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The reduction of IL-5 in 4μ8c treated cells is not due to changes in mRNA levels or stability. D10 cells were treated as in Fig. 1 . a RNA was converted to cDNA and then amplified via qRT-PCR. The results show the relative fold change to the no stimulated sample. The data is an average of six experiments for the PMA and <t>ionomycin</t> stimulated samples (black bars) and five for the plate-bound stimulated ones (white bars). The standard error is graphed. b D10 cells were rested and then stimulated in the presence of 4μ8c for 24 h. The cells were then treated with actinomycin D and harvested at times 0, 10, 30, 60, and 90 min after treatment. RNA was isolated and qRT-PCR performed. The samples were normalized to the time zero point of actinomycin D treatment. The data were graphed on a semi-log scale and are the average of four experiments. The error bars represent the standard error of the mean. c Protein was isolated from cells treated as in A and immunoblotted with GATA-3 and β-actin antibody. The data is representative of three experiments
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1) Product Images from "Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion"

Article Title: Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion

Journal: BMC Immunology

doi: 10.1186/s12865-018-0283-7

The reduction of IL-5 in 4μ8c treated cells is not due to changes in mRNA levels or stability. D10 cells were treated as in Fig. 1 . a RNA was converted to cDNA and then amplified via qRT-PCR. The results show the relative fold change to the no stimulated sample. The data is an average of six experiments for the PMA and ionomycin stimulated samples (black bars) and five for the plate-bound stimulated ones (white bars). The standard error is graphed. b D10 cells were rested and then stimulated in the presence of 4μ8c for 24 h. The cells were then treated with actinomycin D and harvested at times 0, 10, 30, 60, and 90 min after treatment. RNA was isolated and qRT-PCR performed. The samples were normalized to the time zero point of actinomycin D treatment. The data were graphed on a semi-log scale and are the average of four experiments. The error bars represent the standard error of the mean. c Protein was isolated from cells treated as in A and immunoblotted with GATA-3 and β-actin antibody. The data is representative of three experiments
Figure Legend Snippet: The reduction of IL-5 in 4μ8c treated cells is not due to changes in mRNA levels or stability. D10 cells were treated as in Fig. 1 . a RNA was converted to cDNA and then amplified via qRT-PCR. The results show the relative fold change to the no stimulated sample. The data is an average of six experiments for the PMA and ionomycin stimulated samples (black bars) and five for the plate-bound stimulated ones (white bars). The standard error is graphed. b D10 cells were rested and then stimulated in the presence of 4μ8c for 24 h. The cells were then treated with actinomycin D and harvested at times 0, 10, 30, 60, and 90 min after treatment. RNA was isolated and qRT-PCR performed. The samples were normalized to the time zero point of actinomycin D treatment. The data were graphed on a semi-log scale and are the average of four experiments. The error bars represent the standard error of the mean. c Protein was isolated from cells treated as in A and immunoblotted with GATA-3 and β-actin antibody. The data is representative of three experiments

Techniques Used: Amplification, Quantitative RT-PCR, Isolation

IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value
Figure Legend Snippet: IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

2) Product Images from "Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation"

Article Title: Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005066

PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 μM Ionomycin, or DMSO for 6 hours. Induction of HIV transcription was measured using RT-qPCR for the 5’ LTR region or Poly A region of the virus. **, p
Figure Legend Snippet: PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 μM Ionomycin, or DMSO for 6 hours. Induction of HIV transcription was measured using RT-qPCR for the 5’ LTR region or Poly A region of the virus. **, p

Techniques Used: Infection, Isolation, Quantitative RT-PCR

3) Product Images from "PTEN drives Th17 cell differentiation by preventing IL-2 production"

Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20170523

γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P
Figure Legend Snippet: γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P

Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry

4) Product Images from "Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion"

Article Title: Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion

Journal: BMC Immunology

doi: 10.1186/s12865-018-0283-7

IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value
Figure Legend Snippet: IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

5) Product Images from "Autocrine type I IFN signaling in dendritic cells stimulated with fungal β-glucans or lipopolysaccharide promotes CD8 T cell activation"

Article Title: Autocrine type I IFN signaling in dendritic cells stimulated with fungal β-glucans or lipopolysaccharide promotes CD8 T cell activation

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601143

Autocrine type I IFN signaling also regulates DC maturation and CD8 T cell activation following LPS stimulation (A-B) Wild-type and IFNAR1-deficient DCs were stimulated with 100 ng/ml LPS for 24 hours. Surface expression of MHC I-OVA and costimulatory molecules was assessed by flow cytometry (A). For MHC I-OVA measurement, DC cultures were supplemented with 5.5 μM OVA peptide (SIINFEKL) 1 hour prior to LPS stimulation. Cytokine levels in culture supernatants were assessed by ELISA (all except type I IFN) or a luciferase reporter assay (type I IFN) (B; mean plus standard deviation of triplicate culture). (C-E) Wild-type and IFNAR1-deficient DCs were loaded with 1.1 nM OVA peptide (SIINFEKL) and stimulated as above prior to co-culture with CFSE-labeled OT-I CD8 T cells for 3 days. Proliferation was assessed by flow cytometry (C). T cells were also re-stimulated with PMA + ionomycin (6 hour stimulation, with protein export inhibitors for the final 4 hours) to assess IFN-γ and IL-2 production by ELISA (D). Granzyme B production following PMA + ionomycin stimulation was assessed by intracellular flow cytometry; % granzyme B-producing T cells (gated on CD8 + cells) are indicated (E). Data are mean plus standard error of 4-9 independent experiments (A, E), or representative of at least 3 independent experiments (B-D). *p
Figure Legend Snippet: Autocrine type I IFN signaling also regulates DC maturation and CD8 T cell activation following LPS stimulation (A-B) Wild-type and IFNAR1-deficient DCs were stimulated with 100 ng/ml LPS for 24 hours. Surface expression of MHC I-OVA and costimulatory molecules was assessed by flow cytometry (A). For MHC I-OVA measurement, DC cultures were supplemented with 5.5 μM OVA peptide (SIINFEKL) 1 hour prior to LPS stimulation. Cytokine levels in culture supernatants were assessed by ELISA (all except type I IFN) or a luciferase reporter assay (type I IFN) (B; mean plus standard deviation of triplicate culture). (C-E) Wild-type and IFNAR1-deficient DCs were loaded with 1.1 nM OVA peptide (SIINFEKL) and stimulated as above prior to co-culture with CFSE-labeled OT-I CD8 T cells for 3 days. Proliferation was assessed by flow cytometry (C). T cells were also re-stimulated with PMA + ionomycin (6 hour stimulation, with protein export inhibitors for the final 4 hours) to assess IFN-γ and IL-2 production by ELISA (D). Granzyme B production following PMA + ionomycin stimulation was assessed by intracellular flow cytometry; % granzyme B-producing T cells (gated on CD8 + cells) are indicated (E). Data are mean plus standard error of 4-9 independent experiments (A, E), or representative of at least 3 independent experiments (B-D). *p

Techniques Used: Activation Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Standard Deviation, Co-Culture Assay, Labeling

Autocrine type I IFN signaling promotes DC maturation following β-glucan stimulation (A-B) Wild-type and IFNAR1-deficient DCs were stimulated with 100 μg/ml fungal β-glucan particles for 24 hours. Surface expression of MHC I-OVA and co-stimulatory molecules was assessed by flow cytometry (A). For MHC I-OVA measurement, DC cultures were supplemented with 5.5 μM OVA peptide (SIINFEKL) 1 hour prior to β-glucan stimulation. Cytokine levels in culture supernatants were assessed by ELISA (all except type I IFN) or a luciferase reporter assay (type I IFN) (B; mean plus standard deviation of triplicate culture). (C-E) Wild-type DCs were loaded with 1.1 nM OVA peptide (SIINFEKL) and stimulated as above prior to co-culture with naïve CFSE-labeled OT-I CD8 T cells for 3 days in the presence of anti-CD86 or isotype control antibodies (200 μg/ml). Proliferation (CFSE dilution) was assessed by flow cytometry (C). T cells were also re-stimulated with PMA + ionomycin (6 hour stimulation, with protein export inhibitors for the final 4 hours) to assess IFN-γ (D) and granzyme B production (E) by intracellular flow cytometry. % IFN-γ/granzyme B-producing T cells (gated on CD8 + cells) and IFN-γ/granzyme B MFI (gated on IFN-γ/granzyme B-producing CD8 + cells) are indicated (D-E). Data are mean plus standard error of 5-6 independent experiments (A), or representative of at least 3 independent experiments (B-E). *p
Figure Legend Snippet: Autocrine type I IFN signaling promotes DC maturation following β-glucan stimulation (A-B) Wild-type and IFNAR1-deficient DCs were stimulated with 100 μg/ml fungal β-glucan particles for 24 hours. Surface expression of MHC I-OVA and co-stimulatory molecules was assessed by flow cytometry (A). For MHC I-OVA measurement, DC cultures were supplemented with 5.5 μM OVA peptide (SIINFEKL) 1 hour prior to β-glucan stimulation. Cytokine levels in culture supernatants were assessed by ELISA (all except type I IFN) or a luciferase reporter assay (type I IFN) (B; mean plus standard deviation of triplicate culture). (C-E) Wild-type DCs were loaded with 1.1 nM OVA peptide (SIINFEKL) and stimulated as above prior to co-culture with naïve CFSE-labeled OT-I CD8 T cells for 3 days in the presence of anti-CD86 or isotype control antibodies (200 μg/ml). Proliferation (CFSE dilution) was assessed by flow cytometry (C). T cells were also re-stimulated with PMA + ionomycin (6 hour stimulation, with protein export inhibitors for the final 4 hours) to assess IFN-γ (D) and granzyme B production (E) by intracellular flow cytometry. % IFN-γ/granzyme B-producing T cells (gated on CD8 + cells) and IFN-γ/granzyme B MFI (gated on IFN-γ/granzyme B-producing CD8 + cells) are indicated (D-E). Data are mean plus standard error of 5-6 independent experiments (A), or representative of at least 3 independent experiments (B-E). *p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Standard Deviation, Co-Culture Assay, Labeling

Fungal β-glucan particle stimulation of DCs enhances their ability to activate CD8 T cells DCs cultures were supplemented with 1.1 nM OVA peptide (SIINFEKL) for 1 hour prior to stimulation with 100 μg/ml fungal β-glucan particles or 100 ng/ml LPS for 24 hours. DCs were then washed with PBS prior to the addition of CFSE-labeled naïve OT-I CD8 T cells (1:5 DC:T cell ratio). Co-cultures were incubated for 3 days, and T cells were then harvested for assessment of proliferation (CFSE dilution; gated on CD8 + cells) and surface expression of CD44 and CD69 (gated on proliferating CD8 + cells) by flow cytometry (A). T cells were also re-stimulated with PMA + ionomycin to assess cytokine production by ELISA (B; 24 hour supernatants, mean plus standard deviation of triplicate culture) and granzyme B production by intracellular flow cytometry (C; 6 hour stimulation with protein export inhibitors for the final 4 hours, gated on CD8 + cells). % granzyme B-producing T cells and granzyme B MFI (gated on granzyme B-producing T cells) are indicated (C). All data are representative of at least 3 independent experiments. *p
Figure Legend Snippet: Fungal β-glucan particle stimulation of DCs enhances their ability to activate CD8 T cells DCs cultures were supplemented with 1.1 nM OVA peptide (SIINFEKL) for 1 hour prior to stimulation with 100 μg/ml fungal β-glucan particles or 100 ng/ml LPS for 24 hours. DCs were then washed with PBS prior to the addition of CFSE-labeled naïve OT-I CD8 T cells (1:5 DC:T cell ratio). Co-cultures were incubated for 3 days, and T cells were then harvested for assessment of proliferation (CFSE dilution; gated on CD8 + cells) and surface expression of CD44 and CD69 (gated on proliferating CD8 + cells) by flow cytometry (A). T cells were also re-stimulated with PMA + ionomycin to assess cytokine production by ELISA (B; 24 hour supernatants, mean plus standard deviation of triplicate culture) and granzyme B production by intracellular flow cytometry (C; 6 hour stimulation with protein export inhibitors for the final 4 hours, gated on CD8 + cells). % granzyme B-producing T cells and granzyme B MFI (gated on granzyme B-producing T cells) are indicated (C). All data are representative of at least 3 independent experiments. *p

Techniques Used: Labeling, Incubation, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Blockade of IFNAR1 signaling during DC stimulation suppresses CD8 T cell activation by β-glucan-stimulated DCs DCs were loaded with OVA peptide (SIINFEKL; 1.1 nM) and stimulated with 100 μg/ml fungal β-glucan particles for 24 hours. DCs were then washed prior to co-culture with CFSE-labeled naïve OT-I CD8 T cells (1:5 DC:T cell ratio) for 3 days. Cultures were supplemented with anti-IFNAR1 antibodies (or isotype control antibodies; 200 μg/ml) during the DC stimulation stage (αIFNAR DC ), the DC:T cell co-culture stage (αIFNAR co-culture ), or throughout both culture stages (αIFNAR all ; antibodies replaced after DC washing) (A). Proliferation (CFSE dilution; gated on CD8 + cells) and surface expression of CD44 and CD69 (gated on proliferating CD8 + cells) were assessed by flow cytometry (B). T cells were also re-stimulated with PMA + ionomycin to assess cytokine production by ELISA (C; 24 hour supernatants, mean plus standard deviation of triplicate culture), and granzyme B production by intracellular flow cytometry (D; 6 hour stimulation with protein export inhibitors for the final 4 hours, gated on CD8 + cells). % granzyme B-producing T cells and granzyme B MFI (gated on granzyme B-producing T cells) are indicated (D). All data are representative of at least 3 independent experiments. ***p
Figure Legend Snippet: Blockade of IFNAR1 signaling during DC stimulation suppresses CD8 T cell activation by β-glucan-stimulated DCs DCs were loaded with OVA peptide (SIINFEKL; 1.1 nM) and stimulated with 100 μg/ml fungal β-glucan particles for 24 hours. DCs were then washed prior to co-culture with CFSE-labeled naïve OT-I CD8 T cells (1:5 DC:T cell ratio) for 3 days. Cultures were supplemented with anti-IFNAR1 antibodies (or isotype control antibodies; 200 μg/ml) during the DC stimulation stage (αIFNAR DC ), the DC:T cell co-culture stage (αIFNAR co-culture ), or throughout both culture stages (αIFNAR all ; antibodies replaced after DC washing) (A). Proliferation (CFSE dilution; gated on CD8 + cells) and surface expression of CD44 and CD69 (gated on proliferating CD8 + cells) were assessed by flow cytometry (B). T cells were also re-stimulated with PMA + ionomycin to assess cytokine production by ELISA (C; 24 hour supernatants, mean plus standard deviation of triplicate culture), and granzyme B production by intracellular flow cytometry (D; 6 hour stimulation with protein export inhibitors for the final 4 hours, gated on CD8 + cells). % granzyme B-producing T cells and granzyme B MFI (gated on granzyme B-producing T cells) are indicated (D). All data are representative of at least 3 independent experiments. ***p

Techniques Used: Activation Assay, Co-Culture Assay, Labeling, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

IFNAR1-deficient DCs are less efficient than wild-type DCs at activating CD8 T cells following β-glucan stimulation Wild-type and IFNAR1-deficient DCs were loaded with OVA peptide (SIINFEKL; 1.1 nM) and stimulated with 100 μg/ml fungal β-glucan particles for 24 hours. DCs were then washed prior to co-culture with CFSE-labeled naïve OT-I CD8 T cells (1:5 DC:T cell ratio) for 3 days. T cells were then harvested and proliferation (CFSE dilution; gated on CD8 + cells) and surface expression of CD44 and CD69 (gated on proliferating CD8 + cells) were assessed by flow cytometry (A). T cells were also re-stimulated with PMA + ionomycin to assess cytokine production by ELISA (B; 24-hour supernatants, mean plus standard deviation of triplicate culture), and cytokine and granzyme B production by intracellular flow cytometry (C-D; 6-hour stimulation with protein export inhibitors for the final 4 hours, gated on CD8 + cells). % positive cells and MFI (gated on positive cells) are plotted (C-D). Data are representative of at least 3 independent experiments (A-B), or mean plus standard error of 3 independent experiments (C-D). *p
Figure Legend Snippet: IFNAR1-deficient DCs are less efficient than wild-type DCs at activating CD8 T cells following β-glucan stimulation Wild-type and IFNAR1-deficient DCs were loaded with OVA peptide (SIINFEKL; 1.1 nM) and stimulated with 100 μg/ml fungal β-glucan particles for 24 hours. DCs were then washed prior to co-culture with CFSE-labeled naïve OT-I CD8 T cells (1:5 DC:T cell ratio) for 3 days. T cells were then harvested and proliferation (CFSE dilution; gated on CD8 + cells) and surface expression of CD44 and CD69 (gated on proliferating CD8 + cells) were assessed by flow cytometry (A). T cells were also re-stimulated with PMA + ionomycin to assess cytokine production by ELISA (B; 24-hour supernatants, mean plus standard deviation of triplicate culture), and cytokine and granzyme B production by intracellular flow cytometry (C-D; 6-hour stimulation with protein export inhibitors for the final 4 hours, gated on CD8 + cells). % positive cells and MFI (gated on positive cells) are plotted (C-D). Data are representative of at least 3 independent experiments (A-B), or mean plus standard error of 3 independent experiments (C-D). *p

Techniques Used: Co-Culture Assay, Labeling, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

6) Product Images from "Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4"

Article Title: Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4

Journal: Nature Communications

doi: 10.1038/ncomms10857

IL-9 expression correlates with strength of TCR signals. ( a ) Sorted naive WT CD4 + T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml −1 of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. ( b ) Itk-deficient CD4 + T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.
Figure Legend Snippet: IL-9 expression correlates with strength of TCR signals. ( a ) Sorted naive WT CD4 + T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml −1 of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. ( b ) Itk-deficient CD4 + T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.

Techniques Used: Expressing, Staining, Transduction

IL-2 rescues pSTAT5 and pS6 in Itk −/− CD4 T cells. ( a - c ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated for 3 days under Th9 or Th9 plus TL1A conditions in absence or presence of blocking anti-IL-2 plus hIL-2 and analysed for pSTAT5 (MFI are indicated) ( a ) CD25 ( b ) and pS6 ( c ). ( d ) Itk-deficient CD4 + T cells were transduced with control (MIGR) or constitutively active STAT5-expressing retroviruses, differentiated under Th9 plus TL1A conditions and IL-9 production determined by intracellular staining after PMA and Ionomycin restimulation. Results in a - d are representative of one out of at least 3 experiments.
Figure Legend Snippet: IL-2 rescues pSTAT5 and pS6 in Itk −/− CD4 T cells. ( a - c ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated for 3 days under Th9 or Th9 plus TL1A conditions in absence or presence of blocking anti-IL-2 plus hIL-2 and analysed for pSTAT5 (MFI are indicated) ( a ) CD25 ( b ) and pS6 ( c ). ( d ) Itk-deficient CD4 + T cells were transduced with control (MIGR) or constitutively active STAT5-expressing retroviruses, differentiated under Th9 plus TL1A conditions and IL-9 production determined by intracellular staining after PMA and Ionomycin restimulation. Results in a - d are representative of one out of at least 3 experiments.

