15 min or media as a control. Levels of pSTAT6, total STAT6, and α-tubulin protein in restimulated cells are shown. Data are representative of two independent experiments. Sorted T cells were pooled from two to three mice. mW, molecular weight. (C) Proportion and absolute number of Foxp3 YFP+ cells in the spleen of naive Il4ra fl/fl , Il4ra fl/wt , or Il4ra wt/wt fate-reporter mice. Data are representative of two independent experiments. (D) Experimental model. Il4 GFP Foxp3 YFP/Cre R26R FP635 Il4ra fl/fl mice were infected with 200 H. polygyrus larvae. Hp 2° mice were treated with pyrantel embonate on day 14–15 and reinfected on day 28. Mice were harvested at day 7 after infection. (E) Representative FACS plots of Foxp3 FATE expression within CD4 + TCRβ + Il4 GFP+ cells in Il4ra fl/fl or Il4ra wt/wt Hp 2° mice day 7 after infection. (F and G) Proportion (F) and absolute number (G) of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in the spleen. Data represent two independent experiments with three to four mice per group. (H) Experimental model. T reg cells were sort purified from Il4ra wt/wt or Il4ra fl/fl Hp 1° mice day 14 after infection. Il4ra wt/wt or Il4ra fl/fl T reg cells were transferred to Tcra −/− mice. Recipient mice were infected with H. polygyrus , treated with pyrantel embonate at days 14–15, and then infected with H. polygyrus at day 35 and harvested at day 42 after transfer (see experimental model in Fig. 2 A ). (I and J) Representative FACS plots of CD4 + TCRβ + Il4 GFP+ cells (I) and frequency of Il4 GFP+ cells (J) in the spleen and MLN of Il4ra fl/fl or Il4ra wt/wt HpTR-recipient mice day 14 after infection. Data are representative of two experiments with three to five mice per group. Adopt., adoptively. (K) Experimental model. In brief, Il4ra fl/fl or Il4ra wt/wt mice were subjected to a model of HDM/alum sensitization. i.t., intratracheally. (L) Frequency of CD4 + TCRβ + Il4 GFP+ Foxp3 FATE+ cells in mediastinal LNs (medLN) and lungs 24 h after challenge. (M) Proportion of eosinophils in the BAL fluid. Data represent two independent experiments with four to nine mice per group. *, P ≤ 0.05; Mann-Whitney test. Error bars represent SEM.

    Article Snippet: 106 BMDMs were plated in 24-well flat-bottom tissue culture–treated plates and left to rest for 24 h. 105 sort-purified T cells were resuspended in 1% DMEM containing 1 µg/ml soluble CD3 and 10 µg/ml soluble CD28 antibody (Bio X Cell) and cultured with the BMDMs for 24 h. As a control, BMDMs were co-cultured in the presence of 20 ng/ml recombinant IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) or media alone.

    Techniques: In Vivo, Purification, Mouse Assay, Infection, Recombinant, Molecular Weight, FACS, Expressing, MANN-WHITNEY

    Stat6 is activated in the absence of IRS-2. (A) Whole-cell extracts were prepared from enriched T lymphocytes cultured in the presence or absence (−) of IL-4. Activated Stat6 was detected by immunoblotting using an antibody specific for phosphorylated Stat6 (pStat6). The blot was stripped and reprobed with a total Stat6 antibody. The results are representative of three independent experiments. (B) Total splenocytes were cultured overnight in the presence (bold line) or absence (thin line) of IL-4. IL-4Rα expression on B220 − cells and major histocompatibility complex class II expression on B220 + cells were determined by FACS.