Techniques Used: Mouse Assay, Blocking Assay, Transduction, Expressing, Staining

IL-2 rescues Th9 differentiation in Itk −/− CD4 + T cells. ( a - c ) Sorted naïve CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions plus TL1A for 3 days and pSTAT5 ( a ) and CD25 ( b ) were determined by flow cytometry: WT (black), Itk −/− (grey) lines. ( c ) Sorted naive CD4 + T cells were differentiated as in a or under Th17 conditions and IL-2 in supernatants were determined at 48 and 72 h by Luminex. Th9 conditions: black bars. Th17 conditions: hatched bars. ( d , e ) Sorted naïve CD4 + T cells were differentiated for 3 days under Th9 ( d ) or Th9 plus TL1A ( e ) conditions in absence or presence of blocking anti-IL-2 plus hIL-2, restimulated with PMA and Ionomycin and IL-9 analysed by flow cytometry. ( f ) Sorted naive CD4 + T cells from WT and Itk −/− mice were stained with CSFE, differentiated, and restimulated with PMA and Ionomycin to evaluate IL-9 expression. Data in a - f are representative of one out of at least three independent experiments.
Figure Legend Snippet: IL-2 rescues Th9 differentiation in Itk −/− CD4 + T cells. ( a - c ) Sorted naïve CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions plus TL1A for 3 days and pSTAT5 ( a ) and CD25 ( b ) were determined by flow cytometry: WT (black), Itk −/− (grey) lines. ( c ) Sorted naive CD4 + T cells were differentiated as in a or under Th17 conditions and IL-2 in supernatants were determined at 48 and 72 h by Luminex. Th9 conditions: black bars. Th17 conditions: hatched bars. ( d , e ) Sorted naïve CD4 + T cells were differentiated for 3 days under Th9 ( d ) or Th9 plus TL1A ( e ) conditions in absence or presence of blocking anti-IL-2 plus hIL-2, restimulated with PMA and Ionomycin and IL-9 analysed by flow cytometry. ( f ) Sorted naive CD4 + T cells from WT and Itk −/− mice were stained with CSFE, differentiated, and restimulated with PMA and Ionomycin to evaluate IL-9 expression. Data in a - f are representative of one out of at least three independent experiments.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Luminex, Blocking Assay, Staining, Expressing

IL-2 rescues IRF4 expression in Itk −/− CD4 T cells. ( a ) Sorted naive CD4 + T cells differentiated for 3 days under Th9 plus TL1A in absence or presence of blocking anti-IL-2 plus hIL-2, were stained for IRF4 and analysed by flow cytometry. ( b ) mRNA of Irf4 and Maf in cells differentiated as in a evaluated by qRT-PCR. ( c ) Itk-deficient CD4 + T cells were transduced with constitutively active STAT5 (ca-STAT5) or control retroviruses, differentiated under Th9 plus TL1A conditions and intracellular IRF4 determined. ( d ) Sorted naive WT CD4 + T cells were differentiated under Th9 plus TL1A with 0.003 or 0.01 μg ml −1 of anti-CD3, in absence or presence of blocking anti-IL-2 or blocking anti-IL-2 plus hIL-2, then restimulated with PMA and Ionomycin and IL-9 and IRF4 production were analysed by intracellular staining, MFI values for IRF4 are indicated. Data in figures a - d are representative examples from one of at least three independent experiments.
Figure Legend Snippet: IL-2 rescues IRF4 expression in Itk −/− CD4 T cells. ( a ) Sorted naive CD4 + T cells differentiated for 3 days under Th9 plus TL1A in absence or presence of blocking anti-IL-2 plus hIL-2, were stained for IRF4 and analysed by flow cytometry. ( b ) mRNA of Irf4 and Maf in cells differentiated as in a evaluated by qRT-PCR. ( c ) Itk-deficient CD4 + T cells were transduced with constitutively active STAT5 (ca-STAT5) or control retroviruses, differentiated under Th9 plus TL1A conditions and intracellular IRF4 determined. ( d ) Sorted naive WT CD4 + T cells were differentiated under Th9 plus TL1A with 0.003 or 0.01 μg ml −1 of anti-CD3, in absence or presence of blocking anti-IL-2 or blocking anti-IL-2 plus hIL-2, then restimulated with PMA and Ionomycin and IL-9 and IRF4 production were analysed by intracellular staining, MFI values for IRF4 are indicated. Data in figures a - d are representative examples from one of at least three independent experiments.

Techniques Used: Expressing, Blocking Assay, Staining, Flow Cytometry, Cytometry, Quantitative RT-PCR, Transduction

Itk is required for Th9 differentiation. ( a – d ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions (1 μg ml −1 anti-CD3, 3 μg ml −1 anti-CD28, 20 ng ml −1 IL-4, 5 ng ml −1 TGFβ1 with or without 10 ng ml −1 TL1A, in presence of T-depleted splenocytes as APCs) for 3 days, ( a ) cells were restimulated with PMA and Ionomycin and IL-9 and IFN-γ production were analysed by intracellular staining. Representative flow plots from one out of 10 experiments. ( b ) Mean±s.e.m. of 10 independent experiments ** P
Figure Legend Snippet: Itk is required for Th9 differentiation. ( a – d ) Sorted naive CD4 + T cells from WT and Itk −/− mice were differentiated under Th9 conditions (1 μg ml −1 anti-CD3, 3 μg ml −1 anti-CD28, 20 ng ml −1 IL-4, 5 ng ml −1 TGFβ1 with or without 10 ng ml −1 TL1A, in presence of T-depleted splenocytes as APCs) for 3 days, ( a ) cells were restimulated with PMA and Ionomycin and IL-9 and IFN-γ production were analysed by intracellular staining. Representative flow plots from one out of 10 experiments. ( b ) Mean±s.e.m. of 10 independent experiments ** P

Techniques Used: Mouse Assay, Staining, Flow Cytometry

ITK is required for Th9 differentiation of human cells. Purified naive human CD4 + T cells were differentiated under Th9 conditions for 5 days in presence of increasing concentrations of ITK inhibitor. Cells were restimulated with PMA and Ionomycin and stained for IL-9. Data are representative of one of two independent experiments.
Figure Legend Snippet: ITK is required for Th9 differentiation of human cells. Purified naive human CD4 + T cells were differentiated under Th9 conditions for 5 days in presence of increasing concentrations of ITK inhibitor. Cells were restimulated with PMA and Ionomycin and stained for IL-9. Data are representative of one of two independent experiments.

Techniques Used: Purification, Staining

7) Product Images from "Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling"

Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00265

Lipopolysaccharide (LPS) treatment enhances interferon gamma (IFN-γ) production in maternal antibiotic treatment (MAT) effector CD8 + cells in vivo and in vitro . Control (CTRL) and MAT infant mice were infected at day of life (dol) 15 with vaccinia-ovalbumin (Vac-OVA, 1 × 10 4 PFU intraperitoneal) and were treated with Escherichia coli -derived LPS (50 μg o.g.) every other day for 10 days (Vac-OVA + LPS). (A) At day of infection 11, lymphocytes were isolated from the spleens of CTRL and MAT mice infant mice and stimulated in vitro with SIINFEKL peptide, phorbol 12-myristate 13-acetate, and ionomycin for 5 h to analyze the percentage of IFN-γ- and TNF-α-producing CD8 + T effector cells (CD44 + CD62L − , Teff) by flow cytometry (CTRL Vac-OVA, n = 3; CTRL Vac-OVA + LPS, n = 7; MAT Vac-OVA, n = 2; MAT Vac-OVA + LPS, n = 3). (B) Survival curve of CTRL and MAT infant mice following Vac-OVA infection and LPS treatment (CTRL Vac-OVA, n = 5; CTRL Vac-OVA + LPS, n = 5; MAT Vac-OVA, n = 2; MAT Vac-OVA + LPS, n = 11). Comparison of survival curves was performed by log-rank (Mantel–Cox) test. Data are representative of two infection experiments. (C) Pooled CD8 + T cells isolated from the spleens of uninfected dol 15 CTRL ( n = 3) and MAT ( n = 6) infant mice were stimulated with anti-CD3/anti-CD28 with or without Escherichia coli -derived LPS (1 μg/ml) for 72 h and analyzed for the percentage of IFN-γ and TNF-α-producing Teff cells by flow cytometry. Data are representative of three independent experiments.
Figure Legend Snippet: Lipopolysaccharide (LPS) treatment enhances interferon gamma (IFN-γ) production in maternal antibiotic treatment (MAT) effector CD8 + cells in vivo and in vitro . Control (CTRL) and MAT infant mice were infected at day of life (dol) 15 with vaccinia-ovalbumin (Vac-OVA, 1 × 10 4 PFU intraperitoneal) and were treated with Escherichia coli -derived LPS (50 μg o.g.) every other day for 10 days (Vac-OVA + LPS). (A) At day of infection 11, lymphocytes were isolated from the spleens of CTRL and MAT mice infant mice and stimulated in vitro with SIINFEKL peptide, phorbol 12-myristate 13-acetate, and ionomycin for 5 h to analyze the percentage of IFN-γ- and TNF-α-producing CD8 + T effector cells (CD44 + CD62L − , Teff) by flow cytometry (CTRL Vac-OVA, n = 3; CTRL Vac-OVA + LPS, n = 7; MAT Vac-OVA, n = 2; MAT Vac-OVA + LPS, n = 3). (B) Survival curve of CTRL and MAT infant mice following Vac-OVA infection and LPS treatment (CTRL Vac-OVA, n = 5; CTRL Vac-OVA + LPS, n = 5; MAT Vac-OVA, n = 2; MAT Vac-OVA + LPS, n = 11). Comparison of survival curves was performed by log-rank (Mantel–Cox) test. Data are representative of two infection experiments. (C) Pooled CD8 + T cells isolated from the spleens of uninfected dol 15 CTRL ( n = 3) and MAT ( n = 6) infant mice were stimulated with anti-CD3/anti-CD28 with or without Escherichia coli -derived LPS (1 μg/ml) for 72 h and analyzed for the percentage of IFN-γ and TNF-α-producing Teff cells by flow cytometry. Data are representative of three independent experiments.

Techniques Used: In Vivo, In Vitro, Mouse Assay, Infection, Derivative Assay, Isolation, Flow Cytometry, Cytometry

Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p
Figure Legend Snippet: Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p

Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Negative Staining, Fluorescence, Two Tailed Test

8) Product Images from "Myeloperoxidase Attenuates Pathogen Clearance during Plasmodium yoelii Nonlethal Infection"

Article Title: Myeloperoxidase Attenuates Pathogen Clearance during Plasmodium yoelii Nonlethal Infection

Journal: Infection and Immunity

doi: 10.1128/IAI.00475-16

Increased expansion of splenic IFN-γ- and TNF-α-producing T cells in MPO −/− mice during P. yoelii 17NL infection. On selected days splenocytes were isolated and stimulated with phorbol myristate acetate and ionomycin. The
Figure Legend Snippet: Increased expansion of splenic IFN-γ- and TNF-α-producing T cells in MPO −/− mice during P. yoelii 17NL infection. On selected days splenocytes were isolated and stimulated with phorbol myristate acetate and ionomycin. The

Techniques Used: Mouse Assay, Infection, Isolation

9) Product Images from "BJ-3105, a 6-Alkoxypyridin-3-ol Analog, Impairs T Cell Differentiation and Prevents Experimental Autoimmune Encephalomyelitis Disease Progression"

Article Title: BJ-3105, a 6-Alkoxypyridin-3-ol Analog, Impairs T Cell Differentiation and Prevents Experimental Autoimmune Encephalomyelitis Disease Progression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0168942

BJ-3105 decreases the percentage of Th1 and Th17 cells differentiation. (A) Chemical Structure of BJ-3105 (2,4,5-trimethyl-6-(3-phenylpropoxy)pyridin-3-ol). (B) Purified naïve mouse CD4 + T cells from spleens and draining lymph nodes were stimulated in Th1 and Th17 polarizing conditions for 72 h and Treg for 96 h in the presence of BJ-3105 (1 μM) or tofacitinib (1 μM). CD4 + T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. Percentage of IFN-γ + Th1 cells, IL-17A + Th17 cells and Foxp3 + Treg cells were shown in bar diagram. (C) IFN-γ and IL-17A cytokine produced by CD4 + T cells were quantified by cytokine binding assay. (D) Th1, Th17 differentiation with different dose of BJ-3105. (E) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. *p
Figure Legend Snippet: BJ-3105 decreases the percentage of Th1 and Th17 cells differentiation. (A) Chemical Structure of BJ-3105 (2,4,5-trimethyl-6-(3-phenylpropoxy)pyridin-3-ol). (B) Purified naïve mouse CD4 + T cells from spleens and draining lymph nodes were stimulated in Th1 and Th17 polarizing conditions for 72 h and Treg for 96 h in the presence of BJ-3105 (1 μM) or tofacitinib (1 μM). CD4 + T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. Percentage of IFN-γ + Th1 cells, IL-17A + Th17 cells and Foxp3 + Treg cells were shown in bar diagram. (C) IFN-γ and IL-17A cytokine produced by CD4 + T cells were quantified by cytokine binding assay. (D) Th1, Th17 differentiation with different dose of BJ-3105. (E) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. *p

Techniques Used: Purification, Staining, Flow Cytometry, Cytometry, Cell Culture, Produced, Binding Assay

BJ-3105 decreases the percentage of antigen-specific Th1 and Th17 cell differentiation. (A) Naïve CD4 + T cells and antigen presenting cells from spleens and lymph nodes were isolated by immunomagnetic positive selection from 6–10 weeks OT-II mice. CD4 + T cells and irradiated antigen presenting cells were cultured in Th1, Th17 and Treg cells differentiation conditions in the presence of OVA 323–339 (0.1 μM) with BJ-3105 (1 μM) or tofacitinib (1 μM). CD4 + T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. (B) The cells were cultured in Th1 and Th17 cells differentiation conditions for 72 h with different concentration of BJ-3105 and analyzed by flow cytometer. (C) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. *p
Figure Legend Snippet: BJ-3105 decreases the percentage of antigen-specific Th1 and Th17 cell differentiation. (A) Naïve CD4 + T cells and antigen presenting cells from spleens and lymph nodes were isolated by immunomagnetic positive selection from 6–10 weeks OT-II mice. CD4 + T cells and irradiated antigen presenting cells were cultured in Th1, Th17 and Treg cells differentiation conditions in the presence of OVA 323–339 (0.1 μM) with BJ-3105 (1 μM) or tofacitinib (1 μM). CD4 + T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. (B) The cells were cultured in Th1 and Th17 cells differentiation conditions for 72 h with different concentration of BJ-3105 and analyzed by flow cytometer. (C) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. *p

Techniques Used: Cell Differentiation, Isolation, Selection, Mouse Assay, Irradiation, Cell Culture, Staining, Flow Cytometry, Cytometry, Concentration Assay

10) Product Images from "Capability of Neutrophils to Form NETs Is Not Directly Influenced by a CMA-Targeting Peptide"

Article Title: Capability of Neutrophils to Form NETs Is Not Directly Influenced by a CMA-Targeting Peptide

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00016

Plate reader-based quantification of neutrophil extracellular trap formation in mice treated with P140 and scrambled P140 (ScP140) . (A) Relative SYTOX Green fluorescence in neutrophils isolated from the blood of P140- or ScP140-pretreated mice incubated for 2 h with or without 100 ng/mL PMA or 2 µg/mL ionomycin. Graphs show individual values and means, one dot represents one mouse. ns, not significant. (B) Area and fluorescence intensity of SYTOX Green + events in neutrophils isolated from the blood of P140- or ScP140-pretreated mice incubated for 2 h with or without 100 ng/mL PMA or 2 µg/mL ionomycin. Plots show medians and individual values from one representative out of 6–8 mice. *** p
Figure Legend Snippet: Plate reader-based quantification of neutrophil extracellular trap formation in mice treated with P140 and scrambled P140 (ScP140) . (A) Relative SYTOX Green fluorescence in neutrophils isolated from the blood of P140- or ScP140-pretreated mice incubated for 2 h with or without 100 ng/mL PMA or 2 µg/mL ionomycin. Graphs show individual values and means, one dot represents one mouse. ns, not significant. (B) Area and fluorescence intensity of SYTOX Green + events in neutrophils isolated from the blood of P140- or ScP140-pretreated mice incubated for 2 h with or without 100 ng/mL PMA or 2 µg/mL ionomycin. Plots show medians and individual values from one representative out of 6–8 mice. *** p

Techniques Used: Mouse Assay, Fluorescence, Isolation, Incubation

Morphological analysis and quantification of neutrophil extracellular traps (NETs) in P140 and scrambled P140 (ScP140)-treated mice by fluorescence microscopy . Representative immunofluorescence images of SYTOX Green-, neutrophil elastase (NE)-, and citrullinated histone H3 (citH3)-stained neutrophils isolated from ScP140- (A–C) and P140- (D–F) treated mice and incubated without external stimulus (A,D) , with PMA (B,E) or with ionomycin (C,F) . Left panels show staining controls incubated without (wo) primary antibody to NE or citH3. CY5 stands for control Cyanin 5 and OL stands for overlay. (G) Quantitative analysis of NETs. Scatter plots show individual values and mean of %NETs (defined as % PI + /NE + cells with > 3-fold mean nuclear size) from two mice.
Figure Legend Snippet: Morphological analysis and quantification of neutrophil extracellular traps (NETs) in P140 and scrambled P140 (ScP140)-treated mice by fluorescence microscopy . Representative immunofluorescence images of SYTOX Green-, neutrophil elastase (NE)-, and citrullinated histone H3 (citH3)-stained neutrophils isolated from ScP140- (A–C) and P140- (D–F) treated mice and incubated without external stimulus (A,D) , with PMA (B,E) or with ionomycin (C,F) . Left panels show staining controls incubated without (wo) primary antibody to NE or citH3. CY5 stands for control Cyanin 5 and OL stands for overlay. (G) Quantitative analysis of NETs. Scatter plots show individual values and mean of %NETs (defined as % PI + /NE + cells with > 3-fold mean nuclear size) from two mice.

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Immunofluorescence, Staining, Isolation, Incubation

11) Product Images from "Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 (Ape1/Ref-1) Modulates Antigen Presenting Cell-mediated T Helper Cell Type 1 Responses *"

Article Title: Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 (Ape1/Ref-1) Modulates Antigen Presenting Cell-mediated T Helper Cell Type 1 Responses *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.742353

Solid-phase IL-12 promotes the induction of IFN-γ-producing CD4 + T cells. Purified CD4 + T cells were stimulated with various concentrations of plate-bound IL-12, anti-CD3 Ab, and anti-CD28 Ab for 72 h in the presence of IL-2 (10 units/ml). The cells were re-stimulated with PMA, ionomycin, and brefeldin A for the last 4 h of culture and then stained for the cell surface expression of CD3 and CD4 and intracellular expression of IFN-γ. A, flow cytometry profiles of the CD3 + /CD4 + cells, representative of two independent experiments. B, representative graph showing the percentages of IFN-γ-producing CD3 + /CD4 + T cells. Horizontal axis shows the IL-12 concentration used for pretreating the plates.
Figure Legend Snippet: Solid-phase IL-12 promotes the induction of IFN-γ-producing CD4 + T cells. Purified CD4 + T cells were stimulated with various concentrations of plate-bound IL-12, anti-CD3 Ab, and anti-CD28 Ab for 72 h in the presence of IL-2 (10 units/ml). The cells were re-stimulated with PMA, ionomycin, and brefeldin A for the last 4 h of culture and then stained for the cell surface expression of CD3 and CD4 and intracellular expression of IFN-γ. A, flow cytometry profiles of the CD3 + /CD4 + cells, representative of two independent experiments. B, representative graph showing the percentages of IFN-γ-producing CD3 + /CD4 + T cells. Horizontal axis shows the IL-12 concentration used for pretreating the plates.

Techniques Used: Purification, Staining, Expressing, Flow Cytometry, Cytometry, Concentration Assay

E3330 enhances the induction of IFN-γ-producing OT-II T cells by altering antigen presenting cells. Adherent splenocytes ( A ) or BMDCs prepared as described under “Experimental Procedures” ( B ) were pretreated with or without E3330 for 1 h and then stimulated with or without OVA (100 μg/ml) ( A ) or OVA-(323–339) peptide (10 μg/ml) ( B ) and cultured for 24 h. The pretreated cells were washed with PBS to remove the E3330 and then co-cultured with OT-II T cells for 72 h. The OT-II T cells were re-stimulated with PMA, ionomycin, and brefeldin A for the last 4 h of culture and then stained for the cell surface expression of CD3 and CD4 and intracellular expression of IFN-γ. Flow cytometry profiles of the CD3 + /CD4 + cells are representative of two ( A ) and three ( B ) independent experiments. The percentages of the IFN-γ positive cells are indicated.
Figure Legend Snippet: E3330 enhances the induction of IFN-γ-producing OT-II T cells by altering antigen presenting cells. Adherent splenocytes ( A ) or BMDCs prepared as described under “Experimental Procedures” ( B ) were pretreated with or without E3330 for 1 h and then stimulated with or without OVA (100 μg/ml) ( A ) or OVA-(323–339) peptide (10 μg/ml) ( B ) and cultured for 24 h. The pretreated cells were washed with PBS to remove the E3330 and then co-cultured with OT-II T cells for 72 h. The OT-II T cells were re-stimulated with PMA, ionomycin, and brefeldin A for the last 4 h of culture and then stained for the cell surface expression of CD3 and CD4 and intracellular expression of IFN-γ. Flow cytometry profiles of the CD3 + /CD4 + cells are representative of two ( A ) and three ( B ) independent experiments. The percentages of the IFN-γ positive cells are indicated.

Techniques Used: Cell Culture, Staining, Expressing, Flow Cytometry, Cytometry

12) Product Images from "Akt2 Regulates the Differentiation and Function of NKT17 Cells via FoxO-1-ICOS Axis"

Article Title: Akt2 Regulates the Differentiation and Function of NKT17 Cells via FoxO-1-ICOS Axis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01940

Akt2 promotes NKT17 lineage differentiation. Cells were from WT and Akt2 KO mice. (A) Intracellular staining of PLZF, RORγt, T-bet, and GATA3 in iNKT cells from the thymus. Percentages and absolute numbers of NKT1, NKT2, NKT17 cells in the thymus ( n = 6 mice per group) (B) and spleen ( n = 4 mice per group) (C) from WT, KO, WT chimera mice ( n = 3) and Akt2 KO chimera mice ( n = 3). (D–F) Cytokine production by iNKT cells (gated on CD1d-PBS57 + TCRβ + cells) from the thymus ( n = 5 mice per group) and spleen ( n = 5 mice per group) after stimulation with PMA plus ionomycin for 5 h. (G,H) Cytokine production by iNKT cells from WT and Akt2 KO thymocytes stimulated with α-GalCer for 72 h and with PMA plus ionomycin in the last 5 h ( n = 4 mice per group). (I) Critical role of Akt2 for PLZF localization to the nuclear bodies. MACS—enriched and FACS—sorted NKT17 cells from WT and Akt2 KO thymocytes( n = 3) were fixed and stained with a mouse anti-PLZF and Actin as primary antibody, detected with an Goat anti mouse secondary antibody. The nuclei were stained with DAPI. (J) Mean fluorescent intensity of cytoplasm and nucleus. * p
Figure Legend Snippet: Akt2 promotes NKT17 lineage differentiation. Cells were from WT and Akt2 KO mice. (A) Intracellular staining of PLZF, RORγt, T-bet, and GATA3 in iNKT cells from the thymus. Percentages and absolute numbers of NKT1, NKT2, NKT17 cells in the thymus ( n = 6 mice per group) (B) and spleen ( n = 4 mice per group) (C) from WT, KO, WT chimera mice ( n = 3) and Akt2 KO chimera mice ( n = 3). (D–F) Cytokine production by iNKT cells (gated on CD1d-PBS57 + TCRβ + cells) from the thymus ( n = 5 mice per group) and spleen ( n = 5 mice per group) after stimulation with PMA plus ionomycin for 5 h. (G,H) Cytokine production by iNKT cells from WT and Akt2 KO thymocytes stimulated with α-GalCer for 72 h and with PMA plus ionomycin in the last 5 h ( n = 4 mice per group). (I) Critical role of Akt2 for PLZF localization to the nuclear bodies. MACS—enriched and FACS—sorted NKT17 cells from WT and Akt2 KO thymocytes( n = 3) were fixed and stained with a mouse anti-PLZF and Actin as primary antibody, detected with an Goat anti mouse secondary antibody. The nuclei were stained with DAPI. (J) Mean fluorescent intensity of cytoplasm and nucleus. * p

Techniques Used: Mouse Assay, Staining, Magnetic Cell Separation, FACS

13) Product Images from "Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease"

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease

Journal: Nature

doi: 10.1038/s41586-019-1895-7

Correlations of memory T cell populations with cognitive scores. a , Gating strategy for measuring memory T cell populations. b , Linear regression analysis correlating CD8 + T cell populations with cognitive scores indicates a positive relationship between T EM and T CM CD8 + T cells and no relationship with naive cells. The significance of the difference between datasets was measured by ANCOVA. c , The relationship between the percentage of CD8 + T EMRA cells and cognitive score was not influenced by age. The significance of the difference between the two datasets was measured by analysis of covariance (ANCOVA). Pearson’s correlation r values are shown for each group ( b , c ). d , Gating strategy for T cell stimulation experiments. e , Increased intracellular TNF cytokine response in PMA-ionomycin-stimulated CD8 + T cells from patients with MCI or AD. Unpaired two-sided t -test with Welch’s correction ( n = 10 healthy; n = 14 MCI or AD); mean ± s.e.m.
Figure Legend Snippet: Correlations of memory T cell populations with cognitive scores. a , Gating strategy for measuring memory T cell populations. b , Linear regression analysis correlating CD8 + T cell populations with cognitive scores indicates a positive relationship between T EM and T CM CD8 + T cells and no relationship with naive cells. The significance of the difference between datasets was measured by ANCOVA. c , The relationship between the percentage of CD8 + T EMRA cells and cognitive score was not influenced by age. The significance of the difference between the two datasets was measured by analysis of covariance (ANCOVA). Pearson’s correlation r values are shown for each group ( b , c ). d , Gating strategy for T cell stimulation experiments. e , Increased intracellular TNF cytokine response in PMA-ionomycin-stimulated CD8 + T cells from patients with MCI or AD. Unpaired two-sided t -test with Welch’s correction ( n = 10 healthy; n = 14 MCI or AD); mean ± s.e.m.

Techniques Used: Cell Stimulation

Peripheral CD8 + T EMRA cells are increased in AD and are negatively associated with cognition. a , Four cohorts were used to assess adaptive immunity in AD. b , Representative SPADE trees of PBMCs from healthy individuals and patients with MCI or AD in cohort 1 show an increased abundance of a CD8 + cluster (cluster 63) in patients with MCI or AD. Background tree nodes are sized according to cell counts. Insets are coloured according to CD8 expression. c , Quantification of cluster 63 as a percentage of total PBMCs. The percentage of cluster 63 cells is significantly higher in patients with MCI or AD than healthy control individuals. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. d , Marker expression analysis of cluster 63 corresponds to a CD3 + CD8 + CD45RA + CD27 − T EMRA population. Data in c, d were pooled from seven independent experiments with similar results. e , Linear regression showing the inverse correlation between cognitive score and the percentage of CD8 + T EMRA cells in individuals from cohort 2. Pearson’s correlation r values are shown for each group. The significance of the difference between the two data sets was measured by ANCOVA. f , Stimulation with PMA and ionomycin (stim.) induces increased expression of IFN-γ in CD8 + T cells from patients with MCI or AD. Mean ± s.e.m.; unpaired two-sided t -test with Welch’s correction. g , Differential expression analysis (scRNA-seq) of CD8 + T emra cells from healthy individuals ( n = 7) and patients with MCI or AD ( n = 6) shows upregulated TCR signalling. Model-based analysis of single-cell transcriptomics (MAST) differential expression test with Benjamini-Hochberg correction. h , Pathway analysis of differentially expressed genes in CD8 + T EMRA cells from patients with MCI or AD ( n = 6 subjects) versus healthy individuals ( n = 7 subjects) shows increased antigenic stimulation of CD8 + T EMRA cells in patients with MCI or AD. Fisher’s exact test with Benjamini-Hochberg correction. Pathways (circles) with positive z -scores are coloured red; those with negative z -scores are coloured blue. The size of the circle corresponds to the size of the z -score (two-sided).
Figure Legend Snippet: Peripheral CD8 + T EMRA cells are increased in AD and are negatively associated with cognition. a , Four cohorts were used to assess adaptive immunity in AD. b , Representative SPADE trees of PBMCs from healthy individuals and patients with MCI or AD in cohort 1 show an increased abundance of a CD8 + cluster (cluster 63) in patients with MCI or AD. Background tree nodes are sized according to cell counts. Insets are coloured according to CD8 expression. c , Quantification of cluster 63 as a percentage of total PBMCs. The percentage of cluster 63 cells is significantly higher in patients with MCI or AD than healthy control individuals. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. d , Marker expression analysis of cluster 63 corresponds to a CD3 + CD8 + CD45RA + CD27 − T EMRA population. Data in c, d were pooled from seven independent experiments with similar results. e , Linear regression showing the inverse correlation between cognitive score and the percentage of CD8 + T EMRA cells in individuals from cohort 2. Pearson’s correlation r values are shown for each group. The significance of the difference between the two data sets was measured by ANCOVA. f , Stimulation with PMA and ionomycin (stim.) induces increased expression of IFN-γ in CD8 + T cells from patients with MCI or AD. Mean ± s.e.m.; unpaired two-sided t -test with Welch’s correction. g , Differential expression analysis (scRNA-seq) of CD8 + T emra cells from healthy individuals ( n = 7) and patients with MCI or AD ( n = 6) shows upregulated TCR signalling. Model-based analysis of single-cell transcriptomics (MAST) differential expression test with Benjamini-Hochberg correction. h , Pathway analysis of differentially expressed genes in CD8 + T EMRA cells from patients with MCI or AD ( n = 6 subjects) versus healthy individuals ( n = 7 subjects) shows increased antigenic stimulation of CD8 + T EMRA cells in patients with MCI or AD. Fisher’s exact test with Benjamini-Hochberg correction. Pathways (circles) with positive z -scores are coloured red; those with negative z -scores are coloured blue. The size of the circle corresponds to the size of the z -score (two-sided).

Techniques Used: Expressing, Marker, Single-cell Transcriptomics

14) Product Images from "Dendritic Cell-Based Immunization Ameliorates Pulmonary Infection with Highly Virulent Cryptococcus gattii"

Article Title: Dendritic Cell-Based Immunization Ameliorates Pulmonary Infection with Highly Virulent Cryptococcus gattii

Journal: Infection and Immunity

doi: 10.1128/IAI.02827-14

Transferring CAP60Δ/DCs induces IL-17A-producing cells in lungs. Lung leukocytes were obtained from three mice at 1 day after infection and were stimulated with PMA and ionomycin for 3 h. For flow cytometry analysis, gates were set for CD3 + Thy1.2
Figure Legend Snippet: Transferring CAP60Δ/DCs induces IL-17A-producing cells in lungs. Lung leukocytes were obtained from three mice at 1 day after infection and were stimulated with PMA and ionomycin for 3 h. For flow cytometry analysis, gates were set for CD3 + Thy1.2

Techniques Used: Transferring, Mouse Assay, Infection, Flow Cytometry, Cytometry

15) Product Images from "Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation"

Article Title: Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation

Journal: Scientific Reports

doi: 10.1038/s41598-018-26088-y

NTG-A-009 reduces Th1, Th17 cells differentiation in vitro . Naïve CD4 + T cells isolated from spleen and lymph nodes were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) under Th1, Th17 and T reg inducing conditions with NTG-A-009 (1 μM), Tofacitinib (1 μM) or Triamcinolone (1 µM). ( a ) Chemical structure of NTG-A-009. ( b,c ) The percentage of IFN-γ + Th1, IL-17A + Th17 and FoxP3 + T reg cells was determined by flow cytometry. ( d ) Different concentration of NTG-A-009 was used to find its effect in Th1 and Th17 cells in anti-CD3 antibody coating system. ( e,f ) The antigen specific differentiation of Th1, Th17 and T reg cells was done by the isolation of CD4 + T cells and irradiated antigen presenting cells from 6–10 weeks OTII mice and stimulated under Th1, Th17 and T reg polarizing conditions in the presence of OVA 323–339 (0.1 μM) and incubated for 72 hour for Th1 and Th17 and 96 hours for T reg cells differentiation. Cells were then restimulated with PMA, ionomycin and golgistop and analyzed by FACS through intracellular staining. ( g ) Different doses of NTG-A-009 were cultured under Th1, Th17 condition with the cells from OT-II transgenic mice. Data were quantified in bar diagram. Data represent three independent experiments. Mean ± SEM of the triplicates are shown. ***P
Figure Legend Snippet: NTG-A-009 reduces Th1, Th17 cells differentiation in vitro . Naïve CD4 + T cells isolated from spleen and lymph nodes were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) under Th1, Th17 and T reg inducing conditions with NTG-A-009 (1 μM), Tofacitinib (1 μM) or Triamcinolone (1 µM). ( a ) Chemical structure of NTG-A-009. ( b,c ) The percentage of IFN-γ + Th1, IL-17A + Th17 and FoxP3 + T reg cells was determined by flow cytometry. ( d ) Different concentration of NTG-A-009 was used to find its effect in Th1 and Th17 cells in anti-CD3 antibody coating system. ( e,f ) The antigen specific differentiation of Th1, Th17 and T reg cells was done by the isolation of CD4 + T cells and irradiated antigen presenting cells from 6–10 weeks OTII mice and stimulated under Th1, Th17 and T reg polarizing conditions in the presence of OVA 323–339 (0.1 μM) and incubated for 72 hour for Th1 and Th17 and 96 hours for T reg cells differentiation. Cells were then restimulated with PMA, ionomycin and golgistop and analyzed by FACS through intracellular staining. ( g ) Different doses of NTG-A-009 were cultured under Th1, Th17 condition with the cells from OT-II transgenic mice. Data were quantified in bar diagram. Data represent three independent experiments. Mean ± SEM of the triplicates are shown. ***P

Techniques Used: In Vitro, Isolation, Flow Cytometry, Cytometry, Concentration Assay, Irradiation, Mouse Assay, Incubation, FACS, Staining, Cell Culture, Transgenic Assay

NTG-A-009 inhibits Th1 and Th17 cells in vivo and attenuate DSS induced colitis. C57BL/6 mice were orally administered with 2.5% (w/v) DSS in drinking water for 7 days to induce colitis. NTG-A-009 was orally administered every day (n = 6 mice in each group). ( a ) Body weight of water, DSS and NTG-A-009 treated mice was recorded daily and presented as percentage of original body weight. ( b ) Colon length of mice treated with water, DSS and NTG-A-009. ( c ) CD4 + and CD8 + T cells infiltration was detected from the Lymphocytes isolated from colonic lamina propria of water, DSS and NTG-A-009 treated group and analyzed by FACS. ( d ) The percentage of Th1 and Th17 cells was determined by the stimulation of splenocytes from each group with PMA, ionomycin and golgistop for 4 hour and analyzed by FACS through intracellular staining of IFN-γ and IL-17. ( e ) H E staining of colonic section with histological score (magnification 20X). Data represent three independent experiments. Two-way anova (A) or *P
Figure Legend Snippet: NTG-A-009 inhibits Th1 and Th17 cells in vivo and attenuate DSS induced colitis. C57BL/6 mice were orally administered with 2.5% (w/v) DSS in drinking water for 7 days to induce colitis. NTG-A-009 was orally administered every day (n = 6 mice in each group). ( a ) Body weight of water, DSS and NTG-A-009 treated mice was recorded daily and presented as percentage of original body weight. ( b ) Colon length of mice treated with water, DSS and NTG-A-009. ( c ) CD4 + and CD8 + T cells infiltration was detected from the Lymphocytes isolated from colonic lamina propria of water, DSS and NTG-A-009 treated group and analyzed by FACS. ( d ) The percentage of Th1 and Th17 cells was determined by the stimulation of splenocytes from each group with PMA, ionomycin and golgistop for 4 hour and analyzed by FACS through intracellular staining of IFN-γ and IL-17. ( e ) H E staining of colonic section with histological score (magnification 20X). Data represent three independent experiments. Two-way anova (A) or *P

Techniques Used: In Vivo, Mouse Assay, Isolation, FACS, Staining

16) Product Images from "Blocking IL-10 signalling at the time of immunization does not increase unwanted side effects in mice"

Article Title: Blocking IL-10 signalling at the time of immunization does not increase unwanted side effects in mice

Journal: BMC Immunology

doi: 10.1186/s12865-017-0224-x

Blocking IL-10 signalling at the time of long HPV16 E7 peptide/MPLA immunization increases the numbers of IL-10 producing cells. Groups of five C57BL/6 mice were immunized as indicated with 50 μg of E7 peptide/15 μg of Monophosphoryl Lipid A (MPLA), 300 μg of anti-IL10R antibodies or control antibodies s.c on days 0 and 14. Splenocytes and draining lymph node cells from immunized mice were harvested and stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of monensin on 7 days after final immunization. Cells were surface stained for CD3, CD4, and GITR and intracellular stained for IL-10 and IFN-γ. CD3+ cells were gated. Results for cells of draining lymph nodes were shown. A and B: IL-10 secreting CD3 + CD4+ T cells IL-10 ( a ) FACS plot and ( b ) summarised data showing IL-10 expression by CD3 + CD4+ T cells. C and D: CD3 + CD4+ T cells secreting IL-10 and IFN-γ dot plots ( c ) and summarised data from different groups ( d ) E and F: CD4 + GITR+ T cells secreting IL-10: FACS profile ( e ) and summarised data from different groups ( f ). Splenic CD4 + IL-10+ cells were shown in ( g )
Figure Legend Snippet: Blocking IL-10 signalling at the time of long HPV16 E7 peptide/MPLA immunization increases the numbers of IL-10 producing cells. Groups of five C57BL/6 mice were immunized as indicated with 50 μg of E7 peptide/15 μg of Monophosphoryl Lipid A (MPLA), 300 μg of anti-IL10R antibodies or control antibodies s.c on days 0 and 14. Splenocytes and draining lymph node cells from immunized mice were harvested and stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of monensin on 7 days after final immunization. Cells were surface stained for CD3, CD4, and GITR and intracellular stained for IL-10 and IFN-γ. CD3+ cells were gated. Results for cells of draining lymph nodes were shown. A and B: IL-10 secreting CD3 + CD4+ T cells IL-10 ( a ) FACS plot and ( b ) summarised data showing IL-10 expression by CD3 + CD4+ T cells. C and D: CD3 + CD4+ T cells secreting IL-10 and IFN-γ dot plots ( c ) and summarised data from different groups ( d ) E and F: CD4 + GITR+ T cells secreting IL-10: FACS profile ( e ) and summarised data from different groups ( f ). Splenic CD4 + IL-10+ cells were shown in ( g )

Techniques Used: Blocking Assay, Mouse Assay, Staining, FACS, Expressing

17) Product Images from "M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer"

Article Title: M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer

Journal: Journal for Immunotherapy of Cancer

doi: 10.1136/jitc-2020-000979

Functional T-cell exhaustion in the metastatic tumor microenvironment (M-TME) of patients with high-grade serous carcinoma (HGSC). (A and B) Percentage of interferon gamma (IFN-γ + ) and granzyme B (GZMB) + cells in the CD3 + CD8 + population of the paired 15 primary (P-TME) and 15 metastatic tumor microenvironment (M-TME) microenvironment of patients from study group 2 on non-specific stimulation with phorbol 12-myristate13-acetate (PMA) and ionomycin, as determined by flow cytometry. Gating strategy (A) and quantitative results (B) are reported. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (C) Expression levels of IFNG, perforin 1 (PRF1), granulysin (GNLY) and GZMB relative to CD3E in 24 P-TME versus 24 M-TME samples from study group 1, as determined by RNA sequencing. P values are reported. (D) Density of CD8 + T cells and lysosomal-associated membrane protein (DC-LAMP + ) dendritic cells (DCs) in the M-TME of programmed death ligand 1 (PD-L1) − versus PD-L1 + and programmed cell death 1 (PD-1) Lo versus PD-1 Hi patients from study group 1 (n=80), as determined by immunostaining. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (E) Distribution of PD-L1 levels and density of PD-1 + and lymphocyte-activating gene 3 (LAG-3) + cells in paired 80 P-TME and 80 M-TME samples from study group 1, as determined by immunostaining. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (F) Gating strategy of IFN-γ + CD3 + CD8 + T cells in paired 15 P-TME and 15 M-TME samples (study group 2). The percentage of cells in each gate is reported. (G) Fold change of IFN-γ + and GZMB + CD8 + T cells isolated from paired 15 P-TME and 15 M-TME samples (study group 2) after non-specific stimulation with PMA and ionomycin in the context of PD-1 and TIM-3 blockage (Atbs) as determined by flow cytometry. P values are indicated. (H) Hierarchical clustering of 33 genes linked to cytokine/chemokine signaling that were differentially expressed in paired 24 M-TME versus 24 P-TME samples from study group 1. P values are reported. IL, interleukin; ns, not significant.
Figure Legend Snippet: Functional T-cell exhaustion in the metastatic tumor microenvironment (M-TME) of patients with high-grade serous carcinoma (HGSC). (A and B) Percentage of interferon gamma (IFN-γ + ) and granzyme B (GZMB) + cells in the CD3 + CD8 + population of the paired 15 primary (P-TME) and 15 metastatic tumor microenvironment (M-TME) microenvironment of patients from study group 2 on non-specific stimulation with phorbol 12-myristate13-acetate (PMA) and ionomycin, as determined by flow cytometry. Gating strategy (A) and quantitative results (B) are reported. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (C) Expression levels of IFNG, perforin 1 (PRF1), granulysin (GNLY) and GZMB relative to CD3E in 24 P-TME versus 24 M-TME samples from study group 1, as determined by RNA sequencing. P values are reported. (D) Density of CD8 + T cells and lysosomal-associated membrane protein (DC-LAMP + ) dendritic cells (DCs) in the M-TME of programmed death ligand 1 (PD-L1) − versus PD-L1 + and programmed cell death 1 (PD-1) Lo versus PD-1 Hi patients from study group 1 (n=80), as determined by immunostaining. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (E) Distribution of PD-L1 levels and density of PD-1 + and lymphocyte-activating gene 3 (LAG-3) + cells in paired 80 P-TME and 80 M-TME samples from study group 1, as determined by immunostaining. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. (F) Gating strategy of IFN-γ + CD3 + CD8 + T cells in paired 15 P-TME and 15 M-TME samples (study group 2). The percentage of cells in each gate is reported. (G) Fold change of IFN-γ + and GZMB + CD8 + T cells isolated from paired 15 P-TME and 15 M-TME samples (study group 2) after non-specific stimulation with PMA and ionomycin in the context of PD-1 and TIM-3 blockage (Atbs) as determined by flow cytometry. P values are indicated. (H) Hierarchical clustering of 33 genes linked to cytokine/chemokine signaling that were differentially expressed in paired 24 M-TME versus 24 P-TME samples from study group 1. P values are reported. IL, interleukin; ns, not significant.