    Journal: Molecular and Cellular Biology

    Article Title: Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

    doi: 10.1128/MCB.22.1.117-126.2002

    Figure Lengend Snippet: Stat6 is activated in the absence of IRS-2. (A) Whole-cell extracts were prepared from enriched T lymphocytes cultured in the presence or absence (−) of IL-4. Activated Stat6 was detected by immunoblotting using an antibody specific for phosphorylated Stat6 (pStat6). The blot was stripped and reprobed with a total Stat6 antibody. The results are representative of three independent experiments. (B) Total splenocytes were cultured overnight in the presence (bold line) or absence (thin line) of IL-4. IL-4Rα expression on B220 − cells and major histocompatibility complex class II expression on B220 + cells were determined by FACS.

    Article Snippet: Recombinant IL-4 was obtained from Peprotech.

    Techniques: Cell Culture, Expressing, FACS

    IRS-2 and Stat6 are dispensable for the antiapoptotic activities of IL-4. (A) CD4 + /CD62L-high T cells were cultured for 24 to 30 h in the presence or absence of IL-4. The percentage of apoptotic cells in the resulting cultures was determined by propidium iodide staining. Percent rescued from apoptosis compares the percentage of apoptotic cells in the IL-4-treated cultures with the percentage of apoptotic cells in untreated cultures and is presented as the average of four independent experiments. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

    doi: 10.1128/MCB.22.1.117-126.2002

    Figure Lengend Snippet: IRS-2 and Stat6 are dispensable for the antiapoptotic activities of IL-4. (A) CD4 + /CD62L-high T cells were cultured for 24 to 30 h in the presence or absence of IL-4. The percentage of apoptotic cells in the resulting cultures was determined by propidium iodide staining. Percent rescued from apoptosis compares the percentage of apoptotic cells in the IL-4-treated cultures with the percentage of apoptotic cells in untreated cultures and is presented as the average of four independent experiments. *, P

    Article Snippet: Recombinant IL-4 was obtained from Peprotech.

    Techniques: Cell Culture, Staining

    IL-4-induced PI-3 kinase activity is reduced in IRS-2-deficient lymphocytes. Supernatants of homogenates from IL-4-treated and untreated splenocytes were immunoprecipitated with the indicated antibodies and assayed in vitro for PI-3 kinase activity. The results are expressed as fold stimulation of activity above untreated controls and are the average (+ standard deviation) of two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

    doi: 10.1128/MCB.22.1.117-126.2002

    Figure Lengend Snippet: IL-4-induced PI-3 kinase activity is reduced in IRS-2-deficient lymphocytes. Supernatants of homogenates from IL-4-treated and untreated splenocytes were immunoprecipitated with the indicated antibodies and assayed in vitro for PI-3 kinase activity. The results are expressed as fold stimulation of activity above untreated controls and are the average (+ standard deviation) of two independent experiments.

    Article Snippet: Recombinant IL-4 was obtained from Peprotech.

    Techniques: Activity Assay, Immunoprecipitation, In Vitro, Standard Deviation

    Both IRS-2 and Stat6 contribute to IL-4-induced proliferative responses. (A) FACS-purified CD4 + /CD62L-high T cells were cultured in submitogenic PMA (2 ng/ml) supplemented with increasing doses of IL-4 (open symbols) or with plate-bound anti-CD3 and anti-CD28 (solid symbols). The proliferative response was determined by [ 3 H]thymidine incorporation from a 48-h culture. The error bars indicate standard deviation. (B) Enriched T lymphocytes were cultured for 48 h in the presence of submitogenic PMA and 10 ng of IL-4/ml or 50 ng of PMA/ml and 500 ng of ionomycin/ml (P+I). p27kip1 (p27) protein levels were detected in whole-cell extracts by immunoblotting. The blot was stripped and reprobed with antibody specific for cdk2. Unt, untreated. (C) Enriched T lymphocytes were cultured in the presence or absence of submitogenic PMA (2 ng/ml) and 10 ng of IL-4/ml for 1 h. RNA was prepared, and c- myc expression was determined by Northern blot analysis. The blot was stripped and reprobed with γ-actin.