Techniques Used: Functional Assay, Flow Cytometry, Expressing, RNA Sequencing Assay, Immunostaining, Isolation

18) Product Images from "Oxidized Lipids and CD36-Mediated Lipid Peroxidation in CD8 T Cells Suppress Anti-Tumor Immune Responses"

Article Title: Oxidized Lipids and CD36-Mediated Lipid Peroxidation in CD8 T Cells Suppress Anti-Tumor Immune Responses

Journal: bioRxiv

doi: 10.1101/2020.09.03.281691

OxLDL inhibits CD8 + T cell function in a CD36-dependent manner (A) P14 CD8 + T cells were activated in vitro with gp33 peptide plus IL-2 for 24 hrs and then treated with either vehicle control (Ctrl), OxLDL (50 μg/ml), LDL (50 μg/ml), HDL (50 μg/ml), SSO (100 μM), or the combination of OxLDL (50 μg/ml) and SSO (100 μM), for another 48 hrs. TNF, IFNγ and cell viability were then measured upon re-stimulation with gp33 for 6 hours and analyzed by flow cytometry. (B) Human PBMCs were treated with either vehicle control (Ctrl), OxLDL (50 μg/ml), or the combination of OxLDL (50 μg/ml) and SSO (100 μM). TNF, IFNγ and cell viability was measured by flow cytometry 16 hrs after stimulation with Staphylococcal enterotoxin B (SEB). (C) Cd36 +/+ or Cd36 -/- CD8 + TILs isolated from B16 tumors 21 days post implantation were purified by FACS and treated with either vehicle control (Ctrl) or oxLDL (50 μg/ml) for 24 hrs. TNF and IFNγ was measured by flow cytometry 4 hrs after stimulation with PMA/ionomycin. Data shown are mean± SEM and statistical tests were performed by two-tailed unpaired Student’s t-test (A, C), and two-tailed paired Student’s t-test (B), **p
Figure Legend Snippet: OxLDL inhibits CD8 + T cell function in a CD36-dependent manner (A) P14 CD8 + T cells were activated in vitro with gp33 peptide plus IL-2 for 24 hrs and then treated with either vehicle control (Ctrl), OxLDL (50 μg/ml), LDL (50 μg/ml), HDL (50 μg/ml), SSO (100 μM), or the combination of OxLDL (50 μg/ml) and SSO (100 μM), for another 48 hrs. TNF, IFNγ and cell viability were then measured upon re-stimulation with gp33 for 6 hours and analyzed by flow cytometry. (B) Human PBMCs were treated with either vehicle control (Ctrl), OxLDL (50 μg/ml), or the combination of OxLDL (50 μg/ml) and SSO (100 μM). TNF, IFNγ and cell viability was measured by flow cytometry 16 hrs after stimulation with Staphylococcal enterotoxin B (SEB). (C) Cd36 +/+ or Cd36 -/- CD8 + TILs isolated from B16 tumors 21 days post implantation were purified by FACS and treated with either vehicle control (Ctrl) or oxLDL (50 μg/ml) for 24 hrs. TNF and IFNγ was measured by flow cytometry 4 hrs after stimulation with PMA/ionomycin. Data shown are mean± SEM and statistical tests were performed by two-tailed unpaired Student’s t-test (A, C), and two-tailed paired Student’s t-test (B), **p

Techniques Used: Cell Function Assay, In Vitro, Flow Cytometry, Isolation, Purification, FACS, Two Tailed Test

19) Product Images from "Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation"

Article Title: Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20150388

Nur77-GFP reporter expression is induced by IgE/Ag and enhanced by coengagement of Tim-3. BMMCs were generated from Nur77 GFP reporter mice, sensitized with IgE overnight, and stimulated with Ag. In A, Ag (DNP 32 -HSA) was titrated over a range of 10–500 ng/ml. PMA plus ionomycin stimulation was used as a positive control. GFP expression was determined after stimulation for 6 h. (B) Nur77 GFP BMMCs were stimulated with a fixed concentration of Ag, with or without Tim-3 pAb for 6 h. (C) Nur77 GFP BMMCs were stimulated with IgE/Ag and either Tim-3 pAb or Tim4-Fc (which binds to Tim-1), or appropriate isotype controls. (D) Nur77 GFP BMMCs were stimulated with IgE/Ag plus either inhibitor to Src kinases (PP2) or Syk (BAY). (E) Nur77 GFP BMMCs were stimulated with IgE/Ag and the indicated inhibitors to MEK (UO126), Akt (Akt i 1/2), calcineurin (CsA), PI3K (LY294002), or PKC (BIM). GFP signal was quantified by flow cytometry. Results are the average of three independent experiments performed in duplicate. *, P
Figure Legend Snippet: Nur77-GFP reporter expression is induced by IgE/Ag and enhanced by coengagement of Tim-3. BMMCs were generated from Nur77 GFP reporter mice, sensitized with IgE overnight, and stimulated with Ag. In A, Ag (DNP 32 -HSA) was titrated over a range of 10–500 ng/ml. PMA plus ionomycin stimulation was used as a positive control. GFP expression was determined after stimulation for 6 h. (B) Nur77 GFP BMMCs were stimulated with a fixed concentration of Ag, with or without Tim-3 pAb for 6 h. (C) Nur77 GFP BMMCs were stimulated with IgE/Ag and either Tim-3 pAb or Tim4-Fc (which binds to Tim-1), or appropriate isotype controls. (D) Nur77 GFP BMMCs were stimulated with IgE/Ag plus either inhibitor to Src kinases (PP2) or Syk (BAY). (E) Nur77 GFP BMMCs were stimulated with IgE/Ag and the indicated inhibitors to MEK (UO126), Akt (Akt i 1/2), calcineurin (CsA), PI3K (LY294002), or PKC (BIM). GFP signal was quantified by flow cytometry. Results are the average of three independent experiments performed in duplicate. *, P

Techniques Used: Expressing, Generated, Mouse Assay, Positive Control, Concentration Assay, Flow Cytometry, Cytometry

20) Product Images from "Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation"

Article Title: Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation

Journal: Scientific Reports

doi: 10.1038/s41598-018-26088-y

NTG-A-009 reduces Th1, Th17 cells differentiation in vitro . Naïve CD4 + T cells isolated from spleen and lymph nodes were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) under Th1, Th17 and T reg inducing conditions with NTG-A-009 (1 μM), Tofacitinib (1 μM) or Triamcinolone (1 µM). ( a ) Chemical structure of NTG-A-009. ( b,c ) The percentage of IFN-γ + Th1, IL-17A + Th17 and FoxP3 + T reg cells was determined by flow cytometry. ( d ) Different concentration of NTG-A-009 was used to find its effect in Th1 and Th17 cells in anti-CD3 antibody coating system. ( e,f ) The antigen specific differentiation of Th1, Th17 and T reg cells was done by the isolation of CD4 + T cells and irradiated antigen presenting cells from 6–10 weeks OTII mice and stimulated under Th1, Th17 and T reg polarizing conditions in the presence of OVA 323–339 (0.1 μM) and incubated for 72 hour for Th1 and Th17 and 96 hours for T reg cells differentiation. Cells were then restimulated with PMA, ionomycin and golgistop and analyzed by FACS through intracellular staining. ( g ) Different doses of NTG-A-009 were cultured under Th1, Th17 condition with the cells from OT-II transgenic mice. Data were quantified in bar diagram. Data represent three independent experiments. Mean ± SEM of the triplicates are shown. ***P
Figure Legend Snippet: NTG-A-009 reduces Th1, Th17 cells differentiation in vitro . Naïve CD4 + T cells isolated from spleen and lymph nodes were stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) under Th1, Th17 and T reg inducing conditions with NTG-A-009 (1 μM), Tofacitinib (1 μM) or Triamcinolone (1 µM). ( a ) Chemical structure of NTG-A-009. ( b,c ) The percentage of IFN-γ + Th1, IL-17A + Th17 and FoxP3 + T reg cells was determined by flow cytometry. ( d ) Different concentration of NTG-A-009 was used to find its effect in Th1 and Th17 cells in anti-CD3 antibody coating system. ( e,f ) The antigen specific differentiation of Th1, Th17 and T reg cells was done by the isolation of CD4 + T cells and irradiated antigen presenting cells from 6–10 weeks OTII mice and stimulated under Th1, Th17 and T reg polarizing conditions in the presence of OVA 323–339 (0.1 μM) and incubated for 72 hour for Th1 and Th17 and 96 hours for T reg cells differentiation. Cells were then restimulated with PMA, ionomycin and golgistop and analyzed by FACS through intracellular staining. ( g ) Different doses of NTG-A-009 were cultured under Th1, Th17 condition with the cells from OT-II transgenic mice. Data were quantified in bar diagram. Data represent three independent experiments. Mean ± SEM of the triplicates are shown. ***P

Techniques Used: In Vitro, Isolation, Flow Cytometry, Cytometry, Concentration Assay, Irradiation, Mouse Assay, Incubation, FACS, Staining, Cell Culture, Transgenic Assay

NTG-A-009 inhibits Th1 and Th17 cells in vivo and attenuate DSS induced colitis. C57BL/6 mice were orally administered with 2.5% (w/v) DSS in drinking water for 7 days to induce colitis. NTG-A-009 was orally administered every day (n = 6 mice in each group). ( a ) Body weight of water, DSS and NTG-A-009 treated mice was recorded daily and presented as percentage of original body weight. ( b ) Colon length of mice treated with water, DSS and NTG-A-009. ( c ) CD4 + and CD8 + T cells infiltration was detected from the Lymphocytes isolated from colonic lamina propria of water, DSS and NTG-A-009 treated group and analyzed by FACS. ( d ) The percentage of Th1 and Th17 cells was determined by the stimulation of splenocytes from each group with PMA, ionomycin and golgistop for 4 hour and analyzed by FACS through intracellular staining of IFN-γ and IL-17. ( e ) H E staining of colonic section with histological score (magnification 20X). Data represent three independent experiments. Two-way anova (A) or *P
Figure Legend Snippet: NTG-A-009 inhibits Th1 and Th17 cells in vivo and attenuate DSS induced colitis. C57BL/6 mice were orally administered with 2.5% (w/v) DSS in drinking water for 7 days to induce colitis. NTG-A-009 was orally administered every day (n = 6 mice in each group). ( a ) Body weight of water, DSS and NTG-A-009 treated mice was recorded daily and presented as percentage of original body weight. ( b ) Colon length of mice treated with water, DSS and NTG-A-009. ( c ) CD4 + and CD8 + T cells infiltration was detected from the Lymphocytes isolated from colonic lamina propria of water, DSS and NTG-A-009 treated group and analyzed by FACS. ( d ) The percentage of Th1 and Th17 cells was determined by the stimulation of splenocytes from each group with PMA, ionomycin and golgistop for 4 hour and analyzed by FACS through intracellular staining of IFN-γ and IL-17. ( e ) H E staining of colonic section with histological score (magnification 20X). Data represent three independent experiments. Two-way anova (A) or *P

Techniques Used: In Vivo, Mouse Assay, Isolation, FACS, Staining

21) Product Images from "IRF4 Couples Anabolic Metabolism to Th1 Cell Fate Determination"

Article Title: IRF4 Couples Anabolic Metabolism to Th1 Cell Fate Determination

Journal: ImmunoHorizons

doi: 10.4049/immunohorizons.1700012

IRF4 is required for efficient Th1 CD4 + effector cell differentiation ( A ) CD4 + T cells were labeled with CPD, stimulated with anti-CD3/CD28 + IL-2/IL-12, and restimulated with PMA/ionomycin on day 4, followed by FACS analysis. Representative FACS plots of indicated protein versus CPD (left panels). Quantification of the percent positive population in WT and IRF4-KO CD4 + T cells (right panels). ( B ) Representative FACS plots of TCF1 expression versus CPD after 4 d of activation in Th1-inducing conditions (top left and middle panels). Quantification of TCF1-low population in WT and IRF4-KO CD4 + T cells (top right panel). Representative line graphs of T-bet, CD25, and CD62L staining of CD4 + T cells (middle row). Quantification of protein expression in WT and IRF4-KO CD4 + T cells (bottom row). ( C ) Increased T-bet protein expression in WT and IRF4-KO CD4 + T cells following transduction with T-bet–GFP retrovirus (open graph) compared with untransduced cells (shaded graph) at day 4 postactivation (left panels). Expression of TCF1 (upper right panels) and TNF-α (lower right panels) by CD4 + T cells transduced (GFP + ) or untransduced (GFP − ) with T-bet–GFP retrovirus, at day 4 postactivation. Plots are representative of three independent experiments. * p
Figure Legend Snippet: IRF4 is required for efficient Th1 CD4 + effector cell differentiation ( A ) CD4 + T cells were labeled with CPD, stimulated with anti-CD3/CD28 + IL-2/IL-12, and restimulated with PMA/ionomycin on day 4, followed by FACS analysis. Representative FACS plots of indicated protein versus CPD (left panels). Quantification of the percent positive population in WT and IRF4-KO CD4 + T cells (right panels). ( B ) Representative FACS plots of TCF1 expression versus CPD after 4 d of activation in Th1-inducing conditions (top left and middle panels). Quantification of TCF1-low population in WT and IRF4-KO CD4 + T cells (top right panel). Representative line graphs of T-bet, CD25, and CD62L staining of CD4 + T cells (middle row). Quantification of protein expression in WT and IRF4-KO CD4 + T cells (bottom row). ( C ) Increased T-bet protein expression in WT and IRF4-KO CD4 + T cells following transduction with T-bet–GFP retrovirus (open graph) compared with untransduced cells (shaded graph) at day 4 postactivation (left panels). Expression of TCF1 (upper right panels) and TNF-α (lower right panels) by CD4 + T cells transduced (GFP + ) or untransduced (GFP − ) with T-bet–GFP retrovirus, at day 4 postactivation. Plots are representative of three independent experiments. * p

Techniques Used: Cell Differentiation, Labeling, FACS, Expressing, Activation Assay, Staining, Transduction

22) Product Images from "Conditions for the generation of cytotoxic CD4+ Th cells that enhance CD8+ CTL-mediated tumor regression"

Article Title: Conditions for the generation of cytotoxic CD4+ Th cells that enhance CD8+ CTL-mediated tumor regression

Journal: Clinical & Translational Immunology

doi: 10.1038/cti.2016.46

In vitro expanded CD4 + Th cells predominantly express Th1-cytokines, and exhibit antigen-specific cytotoxicity in vivo . Naive CD4 + OT-II cells were stimulated with DC-OVA 323-339, and expanded with IL-2 and IL-7 in A-DMEM/F12. The cells were restimulated with unpulsed DC, DC-OVA 323-339 or PMA/Ionomycin in the presence of Brefeldin A, or left untreated for 5 h at 37 °C/5% CO 2 on day 10 ( a ) and day 20 ( b ). Intracellular expression of T-cell cytokines was measured by intracellular staining and analyzed by flow cytometry. Naive C57BL/6-recipient mice received an i.v. injection of PBS or in vitro expanded CD4 + Th cells. Donor splenocytes consisted of equal numbers of OVA 323-339 -pulsed/CFSE Hi and unpulsed /CFSE Lo cells and were i.v. injected into all recipient mice 24 h post T-cell transfer. The mice were killed 40 h post-target cell injection, and splenocytes were isolated for flow cytometric analysis of specific lysis of the target cell population ( c ). Lysis of MHC-II + cells by CD4 + Th cells expanded with or without anti-IL-4 ( d ); and stimulated with different concentrations of OVA 323–339 -pulsed immature DC or LPS-matured DC ( e ) was measured. Statistical analysis was performed with one-way analysis of variance with Bonferroni's multiple comparison test, *** P
Figure Legend Snippet: In vitro expanded CD4 + Th cells predominantly express Th1-cytokines, and exhibit antigen-specific cytotoxicity in vivo . Naive CD4 + OT-II cells were stimulated with DC-OVA 323-339, and expanded with IL-2 and IL-7 in A-DMEM/F12. The cells were restimulated with unpulsed DC, DC-OVA 323-339 or PMA/Ionomycin in the presence of Brefeldin A, or left untreated for 5 h at 37 °C/5% CO 2 on day 10 ( a ) and day 20 ( b ). Intracellular expression of T-cell cytokines was measured by intracellular staining and analyzed by flow cytometry. Naive C57BL/6-recipient mice received an i.v. injection of PBS or in vitro expanded CD4 + Th cells. Donor splenocytes consisted of equal numbers of OVA 323-339 -pulsed/CFSE Hi and unpulsed /CFSE Lo cells and were i.v. injected into all recipient mice 24 h post T-cell transfer. The mice were killed 40 h post-target cell injection, and splenocytes were isolated for flow cytometric analysis of specific lysis of the target cell population ( c ). Lysis of MHC-II + cells by CD4 + Th cells expanded with or without anti-IL-4 ( d ); and stimulated with different concentrations of OVA 323–339 -pulsed immature DC or LPS-matured DC ( e ) was measured. Statistical analysis was performed with one-way analysis of variance with Bonferroni's multiple comparison test, *** P

Techniques Used: In Vitro, In Vivo, Expressing, Staining, Flow Cytometry, Cytometry, Mouse Assay, Injection, Isolation, Lysis

23) Product Images from "Estrogen receptor α/HDAC/NFAT axis for delphinidin effects on proliferation and differentiation of T lymphocytes from patients with cardiovascular risks"

Article Title: Estrogen receptor α/HDAC/NFAT axis for delphinidin effects on proliferation and differentiation of T lymphocytes from patients with cardiovascular risks

Journal: Scientific Reports

doi: 10.1038/s41598-017-09933-4

Effect of delphinidin on the differentiation of T lymphocytes from healthy subjects. ( A ) Cells were stimulated for 24 h with 10 −2 g/L of delphinidin (Del), 5 µg/mL of PHA or both and stained for T-bet, GATA3, RORγt and FOXP3 transcription factors. Histograms show the percentage of positive cells for T-bet, GATA3, RORγt and FOXP3 expressions. Data are the mean ± SEM ( n = 7). ( B ) T cells were stimulated for 5 h with 10 −2 g/L of delphinidin (Del), 50 ng/mL phorbol-12-myristate-13-acetate (PMA) plus 1 µg/mL ionomycin (Ion) or both, in the presence of 5 µg/mL brefeldin A (BFA) for the last 3 h of culture and stained for IL-2, IFNγ, IL-4, IL-17A and IL-10 cytokines. Histograms show the MFI of positive cells for IL-2, IFNγ, IL-4, IL-17A, and IL-10, respectively. Data are the mean ± SEM ( n = 7). * P
Figure Legend Snippet: Effect of delphinidin on the differentiation of T lymphocytes from healthy subjects. ( A ) Cells were stimulated for 24 h with 10 −2 g/L of delphinidin (Del), 5 µg/mL of PHA or both and stained for T-bet, GATA3, RORγt and FOXP3 transcription factors. Histograms show the percentage of positive cells for T-bet, GATA3, RORγt and FOXP3 expressions. Data are the mean ± SEM ( n = 7). ( B ) T cells were stimulated for 5 h with 10 −2 g/L of delphinidin (Del), 50 ng/mL phorbol-12-myristate-13-acetate (PMA) plus 1 µg/mL ionomycin (Ion) or both, in the presence of 5 µg/mL brefeldin A (BFA) for the last 3 h of culture and stained for IL-2, IFNγ, IL-4, IL-17A and IL-10 cytokines. Histograms show the MFI of positive cells for IL-2, IFNγ, IL-4, IL-17A, and IL-10, respectively. Data are the mean ± SEM ( n = 7). * P

Techniques Used: Staining

Effects of delphinidin on proliferation, cell cycle progression and ERK1/2 pathway activation of T lymphocytes from healthy subjects. Histograms show the percentage of proliferation of cells exposed to T cell activators [10 µg/mL anti-CD3 plus 5 µg/mL anti-CD28, 5 µg/mL PHA or 10 ng/mL PMA plus 1 µM ionomycin (Ion)] in absence or in presence of 10 −2 g/L of delphinidin (Del) for 24 h ( A ) or 48 h ( B ). Data are the mean ± SEM ( n = 5–10). * P
Figure Legend Snippet: Effects of delphinidin on proliferation, cell cycle progression and ERK1/2 pathway activation of T lymphocytes from healthy subjects. Histograms show the percentage of proliferation of cells exposed to T cell activators [10 µg/mL anti-CD3 plus 5 µg/mL anti-CD28, 5 µg/mL PHA or 10 ng/mL PMA plus 1 µM ionomycin (Ion)] in absence or in presence of 10 −2 g/L of delphinidin (Del) for 24 h ( A ) or 48 h ( B ). Data are the mean ± SEM ( n = 5–10). * P

Techniques Used: Activation Assay

24) Product Images from "Immune-checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis"

Article Title: Immune-checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis

Journal: Scientific Reports

doi: 10.1038/s41598-017-01411-1

VISTA suppressed the production of IL-17A by γδ T cells and CD4 + Th17 cells following IMQ treatment. WT and Vsir −/− mice were topically treated on ears with 3.5% IMQ cream daily for 3 days. Cells from ear skin and draining LN were harvested and stimulated in vitro with PMA and Ionomycin. The number of γδ T cells and CD4 + T cells, and their expression of cytokines (IL-17A, IFN-γ, and TNF-α) were examined by surface and intracellular staining, and quantified by flow cytometry. Total cell number of recovered γδ T cells and CD4 + T cells from ear tissue and draining LN are shown ( a ). The percentages of γδ T cells ( b ) and CD4 + T cells ( c ) expressing cytokines IL-17A, IFN-γ, and TNF-α were examined by flow cytometry and are shown as mean ± SEM. Representative flow plots and representative data from three independent experiments are shown.
Figure Legend Snippet: VISTA suppressed the production of IL-17A by γδ T cells and CD4 + Th17 cells following IMQ treatment. WT and Vsir −/− mice were topically treated on ears with 3.5% IMQ cream daily for 3 days. Cells from ear skin and draining LN were harvested and stimulated in vitro with PMA and Ionomycin. The number of γδ T cells and CD4 + T cells, and their expression of cytokines (IL-17A, IFN-γ, and TNF-α) were examined by surface and intracellular staining, and quantified by flow cytometry. Total cell number of recovered γδ T cells and CD4 + T cells from ear tissue and draining LN are shown ( a ). The percentages of γδ T cells ( b ) and CD4 + T cells ( c ) expressing cytokines IL-17A, IFN-γ, and TNF-α were examined by flow cytometry and are shown as mean ± SEM. Representative flow plots and representative data from three independent experiments are shown.

Techniques Used: Mouse Assay, In Vitro, Expressing, Staining, Flow Cytometry, Cytometry

VISTA negatively regulates IMQ-induced activation of DCs and the production of IL-23. WT and Vsir −/− mice were treated on ears with 3.5% IMQ for 3 days. Cells from ear tissues and the ear-draining cervical LNs were harvested. Cells were stimulated with PMA and Ionomycin in vitro for 3 hrs. The expression of IL-23p19 in CD11c + DCs was examined by flow cytometry. The percentages of IL-23p19-expressing DCs were quantified and shown as mean ± SEM (n = 6) in ( a ). The number of total CD11c + DCs from ear tissue and draining LN is shown as mean ± SEM (n = 5) in ( b ). To determine whether ectopic expression of VISTA suppresses TLR7-induced IL-23 production, Vsir −/− BM-derived DC were transduced with lentivirus expressing full-length (FL), or mutant VISTA lacking the cytoplasmic tail (deltaC), or GFP control protein. After culture with GM-CSF (20 ng/ml) for 7 days, cells were stimulated with R848 (5 μg/ml) for 7 hrs. Culture supernatant was harvested and secreted IL-23p19/p40 was examined by ELISA ( c ). To examine TLR7 signaling in DCs, CD11c + DCs were purified from the spleens of naïve Rag1 −/− and Vsir −/− Rag1 −/− mice, and stimulated with R848 (5 μg/ml) for indicated amount of time. Total cell lysates were prepared and examined for the levels of phosphorylated Erk1/2 and Jnk1/2 by western blotting ( d ). The ratio of phosphorylated versus total Erk1/2 and Jnk1/2 was calculated based on the total protein level from the same lysate run on a parallel gel ( d ). The activation of NF-κB signaling was examined by western blotting the level of phosphorylated and total IκB, as well as phosphorylated and total NF-κB p65 subunit ( e ). To determine whether Erk and Jnk were required for the production of IL-23, Splenic DCs were isolated from naïve WT and Vsir −/− mice, and stimulated with R848 (5 μg/ml) in the presence of Erk1/2 inhibitor (SCH772984, 10 μM), or Jnk1/2 inhibitor (SP600125, 10 μM), or DMSO solvent control for overnight. Culture supernatant was collected and secreted IL-23p19/p40 was quantified by ELISA. Values from triplicated cultures are shown as mean ± SEM in ( f ). Representative results from two to three independent experiments were shown.
Figure Legend Snippet: VISTA negatively regulates IMQ-induced activation of DCs and the production of IL-23. WT and Vsir −/− mice were treated on ears with 3.5% IMQ for 3 days. Cells from ear tissues and the ear-draining cervical LNs were harvested. Cells were stimulated with PMA and Ionomycin in vitro for 3 hrs. The expression of IL-23p19 in CD11c + DCs was examined by flow cytometry. The percentages of IL-23p19-expressing DCs were quantified and shown as mean ± SEM (n = 6) in ( a ). The number of total CD11c + DCs from ear tissue and draining LN is shown as mean ± SEM (n = 5) in ( b ). To determine whether ectopic expression of VISTA suppresses TLR7-induced IL-23 production, Vsir −/− BM-derived DC were transduced with lentivirus expressing full-length (FL), or mutant VISTA lacking the cytoplasmic tail (deltaC), or GFP control protein. After culture with GM-CSF (20 ng/ml) for 7 days, cells were stimulated with R848 (5 μg/ml) for 7 hrs. Culture supernatant was harvested and secreted IL-23p19/p40 was examined by ELISA ( c ). To examine TLR7 signaling in DCs, CD11c + DCs were purified from the spleens of naïve Rag1 −/− and Vsir −/− Rag1 −/− mice, and stimulated with R848 (5 μg/ml) for indicated amount of time. Total cell lysates were prepared and examined for the levels of phosphorylated Erk1/2 and Jnk1/2 by western blotting ( d ). The ratio of phosphorylated versus total Erk1/2 and Jnk1/2 was calculated based on the total protein level from the same lysate run on a parallel gel ( d ). The activation of NF-κB signaling was examined by western blotting the level of phosphorylated and total IκB, as well as phosphorylated and total NF-κB p65 subunit ( e ). To determine whether Erk and Jnk were required for the production of IL-23, Splenic DCs were isolated from naïve WT and Vsir −/− mice, and stimulated with R848 (5 μg/ml) in the presence of Erk1/2 inhibitor (SCH772984, 10 μM), or Jnk1/2 inhibitor (SP600125, 10 μM), or DMSO solvent control for overnight. Culture supernatant was collected and secreted IL-23p19/p40 was quantified by ELISA. Values from triplicated cultures are shown as mean ± SEM in ( f ). Representative results from two to three independent experiments were shown.

Techniques Used: Activation Assay, Mouse Assay, In Vitro, Expressing, Flow Cytometry, Cytometry, Derivative Assay, Transduction, Mutagenesis, Enzyme-linked Immunosorbent Assay, Purification, Western Blot, Isolation

25) Product Images from "β7 Integrin Inhibition Can Increase Intestinal Inflammation by Impairing Homing of CD25hiFoxP3+ Regulatory T Cells"

Article Title: β7 Integrin Inhibition Can Increase Intestinal Inflammation by Impairing Homing of CD25hiFoxP3+ Regulatory T Cells

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2019.10.012

β7-deficient Tregs show normal suppression in vitro. ( A and B ) Treg suppression function. Tregs isolated from CD45.2 congenic Itgb7 +/+ Foxp3 GFP (WT Treg) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- Treg) mice were mixed with responder cells at the indicated Treg/responder cell ratios. Responder cells are CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 - conventional T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL2. ( A ) CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry at 72 hours. ( B ) The proliferation index was determined by FlowJo software. ( C ) Intracellular expression of IL10 and TGFβ1 of GFP + Tregs from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Splenocytes were stimulated ex vivo with phorbol myristate acetate and ionomycin in the presence of monensin (for IL10) or brefeldin A (for TGF-β1) for 4 hours at 37°C. Cells were fixed and permeabilized before staining (n = 3). Data represent means ± SEM. Two-tailed t test. ( D ) In vivo competitive homing of Tregs to different lymphoid tissues. GFP + Tregs were sorted from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Lymphoid organs were isolated 3 hours after injection of Tregs before flow cytometry analysis. The ratio of Itgb7 -/- Tregs to Itgb7 +/+ Tregs ( Itgb7 -/- /WT) within various lymphoid tissues is shown ( n = 15). Data represent means ± SEM. One-way analysis of variance with the Bonferroni posttest. WT Tregs, Tregs from Itgb7 +/+ Foxp3 GFP mice; Itgb7 -/- Tregs, Tregs from Itgb7 -/- Foxp3 GFP mice. MFI, mean fluorescence intensity.
Figure Legend Snippet: β7-deficient Tregs show normal suppression in vitro. ( A and B ) Treg suppression function. Tregs isolated from CD45.2 congenic Itgb7 +/+ Foxp3 GFP (WT Treg) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- Treg) mice were mixed with responder cells at the indicated Treg/responder cell ratios. Responder cells are CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 - conventional T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL2. ( A ) CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry at 72 hours. ( B ) The proliferation index was determined by FlowJo software. ( C ) Intracellular expression of IL10 and TGFβ1 of GFP + Tregs from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Splenocytes were stimulated ex vivo with phorbol myristate acetate and ionomycin in the presence of monensin (for IL10) or brefeldin A (for TGF-β1) for 4 hours at 37°C. Cells were fixed and permeabilized before staining (n = 3). Data represent means ± SEM. Two-tailed t test. ( D ) In vivo competitive homing of Tregs to different lymphoid tissues. GFP + Tregs were sorted from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Lymphoid organs were isolated 3 hours after injection of Tregs before flow cytometry analysis. The ratio of Itgb7 -/- Tregs to Itgb7 +/+ Tregs ( Itgb7 -/- /WT) within various lymphoid tissues is shown ( n = 15). Data represent means ± SEM. One-way analysis of variance with the Bonferroni posttest. WT Tregs, Tregs from Itgb7 +/+ Foxp3 GFP mice; Itgb7 -/- Tregs, Tregs from Itgb7 -/- Foxp3 GFP mice. MFI, mean fluorescence intensity.

Techniques Used: In Vitro, Isolation, Mouse Assay, Labeling, Flow Cytometry, Software, Expressing, Ex Vivo, Staining, Two Tailed Test, In Vivo, Injection, Fluorescence

26) Product Images from "Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms"

Article Title: Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aan8292

Neutrophils derived from patients with MPNs are associated with an increase in NET formation and a prothrombotic, NET-rich phenotype (A) NET formation in patients with myeloproliferative neoplasms (MPN) (receiving a JAK inhibitor n=5, receiving other therapy n=14) compared to healthy controls (n=11) when stimulated with 4 μM ionomycin (IO) or DMSO for 2 hours. Patients receiving a JAK inhibitor are indicated by JAKI. Data shown as individual values and medians. (B) Representative immunofluorescence images of human neutrophils after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue and citrullinated histone H3 (H3 cit ) in green. Scale bar=50 μm. (C) The percentages of human neutrophils with evidence of NET formation after stimulation with PMA (10 nM) with and without ruxolitinib pre-treatment (n=4). Neutrophils are derived from controls. (D) Representative images of human neutrophils from healthy controls stimulated with PMA (10 nM) after 150 minutes of ex vivo pre-treatment with DMSO, ruxolitinib (300 nM), or GSK484 (PAD4 inhibitor,10 μM). Scale bar=50 μm. (E) Lung tissue sections from mice expressing the Jak2 V617F mutation as compared to Jak2 WT mice. Scale bar=200 μm. (F) Characterization of clot content in the lungs of Jak2 V617 mice. Hematoxylin and eosin (H E) stain. Scale bar=50 μm. VWF – Von Willebrand factor. (G) Lung tissue sections from mice expressing the Jak2 V617F mutation as compared to Jak2 WT mice. Neutrophil infiltration and NETs are shown by neutrophil-specific Ly6G (red) and H3 cit (green), respectively. Scale bar=100 μm. (H) The percentages of mouse neutrophils with evidence of NET formation by morphological criteria (left) (n=9 for all genotype/treatment combinations) or H3 cit positive staining (right) (n=6 for all genotype/treatment combinations) grouped by genotype after stimulation with 4 μM IO or DMSO for 2 hours. (I) Representative immunofluorescence images of mouse neutrophils derived from Jak2 WT and Jak2 V617F mice after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue and H3 cit in green. Scale bar=50 μm.
Figure Legend Snippet: Neutrophils derived from patients with MPNs are associated with an increase in NET formation and a prothrombotic, NET-rich phenotype (A) NET formation in patients with myeloproliferative neoplasms (MPN) (receiving a JAK inhibitor n=5, receiving other therapy n=14) compared to healthy controls (n=11) when stimulated with 4 μM ionomycin (IO) or DMSO for 2 hours. Patients receiving a JAK inhibitor are indicated by JAKI. Data shown as individual values and medians. (B) Representative immunofluorescence images of human neutrophils after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue and citrullinated histone H3 (H3 cit ) in green. Scale bar=50 μm. (C) The percentages of human neutrophils with evidence of NET formation after stimulation with PMA (10 nM) with and without ruxolitinib pre-treatment (n=4). Neutrophils are derived from controls. (D) Representative images of human neutrophils from healthy controls stimulated with PMA (10 nM) after 150 minutes of ex vivo pre-treatment with DMSO, ruxolitinib (300 nM), or GSK484 (PAD4 inhibitor,10 μM). Scale bar=50 μm. (E) Lung tissue sections from mice expressing the Jak2 V617F mutation as compared to Jak2 WT mice. Scale bar=200 μm. (F) Characterization of clot content in the lungs of Jak2 V617 mice. Hematoxylin and eosin (H E) stain. Scale bar=50 μm. VWF – Von Willebrand factor. (G) Lung tissue sections from mice expressing the Jak2 V617F mutation as compared to Jak2 WT mice. Neutrophil infiltration and NETs are shown by neutrophil-specific Ly6G (red) and H3 cit (green), respectively. Scale bar=100 μm. (H) The percentages of mouse neutrophils with evidence of NET formation by morphological criteria (left) (n=9 for all genotype/treatment combinations) or H3 cit positive staining (right) (n=6 for all genotype/treatment combinations) grouped by genotype after stimulation with 4 μM IO or DMSO for 2 hours. (I) Representative immunofluorescence images of mouse neutrophils derived from Jak2 WT and Jak2 V617F mice after stimulation with 4 μM IO or DMSO for 2 hours. DAPI is shown in blue and H3 cit in green. Scale bar=50 μm.

Techniques Used: Derivative Assay, Immunofluorescence, Ex Vivo, Mouse Assay, Expressing, Mutagenesis, H&E Stain, Staining

27) Product Images from "Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression"

Article Title: Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01617

CD49d high CD4 + T cells rapidly secreted Th1 cytokines. (A,B) Representative flow cytometric plots of intracellular staining for various cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-17, and IL-21). (A) Isolated peritoneal cells and (B) PB mononuclear cells were stimulated with PMA and ionomycin for 4 h. Cytokine expression in CD49d high CD4 + and CD49d low CD4 + T cells was analyzed by flow cytometry. Data are representative of three independent experiments (left panel). Proportions of TNF-α, IFN-γ, IL-2, and IL-21 positive CD4 + T cells in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white) in the (A) PEC and (B) blood (right panel). (C) CD49d high CD4 + and CD49d low CD4 + T cells isolated from the PEC or PB were sorted and labeled with CTV, then stimulated for 72 h with immobilized anti-CD3 and anti-CD28 antibodies. The proliferation of CD49d high CD4 + T cells (solid lines) and CD49d low CD4 + T cells (gray filled) is shown, along with total mitotic events in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white). Data are expressed as the mean ± SEM ( n = 3 donors per each group). * P
Figure Legend Snippet: CD49d high CD4 + T cells rapidly secreted Th1 cytokines. (A,B) Representative flow cytometric plots of intracellular staining for various cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-17, and IL-21). (A) Isolated peritoneal cells and (B) PB mononuclear cells were stimulated with PMA and ionomycin for 4 h. Cytokine expression in CD49d high CD4 + and CD49d low CD4 + T cells was analyzed by flow cytometry. Data are representative of three independent experiments (left panel). Proportions of TNF-α, IFN-γ, IL-2, and IL-21 positive CD4 + T cells in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white) in the (A) PEC and (B) blood (right panel). (C) CD49d high CD4 + and CD49d low CD4 + T cells isolated from the PEC or PB were sorted and labeled with CTV, then stimulated for 72 h with immobilized anti-CD3 and anti-CD28 antibodies. The proliferation of CD49d high CD4 + T cells (solid lines) and CD49d low CD4 + T cells (gray filled) is shown, along with total mitotic events in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white). Data are expressed as the mean ± SEM ( n = 3 donors per each group). * P

Techniques Used: Flow Cytometry, Staining, Isolation, Expressing, Cytometry, Labeling

28) Product Images from "A Novel, Rapid Method to Quantify Intraplatelet Calcium Dynamics by Ratiometric Flow Cytometry"

Article Title: A Novel, Rapid Method to Quantify Intraplatelet Calcium Dynamics by Ratiometric Flow Cytometry

Journal: PLoS ONE

doi: 10.1371/journal.pone.0122527

Overview of the test procedure. (A) PRP or (B) citrated whole blood can be used for the assay. Whole blood has to be incubated with anti-CD61 antibody to allow detection of platelets. 300μl of the respective cell suspension is incubated with 1μl Fluo-4 (1mg/ml) and 2 μl Fura Red (1mg/ml) for 15 min at 37°C and 5% CO 2 at dark. 10 μl of cell suspension are then transferred into FACS tubes containing 490μl calibration buffer A (10 mM CaEGTA, 3 μg/ml ionomycin, 2 μg/ml nigericin and 10 μM CCCP) or calibration buffer B (10 mM K 2 EGTA, 3 μg/ml ionomycin, 2 μg/ml nigericin and 10 μM CCCP), respectively. 10 μl PRP or whole blood diluted in 490 μl HEPES-Tyrode buffer can then be used for each sample analysis. Basal levels of [Ca 2+ ] i should be recorded for approximately 50 seconds, followed by the addition of the agonist and further recording for changes in FL1 and FL3 over at least a further 100 seconds. This will yield in FL1/FL3 ratios over time (r i ), which can be calculated e.g. by FlowJo and allows calculation of absolute [Ca 2+ ] i levels by the depicted algorism in e.g. Sigma Plot given that time data are exported in the 1st column and FL1/FL3 ratios into the 2nd column. Blue values (R min and FL3 max ) are obtained by calibration in Ca 2+ free buffer, green values (R max and FL3 min ) are obtained by measuring maximal calcium concentrations, golden values (r, i) are obtained from the actual sample measured and red values (kd1 and kd2) represent values that have been determined within this manuscript.
Figure Legend Snippet: Overview of the test procedure. (A) PRP or (B) citrated whole blood can be used for the assay. Whole blood has to be incubated with anti-CD61 antibody to allow detection of platelets. 300μl of the respective cell suspension is incubated with 1μl Fluo-4 (1mg/ml) and 2 μl Fura Red (1mg/ml) for 15 min at 37°C and 5% CO 2 at dark. 10 μl of cell suspension are then transferred into FACS tubes containing 490μl calibration buffer A (10 mM CaEGTA, 3 μg/ml ionomycin, 2 μg/ml nigericin and 10 μM CCCP) or calibration buffer B (10 mM K 2 EGTA, 3 μg/ml ionomycin, 2 μg/ml nigericin and 10 μM CCCP), respectively. 10 μl PRP or whole blood diluted in 490 μl HEPES-Tyrode buffer can then be used for each sample analysis. Basal levels of [Ca 2+ ] i should be recorded for approximately 50 seconds, followed by the addition of the agonist and further recording for changes in FL1 and FL3 over at least a further 100 seconds. This will yield in FL1/FL3 ratios over time (r i ), which can be calculated e.g. by FlowJo and allows calculation of absolute [Ca 2+ ] i levels by the depicted algorism in e.g. Sigma Plot given that time data are exported in the 1st column and FL1/FL3 ratios into the 2nd column. Blue values (R min and FL3 max ) are obtained by calibration in Ca 2+ free buffer, green values (R max and FL3 min ) are obtained by measuring maximal calcium concentrations, golden values (r, i) are obtained from the actual sample measured and red values (kd1 and kd2) represent values that have been determined within this manuscript.