    Journal: Molecular and Cellular Biology

    Article Title: Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis

    doi: 10.1128/MCB.22.1.117-126.2002

    Figure Lengend Snippet: Both IRS-2 and Stat6 contribute to IL-4-induced proliferative responses. (A) FACS-purified CD4 + /CD62L-high T cells were cultured in submitogenic PMA (2 ng/ml) supplemented with increasing doses of IL-4 (open symbols) or with plate-bound anti-CD3 and anti-CD28 (solid symbols). The proliferative response was determined by [ 3 H]thymidine incorporation from a 48-h culture. The error bars indicate standard deviation. (B) Enriched T lymphocytes were cultured for 48 h in the presence of submitogenic PMA and 10 ng of IL-4/ml or 50 ng of PMA/ml and 500 ng of ionomycin/ml (P+I). p27kip1 (p27) protein levels were detected in whole-cell extracts by immunoblotting. The blot was stripped and reprobed with antibody specific for cdk2. Unt, untreated. (C) Enriched T lymphocytes were cultured in the presence or absence of submitogenic PMA (2 ng/ml) and 10 ng of IL-4/ml for 1 h. RNA was prepared, and c- myc expression was determined by Northern blot analysis. The blot was stripped and reprobed with γ-actin.

    Article Snippet: Recombinant IL-4 was obtained from Peprotech.

    Techniques: FACS, Purification, Cell Culture, Standard Deviation, Expressing, Northern Blot

    IL-4 induces IL-12 and inhibits IL-10 secretion by conventional dendritic cells stimulated with C. neoformans . Conventional BMDCs were generated from bone marrow cells by incubation for 8 to 10 days in the presence of GM-CSF. After harvesting, the cells were stimulated with C. neoformans 1841 (MOI 10) in the presence or absence of IL-4 (25 U/ml) for 48 h in duplicates. Supernatants were collected and analyzed for the production of IL-12p70 (n = 3) and IL-10 (n = 4) by sandwich ELISA. The mean of the duplicates without IL-4 was set at 100% and the percentage of each value was calculated in relation to this mean. In a representative experiment 199.5 m±15.5 pg/ml IL-12p70 and 857.5±7.5 pg/ml IL-10 vs. 391.5±2.5 pg/ml IL-12p70 and 696±21 pg/ml IL-10 (mean ± SD) were produced after stimulation with C. neoformans in the absence or presence of IL-4, respectively. When incubating cells only in the presence of IL-4 neither IL-12p70 nor IL-10 was detectable (not shown). Data are expressed as the mean ± S.E.M. Statistical analysis was performed by using the unpaired Student's t-test. *** P

    Journal: PLoS ONE

    Article Title: IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

    doi: 10.1371/journal.pone.0087341

    Figure Lengend Snippet: IL-4 induces IL-12 and inhibits IL-10 secretion by conventional dendritic cells stimulated with C. neoformans . Conventional BMDCs were generated from bone marrow cells by incubation for 8 to 10 days in the presence of GM-CSF. After harvesting, the cells were stimulated with C. neoformans 1841 (MOI 10) in the presence or absence of IL-4 (25 U/ml) for 48 h in duplicates. Supernatants were collected and analyzed for the production of IL-12p70 (n = 3) and IL-10 (n = 4) by sandwich ELISA. The mean of the duplicates without IL-4 was set at 100% and the percentage of each value was calculated in relation to this mean. In a representative experiment 199.5 m±15.5 pg/ml IL-12p70 and 857.5±7.5 pg/ml IL-10 vs. 391.5±2.5 pg/ml IL-12p70 and 696±21 pg/ml IL-10 (mean ± SD) were produced after stimulation with C. neoformans in the absence or presence of IL-4, respectively. When incubating cells only in the presence of IL-4 neither IL-12p70 nor IL-10 was detectable (not shown). Data are expressed as the mean ± S.E.M. Statistical analysis was performed by using the unpaired Student's t-test. *** P

    Article Snippet: After harvesting, the cells were adjusted to 5×105 /ml and stimulated with C. neoformans , strain 1841 in the presence or absence of 25 U/ml recombinant IL-4 (Peprotech, Hamburg, Germany).