Techniques Used: Incubation, FACS

29) Product Images from "PTEN drives Th17 cell differentiation by preventing IL-2 production"

Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20170523

γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P
Figure Legend Snippet: γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P

Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry

30) Product Images from "A rare subset of skin-tropic regulatory T cells expressing Il10/Gzmb inhibits the cutaneous immune response"

Article Title: A rare subset of skin-tropic regulatory T cells expressing Il10/Gzmb inhibits the cutaneous immune response

Journal: Scientific Reports

doi: 10.1038/srep35002

CD43 + CCR5 + Tregs are a major subset of Gzmb -/ IL10 -expressing Tregs. ( A ) Flow cytometry gating strategy for five Treg subsets (CD43 − and CD43 + CCR5 +/− CXCR3 +/− Tregs) isolated from the dLN of CHS mice. ( B ) The population-wide expression levels of the genes Il10 , Gzmb , Tgfb1 and Ctla4 genes within CD43 − and CD43 + CXCR3 +/− CCR5 +/− Treg subsets, relative to expression levels in naive T cells ( n = 3). ( C ) Violin plots showing expression levels of the genes Il10 , Gzmb , Tgfb1 and Ctla4 genes in individual Tregs in the CD43 + CCR5 + CXCR3 − or CXCR3 + subsets ( n = 37 each). Bars indicate average expression levels. Percentages under each plot indicate the proportion of cells that expressed the gene. Figures 4C and 5A show scqPCR data for CD43 + CCR5 + CXCR3 − or CXCR3 + Tregs. ( D,E ) The proportions of IL-10-producing cells in Treg subsets ( n = 3) stimulated with DNBS ( D ) or PMA/ionomycin ( E ). Flow cytometry dot plots indicate IL-10 expression in the CD43 + CXCR3 +/− CCR5 +/− Treg subsets. Flow cytometry data are representative of three independent experiments; values on the plots indicate the percentage of the parent population. Data in bar graphs represent means ± SEM. Statistical comparisons were performed by one-way ANOVA with Tukey’s post-hoc test (* p
Figure Legend Snippet: CD43 + CCR5 + Tregs are a major subset of Gzmb -/ IL10 -expressing Tregs. ( A ) Flow cytometry gating strategy for five Treg subsets (CD43 − and CD43 + CCR5 +/− CXCR3 +/− Tregs) isolated from the dLN of CHS mice. ( B ) The population-wide expression levels of the genes Il10 , Gzmb , Tgfb1 and Ctla4 genes within CD43 − and CD43 + CXCR3 +/− CCR5 +/− Treg subsets, relative to expression levels in naive T cells ( n = 3). ( C ) Violin plots showing expression levels of the genes Il10 , Gzmb , Tgfb1 and Ctla4 genes in individual Tregs in the CD43 + CCR5 + CXCR3 − or CXCR3 + subsets ( n = 37 each). Bars indicate average expression levels. Percentages under each plot indicate the proportion of cells that expressed the gene. Figures 4C and 5A show scqPCR data for CD43 + CCR5 + CXCR3 − or CXCR3 + Tregs. ( D,E ) The proportions of IL-10-producing cells in Treg subsets ( n = 3) stimulated with DNBS ( D ) or PMA/ionomycin ( E ). Flow cytometry dot plots indicate IL-10 expression in the CD43 + CXCR3 +/− CCR5 +/− Treg subsets. Flow cytometry data are representative of three independent experiments; values on the plots indicate the percentage of the parent population. Data in bar graphs represent means ± SEM. Statistical comparisons were performed by one-way ANOVA with Tukey’s post-hoc test (* p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Mouse Assay

31) Product Images from "Foxo1 and Foxp1 play opposing roles in regulating the differentiation and antitumor activity of TH9 cells programmed by IL-7"

Article Title: Foxo1 and Foxp1 play opposing roles in regulating the differentiation and antitumor activity of TH9 cells programmed by IL-7

Journal: Science signaling

doi: 10.1126/scisignal.aak9741

Pretreatment of naïve CD4 + T cells with IL-7 enhances their differentiation into T H 9 cells ( A to C ) Naïve OT-II CD4 + T cells were cultured with the indicated concentrations of IL-7 for 2 days, washed twice with T cell medium, and then cultured for 4 days under the T H 9-polarizing conditions. Freshly isolated naïve CD4 + T cells that were not pretreated with IL-7 were used as a control. The cells were then harvested and restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. The cells were then analyzed by flow cytometry to detect IL-9 and IL-21 (A, dot plots), to determine the percentages of IL-9–expressing cells (A, bar graph), and to calculate the MFI of IL-9 (B). Alternatively, the cells were stimulated with anti-CD3 (1 μg/ml) for 12 hours, and the cell culture medium was analyzed by ELISA to determine the amount of secreted IL-9 (C). ( D ) T H 9 cells (open squares) and IL-7–T H 9 cells (open triangles) were harvested on the indicated days of T H 9 cell differentiation, and RNA was extracted and used for real-time PCR analysis of the relative abundances of Il9 and Il21 mRNAs, which were normalized to that of mouse actb mRNA. ( E ) Naïve OT-II CD4 + T cells were pretreated with the indicated γC family cytokines for 2 days, washed twice, and then cultured under T H 9-polarizing conditions for 4 days. The cells were then analyzed by flow cytometry to detect IL-9 and IL-21 (dot plots) and to determine the percentages of IL-9–expressing cells (bar graph). ( F ) T H 9 and IL-7–T H 9 cells were collected on day 3, and RNA was extracted for gene microarray analysis. The expression of T H 9 cell–related genes, including those encoding cytokines and transcription factors, is shown. R1 to R3 indicate three replicates. Data in (A) to (E) are means ± SD of three independent experiments. * P
Figure Legend Snippet: Pretreatment of naïve CD4 + T cells with IL-7 enhances their differentiation into T H 9 cells ( A to C ) Naïve OT-II CD4 + T cells were cultured with the indicated concentrations of IL-7 for 2 days, washed twice with T cell medium, and then cultured for 4 days under the T H 9-polarizing conditions. Freshly isolated naïve CD4 + T cells that were not pretreated with IL-7 were used as a control. The cells were then harvested and restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. The cells were then analyzed by flow cytometry to detect IL-9 and IL-21 (A, dot plots), to determine the percentages of IL-9–expressing cells (A, bar graph), and to calculate the MFI of IL-9 (B). Alternatively, the cells were stimulated with anti-CD3 (1 μg/ml) for 12 hours, and the cell culture medium was analyzed by ELISA to determine the amount of secreted IL-9 (C). ( D ) T H 9 cells (open squares) and IL-7–T H 9 cells (open triangles) were harvested on the indicated days of T H 9 cell differentiation, and RNA was extracted and used for real-time PCR analysis of the relative abundances of Il9 and Il21 mRNAs, which were normalized to that of mouse actb mRNA. ( E ) Naïve OT-II CD4 + T cells were pretreated with the indicated γC family cytokines for 2 days, washed twice, and then cultured under T H 9-polarizing conditions for 4 days. The cells were then analyzed by flow cytometry to detect IL-9 and IL-21 (dot plots) and to determine the percentages of IL-9–expressing cells (bar graph). ( F ) T H 9 and IL-7–T H 9 cells were collected on day 3, and RNA was extracted for gene microarray analysis. The expression of T H 9 cell–related genes, including those encoding cytokines and transcription factors, is shown. R1 to R3 indicate three replicates. Data in (A) to (E) are means ± SD of three independent experiments. * P

Techniques Used: Cell Culture, Isolation, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Cell Differentiation, Real-time Polymerase Chain Reaction, Microarray

IL-7–T H 9 cells have enhanced antitumor activity ( A to G ) C57BL/6 mice (five mice per group) were challenged with 2 × 10 5 B16-OVA cells, which was followed by the transfer of 2 × 10 6 T H 1, T H 9, or IL-7–T H 9 cells from CD45.1 OT-II mice on the same day. On day 14, the mice were sacrificed and tissues were harvested for different assays. (A) Schematic showing the time course of the prophylactic model. (B) Images of lungs showing tumor development and foci in the different treatment groups. PBS, phosphate-buffered saline. (C) Lung tumor foci numbers according to the indicated treatment. (D) Numbers of adoptively transferred CD4 + T cells in the lungs of the recipient mice. (E) Numbers of CD8 + T cells that infiltrated the lungs for the indicated treatments. (F and G) Donor-derived CD4 + T cells from lung lymph nodes in the T H 9 and IL-7–T H 9 groups were sorted and stimulated with PMA and ionomycin. The amounts of IL-9 (F) and IL-21 (G) in the cell culture medium were determined by ELISA. ( H ) Therapeutic effect of T H cells in the B16-OVA lung model. Similarly to the prophylactic model, T cells were transferred to mice (four to five mice per group) on day 4 after tumor injection. ( I ) Numbers of tumor foci in the lungs according to the treatment group. In the therapeutic model (five mice per group), anti–IL-9 and anti–IL-21R antibodies (both at 200 μg per mice per injection) were injected intraperitoneally every other day, beginning 1 day before the T H 9 cells were transferred, and tumor foci were analyzed on day 14. IgG, immunoglobulin G. ( J to L ) C57BL/6 mice (four to five mice per group) were injected with B16 tumor cells and received IL-7 (5 μg per injection) or PBS daily from the same day for 14 days. Anti–IL-9 or anti–IL-21R antibodies were injected every other day, and 1 day before IL-7 injection in the indicated groups. Then, mice were sacrificed on day 14, lung lymph nodes were collected from the PBS and IL-7 only groups, and total CD4 + T cells were isolated and stimulated with PMA and ionomycin before the amounts of IL-9 (J) and IL-21 (K) released into the cell culture medium were determined by ELISA. (L) The numbers of tumor foci were counted in all groups. Data in (B) to (L) were from three independent experiments and are presented as means ± SD. * P
Figure Legend Snippet: IL-7–T H 9 cells have enhanced antitumor activity ( A to G ) C57BL/6 mice (five mice per group) were challenged with 2 × 10 5 B16-OVA cells, which was followed by the transfer of 2 × 10 6 T H 1, T H 9, or IL-7–T H 9 cells from CD45.1 OT-II mice on the same day. On day 14, the mice were sacrificed and tissues were harvested for different assays. (A) Schematic showing the time course of the prophylactic model. (B) Images of lungs showing tumor development and foci in the different treatment groups. PBS, phosphate-buffered saline. (C) Lung tumor foci numbers according to the indicated treatment. (D) Numbers of adoptively transferred CD4 + T cells in the lungs of the recipient mice. (E) Numbers of CD8 + T cells that infiltrated the lungs for the indicated treatments. (F and G) Donor-derived CD4 + T cells from lung lymph nodes in the T H 9 and IL-7–T H 9 groups were sorted and stimulated with PMA and ionomycin. The amounts of IL-9 (F) and IL-21 (G) in the cell culture medium were determined by ELISA. ( H ) Therapeutic effect of T H cells in the B16-OVA lung model. Similarly to the prophylactic model, T cells were transferred to mice (four to five mice per group) on day 4 after tumor injection. ( I ) Numbers of tumor foci in the lungs according to the treatment group. In the therapeutic model (five mice per group), anti–IL-9 and anti–IL-21R antibodies (both at 200 μg per mice per injection) were injected intraperitoneally every other day, beginning 1 day before the T H 9 cells were transferred, and tumor foci were analyzed on day 14. IgG, immunoglobulin G. ( J to L ) C57BL/6 mice (four to five mice per group) were injected with B16 tumor cells and received IL-7 (5 μg per injection) or PBS daily from the same day for 14 days. Anti–IL-9 or anti–IL-21R antibodies were injected every other day, and 1 day before IL-7 injection in the indicated groups. Then, mice were sacrificed on day 14, lung lymph nodes were collected from the PBS and IL-7 only groups, and total CD4 + T cells were isolated and stimulated with PMA and ionomycin before the amounts of IL-9 (J) and IL-21 (K) released into the cell culture medium were determined by ELISA. (L) The numbers of tumor foci were counted in all groups. Data in (B) to (L) were from three independent experiments and are presented as means ± SD. * P

Techniques Used: Activity Assay, Mouse Assay, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Injection, Isolation

32) Product Images from "Efficient ex vivo analysis of CD4+ T-cell responses using combinatorial HLA class II tetramer staining"

Article Title: Efficient ex vivo analysis of CD4+ T-cell responses using combinatorial HLA class II tetramer staining

Journal: Nature Communications

doi: 10.1038/ncomms12614

T-helper lineage analysis of the antigen-specific CD4+ T cells. Phenotypic lineage analysis of the tetramer-positive memory (CD45RA − ) cells from the combinatorial staining. ( a ) Combined quantification of the lineage composition of tetramer-specific T cells against all epitopes and from all subjects demonstrates the predominance of Th1 cells at either time point. Data are shown as box plots with tukey bars to indicate the distribution of the data. ( b ) Analysis of Th1 and Th2 frequencies for the different epitopes after vaccination. T cells specific to TxHA-321 and CaHA-265 appear to trend towards lower and higher levels of Th1 and Th2, respectively, while cells from subject 1 (red star) consistently displayed increased frequencies of Th2 cells. ( c ) Quantification of the Th2 frequencies for all epitopes combined for the differed subjects. ( d ) Intracellular expression of IFN-γ, IL-4 and IL-17 in cells from cells stained with single tetramers for CaHA-265, H1HA-393 and MP-97 for three of the study subjects after vaccination following short stimulation with phorbol myristate acetate/ionomycin. Tetramer+ cells were gated (dark gate) and cytokine expression quantified, demonstrating dominance of IFN-γ expression and significantly higher expression in MP-97 positive cells (red triangle) compared to the other populations. Selected statistical significances are displayed from one-way analysis of variance with Holm–Sidak correction (upper values) and Kruskal–Wallis test with Dunn's correction (lower values) after comparison of all columns. P values ≥0.05 are indicated by parenthesis.
Figure Legend Snippet: T-helper lineage analysis of the antigen-specific CD4+ T cells. Phenotypic lineage analysis of the tetramer-positive memory (CD45RA − ) cells from the combinatorial staining. ( a ) Combined quantification of the lineage composition of tetramer-specific T cells against all epitopes and from all subjects demonstrates the predominance of Th1 cells at either time point. Data are shown as box plots with tukey bars to indicate the distribution of the data. ( b ) Analysis of Th1 and Th2 frequencies for the different epitopes after vaccination. T cells specific to TxHA-321 and CaHA-265 appear to trend towards lower and higher levels of Th1 and Th2, respectively, while cells from subject 1 (red star) consistently displayed increased frequencies of Th2 cells. ( c ) Quantification of the Th2 frequencies for all epitopes combined for the differed subjects. ( d ) Intracellular expression of IFN-γ, IL-4 and IL-17 in cells from cells stained with single tetramers for CaHA-265, H1HA-393 and MP-97 for three of the study subjects after vaccination following short stimulation with phorbol myristate acetate/ionomycin. Tetramer+ cells were gated (dark gate) and cytokine expression quantified, demonstrating dominance of IFN-γ expression and significantly higher expression in MP-97 positive cells (red triangle) compared to the other populations. Selected statistical significances are displayed from one-way analysis of variance with Holm–Sidak correction (upper values) and Kruskal–Wallis test with Dunn's correction (lower values) after comparison of all columns. P values ≥0.05 are indicated by parenthesis.

Techniques Used: Staining, Expressing

33) Product Images from "Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T Lymphocyte Jurkat Cell Line"

Article Title: Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T Lymphocyte Jurkat Cell Line

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18071409

Effects of I3C, DIM on nuclear factor of activated T-cells 1 (NFAT1) dephosphorylation in Jurkat cells. Jurkat cells were treated with 50 μM of I3C or 10 μM of DIM for 48 h and then stimulated with PMA+ionomycin for 10 or 30 min. Nuclear extracts were prepared following cell harvest and dephosphorylation of NFAT1 was analyzed by western blot in: ( A ) I3C-pretreated cells; and ( B ) DIM-pretreated cells. Protein expression was normalized to levels of housekeeping gene (Histone H3). The inset shows representative Western blots. Results expressed as mean ± SD ( n = 3). * indicate significantly different from control at p
Figure Legend Snippet: Effects of I3C, DIM on nuclear factor of activated T-cells 1 (NFAT1) dephosphorylation in Jurkat cells. Jurkat cells were treated with 50 μM of I3C or 10 μM of DIM for 48 h and then stimulated with PMA+ionomycin for 10 or 30 min. Nuclear extracts were prepared following cell harvest and dephosphorylation of NFAT1 was analyzed by western blot in: ( A ) I3C-pretreated cells; and ( B ) DIM-pretreated cells. Protein expression was normalized to levels of housekeeping gene (Histone H3). The inset shows representative Western blots. Results expressed as mean ± SD ( n = 3). * indicate significantly different from control at p

Techniques Used: De-Phosphorylation Assay, Western Blot, Expressing

Effects of I3C, DIM on IL-2, IL-8 and TNF-α protein secretion in Jurkat cells. Jurkat cells were treated with 5, 25 or 50 μM of I3C or 5, 10 or 25 μM of DIM for 48 h and then stimulated with PMA + ionomycin for 24 h. Media were harvested and: ( A ) IL-2; ( B ) IL-8; and ( C ) TNF-α protein determined using enzyme linked immunosorbent assay (ELISA). Results expressed as mean ± SD ( n = 3) from three independent experiments. * indicates significantly different from control at p
Figure Legend Snippet: Effects of I3C, DIM on IL-2, IL-8 and TNF-α protein secretion in Jurkat cells. Jurkat cells were treated with 5, 25 or 50 μM of I3C or 5, 10 or 25 μM of DIM for 48 h and then stimulated with PMA + ionomycin for 24 h. Media were harvested and: ( A ) IL-2; ( B ) IL-8; and ( C ) TNF-α protein determined using enzyme linked immunosorbent assay (ELISA). Results expressed as mean ± SD ( n = 3) from three independent experiments. * indicates significantly different from control at p

Techniques Used: Enzyme-linked Immunosorbent Assay

Effects of I3C, DIM on Nuclear Factor κB (NF-κB) activation in Jurkat cells. Jurkat cells were treated with 50 μM of I3C or 10 μM of DIM for 48 h and then stimulated with PMA + ionomycin for 10 or 30 min. Cells were harvested for protein. ( A , C ) Phosphorylation levels of Iκ-Bα (ser32); and ( B , D ) Phosphorylation levels of Nuclear Factor κB-p65 (NF-κB-p65) (ser536) were determined by western blot analysis. Protein expression was normalized to levels of housekeeping gene (β-actin). The inset shows representative Western blots. * indicates significantly different from control at p
Figure Legend Snippet: Effects of I3C, DIM on Nuclear Factor κB (NF-κB) activation in Jurkat cells. Jurkat cells were treated with 50 μM of I3C or 10 μM of DIM for 48 h and then stimulated with PMA + ionomycin for 10 or 30 min. Cells were harvested for protein. ( A , C ) Phosphorylation levels of Iκ-Bα (ser32); and ( B , D ) Phosphorylation levels of Nuclear Factor κB-p65 (NF-κB-p65) (ser536) were determined by western blot analysis. Protein expression was normalized to levels of housekeeping gene (β-actin). The inset shows representative Western blots. * indicates significantly different from control at p

Techniques Used: Activation Assay, Western Blot, Expressing

Effects of I3C, DIM on Interleukin-2 (IL-2), Interleukin-8 (IL-8) and Tumor Necrosis Factor-α (TNF-α) mRNA levels in Jurkat cells. Jurkat cells were treated with 10, 25 or 50 μM of I3C or 5, 10 or 25 μM of DIM for 48 h and then stimulated with phorbol-12-myristate-13-acetate (PMA) + anti-cluster of differentiation 3 (CD3) antibody or PMA + ionomycin for 6 h. Genes expression determinations of: ( A ) IL-2; ( B ) IL-8; and ( C ) TNF-α were analyzed using real time polymerase chain reaction (RT-PCR). Results expressed as mean ± SD ( n = 3) from three independent experiments. * indicates significantly different from control at p
Figure Legend Snippet: Effects of I3C, DIM on Interleukin-2 (IL-2), Interleukin-8 (IL-8) and Tumor Necrosis Factor-α (TNF-α) mRNA levels in Jurkat cells. Jurkat cells were treated with 10, 25 or 50 μM of I3C or 5, 10 or 25 μM of DIM for 48 h and then stimulated with phorbol-12-myristate-13-acetate (PMA) + anti-cluster of differentiation 3 (CD3) antibody or PMA + ionomycin for 6 h. Genes expression determinations of: ( A ) IL-2; ( B ) IL-8; and ( C ) TNF-α were analyzed using real time polymerase chain reaction (RT-PCR). Results expressed as mean ± SD ( n = 3) from three independent experiments. * indicates significantly different from control at p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

34) Product Images from "PTEN drives Th17 cell differentiation by preventing IL-2 production"

Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20170523

γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P
Figure Legend Snippet: γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P

Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry

35) Product Images from "Critical Role of Myeloid-Derived Suppressor Cells in Tumor-Induced Liver Immune Suppression through Inhibition of NKT Cell Function"

Article Title: Critical Role of Myeloid-Derived Suppressor Cells in Tumor-Induced Liver Immune Suppression through Inhibition of NKT Cell Function

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00129

Myeloid-derived suppressor cells (MDSCs) inhibited IFN-γ production of NKT cells via membrane-bounded transforming growth factor β (TGF-β) in a cell contact-dependent manner . (A) Membrane-bound TGF-β is the main factor for suppression of NKT cell IFN-γ secretion. NKT cells (CD3 + NK1.1 + ) were isolated from liver tissues of wild-type (WT) mice, co-cultured with MDSCs sorted from livers of tumor-bearing (TB) mice at the ratio of 1:1 under various conditions, including either transwell (0.4 µm) separation or in the presence of 10 ng/ml anti-TGF-β1 mAb or 0.5 ng/ml rmTGF-β1 for 6 h, and then cells were then stimulated with PMA (25 ng/ml) plus ionomycin (1 µg/ml). Eighteen hours later, supernatants were harvested for determination of IFN-γ levels using ELISA kit. Results (mean ± SEM) of triplicated wells from one representative experiment are shown. (B) Expression levels of arginase 1 (Arg-1) and inducible nitric oxide synthase (iNOS) were not changed in liver tissues of TB mice. Livers were isolated from either WT or TB mice (day 15 posttumor inoculation), and mRNA levels of Arg-1 and iNOS were determined via quantitative real-time PCR ( n = 3). Results from one representative experiment are shown. (C) Reactive oxygen species (ROS) expression was increased in the liver of TB mice. Livers were isolated from either WT or TB mice 15 days posttumor inoculation. ROS levels were determined via ROS assay kit as described in Section “ Materials and Methods ” ( n = 3). Results from one representative experiment are shown. (D) Arg-1, iNOS, and ROS inhibitors failed to restore IFN production of NKT cells in vitro . NKT cells were co-cultured with MDSCs isolated from the liver of TB mice at the ratio of 1:1 as described above in the absence or presence of various inhibitors, including N -hydroxy-nor-arginine (1 mM), catalase (1,000 U/ml), or l -NIL (0.5 µM) for 6 h. Then cells were stimulated with PMA plus ionomycin, and IFN-γ levels at the supernatant were determined using ELISA kit. Results from triplicated wells (mean ± SEM) are shown.
Figure Legend Snippet: Myeloid-derived suppressor cells (MDSCs) inhibited IFN-γ production of NKT cells via membrane-bounded transforming growth factor β (TGF-β) in a cell contact-dependent manner . (A) Membrane-bound TGF-β is the main factor for suppression of NKT cell IFN-γ secretion. NKT cells (CD3 + NK1.1 + ) were isolated from liver tissues of wild-type (WT) mice, co-cultured with MDSCs sorted from livers of tumor-bearing (TB) mice at the ratio of 1:1 under various conditions, including either transwell (0.4 µm) separation or in the presence of 10 ng/ml anti-TGF-β1 mAb or 0.5 ng/ml rmTGF-β1 for 6 h, and then cells were then stimulated with PMA (25 ng/ml) plus ionomycin (1 µg/ml). Eighteen hours later, supernatants were harvested for determination of IFN-γ levels using ELISA kit. Results (mean ± SEM) of triplicated wells from one representative experiment are shown. (B) Expression levels of arginase 1 (Arg-1) and inducible nitric oxide synthase (iNOS) were not changed in liver tissues of TB mice. Livers were isolated from either WT or TB mice (day 15 posttumor inoculation), and mRNA levels of Arg-1 and iNOS were determined via quantitative real-time PCR ( n = 3). Results from one representative experiment are shown. (C) Reactive oxygen species (ROS) expression was increased in the liver of TB mice. Livers were isolated from either WT or TB mice 15 days posttumor inoculation. ROS levels were determined via ROS assay kit as described in Section “ Materials and Methods ” ( n = 3). Results from one representative experiment are shown. (D) Arg-1, iNOS, and ROS inhibitors failed to restore IFN production of NKT cells in vitro . NKT cells were co-cultured with MDSCs isolated from the liver of TB mice at the ratio of 1:1 as described above in the absence or presence of various inhibitors, including N -hydroxy-nor-arginine (1 mM), catalase (1,000 U/ml), or l -NIL (0.5 µM) for 6 h. Then cells were stimulated with PMA plus ionomycin, and IFN-γ levels at the supernatant were determined using ELISA kit. Results from triplicated wells (mean ± SEM) are shown.

Techniques Used: Derivative Assay, Isolation, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, ROS Assay, In Vitro

36) Product Images from "Distinct modes of mitochondrial metabolism uncouple T helper cell differentiation and function"

Article Title: Distinct modes of mitochondrial metabolism uncouple T helper cell differentiation and function

Journal: Nature

doi: 10.1038/s41586-019-1311-3

CD4 T cells from either IL-4 (4GET) or IL17-GFP reporter mice were activated under Th2 and Th17 culture conditions, respectively. Cells were then treated overnight with either DMSO, rotenone, dimethyl malonate (DMM), antimycin A, or oligomycin. On day 5, cells were re-stimulated with PMA and ionomycin and reporter activity was measured by flow cytometry for a , Th2 culture conditions, and b , Th17 culture conditions.
Figure Legend Snippet: CD4 T cells from either IL-4 (4GET) or IL17-GFP reporter mice were activated under Th2 and Th17 culture conditions, respectively. Cells were then treated overnight with either DMSO, rotenone, dimethyl malonate (DMM), antimycin A, or oligomycin. On day 5, cells were re-stimulated with PMA and ionomycin and reporter activity was measured by flow cytometry for a , Th2 culture conditions, and b , Th17 culture conditions.

Techniques Used: Mouse Assay, Activity Assay, Flow Cytometry, Cytometry

The TCA cycle promotes Th1 cell effector function and limits proliferation through the activity of ETC Complex II. a , CD4 T cells from Ifng -Katushka mice were activated under Th1 culture conditions in the presence of serially diluted 2DG or NaFlAc. After 5 days, cells were re-stimulated with PMA and ionomycin and Ifng -Katushka reporter activity was measured by flow cytometry. b , WT CD4 T cells were activated under Th1 conditions and treated overnight from day 4 to day 5 with either DMSO, rotenone, dimethyl malonate (DMM), antimycin A, oligomycin, or BMS-303141. Per cell intracellular IFNγ protein expression was quantified by FACS on day 5 in restimulated T cells. c , WT CD4 T cells were activated under Th1 conditions and treated overnight from day 4 to day 5 with either DMSO, DMM, 3-nitropropionic acid (3NP), thenoyltrifluoroaceton (TTFA), or atpenin A5. Per cell intracellular IFNγ protein expression was quantified by FACS on day 5 in restimulated T cells. d , CD4 T cells from either doxycycline-treated Sdhcfl/fl TetO-Cre−/+ R26rtTA/+ (Sdhc cKO) or Sdhc+/+ TetO-Cre−/+ R26rtTA/+ mice were CTV labeled and activated under Th1 conditions. On day 5, cells were restimulated. Cells were analyzed by flow cytometry for IFNγ production and e , cell proliferation. Plots shown are from five combined experiments, control n = 13, Sdhc cKO = 14. g , Tbet protein expression in day 5 CD4 T cells cultured under Th1 conditions from control (n = 4) or Sdhc-cKO mice (n = 3). RNA-seq analysis was performed on CD4 T cells cultured under Th1 conditions from control (n = 3) or Sdhc-cKO mice (n = 3) and evaluated by g , DAVID GO pathway analysis. h , Heatmap of gene expression from RNA-seq results for the Cytokine Production GO Pathway. Representative plots and a graph summarizing the results of at least two independent experiments are shown, except where noted otherwise. When applicable, mean and standard deviation of replicates are presented on summarized plots and unpaired t -test used to determine significance (* p
Figure Legend Snippet: The TCA cycle promotes Th1 cell effector function and limits proliferation through the activity of ETC Complex II. a , CD4 T cells from Ifng -Katushka mice were activated under Th1 culture conditions in the presence of serially diluted 2DG or NaFlAc. After 5 days, cells were re-stimulated with PMA and ionomycin and Ifng -Katushka reporter activity was measured by flow cytometry. b , WT CD4 T cells were activated under Th1 conditions and treated overnight from day 4 to day 5 with either DMSO, rotenone, dimethyl malonate (DMM), antimycin A, oligomycin, or BMS-303141. Per cell intracellular IFNγ protein expression was quantified by FACS on day 5 in restimulated T cells. c , WT CD4 T cells were activated under Th1 conditions and treated overnight from day 4 to day 5 with either DMSO, DMM, 3-nitropropionic acid (3NP), thenoyltrifluoroaceton (TTFA), or atpenin A5. Per cell intracellular IFNγ protein expression was quantified by FACS on day 5 in restimulated T cells. d , CD4 T cells from either doxycycline-treated Sdhcfl/fl TetO-Cre−/+ R26rtTA/+ (Sdhc cKO) or Sdhc+/+ TetO-Cre−/+ R26rtTA/+ mice were CTV labeled and activated under Th1 conditions. On day 5, cells were restimulated. Cells were analyzed by flow cytometry for IFNγ production and e , cell proliferation. Plots shown are from five combined experiments, control n = 13, Sdhc cKO = 14. g , Tbet protein expression in day 5 CD4 T cells cultured under Th1 conditions from control (n = 4) or Sdhc-cKO mice (n = 3). RNA-seq analysis was performed on CD4 T cells cultured under Th1 conditions from control (n = 3) or Sdhc-cKO mice (n = 3) and evaluated by g , DAVID GO pathway analysis. h , Heatmap of gene expression from RNA-seq results for the Cytokine Production GO Pathway. Representative plots and a graph summarizing the results of at least two independent experiments are shown, except where noted otherwise. When applicable, mean and standard deviation of replicates are presented on summarized plots and unpaired t -test used to determine significance (* p

Techniques Used: Activity Assay, Mouse Assay, Flow Cytometry, Cytometry, Expressing, FACS, Labeling, Cell Culture, RNA Sequencing Assay, Standard Deviation

a , IFNγ protein production in day 5 Th1 cells treated with 10 mM diethyl succinate (DES) overnight for 16 hours, restimulated in PMA and ionomycin and analyzed by intracellular flow cytometry. b , CD4 T cells were activated under Th1 conditions and retrovirally transduced with three individual sgRNA against either Sdha or with vector control, on day 1 post-activation. On day 5, IFNγ was analyzed by intracellular flow cytometry. CD4 T cells from control or Sdhc cKO mice were activated and cultured under Th1 conditions. On day 5, cells were transferred to media containing dialyzed FBS for 4 hours and then processed for LC-MS analysis. Shown are quantification of cellular c , succinate and d , α-ketoglutarate.
Figure Legend Snippet: a , IFNγ protein production in day 5 Th1 cells treated with 10 mM diethyl succinate (DES) overnight for 16 hours, restimulated in PMA and ionomycin and analyzed by intracellular flow cytometry. b , CD4 T cells were activated under Th1 conditions and retrovirally transduced with three individual sgRNA against either Sdha or with vector control, on day 1 post-activation. On day 5, IFNγ was analyzed by intracellular flow cytometry. CD4 T cells from control or Sdhc cKO mice were activated and cultured under Th1 conditions. On day 5, cells were transferred to media containing dialyzed FBS for 4 hours and then processed for LC-MS analysis. Shown are quantification of cellular c , succinate and d , α-ketoglutarate.

Techniques Used: Flow Cytometry, Cytometry, Transduction, Plasmid Preparation, Activation Assay, Mouse Assay, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Pooled CRISPR screen for the functional annotation of the T cell metabolome. a , CD4 T cells from Cas9tg mice were stimulated with anti-CD3 and anti-CD28 beads in IL-2 (5 ng/mL), anti-IL-4 (10 ug/mL), and IL-12 (2 ng/mL) and retrovirally transduced 24 hours after activation with either empty MG-guide (shaded blue) or MG-guide expressing a sgRNA against Tbx21 (outline). T-bet protein expression was measured by intracellular flow cytometry on day 3. b , Cas9tg CD4 T cells were cultured as above and infected with either MGguide, a sgRNA against Tbx21 , or a sgRNA against Il12rb . IFNγ protein was measured by intracellular flow cytometry on day 5 after restimulation with PMA (20 ng/ml) and ionomycin (1 ug/ml). c , Schematic of Th1 metabolome CRISPR screen. CD4 T cells from Cas9tg Ifng -Katushka reporter (Cas9tg Ifng -Kat) mice were activated as described and then transduced 24 hours after activation with the metabolome sgRNA retroviral library. At day 5 post-activation, cells were re-stimulated with PMA and ionomycin and sorted based on quartiles of Ifng -Katushka reporter activity. d , Volcano plot and histogram of results from three independent replicates of the metabolome CRISPR screen. Shown are values for targeting sgRNA from a comparison of Q1 vs Q4 (red) and Q2 vs Q4 (blue), as well as non-targeting control sgRNA (light gray). Positive control sgRNAs against Tbx21 (orange) and Il12rb (green) are also displayed. e , Binned Z-scores of Q1 / Q4 sgRNA ratios (y-axis) versus sgRNA mean abundances in Q1 and Q4 (x-axis). f , Summary of KEGG pathway and module analysis.
Figure Legend Snippet: Pooled CRISPR screen for the functional annotation of the T cell metabolome. a , CD4 T cells from Cas9tg mice were stimulated with anti-CD3 and anti-CD28 beads in IL-2 (5 ng/mL), anti-IL-4 (10 ug/mL), and IL-12 (2 ng/mL) and retrovirally transduced 24 hours after activation with either empty MG-guide (shaded blue) or MG-guide expressing a sgRNA against Tbx21 (outline). T-bet protein expression was measured by intracellular flow cytometry on day 3. b , Cas9tg CD4 T cells were cultured as above and infected with either MGguide, a sgRNA against Tbx21 , or a sgRNA against Il12rb . IFNγ protein was measured by intracellular flow cytometry on day 5 after restimulation with PMA (20 ng/ml) and ionomycin (1 ug/ml). c , Schematic of Th1 metabolome CRISPR screen. CD4 T cells from Cas9tg Ifng -Katushka reporter (Cas9tg Ifng -Kat) mice were activated as described and then transduced 24 hours after activation with the metabolome sgRNA retroviral library. At day 5 post-activation, cells were re-stimulated with PMA and ionomycin and sorted based on quartiles of Ifng -Katushka reporter activity. d , Volcano plot and histogram of results from three independent replicates of the metabolome CRISPR screen. Shown are values for targeting sgRNA from a comparison of Q1 vs Q4 (red) and Q2 vs Q4 (blue), as well as non-targeting control sgRNA (light gray). Positive control sgRNAs against Tbx21 (orange) and Il12rb (green) are also displayed. e , Binned Z-scores of Q1 / Q4 sgRNA ratios (y-axis) versus sgRNA mean abundances in Q1 and Q4 (x-axis). f , Summary of KEGG pathway and module analysis.