    Techniques: Generated, Incubation, Sandwich ELISA, Produced

    Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Journal: Frontiers in Immunology

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    doi: 10.3389/fimmu.2019.02816

    Figure Lengend Snippet: Ex vivo expansion and characterization of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells cultured in optimized NK cell medium. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with solely IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. IL-21 was added as a cytokine boost 2 days prior to harvest. NK cells were cultured in X-VIVO TM 10 medium ( ) or NK MACS® medium ( ). (B) NK cells in X-VIVO TM 10 medium expanded 13.2-fold (IL-15 low ) and 9.6-fold (IL-15 low + IL-21 ). Expansion rates were significantly higher in NK MACS® medium at 26.4-fold (IL-15 low ) and 24.4-fold (IL-15 low +IL-21 ). (C) All cell products showed a high viability with a median 96.5% following the purification procedure on day 0 (white symbols gray background) and remained > 90% during the expansion procedure, independent of the cytokine additive. Ex vivo cultivation in NK MACS® medium even led to viability > 96%. (D) Purified CD3/CD19-depleted cells on day 0 contained a median 54.4% NK cells. Upon cytokine stimulation for 15 days NK cell purity significantly increased in CD3/CD19-depleted cell products to > 95% in all protocols. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation. In total, 42.7% (IL-15 low ) and 42.6% (IL-15 low + IL-21 ) of NK cells were CD16 − after cultivation in X-VIVO TM 10 medium. Percentages of CD16 − cells were significantly higher after cultivation in NK MACS® medium: 69.5% (IL-15 low ) and 71.6% (IL-15 low + IL-21 ) ( n = 6 independent experiments, (B) median fold expansion rate day 15 compared to day 0, gated on: (C) viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Article Snippet: Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis.

    Techniques: Ex Vivo, Cell Culture, Purification, Magnetic Cell Separation, Whisker Assay

    Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Journal: Frontiers in Immunology

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    doi: 10.3389/fimmu.2019.02816

    Figure Lengend Snippet: Phenotype analyses of NK cells and CD16 − and CD16 + subpopulations. (A) Expression of various surface markers on NK cells including CD16 − (B) and CD16 + (C) NK cell subpopulations on the day of harvest. No significant differences between stimulation with IL-15 solely ( ) or in combination with IL-21 ( ) could be seen. The activating receptors NKp44 and NKG2D and the activation marker CD69 showed higher expression on NK cells cultured in X-VIVO TM 10 medium ( ) compared to ones cultured in NK MACS® medium ( ). While the CD16 + NK cell population expressed higher levels of maturation marker CD57, the inhibitory receptor NKG2A and the death receptor FASL, CD16 − NK cells expressed the activating receptors NKp44 and NKp46 as well as the α-chain of the IL-2/IL-15 receptor CD25 to a higher extend (statistically not relevant differences). n = 4, independent results, median fluorescence intensity (MFI), bar graphs show median and interquartile range, gated on viable 7-AAD − NK cells using FMO (fluorescence minus one) controls for each antigen.

    Article Snippet: Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis.

    Techniques: Expressing, Activation Assay, Marker, Cell Culture, Magnetic Cell Separation, Fluorescence

    CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Journal: Frontiers in Immunology

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    doi: 10.3389/fimmu.2019.02816

    Figure Lengend Snippet: CD107a degranulation and IFN-γ production. (A) Intracellular IFN-γ expression and degranulation potential indicated by CD107a expression of cytokine stimulated NK cells cultured in X-VIVO™10 or NK MACS® media was assessed after 15 days of cultivation with the IL-15 low or IL-15 low +IL-21 stimulation protocol. Cells were either co-incubated with SK-N-AS NB target cells (E:T ratio 1:1) or stimulated with IL-12+IL-18 mimicking stimulation by dendritic cells. After the total incubation time of 4 h, cells were stained and measured by flow cytometry. IFN-γ and CD107a expression was compared to unstimulated cells in each cultivation setting used as negative control. Both NK cell subsets produced IFN-γ upon cytokine stimulation and target cell co-incubation, with higher levels after the cytokine stimulus, which was statistically significant for the CD16 − subset. Similar effects were seen in both media, except CD16 − NK cells grown in X-VIVO™10 produced significantly more IFN-γ upon IL-12+IL-18 cytokine stimulation. Target cell co-incubation and cytokine stimulation led to a high CD107a expression in both NK cell subpopulations, especially within the CD16 − NK cell population. Only small differences were seen between both cell culture media. Throughout all experiments, the additional IL-21 boost during NK cell cultivation enhanced IFN-γ and CD107a expression, which was even statistically significant in n = 2 settings (light gray vs. dark gray bars). Summary data show mean and SEM percentage of CD107a + and IFN-γ + NK cells ( n = 4 independent results). (B) FACS plots show IFN-γ and CD107a expression in both CD16 − and CD16 + NK cell subpopulations of IL-15 low +IL-21 NK cells grown 15 days in NK MACS® media. This stimulation protocol led to an outgrowth of the CD16 − NK cell subpopulation resulting in an inverse CD16 − /CD16 + distribution. The short-term stimulus of IL-12+IL-15 and target cell co-incubation, demonstrated that both NK cell subpopulations are capable of IFN-γ production and CD107a expression. Thereby CD107a expression was higher present on CD16 − NK cells, while IFN-γ was produced equally by both subsets. FACS plots gated on viable Zombie Violet − CD3 − CD56 + NK cells (density plots show one representative result from n = 4 independent experiments). Differences were considered significant for p

    Article Snippet: Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis.

    Techniques: Expressing, Cell Culture, Magnetic Cell Separation, Incubation, Staining, Flow Cytometry, Cytometry, Negative Control, Produced, FACS

    Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Journal: Frontiers in Immunology

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    doi: 10.3389/fimmu.2019.02816

    Figure Lengend Snippet: Ex vivo expansion and characterization of IL-15 and IL-15+IL-21 stimulated NK cells following CD3/CD19-depletion. (A) NK cells were purified by CD3/CD19-depletion and ex vivo stimulated with IL-15 ( ) or with a combination of IL-15 and IL-21 ( ) for 15 days. On day 11, cells were either treated with IL-15 (IL-15 low ) or the supernatant was removed and no cytokines were added (IL-15 gap ). IL-21 was added as a cytokine boost 48 h prior to harvest. (B) NK cells in the IL-15 low protocol ) expanded 6.8-fold. An IL-21 boost was able to further enhance proliferation, irrespective of gap or continuous treatment. Expansion rates reached 7.1-fold in the IL-15 gap + IL-21 protocol ( ) and 16.5-fold in the IL-15 low + IL-21 protocol ( ) (statistically not significant differences). (C) All cell products showed a high viability of median 97.5% following the purification procedure on day 0 (white symbols gray background) and remained > 80% during the expansion procedure independent of the cytokine additive. However, the gap treatment led to the lowest viability ( ). (D) Purified CD3/CD19-depleted cells on day 0 contained a median 53.1% NK cells. Upon cytokine stimulation for 15 days, NK cell purity significantly increased in CD3/CD19-depleted cell products regardless of the cytokine combination. (E) The frequency of the CD16 − NK cell subpopulation significantly increased during ex vivo stimulation within all protocols ( n = 5–6 independent experiments, (B) median fold expansion rate on day 15 compared to day 0, (C) gated on viable 7-AAD − CD45 + cells, (D) CD56 + CD3 − NK cells, (E) CD16 − NK cells. Box-and-whisker plots show median, 25th−75th percentiles, Min-Max. Bar graphs show median and interquartile range. Differences were considered significant for p

    Article Snippet: Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis.

    Techniques: Ex Vivo, Purification, Whisker Assay

    Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Journal: Frontiers in Immunology

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    doi: 10.3389/fimmu.2019.02816

    Figure Lengend Snippet: Cytotoxic potential of IL-15+IL-21 stimulated CD3/CD19-depleted NK cells against NB target cells. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol ( ) was 69. Five percent for SK-N-SH and 53.5% for SK-N-AS cells (E:T ratio 10:1). An additional IL-21 boost elevated cytotoxic activity of IL15-stimulated NK cells to a median cell lysis of 71.5% (IL-15 low + IL-21 ) and 77.6% (IL-15 gap +IL-21 ) for SK-N-SH and 54.4% (IL-15 low +IL-21) and 63.3% (IL-15 gap +IL-21) for SK-N-AS cells (all E:T ratio 10:1) (statistically not significant differences). Effector to target (E:T) ratios 10:1, 5:1, 1:1, and 0.5:1, n = 5–6 independent results, experiments performed in triplicate, incubation time: 3 h, box-and-whisker plots show median, 25th−75th percentiles, Min-Max.

    Article Snippet: Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis.

    Techniques: Lysis, Release Assay, Activity Assay, Incubation, Whisker Assay

    Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Journal: Frontiers in Immunology

    Article Title: The Synergistic Use of IL-15 and IL-21 for the Generation of NK Cells From CD3/CD19-Depleted Grafts Improves Their ex vivo Expansion and Cytotoxic Potential Against Neuroblastoma: Perspective for Optimized Immunotherapy Post Haploidentical Stem Cell Transplantation

    doi: 10.3389/fimmu.2019.02816

    Figure Lengend Snippet: Cytotoxic potential and long-term cytotoxicity after optimization of NK cell cultivation. Specific lysis of the NB cell lines SK-N-SH (A) and SK-N-AS (B) was evaluated by Europium release assay. Both NB cell lines were efficiently lysed by CD3/CD19-depleted NK cells. Median target cell lysis of NK cells treated in the IL-15 low protocol in X-VIVO TM 10 ( ) was 73.6% for SK-N-SH and 57.4% for SK-N-AS. An IL-21 boost ( ) significantly elevated the cytotoxic activity of IL15-stimulated NK cells in X-VIVO TM 10 medium to a median cell lysis of 94.92% for SK-N-SH and 68.09% for SK-N-AS cells. IL-21 also significantly increased target cell lysis in NK MACS® medium. Cultivation in NK MACS® medium resulted in slightly lower cytotoxic activity with median cell lysis of 70.3% (IL-15 low ) and 80.1% (IL-15 low + IL-21 ) against SK-N-SH and 50.7 and 57.8% against SK-N-AS (all E:T ratio 10:1). E:T ratios 10:1, 5:1, 1:1, and 0.5:1, n = 6 independent results, experiments performed in triplicate, incubation time: 3 hours, box-and-whisker plots show median, 25th−75th percentiles, Min-Max. (C) Tumor spheroids were produced from 10,000 SK-N-AS cells and co-incubated with 200,000 NK cells. As a control the dynamics of tumor spheroids without effector cells were observed in both cell culture media. The cultures were imaged via a Celigo cell cytometer after 6 h, 24 h, 3, 5, 8 and up to 10 days. IL-15 low +IL-21 stimulated NK cells grown in both cell culture media were able to completely eradicate tumor spheroids in this 10 day long-term cytotoxicity assay ( n = 1 representative of 3 independent experiments). Differences were considered significant for p

    Article Snippet: Here, even at an E:T ratio of 0.5:1, the addition of IL-21 led to an significant increase in target cell lysis.

    Techniques: Lysis, Release Assay, Activity Assay, Magnetic Cell Separation, Incubation, Whisker Assay, Produced, Cell Culture, Cytometry, Cytotoxicity Assay