Techniques Used: CRISPR, Functional Assay, Mouse Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry, Cell Culture, Infection, Activity Assay, Positive Control

The malate-aspartate shuttle and mitochondrial citrate export are required for histone acetylation and proliferation in differentiating Th1 cells. a , Schematic of the malate-aspartate shuttle and mitochondrial citrate export. b , CD4 T cells were activated under Th1 culture conditions and retrovirally transduced with three distinct individual sgRNAs against each of the enzymes and transporters that participate in the malate-aspartate shuttle, or specific to citrate export. T cells were restimulated with PMA and ionomycin on day 5 post-activation and IFNγ protein was evaluated by intracellular flow cytometry. Graphs show individual sgRNA for each gene as well as the average for all three sgRNAs. c,d , Cas9 CD4 T cells were activated in Th1 conditions and transduced with control vector or sgRNA against Acly , Slc25a1 , Mdh1 , Slc25a11 , or Slc1a3 on day 1 post-activation. One day after transduction, cells were supplemented with 5mM Acetate (light blue bars), 20mM acetate (dark blue bars), or a vehicle DMSO control (clear bars). On day 4, cells were assayed by FACS for H3K9Ac. CD4 T cells from Cas9 mice were activated under Th1 culture conditions and transduced with either control vector or sgRNA targeting Slc25a11 and subjected to H3K9Ac ChIP-seq and RNA-seq analysis. e , Pie chart summarizing the percentage of differentially acetylated peaks exhibiting increased or decreased H3K9Ac labeling in Slc25a11 sgRNA expressing Th1 cells versus control. f, Heatmap summarizing RNA-seq results, overlaid with peaks significantly decreased in H3K9Ac (black bar), and genes belonging to the GO Metabolism Pathway. Representative plots and a graph summarizing the results of at least two independent experiments are shown. When applicable, mean and standard deviation of replicates are presented on summarized plots and unpaired t -test used to determine significance (* p
Figure Legend Snippet: The malate-aspartate shuttle and mitochondrial citrate export are required for histone acetylation and proliferation in differentiating Th1 cells. a , Schematic of the malate-aspartate shuttle and mitochondrial citrate export. b , CD4 T cells were activated under Th1 culture conditions and retrovirally transduced with three distinct individual sgRNAs against each of the enzymes and transporters that participate in the malate-aspartate shuttle, or specific to citrate export. T cells were restimulated with PMA and ionomycin on day 5 post-activation and IFNγ protein was evaluated by intracellular flow cytometry. Graphs show individual sgRNA for each gene as well as the average for all three sgRNAs. c,d , Cas9 CD4 T cells were activated in Th1 conditions and transduced with control vector or sgRNA against Acly , Slc25a1 , Mdh1 , Slc25a11 , or Slc1a3 on day 1 post-activation. One day after transduction, cells were supplemented with 5mM Acetate (light blue bars), 20mM acetate (dark blue bars), or a vehicle DMSO control (clear bars). On day 4, cells were assayed by FACS for H3K9Ac. CD4 T cells from Cas9 mice were activated under Th1 culture conditions and transduced with either control vector or sgRNA targeting Slc25a11 and subjected to H3K9Ac ChIP-seq and RNA-seq analysis. e , Pie chart summarizing the percentage of differentially acetylated peaks exhibiting increased or decreased H3K9Ac labeling in Slc25a11 sgRNA expressing Th1 cells versus control. f, Heatmap summarizing RNA-seq results, overlaid with peaks significantly decreased in H3K9Ac (black bar), and genes belonging to the GO Metabolism Pathway. Representative plots and a graph summarizing the results of at least two independent experiments are shown. When applicable, mean and standard deviation of replicates are presented on summarized plots and unpaired t -test used to determine significance (* p

Techniques Used: Transduction, Activation Assay, Flow Cytometry, Cytometry, Plasmid Preparation, FACS, Mouse Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Labeling, Expressing, Standard Deviation

37) Product Images from "Microwave ablation combined with OK-432 induces Th1-type response and specific antitumor immunity in a murine model of breast cancer"

Article Title: Microwave ablation combined with OK-432 induces Th1-type response and specific antitumor immunity in a murine model of breast cancer

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-017-1124-9

MWA plus OK-432 polarized T-cell responses to Th1 dominance. Mononuclear cells were prepared from spleen and stimulated with PMA, ionomycin, and brefeldin A for 5 h before flow cytometric analysis. a Representative flow cytometric plots showing CD4 + IFN-γ + and CD4 + IL-4 + cells on days 7 after MWA. b , c , the percentage of IFN-γ secreting Th1 and IL-4 secreting Th2 cells in CD4 + Th cells. d The ratio of Th1 to Th2 cells was calculated for each mouse. Column, mean; error bars , SEM. *P
Figure Legend Snippet: MWA plus OK-432 polarized T-cell responses to Th1 dominance. Mononuclear cells were prepared from spleen and stimulated with PMA, ionomycin, and brefeldin A for 5 h before flow cytometric analysis. a Representative flow cytometric plots showing CD4 + IFN-γ + and CD4 + IL-4 + cells on days 7 after MWA. b , c , the percentage of IFN-γ secreting Th1 and IL-4 secreting Th2 cells in CD4 + Th cells. d The ratio of Th1 to Th2 cells was calculated for each mouse. Column, mean; error bars , SEM. *P

Techniques Used: Flow Cytometry

38) Product Images from "Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression"

Article Title: Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01617

CD49d high CD4 + T cells rapidly secreted Th1 cytokines. (A,B) Representative flow cytometric plots of intracellular staining for various cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-17, and IL-21). (A) Isolated peritoneal cells and (B) PB mononuclear cells were stimulated with PMA and ionomycin for 4 h. Cytokine expression in CD49d high CD4 + and CD49d low CD4 + T cells was analyzed by flow cytometry. Data are representative of three independent experiments (left panel). Proportions of TNF-α, IFN-γ, IL-2, and IL-21 positive CD4 + T cells in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white) in the (A) PEC and (B) blood (right panel). (C) CD49d high CD4 + and CD49d low CD4 + T cells isolated from the PEC or PB were sorted and labeled with CTV, then stimulated for 72 h with immobilized anti-CD3 and anti-CD28 antibodies. The proliferation of CD49d high CD4 + T cells (solid lines) and CD49d low CD4 + T cells (gray filled) is shown, along with total mitotic events in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white). Data are expressed as the mean ± SEM ( n = 3 donors per each group). * P
Figure Legend Snippet: CD49d high CD4 + T cells rapidly secreted Th1 cytokines. (A,B) Representative flow cytometric plots of intracellular staining for various cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-17, and IL-21). (A) Isolated peritoneal cells and (B) PB mononuclear cells were stimulated with PMA and ionomycin for 4 h. Cytokine expression in CD49d high CD4 + and CD49d low CD4 + T cells was analyzed by flow cytometry. Data are representative of three independent experiments (left panel). Proportions of TNF-α, IFN-γ, IL-2, and IL-21 positive CD4 + T cells in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white) in the (A) PEC and (B) blood (right panel). (C) CD49d high CD4 + and CD49d low CD4 + T cells isolated from the PEC or PB were sorted and labeled with CTV, then stimulated for 72 h with immobilized anti-CD3 and anti-CD28 antibodies. The proliferation of CD49d high CD4 + T cells (solid lines) and CD49d low CD4 + T cells (gray filled) is shown, along with total mitotic events in CD49d high CD4 + (black) and CD49d low CD4 + T cells (white). Data are expressed as the mean ± SEM ( n = 3 donors per each group). * P

Techniques Used: Flow Cytometry, Staining, Isolation, Expressing, Cytometry, Labeling

39) Product Images from "RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses"

Article Title: RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

Journal: Cell

doi: 10.1016/j.cell.2018.08.064

Overexpression of Rab11Fip5 in NK-92 Cells Increases Cytokine and Granzyme Release (A) Effect of Rab11Fip5 overexpression on IFN-γ production and degranulation (CD107a expression) of NK-92 cells in response to stimulation with PMA/ionomycin. NK-92/Rab11Fip5 (red symbols) and NK-92/ZsGreen cells (blue symbols) were stimulated with 500 ng/mL PMA and 5 μg/mL ionomycin for 2 hr in the presence of a CD107a antibody and the protein transport inhibitor monensin. Dots represent MFIs from 6 replicate wells. Significance determined by Wilcoxon-Mann-Whitney (ns, not significant; ∗∗ p
Figure Legend Snippet: Overexpression of Rab11Fip5 in NK-92 Cells Increases Cytokine and Granzyme Release (A) Effect of Rab11Fip5 overexpression on IFN-γ production and degranulation (CD107a expression) of NK-92 cells in response to stimulation with PMA/ionomycin. NK-92/Rab11Fip5 (red symbols) and NK-92/ZsGreen cells (blue symbols) were stimulated with 500 ng/mL PMA and 5 μg/mL ionomycin for 2 hr in the presence of a CD107a antibody and the protein transport inhibitor monensin. Dots represent MFIs from 6 replicate wells. Significance determined by Wilcoxon-Mann-Whitney (ns, not significant; ∗∗ p

Techniques Used: Over Expression, Expressing, MANN-WHITNEY

Transcript Expression from Bulk NK Cell RNA-Seq and Cytokine Secretion from Rab11Fip5-Expressing NK-92 Cells Measured by Luminex Assay Together with Analysis of Their Cytolytic Activity, Related to Figures 6 and 7 (A) Median unique molecular identifiers (UMIs) and genes detected per cell in the scRNA-seq datasets. (B) Single-cell RNA-seq analysis was performed on CD56bright, dim and neg NK subsets isolated from a single donor by cell sorting. Violin plots of transcripts significantly upregulated in RAB11FIP5 -expressing cells (red) compared to cells not expressing RAB11FIP5 (blue). Data for each NK cell subset is shown separately for significant genes (determined by likelihood ratio test and p ≤ 0.05). Normalized transcript expression is shown on the y axis. (C) NK cells from three HIV-infected donors were sorted into subsets on the basis of CD56 expression and subjected to bulk RNA-seq. Reads were aligned to the human genome (Hg38) and Fragments Per Kilobases of transcript per Million mapped reads (FPKM) were determined for each donor in each subset for IFNG transcript expression. (D) NK-92/ RAB11FIP5 transduced cells and NK-92/empty vector control cells were stimulated with 500 ng/ml PMA and 5 μg/ml ionomycin for 2 hours. Supernatants were harvested and levels of GM-CSF, sCD137, IFN-γ, sFas, sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α and perforin were analyzed by Luminex assay. All the cytokines in range of the standard curves are shown. Error bars represent standard deviation of quadruplicate wells. Wilcoxon-Mann-Whitney was utilized for statistical analysis. ns, not significant; ∗∗∗ p ≤ 0.001. (E) Granzyme B (GzB)-based cytotoxicity assays performed using NK-92/ RAB11FIP5 (red) or NK-92/ZsGreen control (blue) cells as effectors and K562 cells as targets at different effector:target ratios. The percentage of cells positive for proteolytically active GzB is represented as % GzB activity. Data from 3 individual experiments, each of which was were performed in triplicate, are shown.
Figure Legend Snippet: Transcript Expression from Bulk NK Cell RNA-Seq and Cytokine Secretion from Rab11Fip5-Expressing NK-92 Cells Measured by Luminex Assay Together with Analysis of Their Cytolytic Activity, Related to Figures 6 and 7 (A) Median unique molecular identifiers (UMIs) and genes detected per cell in the scRNA-seq datasets. (B) Single-cell RNA-seq analysis was performed on CD56bright, dim and neg NK subsets isolated from a single donor by cell sorting. Violin plots of transcripts significantly upregulated in RAB11FIP5 -expressing cells (red) compared to cells not expressing RAB11FIP5 (blue). Data for each NK cell subset is shown separately for significant genes (determined by likelihood ratio test and p ≤ 0.05). Normalized transcript expression is shown on the y axis. (C) NK cells from three HIV-infected donors were sorted into subsets on the basis of CD56 expression and subjected to bulk RNA-seq. Reads were aligned to the human genome (Hg38) and Fragments Per Kilobases of transcript per Million mapped reads (FPKM) were determined for each donor in each subset for IFNG transcript expression. (D) NK-92/ RAB11FIP5 transduced cells and NK-92/empty vector control cells were stimulated with 500 ng/ml PMA and 5 μg/ml ionomycin for 2 hours. Supernatants were harvested and levels of GM-CSF, sCD137, IFN-γ, sFas, sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α and perforin were analyzed by Luminex assay. All the cytokines in range of the standard curves are shown. Error bars represent standard deviation of quadruplicate wells. Wilcoxon-Mann-Whitney was utilized for statistical analysis. ns, not significant; ∗∗∗ p ≤ 0.001. (E) Granzyme B (GzB)-based cytotoxicity assays performed using NK-92/ RAB11FIP5 (red) or NK-92/ZsGreen control (blue) cells as effectors and K562 cells as targets at different effector:target ratios. The percentage of cells positive for proteolytically active GzB is represented as % GzB activity. Data from 3 individual experiments, each of which was were performed in triplicate, are shown.

Techniques Used: Expressing, RNA Sequencing Assay, Luminex, Activity Assay, Isolation, FACS, Infection, Plasmid Preparation, Standard Deviation, MANN-WHITNEY

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Isolation:

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Cytometry:

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Cell Culture:

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Enzyme-linked Immunosorbent Assay:

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Activation Assay:

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Incubation:

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Staining:

Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production
Article Snippet: .. Mononuclear cells were isolated by gradient centrifugation on a 30/70% Percoll gradient (GE Healthcare) at 670 g for 30 min. Isolated cells were stained for ILC3 isolation or stimulated with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend) for 4 h and then stained with PerCP/Cy5.5-conjugated IL-17A, and APC-conjugated anti-CD4 antibodies. .. For ILC3 isolation, cells were stained with FITC-conjugated anti-Lineage cocktail, APC-conjugated anti-CD117, and PerCP/Cy5.5-conjugated anti-NKp46 antibodies, and then Lineage− NKp46− CD117+ cells were sorted.

Gradient Centrifugation:

Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production
Article Snippet: .. Mononuclear cells were isolated by gradient centrifugation on a 30/70% Percoll gradient (GE Healthcare) at 670 g for 30 min. Isolated cells were stained for ILC3 isolation or stimulated with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend) for 4 h and then stained with PerCP/Cy5.5-conjugated IL-17A, and APC-conjugated anti-CD4 antibodies. .. For ILC3 isolation, cells were stained with FITC-conjugated anti-Lineage cocktail, APC-conjugated anti-CD117, and PerCP/Cy5.5-conjugated anti-NKp46 antibodies, and then Lineage− NKp46− CD117+ cells were sorted.

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    The reduction of IL-5 in 4μ8c treated cells is not due to changes in mRNA levels or stability. D10 cells were treated as in Fig. 1 . a RNA was converted to cDNA and then amplified via qRT-PCR. The results show the relative fold change to the no stimulated sample. The data is an average of six experiments for the PMA and <t>ionomycin</t> stimulated samples (black bars) and five for the plate-bound stimulated ones (white bars). The standard error is graphed. b D10 cells were rested and then stimulated in the presence of 4μ8c for 24 h. The cells were then treated with actinomycin D and harvested at times 0, 10, 30, 60, and 90 min after treatment. RNA was isolated and qRT-PCR performed. The samples were normalized to the time zero point of actinomycin D treatment. The data were graphed on a semi-log scale and are the average of four experiments. The error bars represent the standard error of the mean. c Protein was isolated from cells treated as in A and immunoblotted with GATA-3 and β-actin antibody. The data is representative of three experiments
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    The reduction of IL-5 in 4μ8c treated cells is not due to changes in mRNA levels or stability. D10 cells were treated as in Fig. 1 . a RNA was converted to cDNA and then amplified via qRT-PCR. The results show the relative fold change to the no stimulated sample. The data is an average of six experiments for the PMA and ionomycin stimulated samples (black bars) and five for the plate-bound stimulated ones (white bars). The standard error is graphed. b D10 cells were rested and then stimulated in the presence of 4μ8c for 24 h. The cells were then treated with actinomycin D and harvested at times 0, 10, 30, 60, and 90 min after treatment. RNA was isolated and qRT-PCR performed. The samples were normalized to the time zero point of actinomycin D treatment. The data were graphed on a semi-log scale and are the average of four experiments. The error bars represent the standard error of the mean. c Protein was isolated from cells treated as in A and immunoblotted with GATA-3 and β-actin antibody. The data is representative of three experiments

    Journal: BMC Immunology

    Article Title: Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion

    doi: 10.1186/s12865-018-0283-7

    Figure Lengend Snippet: The reduction of IL-5 in 4μ8c treated cells is not due to changes in mRNA levels or stability. D10 cells were treated as in Fig. 1 . a RNA was converted to cDNA and then amplified via qRT-PCR. The results show the relative fold change to the no stimulated sample. The data is an average of six experiments for the PMA and ionomycin stimulated samples (black bars) and five for the plate-bound stimulated ones (white bars). The standard error is graphed. b D10 cells were rested and then stimulated in the presence of 4μ8c for 24 h. The cells were then treated with actinomycin D and harvested at times 0, 10, 30, 60, and 90 min after treatment. RNA was isolated and qRT-PCR performed. The samples were normalized to the time zero point of actinomycin D treatment. The data were graphed on a semi-log scale and are the average of four experiments. The error bars represent the standard error of the mean. c Protein was isolated from cells treated as in A and immunoblotted with GATA-3 and β-actin antibody. The data is representative of three experiments

    Article Snippet: For flow cytometry experiments, D10 cells were treated as explained above with PMA and ionomycin or plate-bound α-CD3 and α-CD28 for 20 h when monensin (Biolegend) was added.

    Techniques: Amplification, Quantitative RT-PCR, Isolation

    IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value

    Journal: BMC Immunology

    Article Title: Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion

    doi: 10.1186/s12865-018-0283-7

    Figure Lengend Snippet: IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value

    Article Snippet: For flow cytometry experiments, D10 cells were treated as explained above with PMA and ionomycin or plate-bound α-CD3 and α-CD28 for 20 h when monensin (Biolegend) was added.

    Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 μM Ionomycin, or DMSO for 6 hours. Induction of HIV transcription was measured using RT-qPCR for the 5’ LTR region or Poly A region of the virus. **, p

    Journal: PLoS Pathogens

    Article Title: Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation

    doi: 10.1371/journal.ppat.1005066

    Figure Lengend Snippet: PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 μM Ionomycin, or DMSO for 6 hours. Induction of HIV transcription was measured using RT-qPCR for the 5’ LTR region or Poly A region of the virus. **, p

    Article Snippet: To measure changes in the cell activation status of CD4+ and CD8+ T cell subsets, PBMCs were isolated from uninfected controls and 2x106 cells were incubated with DMSO, 200 ng/ml PMA plus 2 μM Ionomycin, 6–12 nM PEP005 for 24 hrs or 72 hrs, and immunostained with anti-CD3, anti-CD38, anti-CD69, or anti-HLA-DR antibodies (Biolegend) for 20 min at 4°C.

    Techniques: Infection, Isolation, Quantitative RT-PCR

    γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: PTEN drives Th17 cell differentiation by preventing IL-2 production

    doi: 10.1084/jem.20170523

    Figure Lengend Snippet: γ:δ T cells are not altered in Pten cKO mice. (A) Pten mRNA expression in CD4 + T cells, neutrophils, γ:δ T cells, dendritic cells, and ILC3 cells was measured by qRT-PCR. Each cell type was isolated from WT ( Pten fl/fl ) and cKO ( Pten fl/fl Il17a cre ) mice. Data were normalized to Gapdh expression. Data were pooled from three individual experiments. (B) Development of γ:δ T cells in the spleen, pLNs, and thymus was measured by flow cytometry after cells were isolated from WT and cKO mice. (C) Splenocytes from WT and cKO mice were stimulated with PMA/ionomycin for 4 h, and gated on CD3ε + CD4 + population (top) or CD3ε + γ:δ TCR + population (bottom), and IL-17A expression was measured. Data were pooled from three individual experiments (right). (D) Mononuclear cells were isolated from colonic lamina propria. Cells were stimulated with PMA/ionomycin for 4 h, and IL-17A expression by CD4 + T cells was measured by flow cytometry. Data were pooled from three individual experiments (right). Dot plot data in B–D are representative of three individual experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P

    Article Snippet: Mononuclear cells were isolated by gradient centrifugation on a 30/70% Percoll gradient (GE Healthcare) at 670 g for 30 min. Isolated cells were stained for ILC3 isolation or stimulated with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend) for 4 h and then stained with PerCP/Cy5.5-conjugated IL-17A, and APC-conjugated anti-CD4 antibodies.

    Techniques: Mouse Assay, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry

    IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value

    Journal: BMC Immunology

    Article Title: Treatment of established TH2 cells with 4μ8c, an inhibitor of IRE1α, blocks IL-5 but not IL-4 secretion

    doi: 10.1186/s12865-018-0283-7

    Figure Lengend Snippet: IL-5 is reduced in established mouse TH2 cells upon treatment with 4μ8c. D10 cells were rested in complete T cell media for 24 h at 37 °C. The cells were then left un-stimulated (NS) or stimulated with PMA and ionomycin (PI) or plate-bound α-CD3 and α-CD28 in the presence or absence (−) of 4μ8c for 24 h. a As a control the level of spliced xbp1 mRNA was measured by qRT-PCR, as 4μ8c blocks the ability of IRE1α to cleave xbp1 . The data shown is the fold change in reduction of treated vs. untreated after normalizing to the ns control for five experiments. The supernatants were harvested, and ELISA was performed from these samples as shown in B and C. b The data shown is from six experiments where cells were re-stimulated with PMA and ionomycin in the presence or absence (−) of 4μ8c. c The data shown is for five experiments where the cells were re-stimulated with plate-bound antibodies in the presence or absence (−) of 4μ8c. The standard error, upper and lower bars, and the mean, middle bar, is shown in all graphs. Hypothesis testing was done by Student’s T test unpaired, Welch’s correction ( p value

    Article Snippet: For flow cytometry experiments, D10 cells were treated as explained above with PMA and ionomycin or plate-bound α-CD3 and α-CD28 for 20 h when monensin (Biolegend) was added.

    Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay