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Becton Dickinson brefeldin a
Tim-3 +  gB-CD8 +  T cells are multifunctional after  ex vivo  stimulation. TGs from latently infected mice (33 to 35 dpi) were removed, processed into single-cell suspensions, stained with anti-Tim-3 antibody, and stimulated for 6 h with gB peptide-pulsed B6WT3 cells in the presence of anti-CD107a and brefeldin A. Cells were then collected and stained for viability, CD45, CD8, IFN-γ, and TNF-α. In graphs, bars represent means ± SEM ( n  = 24), from two pooled independent experiments. (A) Representative dot plots of IFN-γ and TNF-α responses from an individual TG that received no peptide stimulation (3rd plot from the left) and an individual TG that received gB peptide stimulation (4th plot from the left). Shaded quadrants make up the “responding CD8 +  T cells.” Gating for CD107a is shown at the far right, with FMO represented in black and a representative sample in gray. (B) Percentage of responding CD8 +  T cells that are positive for Tim-3. (C) Percentage of Tim-3 +  responding CD8 +  T cells that are multifunctional (IFN-γ +  TNF-α +  CD107a + ).
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1) Product Images from "Differential Expression of Immune Checkpoint Molecules on CD8+ T Cells Specific for Immunodominant and Subdominant Herpes Simplex Virus 1 Epitopes"

Article Title: Differential Expression of Immune Checkpoint Molecules on CD8+ T Cells Specific for Immunodominant and Subdominant Herpes Simplex Virus 1 Epitopes

Journal: Journal of Virology

doi: 10.1128/JVI.01132-19

Tim-3 +  gB-CD8 +  T cells are multifunctional after  ex vivo  stimulation. TGs from latently infected mice (33 to 35 dpi) were removed, processed into single-cell suspensions, stained with anti-Tim-3 antibody, and stimulated for 6 h with gB peptide-pulsed B6WT3 cells in the presence of anti-CD107a and brefeldin A. Cells were then collected and stained for viability, CD45, CD8, IFN-γ, and TNF-α. In graphs, bars represent means ± SEM ( n  = 24), from two pooled independent experiments. (A) Representative dot plots of IFN-γ and TNF-α responses from an individual TG that received no peptide stimulation (3rd plot from the left) and an individual TG that received gB peptide stimulation (4th plot from the left). Shaded quadrants make up the “responding CD8 +  T cells.” Gating for CD107a is shown at the far right, with FMO represented in black and a representative sample in gray. (B) Percentage of responding CD8 +  T cells that are positive for Tim-3. (C) Percentage of Tim-3 +  responding CD8 +  T cells that are multifunctional (IFN-γ +  TNF-α +  CD107a + ).
Figure Legend Snippet: Tim-3 + gB-CD8 + T cells are multifunctional after ex vivo stimulation. TGs from latently infected mice (33 to 35 dpi) were removed, processed into single-cell suspensions, stained with anti-Tim-3 antibody, and stimulated for 6 h with gB peptide-pulsed B6WT3 cells in the presence of anti-CD107a and brefeldin A. Cells were then collected and stained for viability, CD45, CD8, IFN-γ, and TNF-α. In graphs, bars represent means ± SEM ( n  = 24), from two pooled independent experiments. (A) Representative dot plots of IFN-γ and TNF-α responses from an individual TG that received no peptide stimulation (3rd plot from the left) and an individual TG that received gB peptide stimulation (4th plot from the left). Shaded quadrants make up the “responding CD8 + T cells.” Gating for CD107a is shown at the far right, with FMO represented in black and a representative sample in gray. (B) Percentage of responding CD8 + T cells that are positive for Tim-3. (C) Percentage of Tim-3 + responding CD8 + T cells that are multifunctional (IFN-γ + TNF-α + CD107a + ).

Techniques Used: Ex Vivo, Infection, Mouse Assay, Staining

2) Product Images from "Costimulation through TLR2 drives polyfunctional CD8+ T cell responses"

Article Title: Costimulation through TLR2 drives polyfunctional CD8+ T cell responses

Journal: bioRxiv

doi: 10.1101/375840

TLR2-mediated costimulation augments Ifng mRNA expression levels ( A ) In vitro activated OT-I T cells (rested for 5-7 days) were stimulated with OVA int , OVA int plus Pam3 or OVA hi for indicated time points. 1μg/ml brefeldin A was added during the last 2h of activation and intracellular cytokine staining was performed to analyze protein production kinetics. Graphs indicate the total percentage of IFN-γ + , TNF-α + and IL-2 + T cells (mean±SD) pooled from 3 mice and 3 independently performed experiments. ( B-C ) Ifng mRNA expression and IFN-γ protein production was determined by Flow-FISH upon 2, 4, or 6h T cell activation as described above. Unstimulated T cells were used as negative control. (C) Graph indicates the total percentage of Ifng mRNA + T cells (top) and fold increase of Ifng mRNA MFI upon activation with indicated stimuli compared to unstimulated T cells (time 0; bottom). Data are presented as mean±SD of 3-5 mice and 2-4 independently performed experiments.
Figure Legend Snippet: TLR2-mediated costimulation augments Ifng mRNA expression levels ( A ) In vitro activated OT-I T cells (rested for 5-7 days) were stimulated with OVA int , OVA int plus Pam3 or OVA hi for indicated time points. 1μg/ml brefeldin A was added during the last 2h of activation and intracellular cytokine staining was performed to analyze protein production kinetics. Graphs indicate the total percentage of IFN-γ + , TNF-α + and IL-2 + T cells (mean±SD) pooled from 3 mice and 3 independently performed experiments. ( B-C ) Ifng mRNA expression and IFN-γ protein production was determined by Flow-FISH upon 2, 4, or 6h T cell activation as described above. Unstimulated T cells were used as negative control. (C) Graph indicates the total percentage of Ifng mRNA + T cells (top) and fold increase of Ifng mRNA MFI upon activation with indicated stimuli compared to unstimulated T cells (time 0; bottom). Data are presented as mean±SD of 3-5 mice and 2-4 independently performed experiments.

Techniques Used: Expressing, In Vitro, Activation Assay, Staining, Mouse Assay, Fluorescence In Situ Hybridization, Negative Control

3) Product Images from "RNA binding protein PCBP1 is an intracellular immune checkpoint for shaping T cell responses in cancer immunity"

Article Title: RNA binding protein PCBP1 is an intracellular immune checkpoint for shaping T cell responses in cancer immunity

Journal: Science Advances

doi: 10.1126/sciadv.aaz3865

Loss of PCBP1 causes optimal T reg cell development but is not required for the maintenance of T regs . ( A and B ) Intracellular FoxP3 in CD4 + CD8 − thymocytes (Thy) and peripheral T cells from the spleen (Spl) and mesenteric lymph nodes (MLN) (A) and quantification (B). n = 7. ( C and D ) Frequencies of CD25-, Helios-, and NRP1-expressing T cells among CD4 + FoxP3 + T regs (C) and CD4 + FoxP3 − T cells (D) from the spleen of Pcbp1 +/+ C d4 -Cre and Pcbp1 fl/fl C d4 -Cre mice. n = 5. ( E and F ) Representative flow cytometry plots of CD44 and CD62L (left) and quantification (right) in splenic CD4 + (E) and CD8 + (F) T cells from WT and Pcbp1 fl/fl C d4 -Cre mice ( n = 6). ( G and H ) Percentage IFN-γ + TNF-α + –producing T cells (left) and quantification (right) of CD4 + (G) and CD8 + (H) T cells stimulated with phorbol 12-myristate-13-acetate (PMA)/ionomycin in the presence of brefeldin A for 2 hours (WT, n = 3; KO, n = 4). ( I ) Histograms of CD25, CTLA-4, NRP1, ICOS, GITR, and PD-1 MFI (top) and quantification (bottom) in splenic T regs from Pcbp1 fl/fl Foxp3 /YFP-Cre +/− chimera female mice. n = 3. (B to I) Error bars represent means ± SE. * P
Figure Legend Snippet: Loss of PCBP1 causes optimal T reg cell development but is not required for the maintenance of T regs . ( A and B ) Intracellular FoxP3 in CD4 + CD8 − thymocytes (Thy) and peripheral T cells from the spleen (Spl) and mesenteric lymph nodes (MLN) (A) and quantification (B). n = 7. ( C and D ) Frequencies of CD25-, Helios-, and NRP1-expressing T cells among CD4 + FoxP3 + T regs (C) and CD4 + FoxP3 − T cells (D) from the spleen of Pcbp1 +/+ C d4 -Cre and Pcbp1 fl/fl C d4 -Cre mice. n = 5. ( E and F ) Representative flow cytometry plots of CD44 and CD62L (left) and quantification (right) in splenic CD4 + (E) and CD8 + (F) T cells from WT and Pcbp1 fl/fl C d4 -Cre mice ( n = 6). ( G and H ) Percentage IFN-γ + TNF-α + –producing T cells (left) and quantification (right) of CD4 + (G) and CD8 + (H) T cells stimulated with phorbol 12-myristate-13-acetate (PMA)/ionomycin in the presence of brefeldin A for 2 hours (WT, n = 3; KO, n = 4). ( I ) Histograms of CD25, CTLA-4, NRP1, ICOS, GITR, and PD-1 MFI (top) and quantification (bottom) in splenic T regs from Pcbp1 fl/fl Foxp3 /YFP-Cre +/− chimera female mice. n = 3. (B to I) Error bars represent means ± SE. * P

Techniques Used: Expressing, Mouse Assay, Flow Cytometry

4) Product Images from "Exacerbation of autoimmune neuroinflammation by dietary sodium is genetically controlled and sex specific"

Article Title: Exacerbation of autoimmune neuroinflammation by dietary sodium is genetically controlled and sex specific

Journal: The FASEB Journal

doi: 10.1096/fj.15-272542

Dietary sodium does not augment encephalitogenic T-cell responses in the CNS. Female B6 mice were immunized using 2 × MOG 35-55 /CFA. On day 21 after immunization, cells were isolated from the draining lymph nodes (dLN) or the CNS using a Percoll gradient (as described in Materials and Methods) and counted ( A ). CNS cells were stimulated for 4 h  ex vivo  with 50 µg/ml MOG 35-55  in the presence of brefeldin A, then surface stained and fixed, followed by permeabilization, staining for cytokines, and flow cytometry analysis ( B ). Cells were gated on live TCRβ +  and CD4 +  cells, and the percentages of cells positive for IL-17, IFN-γ, and GM-CSF were calculated. Data were analyzed by 2-way ANOVA and  post hoc  comparisons for effect of diet;  n  = 10 for each group. Representative flow cytometry plots are shown ( C ).
Figure Legend Snippet: Dietary sodium does not augment encephalitogenic T-cell responses in the CNS. Female B6 mice were immunized using 2 × MOG 35-55 /CFA. On day 21 after immunization, cells were isolated from the draining lymph nodes (dLN) or the CNS using a Percoll gradient (as described in Materials and Methods) and counted ( A ). CNS cells were stimulated for 4 h ex vivo with 50 µg/ml MOG 35-55 in the presence of brefeldin A, then surface stained and fixed, followed by permeabilization, staining for cytokines, and flow cytometry analysis ( B ). Cells were gated on live TCRβ + and CD4 + cells, and the percentages of cells positive for IL-17, IFN-γ, and GM-CSF were calculated. Data were analyzed by 2-way ANOVA and post hoc comparisons for effect of diet; n = 10 for each group. Representative flow cytometry plots are shown ( C ).

Techniques Used: Mouse Assay, Isolation, Ex Vivo, Staining, Flow Cytometry, Cytometry

Dietary sodium does not augment generation of peripheral encephalitogenic T-cell responses. Male (M) and female (F) B6 mice ( A–C ) were immunized using 2 × MOG 35-55 /CFA. SJL mice ( D–F ) were immunized using 2 × PLP 135-151 /CFA. On day 10 after immunization, cells were isolated from the draining lymph nodes ( A  and  D ), spleen ( B  and  E ), or mesenteric lymph nodes ( C  and  F ), then stimulated for 4 h  ex vivo  with PMA and ionomycin in the presence of brefeldin A. Cells were then surface stained and fixed, followed by permeabilization, staining for cytokines, and flow cytometry analysis. Cells were gated on live TCRβ +  and CD4 +  cells, and the percentages of cells positive for IL-17, IFN-γ, and GM-CSF were calculated. Data were analyzed by 2-way ANOVA and  post hoc  comparisons for effect of diet;  n  = 8 for each group. Representative flow cytometry plots of B6 male spleen cells are shown ( G ).
Figure Legend Snippet: Dietary sodium does not augment generation of peripheral encephalitogenic T-cell responses. Male (M) and female (F) B6 mice ( A–C ) were immunized using 2 × MOG 35-55 /CFA. SJL mice ( D–F ) were immunized using 2 × PLP 135-151 /CFA. On day 10 after immunization, cells were isolated from the draining lymph nodes ( A and D ), spleen ( B and E ), or mesenteric lymph nodes ( C and F ), then stimulated for 4 h ex vivo with PMA and ionomycin in the presence of brefeldin A. Cells were then surface stained and fixed, followed by permeabilization, staining for cytokines, and flow cytometry analysis. Cells were gated on live TCRβ + and CD4 + cells, and the percentages of cells positive for IL-17, IFN-γ, and GM-CSF were calculated. Data were analyzed by 2-way ANOVA and post hoc comparisons for effect of diet; n = 8 for each group. Representative flow cytometry plots of B6 male spleen cells are shown ( G ).

Techniques Used: Mouse Assay, Plasmid Purification, Isolation, Ex Vivo, Staining, Flow Cytometry, Cytometry

5) Product Images from "Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)"

Article Title: Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)

Journal: Clinical immunology (Orlando, Fla.)

doi: 10.1016/j.clim.2014.01.009

A.  Stimulation of sorted CD8+CD146+ T cells and CD8+CD146− T cells from healthy donors for 5 days in vitro with CD3/CD28 without the addition of any exogenous polarizing cytokines. PMA, ionomycin ,  and brefeldin A was added for the last 4 hours
Figure Legend Snippet: A. Stimulation of sorted CD8+CD146+ T cells and CD8+CD146− T cells from healthy donors for 5 days in vitro with CD3/CD28 without the addition of any exogenous polarizing cytokines. PMA, ionomycin , and brefeldin A was added for the last 4 hours

Techniques Used: In Vitro

Production of intracellular IFN-γ and IL-17A in CD8+ T cells in relationship to expression of CD146 in a representative sample of fresh peripheral blood from a healthy donor stimulated for 6 hours with PMA, ionomycin, and brefeldin A.
Figure Legend Snippet: Production of intracellular IFN-γ and IL-17A in CD8+ T cells in relationship to expression of CD146 in a representative sample of fresh peripheral blood from a healthy donor stimulated for 6 hours with PMA, ionomycin, and brefeldin A.

Techniques Used: Expressing

6) Product Images from "iNKT Cell Production of GM-CSF Controls Mycobacterium tuberculosis"

Article Title: iNKT Cell Production of GM-CSF Controls Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003805

iNKT cells produce GM-CSF during Mtb infection in a CD1d-dependent manner and it is critical for controlling Mtb growth. (A, B) IFNγ −/− iNKT cells added to uninfected or H37Rv-infected WT (A, B) and CD1d −/− mϕ (B). Murine GM-CSF measured in supernatant harvested after 24 hours. (C) % CFU reduction calculated from CFU assays for H37Rv-infected WT mϕ with IFNγ −/− iNKT cells added on d1 in the presence of anti-GM-CSF blocking antibody (10–50 µg/ml) or isotype control. (D) J3N.5 human iNKT cell clone added to uninfected or H37Rv-infected U937 cells in the presence of anti-human-CD1d blocking antibody or isotype control. Human GM-CSF measured in supernatant harvested after 24 hours. (E, F) Lung mononuclear cells from WT Mtb-infected mice were incubated with brefeldin A for four hours at 37°C and then stained. iNKT cells were identified as TCRβ + and CD1d-tetramer + . Percentage (E) and number (F) of iNKT cells producing GM-CSF and IFNγ. (G) % GM-CSF + , % IFNγ + , and CD69 MFI for iNKT cells transferred iv into WT or CD1d −/− Mtb-infected hosts and iNKT cells cultured for 20 hours in basic media. Lung mononuclear cells were treated and stained as in (E, F). Transferred iNKT cells were distinguished from host cells by eFluor 450 staining. Error bars indicate mean ± SEM. +P
Figure Legend Snippet: iNKT cells produce GM-CSF during Mtb infection in a CD1d-dependent manner and it is critical for controlling Mtb growth. (A, B) IFNγ −/− iNKT cells added to uninfected or H37Rv-infected WT (A, B) and CD1d −/− mϕ (B). Murine GM-CSF measured in supernatant harvested after 24 hours. (C) % CFU reduction calculated from CFU assays for H37Rv-infected WT mϕ with IFNγ −/− iNKT cells added on d1 in the presence of anti-GM-CSF blocking antibody (10–50 µg/ml) or isotype control. (D) J3N.5 human iNKT cell clone added to uninfected or H37Rv-infected U937 cells in the presence of anti-human-CD1d blocking antibody or isotype control. Human GM-CSF measured in supernatant harvested after 24 hours. (E, F) Lung mononuclear cells from WT Mtb-infected mice were incubated with brefeldin A for four hours at 37°C and then stained. iNKT cells were identified as TCRβ + and CD1d-tetramer + . Percentage (E) and number (F) of iNKT cells producing GM-CSF and IFNγ. (G) % GM-CSF + , % IFNγ + , and CD69 MFI for iNKT cells transferred iv into WT or CD1d −/− Mtb-infected hosts and iNKT cells cultured for 20 hours in basic media. Lung mononuclear cells were treated and stained as in (E, F). Transferred iNKT cells were distinguished from host cells by eFluor 450 staining. Error bars indicate mean ± SEM. +P

Techniques Used: Infection, Blocking Assay, Mouse Assay, Incubation, Staining, Cell Culture

7) Product Images from "Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes"

Article Title: Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004287

HDAC inhibitors impair IFN-γ production from PMA/ionomycin stimulated CD4 +  and CD8 +  T-cells. A, B . PBMC from subject OM292 were exposed to HDACis for 4 hours. Cells were then washed and cultured in fresh medium with PMA/ionomycin for an additional 5 hours in the presence of brefeldin A.  A . IFN-γ production in CD4 +  and CD8 +  T-cells was measured by intracellular cytokine staining flow cytometry. Shown are mean ± SEM values. P values were calculated by two-way ANOVA with Dunnett's multiple comparison test (comparing to the no drug control) * p
Figure Legend Snippet: HDAC inhibitors impair IFN-γ production from PMA/ionomycin stimulated CD4 + and CD8 + T-cells. A, B . PBMC from subject OM292 were exposed to HDACis for 4 hours. Cells were then washed and cultured in fresh medium with PMA/ionomycin for an additional 5 hours in the presence of brefeldin A. A . IFN-γ production in CD4 + and CD8 + T-cells was measured by intracellular cytokine staining flow cytometry. Shown are mean ± SEM values. P values were calculated by two-way ANOVA with Dunnett's multiple comparison test (comparing to the no drug control) * p

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

8) Product Images from "Human T cells employ conserved AU‐rich elements to fine‐tune IFN‐γ production"

Article Title: Human T cells employ conserved AU‐rich elements to fine‐tune IFN‐γ production

Journal: European Journal of Immunology

doi: 10.1002/eji.201948458

Enhanced IFN‐γ production by ARE‐Del T cells upon target cell recognition. MART‐1 TCR‐engineered T cells were co‐cultured with MART‐1 + and MART‐1 − cell lines expressing MART‐1 peptide for indicated time points. Brefeldin A was added 2 h prior to IFN‐γ production assessment. To define IFN‐γ producing T cells, MART‐1 TCR + T cells co‐cultured with MART‐1 − cells were used. Control T cells are depicted in gray, and ARE‐Del T cells are depicted in black. (A) Representative concatenated flow cytometry dot plots of IFN‐γ production of control and ARE‐Del T cells after 6, 24, and 48 h of co‐culture with MART‐1 + and MART‐1 − tumor cells. Separately measured samples containing control or ARE‐Del T cells were gated on live CD8 + T MART‐1 TCR + T cells (as depicted in Supporting Information Fig. 1B), exported and subsequently concatenated into one dot plot. IFN‐γ production is shown on the y ‐axis; samples are separated on the x ‐axis based on SampleID. Control T cells are depicted in gray, and ARE‐Del T cells are depicted in black. (B) Compiled flow cytometry data from n = 4 donors from three independent experiments (paired Student's t ‐test; * p
Figure Legend Snippet: Enhanced IFN‐γ production by ARE‐Del T cells upon target cell recognition. MART‐1 TCR‐engineered T cells were co‐cultured with MART‐1 + and MART‐1 − cell lines expressing MART‐1 peptide for indicated time points. Brefeldin A was added 2 h prior to IFN‐γ production assessment. To define IFN‐γ producing T cells, MART‐1 TCR + T cells co‐cultured with MART‐1 − cells were used. Control T cells are depicted in gray, and ARE‐Del T cells are depicted in black. (A) Representative concatenated flow cytometry dot plots of IFN‐γ production of control and ARE‐Del T cells after 6, 24, and 48 h of co‐culture with MART‐1 + and MART‐1 − tumor cells. Separately measured samples containing control or ARE‐Del T cells were gated on live CD8 + T MART‐1 TCR + T cells (as depicted in Supporting Information Fig. 1B), exported and subsequently concatenated into one dot plot. IFN‐γ production is shown on the y ‐axis; samples are separated on the x ‐axis based on SampleID. Control T cells are depicted in gray, and ARE‐Del T cells are depicted in black. (B) Compiled flow cytometry data from n = 4 donors from three independent experiments (paired Student's t ‐test; * p

Techniques Used: Cell Culture, Expressing, Flow Cytometry, Co-Culture Assay

9) Product Images from "IL-17-committed V?4+ ?? T cell deficiency in a spontaneous Sox13 mutant CD45.1 congenic mouse substrain protects from dermatitis"

Article Title: IL-17-committed V?4+ ?? T cell deficiency in a spontaneous Sox13 mutant CD45.1 congenic mouse substrain protects from dermatitis

Journal: Nature immunology

doi: 10.1038/ni.2585

Sox13 mut/mut  (B6.SJL/NCI) mice are protected from psoriasis-like dermatitis ( a ) Quantification of ear skin thickness, plotted as fraction increase relative to baseline (day 0), of WT (B6/NCI) and  Sox13 mut/mut  (B6.SJL/NCI) mice treated with imiquimod or control cream daily for 5 days. Boxes represent the mean (± s.d.). ( b ) H  E staining of ear skin from WT and  Sox13 mut/mut  mice treated per ( a ) for 5 days. ( c ) Quantification of Ly6G + CD11b +  neutrophils in ear skin cell suspensions from WT and  Sox13 mut/mut  mice treated per ( a ) for 3 or 5 days. Each symbol represents an individual mouse; horizontal bars represent the mean (± s.d.). ( d ) RT-PCR quantification of ear skin mRNA from WT and  Sox13 mut/mut  mice treated with control (−) or imiquimod cream for 3 or 5 days. Boxes represent the mean (± s.d.). ( e ) Intracellular IL-17A staining of ear skin cell suspensions from WT and  Sox13 mut/mut  mice treated as in ( a ) for 3 days and digested in the presence of Brefeldin A, gated on total γδ T cells. Mean (± s.d.) is indicated. ( f ) Transwell assay of neutrophil migration to ear skin supernatants prepared from WT and  Sox13 mut/mut  mice treated as in ( a ) for 3 days. Each symbol represents migration from an individual transwell, horizontal bars represent the mean (± s.d.). * P ≤0.05, ** P ≤0.01. Data are representative of three experiments with 3–6 mice ( a–d ), two experiments with 2–5 mice ( e ), and four experiments with 9 mice ( f ).
Figure Legend Snippet: Sox13 mut/mut (B6.SJL/NCI) mice are protected from psoriasis-like dermatitis ( a ) Quantification of ear skin thickness, plotted as fraction increase relative to baseline (day 0), of WT (B6/NCI) and Sox13 mut/mut (B6.SJL/NCI) mice treated with imiquimod or control cream daily for 5 days. Boxes represent the mean (± s.d.). ( b ) H E staining of ear skin from WT and Sox13 mut/mut mice treated per ( a ) for 5 days. ( c ) Quantification of Ly6G + CD11b + neutrophils in ear skin cell suspensions from WT and Sox13 mut/mut mice treated per ( a ) for 3 or 5 days. Each symbol represents an individual mouse; horizontal bars represent the mean (± s.d.). ( d ) RT-PCR quantification of ear skin mRNA from WT and Sox13 mut/mut mice treated with control (−) or imiquimod cream for 3 or 5 days. Boxes represent the mean (± s.d.). ( e ) Intracellular IL-17A staining of ear skin cell suspensions from WT and Sox13 mut/mut mice treated as in ( a ) for 3 days and digested in the presence of Brefeldin A, gated on total γδ T cells. Mean (± s.d.) is indicated. ( f ) Transwell assay of neutrophil migration to ear skin supernatants prepared from WT and Sox13 mut/mut mice treated as in ( a ) for 3 days. Each symbol represents migration from an individual transwell, horizontal bars represent the mean (± s.d.). * P ≤0.05, ** P ≤0.01. Data are representative of three experiments with 3–6 mice ( a–d ), two experiments with 2–5 mice ( e ), and four experiments with 9 mice ( f ).

Techniques Used: Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Transwell Assay, Migration

10) Product Images from "Monocyte Trafficking to Hepatic Sites of Bacterial Infection Is Chemokine Independent and Directed by Focal Intercellular Adhesion Molecule-1 Expression"

Article Title: Monocyte Trafficking to Hepatic Sites of Bacterial Infection Is Chemokine Independent and Directed by Focal Intercellular Adhesion Molecule-1 Expression

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

doi: 10.4049/jimmunol.0904160

Ly6C high monocytes differentiate into TipDCs at foci of infection. Liver nonparenchymal cells were collected from C57BL/6 mice 3 d following L. monocytogenes infection and incubated with brefeldin A for 4 h. A , CD11b expression and TNF production by cells
Figure Legend Snippet: Ly6C high monocytes differentiate into TipDCs at foci of infection. Liver nonparenchymal cells were collected from C57BL/6 mice 3 d following L. monocytogenes infection and incubated with brefeldin A for 4 h. A , CD11b expression and TNF production by cells

Techniques Used: Infection, Mouse Assay, Incubation, Expressing

11) Product Images from "A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy"

Article Title: A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000948

Primary CD8 + T-cell responses against FrCas E -infected cells. Mice were infected and treated, or not, as in Figure 1 . (A) Kinetics of the primary anti-FrCas E CTL response . Spleen cells from 2 mice of each group per time point (days 8, 22 and 28 post-infection) and from 5 mice per group at day 15 post-infection were stained with an anti-CD8 mAb and the D b -GagL tetramer and analyzed by flow cytometry. (B–D) Phenotypic characterization of primary CD8 + T-cell responses in infected/treated animals. Mice were infected and treated as in Figure 1 . CD3 + cells were isolated by negative selection from the spleens of 4 infected/treated- and 2 infected/non-treated mice on day 56 post-infection. Half of the CD3 + cells were then stained with the D b -GagL tetramer and anti-CD8, anti-CD44, anti-CD62L and anti-CD25 mAb and analyzed by flow cytometry. In parallel, the other half of the CD3 + cells were incubated with PMA-ionomycin plus brefeldin A and IFN-γ production by tetramer + CD8 + T cells was assessed by intracellular staining. The statistical significance of data between infected/treated- and infected/non-treated mice was established using the Student's t test (* P = 0,045). (B) Assay of GagL-specific CD8 + T cells in infected/treated and infected/non-treated mice on day 56 post-infection. (C–D) Phenotypic characterization of GagL-specific CD8 + T cells on day 56 post-infection. Data from 1 age-matched non-infected/non-treated (naive) mouse and 4 infected/treated mice are presented.
Figure Legend Snippet: Primary CD8 + T-cell responses against FrCas E -infected cells. Mice were infected and treated, or not, as in Figure 1 . (A) Kinetics of the primary anti-FrCas E CTL response . Spleen cells from 2 mice of each group per time point (days 8, 22 and 28 post-infection) and from 5 mice per group at day 15 post-infection were stained with an anti-CD8 mAb and the D b -GagL tetramer and analyzed by flow cytometry. (B–D) Phenotypic characterization of primary CD8 + T-cell responses in infected/treated animals. Mice were infected and treated as in Figure 1 . CD3 + cells were isolated by negative selection from the spleens of 4 infected/treated- and 2 infected/non-treated mice on day 56 post-infection. Half of the CD3 + cells were then stained with the D b -GagL tetramer and anti-CD8, anti-CD44, anti-CD62L and anti-CD25 mAb and analyzed by flow cytometry. In parallel, the other half of the CD3 + cells were incubated with PMA-ionomycin plus brefeldin A and IFN-γ production by tetramer + CD8 + T cells was assessed by intracellular staining. The statistical significance of data between infected/treated- and infected/non-treated mice was established using the Student's t test (* P = 0,045). (B) Assay of GagL-specific CD8 + T cells in infected/treated and infected/non-treated mice on day 56 post-infection. (C–D) Phenotypic characterization of GagL-specific CD8 + T cells on day 56 post-infection. Data from 1 age-matched non-infected/non-treated (naive) mouse and 4 infected/treated mice are presented.

Techniques Used: Infection, Mouse Assay, CTL Assay, Staining, Flow Cytometry, Cytometry, Isolation, Selection, Incubation

12) Product Images from "Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy"

Article Title: Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1601279

Cytokine secretion by dNK cells in response to KIR2DS4 activation. ( A  and  B ) A semiquantitative fluorescent chip-based sandwich ELISA was used to screen for 120 cytokines in supernatants taken from mixed decidual leukocytes of KIR2DS4 +  donors (see   Supplemental Table I ). Leukocytes were cultured on Ab-coated plastic for 12–24 h, where the only cells to express KIR2DS4 were the dNK cells. Fluorescent spots for cytokines of interest are highlighted in (A). The cropped regions of interest are taken from different chips and different donors. They are grouped according to whether they show a  > 1.5-fold increase in secretion on average across all donors (Increase); secretion that was already high within the isotype control stimulation, so the screen was insensitive (Ambiguous); and control spots (Control). (B) The cytokines that were upregulated  > 1.25-fold upon KIR2DS4 activation in at least one of four donors tested are listed in the table. The mean fold change across all four donors is shown to the right. Values  > 1.25-fold are highlighted in gray. ( C – E ) Mixed decidual leukocytes were cultured on plastic coated with either anti-KIR2DS4 Ab or an isotype control (IgG2a) in the presence of monensin and brefeldin A. After 5 h, cells were fixed and live CD56 + CD9 + KIR2DS4 +  dNK cells were identified by flow cytometry (C). Although KIR2DS4 expression reduced upon cross-linking (C), most retained KIR2DS4 expression. These KIR2DS4 +  dNK cells were assessed for intracellular cytokines: (D) GM-CSF ( n  = 7) and (E) CCL3 ( n  = 7). ( F ) When Abs for flow cytometry were not available, purified dNK cells were cultured on Ab-coated plastic for 12–48 h and the production of CCL1 and XCL1 ( n  = 8) was detected in supernatants by commercial sandwich ELISA. Results are color coded according to donor. * p
Figure Legend Snippet: Cytokine secretion by dNK cells in response to KIR2DS4 activation. ( A and B ) A semiquantitative fluorescent chip-based sandwich ELISA was used to screen for 120 cytokines in supernatants taken from mixed decidual leukocytes of KIR2DS4 + donors (see Supplemental Table I ). Leukocytes were cultured on Ab-coated plastic for 12–24 h, where the only cells to express KIR2DS4 were the dNK cells. Fluorescent spots for cytokines of interest are highlighted in (A). The cropped regions of interest are taken from different chips and different donors. They are grouped according to whether they show a > 1.5-fold increase in secretion on average across all donors (Increase); secretion that was already high within the isotype control stimulation, so the screen was insensitive (Ambiguous); and control spots (Control). (B) The cytokines that were upregulated > 1.25-fold upon KIR2DS4 activation in at least one of four donors tested are listed in the table. The mean fold change across all four donors is shown to the right. Values > 1.25-fold are highlighted in gray. ( C – E ) Mixed decidual leukocytes were cultured on plastic coated with either anti-KIR2DS4 Ab or an isotype control (IgG2a) in the presence of monensin and brefeldin A. After 5 h, cells were fixed and live CD56 + CD9 + KIR2DS4 + dNK cells were identified by flow cytometry (C). Although KIR2DS4 expression reduced upon cross-linking (C), most retained KIR2DS4 expression. These KIR2DS4 + dNK cells were assessed for intracellular cytokines: (D) GM-CSF ( n = 7) and (E) CCL3 ( n = 7). ( F ) When Abs for flow cytometry were not available, purified dNK cells were cultured on Ab-coated plastic for 12–48 h and the production of CCL1 and XCL1 ( n = 8) was detected in supernatants by commercial sandwich ELISA. Results are color coded according to donor. * p

Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Sandwich ELISA, Cell Culture, Flow Cytometry, Cytometry, Expressing, Purification

13) Product Images from "Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia"

Article Title: Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia

Journal: Cellular and Molecular Immunology

doi: 10.1038/cmi.2016.69

Itch −/−  CD4 +  T cells are sufficient to drive loss of alveolar macrophages in an IL-4-dependent manner. ( a ) Diagram describing experimental design. A total of 4 × 10 6  purified spleen CD4 +  T cells from WT, Itch −/−  or Itch −/− IL4 −/−  mice were transferred intravenously to Rag −/−  mice, then BAL and lungs were collected 8 weeks later. ( b ) Lung cell suspensions were stimulated with PMA and ionomycin for 4 h in the presence of Brefeldin A, and then cells were stained for surface markers and intracellular cytokines. Representative flow cytometry plots and quantifications of absolute numbers are shown. Cells are gated on live, singlet, CD3 + CD4 + . Dots represent individual mice ( n =5–6). ( c ) Alveolar macrophages were identified from lung cell suspensions by flow cytometry. Representative flow cytometry plots and quantification of alveolar macrophages are shown. Flow plots were gated on live, singlet, CD45 + , and alveolar macrophages were CD11c + CD11b lo SiglecF + . ( d ) The mean fluorescence intensity of the forward- and side-scatter parameters (FSC and SSC, respectively) was calculated for alveolar macrophages using the geometric mean formula on FlowJo software. Alveolar macrophage numbers, FSC and SSC were divided by the average WT numbers within each experiment to calculate fold change per experiment, normalizing for experimental variability ( n =5–6, compiled from two independent experiments). ( e ) Representative BAL cytospins stained with modified Giemsa stain and visualized at × 40. Significance was calculated using a one-way analysis of variance. * or ** denote  P ≤0.05 or  P ≤0.01, respectively.
Figure Legend Snippet: Itch −/− CD4 + T cells are sufficient to drive loss of alveolar macrophages in an IL-4-dependent manner. ( a ) Diagram describing experimental design. A total of 4 × 10 6 purified spleen CD4 + T cells from WT, Itch −/− or Itch −/− IL4 −/− mice were transferred intravenously to Rag −/− mice, then BAL and lungs were collected 8 weeks later. ( b ) Lung cell suspensions were stimulated with PMA and ionomycin for 4 h in the presence of Brefeldin A, and then cells were stained for surface markers and intracellular cytokines. Representative flow cytometry plots and quantifications of absolute numbers are shown. Cells are gated on live, singlet, CD3 + CD4 + . Dots represent individual mice ( n =5–6). ( c ) Alveolar macrophages were identified from lung cell suspensions by flow cytometry. Representative flow cytometry plots and quantification of alveolar macrophages are shown. Flow plots were gated on live, singlet, CD45 + , and alveolar macrophages were CD11c + CD11b lo SiglecF + . ( d ) The mean fluorescence intensity of the forward- and side-scatter parameters (FSC and SSC, respectively) was calculated for alveolar macrophages using the geometric mean formula on FlowJo software. Alveolar macrophage numbers, FSC and SSC were divided by the average WT numbers within each experiment to calculate fold change per experiment, normalizing for experimental variability ( n =5–6, compiled from two independent experiments). ( e ) Representative BAL cytospins stained with modified Giemsa stain and visualized at × 40. Significance was calculated using a one-way analysis of variance. * or ** denote P ≤0.05 or P ≤0.01, respectively.

Techniques Used: Purification, Mouse Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, Software, Modification, Giemsa Stain

14) Product Images from "Cytomegalovirus Evades TRAIL-Mediated Innate Lymphoid Cell 1 Defenses"

Article Title: Cytomegalovirus Evades TRAIL-Mediated Innate Lymphoid Cell 1 Defenses

Journal: Journal of Virology

doi: 10.1128/JVI.00617-19

TRAIL-expressing group I ILCs display characteristic phenotypic markers of ILC1. (A) Expression of CD49a, CD69, CD61, CD62L, CD11b, and KLRG1 was assessed in total TRAIL + group I ILCs, ILC1, and cNK at 36 h after infection. (B, C) Expression of CD49a, CD69, CD61, CD62L, CD11b, and KLRG1. (B) Expression of each marker by ILC1, cNK, and TRAIL + group I ILC subsets from naive mice. (C) Expression of each marker by cNK and TRAIL + group I ILCs at 36 h after infection with the MCMV WT or m166 stop . (D) ILC1 and cNK from naive mice and mice infected for 36 h were analyzed directly ex vivo for their expression of IFN-γ after 5 h of incubation with brefeldin A and monensin with or without PMA and ionomycin. No Stim, no stimulation; FSCA, forward scatter area. *, P
Figure Legend Snippet: TRAIL-expressing group I ILCs display characteristic phenotypic markers of ILC1. (A) Expression of CD49a, CD69, CD61, CD62L, CD11b, and KLRG1 was assessed in total TRAIL + group I ILCs, ILC1, and cNK at 36 h after infection. (B, C) Expression of CD49a, CD69, CD61, CD62L, CD11b, and KLRG1. (B) Expression of each marker by ILC1, cNK, and TRAIL + group I ILC subsets from naive mice. (C) Expression of each marker by cNK and TRAIL + group I ILCs at 36 h after infection with the MCMV WT or m166 stop . (D) ILC1 and cNK from naive mice and mice infected for 36 h were analyzed directly ex vivo for their expression of IFN-γ after 5 h of incubation with brefeldin A and monensin with or without PMA and ionomycin. No Stim, no stimulation; FSCA, forward scatter area. *, P

Techniques Used: Expressing, Infection, Marker, Mouse Assay, Ex Vivo, Incubation

IFN-γ hyporesponsiveness in the SG is group I ILC specific. T cells (CD3 + TCRβ + ) isolated from the SG of naive mice and mice infected for 8 days were analyzed ex vivo for their expression of IFN-γ after 5 h of incubation with brefeldin A and monensin with or without PMA and ionomycin.
Figure Legend Snippet: IFN-γ hyporesponsiveness in the SG is group I ILC specific. T cells (CD3 + TCRβ + ) isolated from the SG of naive mice and mice infected for 8 days were analyzed ex vivo for their expression of IFN-γ after 5 h of incubation with brefeldin A and monensin with or without PMA and ionomycin.

Techniques Used: Isolation, Mouse Assay, Infection, Ex Vivo, Expressing, Incubation

ILC1 are the major TRAIL-expressing cells in MCMV-infected salivary glands. The panels present the results of analysis of total SG mononuclear cells isolated from naive WT or Trail −/− mice and at 8 days postinfection with MCMV WT or m166 stop . (A) Group I ILCs from mice infected with the MCMV WT for 8 days were divided according to their expression of CD61. Cell surface expression of CD49a, CD69, CD62L, CD11b, and KLRG1 was assessed on CD61 + , CD61 − , and TRAIL + total group I ILCs. (B) Absolute number of ILC1 (CD61 + ) and cNK (CD61 − ) group I ILCs from naive mice (yellow) and at 8 days after infection with either the MCMV WT (purple) or m166 stop (green). (C) TRAIL expression by cNK (CD61 − group I ILCs) and ILC1 (CD61 + group I ILCs) from naive mice and mice infected for 8 days. (D) The proportion of TRAIL + ILC1 and cNK from naive mice (yellow) and at 8 days after infection with MCMV WT (purple) or m166 stop (green). (E) ILC1 (CD61 + group I ILCs, blue) and cNK (CD61 − group I ILCs, gray) from naive mice and mice infected for 8 days were analyzed ex vivo for their expression of IFN-γ after 5 h of incubation with brefeldin A and monensin with or without PMA and ionomycin. *, P
Figure Legend Snippet: ILC1 are the major TRAIL-expressing cells in MCMV-infected salivary glands. The panels present the results of analysis of total SG mononuclear cells isolated from naive WT or Trail −/− mice and at 8 days postinfection with MCMV WT or m166 stop . (A) Group I ILCs from mice infected with the MCMV WT for 8 days were divided according to their expression of CD61. Cell surface expression of CD49a, CD69, CD62L, CD11b, and KLRG1 was assessed on CD61 + , CD61 − , and TRAIL + total group I ILCs. (B) Absolute number of ILC1 (CD61 + ) and cNK (CD61 − ) group I ILCs from naive mice (yellow) and at 8 days after infection with either the MCMV WT (purple) or m166 stop (green). (C) TRAIL expression by cNK (CD61 − group I ILCs) and ILC1 (CD61 + group I ILCs) from naive mice and mice infected for 8 days. (D) The proportion of TRAIL + ILC1 and cNK from naive mice (yellow) and at 8 days after infection with MCMV WT (purple) or m166 stop (green). (E) ILC1 (CD61 + group I ILCs, blue) and cNK (CD61 − group I ILCs, gray) from naive mice and mice infected for 8 days were analyzed ex vivo for their expression of IFN-γ after 5 h of incubation with brefeldin A and monensin with or without PMA and ionomycin. *, P

Techniques Used: Expressing, Infection, Isolation, Mouse Assay, Ex Vivo, Incubation

15) Product Images from "Cutting Edge: A Single MHC Class Ia Is Sufficient for CD8 Memory T Cell Differentiation 1"

Article Title: Cutting Edge: A Single MHC Class Ia Is Sufficient for CD8 Memory T Cell Differentiation 1

Journal:

doi:

Memory cells from ScD d β 2 M −/−  mice express normal levels of IL-7R α  and IL-2. At 90 days p.i., splenocytes were assessed for the expression of IFN- γ  following a 4-h restimulation in the presence of Brefeldin A. Cells
Figure Legend Snippet: Memory cells from ScD d β 2 M −/− mice express normal levels of IL-7R α and IL-2. At 90 days p.i., splenocytes were assessed for the expression of IFN- γ following a 4-h restimulation in the presence of Brefeldin A. Cells

Techniques Used: Mouse Assay, Expressing

16) Product Images from "Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome"

Article Title: Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome

Journal: Blood

doi: 10.1182/blood-2010-05-285080

PD-1 expressing cells produce more IFN-γ in response to polyclonal stimulation in comparison to PD-1 − CD4 + T cells . Cryopreserved PBMCs were thawed and cultured for 5 hours in the presence of PMA, ionomycin, and Brefeldin-A. The percentage of IFN-γ (A), TNF-α (B), IL-2 (C), IL-4 (D), and IL-17 (E) expression within PD-1 + (filled symbols) and PD-1 − (open symbols) CD4 + T lymphocytes in HIV + (controls and IRIS) patients were measured before and 3 months after ART initiation, as well as in healthy donors (HD). Symbols represent frequencies of cytokine-producing cells after PMA/ionomycin stimulation minus background cytokine production in unstimulated controls.
Figure Legend Snippet: PD-1 expressing cells produce more IFN-γ in response to polyclonal stimulation in comparison to PD-1 − CD4 + T cells . Cryopreserved PBMCs were thawed and cultured for 5 hours in the presence of PMA, ionomycin, and Brefeldin-A. The percentage of IFN-γ (A), TNF-α (B), IL-2 (C), IL-4 (D), and IL-17 (E) expression within PD-1 + (filled symbols) and PD-1 − (open symbols) CD4 + T lymphocytes in HIV + (controls and IRIS) patients were measured before and 3 months after ART initiation, as well as in healthy donors (HD). Symbols represent frequencies of cytokine-producing cells after PMA/ionomycin stimulation minus background cytokine production in unstimulated controls.

Techniques Used: Expressing, Cell Culture

17) Product Images from "Mononuclear Phagocyte-Derived Interleukin-10 Suppresses the Innate Pulmonary Granuloma Cytokine Response in Aged Mice"

Article Title: Mononuclear Phagocyte-Derived Interleukin-10 Suppresses the Innate Pulmonary Granuloma Cytokine Response in Aged Mice

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2007.061122

Blood monocytes and lung mononuclear phagocytes of young and aged mice display similar ex vivo IL-10 responses to PPD challenge. Pooled peripheral blood leukocytes from naïve young (4 months) and aged (22 months) C57BL/6 mice were collected and washed by centrifugation before culture. Individual lungs from naïve young (4 months) and aged (22 months) C57BL/6 mice were harvested, and single-cell suspensions from digested whole lung tissues were washed by centrifugation before culture. Blood and lung preparations were incubated for 3 hours in complete medium in the presence of brefeldin A with or without PPD (5 μg/ml). Cells were then stained for expression of CD11b, Gr-1, and intracellular IL-10 and then analyzed by flow cytometry. A: Scattergrams comparing IL-10-producing CD11b + Gr1 lo cells in control and PPD-stimulated blood leukocyte cultures of young and aged mice. B: Bar graph showing that the frequency of IL-10-producing cells in the blood does not change with age. C: Scattergrams comparing IL-10-producing CD11b + Gr1 lo cells in control and PPD-stimulated lung cell cultures of young and aged mice. D: Bar graphs showing the proportion and absolute number of IL-10-producing cells in the lung cells in young versus aged mice. * P
Figure Legend Snippet: Blood monocytes and lung mononuclear phagocytes of young and aged mice display similar ex vivo IL-10 responses to PPD challenge. Pooled peripheral blood leukocytes from naïve young (4 months) and aged (22 months) C57BL/6 mice were collected and washed by centrifugation before culture. Individual lungs from naïve young (4 months) and aged (22 months) C57BL/6 mice were harvested, and single-cell suspensions from digested whole lung tissues were washed by centrifugation before culture. Blood and lung preparations were incubated for 3 hours in complete medium in the presence of brefeldin A with or without PPD (5 μg/ml). Cells were then stained for expression of CD11b, Gr-1, and intracellular IL-10 and then analyzed by flow cytometry. A: Scattergrams comparing IL-10-producing CD11b + Gr1 lo cells in control and PPD-stimulated blood leukocyte cultures of young and aged mice. B: Bar graph showing that the frequency of IL-10-producing cells in the blood does not change with age. C: Scattergrams comparing IL-10-producing CD11b + Gr1 lo cells in control and PPD-stimulated lung cell cultures of young and aged mice. D: Bar graphs showing the proportion and absolute number of IL-10-producing cells in the lung cells in young versus aged mice. * P

Techniques Used: Mouse Assay, Ex Vivo, Centrifugation, Incubation, Staining, Expressing, Flow Cytometry, Cytometry

Numbers of IL-10-producing CD11b + Gr1 lo mononuclear cells increase in aged mice during innate pulmonary PPD bead granuloma formation. Young (4 months) and aged (22 months) C57BL/6 mice were injected intravenously with PPD beads on day 0. Lungs were harvested on day 2. Single-cell suspensions from digested whole lung tissues were incubated for 3 hours in complete medium in the presence of brefeldin A without stimulation to allow intracellular accumulation of spontaneous production of cytokines. The cells were then stained for expression of CD11b, Gr-1, and intracellular IL-10 and analyzed by flow cytometry. A: Scattergram demonstrating IL-10 expression by CD11b + cells in the lung. B: Scattergram demonstrating that IL-10 is not expressed by Gr-1 hi (neutrophils) but by a subset of Gr-1 lo (mononuclear phagocytes) among gated CD11b + lung cells. C shows the proportion and number of IL-10-producing cells in the lung and the mean fluorescent intensity (MFI) of IL-10 + cells in young versus aged mice. * P
Figure Legend Snippet: Numbers of IL-10-producing CD11b + Gr1 lo mononuclear cells increase in aged mice during innate pulmonary PPD bead granuloma formation. Young (4 months) and aged (22 months) C57BL/6 mice were injected intravenously with PPD beads on day 0. Lungs were harvested on day 2. Single-cell suspensions from digested whole lung tissues were incubated for 3 hours in complete medium in the presence of brefeldin A without stimulation to allow intracellular accumulation of spontaneous production of cytokines. The cells were then stained for expression of CD11b, Gr-1, and intracellular IL-10 and analyzed by flow cytometry. A: Scattergram demonstrating IL-10 expression by CD11b + cells in the lung. B: Scattergram demonstrating that IL-10 is not expressed by Gr-1 hi (neutrophils) but by a subset of Gr-1 lo (mononuclear phagocytes) among gated CD11b + lung cells. C shows the proportion and number of IL-10-producing cells in the lung and the mean fluorescent intensity (MFI) of IL-10 + cells in young versus aged mice. * P

Techniques Used: Mouse Assay, Injection, Incubation, Staining, Expressing, Flow Cytometry, Cytometry

18) Product Images from "Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells"

Article Title: Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-2-41

Hyperexpanded CD4 + and CD8 + T cells produce IFN-γ in response to tumor stimulation. CD62L low TDLN cells were culture activated with anti-CD3 and IL-2 plus IL-7 for 23 days then were restimulated with anti-CD3 every 7 days. T cells were removed from culture on day 8, on day 36 for CD8 + cultures, or day 43 for CD4 + cultures. T cells were incubated without additional stimulus to determine spontaneous production of IFN-γ or with single cell digest of MCA205 or MCA207 tumors or with immobilized anti-CD3 mAb and Brefeldin A was added at 5 hrs and cells were harvested after 14 hrs. Intracellular IFN-γ was determined by FACS and the percentage of T cells is indicated.
Figure Legend Snippet: Hyperexpanded CD4 + and CD8 + T cells produce IFN-γ in response to tumor stimulation. CD62L low TDLN cells were culture activated with anti-CD3 and IL-2 plus IL-7 for 23 days then were restimulated with anti-CD3 every 7 days. T cells were removed from culture on day 8, on day 36 for CD8 + cultures, or day 43 for CD4 + cultures. T cells were incubated without additional stimulus to determine spontaneous production of IFN-γ or with single cell digest of MCA205 or MCA207 tumors or with immobilized anti-CD3 mAb and Brefeldin A was added at 5 hrs and cells were harvested after 14 hrs. Intracellular IFN-γ was determined by FACS and the percentage of T cells is indicated.

Techniques Used: Incubation, FACS

19) Product Images from "Oligonucleotide Motifs That Disappear during the Evolution of Influenza Virus in Humans Increase Alpha Interferon Secretion by Plasmacytoid Dendritic Cells ▿"

Article Title: Oligonucleotide Motifs That Disappear during the Evolution of Influenza Virus in Humans Increase Alpha Interferon Secretion by Plasmacytoid Dendritic Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01908-10

CpG motifs and in a U-rich context determine the ssRNA capacity to induce TNF-α production by pDCs. Pure pDCs were stimulated with the indicated CpG/GpC RNA sequences for 18 h in the presence of brefeldin A. Intracellular detection of TNF-α was carried out using specific antibodies for the cytokine in previously gated pDCs (CD123 +  cells). (A) Cytometry panels show TNF-α production in stimulated pDCs from one donor after CpG/GpC RNA stimulation. (B) TNF-α-induced production comparing GpC RNA and CpG RNA sequences is shown. The value for CpG RNA-induced production was set at 100%. The MFI for TNF-α is represented ( n  = 4, in duplicate). Statistical analysis was done using unpaired Student's  t  test (2-tailed), comparing in each sequence CpG RNA- and A RNA- versus GpC-induced cytokine production. *,  P
Figure Legend Snippet: CpG motifs and in a U-rich context determine the ssRNA capacity to induce TNF-α production by pDCs. Pure pDCs were stimulated with the indicated CpG/GpC RNA sequences for 18 h in the presence of brefeldin A. Intracellular detection of TNF-α was carried out using specific antibodies for the cytokine in previously gated pDCs (CD123 + cells). (A) Cytometry panels show TNF-α production in stimulated pDCs from one donor after CpG/GpC RNA stimulation. (B) TNF-α-induced production comparing GpC RNA and CpG RNA sequences is shown. The value for CpG RNA-induced production was set at 100%. The MFI for TNF-α is represented ( n = 4, in duplicate). Statistical analysis was done using unpaired Student's t test (2-tailed), comparing in each sequence CpG RNA- and A RNA- versus GpC-induced cytokine production. *, P

Techniques Used: Gel Permeation Chromatography, Cytometry, Sequencing

20) Product Images from "Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle"

Article Title: Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle

Journal: Scientific Reports

doi: 10.1038/s41598-020-75410-0

The protection against L. longipalpis sand fly-transmitted L. major infection in BALB/c mice is dependent on high IFN-γ/IL-10 and IFN-γ/IL-4 ratios. BALB/C mice were immunized intradermally in the right ear three times at 2 weeks intervals with 5 µg of VR-2001-SfSPChimera plasmid (crossed circles), or with the empty vector (control animals; black circles). One month after the last immunization mice were challenged on the contralateral ear via sand fly bites—10 L. longipalpis sand flies carrying mature L. major infections were used per animal. Two weeks later animals were euthanized and their ears collected and processed. Parasite burdens were determined by limiting dilution (Fig. 5 F) and T cell cytokine secretion was analysed by flow cytometry after an overnight culture of total ear cells in the presence of L. major SLA, and additionally PMA, Ionomycin and Brefeldin A during the last 4 h of culture. For each animal, including controls, parasite burden values were plotted against the frequencies of CD4 + T cells secreting IFN-γ, IL-10 or IL-4, in a way to assess their correlation ( A ). These frequencies are represented for VR-2001-SfSPChimera immunized and control animals, sub-grouped (Fig. 5 F) according to parasite burden values ( B ). IFN-γ/IL-10 and IFN-γ/IL-4 ratios were calculated and are represented using the same group division criteria ( C ). The correlation between these ratio values and the parasite burdens was also assessed, now only considering the animals immunized with VR-2001-SfSPChimera DNA vaccine ( C ). Results from two independent experiments are represented. Each dot represents one animal. Average and SD of the values within each group are shown. Statistical differences are properly identified (Mann–Whitney test: * p ≤ 0.05 and ** p ≤ 0.01) and refer to differences between sub-groups (black bars), or differences between VR-2001-SfSPChimera vaccinated and control groups (grey bars). Pearson correlation coefficients (r) and significance are also represented.
Figure Legend Snippet: The protection against L. longipalpis sand fly-transmitted L. major infection in BALB/c mice is dependent on high IFN-γ/IL-10 and IFN-γ/IL-4 ratios. BALB/C mice were immunized intradermally in the right ear three times at 2 weeks intervals with 5 µg of VR-2001-SfSPChimera plasmid (crossed circles), or with the empty vector (control animals; black circles). One month after the last immunization mice were challenged on the contralateral ear via sand fly bites—10 L. longipalpis sand flies carrying mature L. major infections were used per animal. Two weeks later animals were euthanized and their ears collected and processed. Parasite burdens were determined by limiting dilution (Fig. 5 F) and T cell cytokine secretion was analysed by flow cytometry after an overnight culture of total ear cells in the presence of L. major SLA, and additionally PMA, Ionomycin and Brefeldin A during the last 4 h of culture. For each animal, including controls, parasite burden values were plotted against the frequencies of CD4 + T cells secreting IFN-γ, IL-10 or IL-4, in a way to assess their correlation ( A ). These frequencies are represented for VR-2001-SfSPChimera immunized and control animals, sub-grouped (Fig. 5 F) according to parasite burden values ( B ). IFN-γ/IL-10 and IFN-γ/IL-4 ratios were calculated and are represented using the same group division criteria ( C ). The correlation between these ratio values and the parasite burdens was also assessed, now only considering the animals immunized with VR-2001-SfSPChimera DNA vaccine ( C ). Results from two independent experiments are represented. Each dot represents one animal. Average and SD of the values within each group are shown. Statistical differences are properly identified (Mann–Whitney test: * p ≤ 0.05 and ** p ≤ 0.01) and refer to differences between sub-groups (black bars), or differences between VR-2001-SfSPChimera vaccinated and control groups (grey bars). Pearson correlation coefficients (r) and significance are also represented.

Techniques Used: Infection, Mouse Assay, Plasmid Preparation, Flow Cytometry, MANN-WHITNEY

21) Product Images from "Oncolytic vesicular stomatitis virus quantitatively and qualitatively improves primary CD8+ T-cell responses to anticancer vaccines"

Article Title: Oncolytic vesicular stomatitis virus quantitatively and qualitatively improves primary CD8+ T-cell responses to anticancer vaccines

Journal: Oncoimmunology

doi: 10.4161/onci.26013

Quantitative assessment of adenovirus-primed and VSV-boosted CD8+ T cell responses. ( A ) C57BL/6 mice were primed by intramuscular injection of 1 × 108 plaque-forming units (PFUs) of Ad-hDCT. Blood samples were drawn on various days post-priming to quantify CD8+ T-cell response to the immunodominant epitope DCT180–188 by cytofluorometric detection of intracellular interferon γ (IFNγ) upon in vitro stimulation with the cognate peptide in the presence of brefeldin A. Data were pooled to plot the kinetics of the response. ( B ) Mice primed with Ad-hDCT were boosted after a 14-d interval by intravenous injection of 1 × 109 PFUs of VSV-hDCT. Transgene-specific CD8+ T-cell responses were measured in the blood at various days post-boosting to establish the kinetics of secondary responses. ( C and D ) Mice were primed by intramuscular injection of 1 × 108 PFUs of Ad-hDCT or Ad-SIINFEKL. Half of these mice were then boosted by intravenous injection of 1 × 109 PFUs of VSV-hDCT or VSV-SIINFEKL. The vaccinations were offset such that the peak of primary transgene-specific CD8+ T-cell responses in mice subjected to priming only (11 d post-priming) coincided with the peak of secondary responses in VSV-boosted animals (5 d post-boosting). The frequency of circulating CD8+ T cells specific for DCT180–188 ( C ) and SIINFEKL ( D ) was quantified by flow cytometry in terms of IFNγ-expressing cells upon in vitro antigenic stimulation. In all cases, n = 5 animals/group; data are reported as means ± SEM and are representative of two experiments. ν adenovirus-induced primary response; ϒ adenovirus-primed, VSV-boosted secondary response. Statistical significance was determined by two-way ANOVA: **p
Figure Legend Snippet: Quantitative assessment of adenovirus-primed and VSV-boosted CD8+ T cell responses. ( A ) C57BL/6 mice were primed by intramuscular injection of 1 × 108 plaque-forming units (PFUs) of Ad-hDCT. Blood samples were drawn on various days post-priming to quantify CD8+ T-cell response to the immunodominant epitope DCT180–188 by cytofluorometric detection of intracellular interferon γ (IFNγ) upon in vitro stimulation with the cognate peptide in the presence of brefeldin A. Data were pooled to plot the kinetics of the response. ( B ) Mice primed with Ad-hDCT were boosted after a 14-d interval by intravenous injection of 1 × 109 PFUs of VSV-hDCT. Transgene-specific CD8+ T-cell responses were measured in the blood at various days post-boosting to establish the kinetics of secondary responses. ( C and D ) Mice were primed by intramuscular injection of 1 × 108 PFUs of Ad-hDCT or Ad-SIINFEKL. Half of these mice were then boosted by intravenous injection of 1 × 109 PFUs of VSV-hDCT or VSV-SIINFEKL. The vaccinations were offset such that the peak of primary transgene-specific CD8+ T-cell responses in mice subjected to priming only (11 d post-priming) coincided with the peak of secondary responses in VSV-boosted animals (5 d post-boosting). The frequency of circulating CD8+ T cells specific for DCT180–188 ( C ) and SIINFEKL ( D ) was quantified by flow cytometry in terms of IFNγ-expressing cells upon in vitro antigenic stimulation. In all cases, n = 5 animals/group; data are reported as means ± SEM and are representative of two experiments. ν adenovirus-induced primary response; ϒ adenovirus-primed, VSV-boosted secondary response. Statistical significance was determined by two-way ANOVA: **p

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

22) Product Images from "Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition"

Article Title: Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0801968105

A codon-modified GAr sequence enhances the presentation of CD8 + T cell epitopes. ( A ) Expression of surface H-2K b –SIIN complexes was assessed by flow cytometry on 293KbC2 cells transfected with EBNA1-SIIN-GFP constructs encoding native or codon-modified GAr sequences (as shown). Data are the percent of cells positive for H-2K b –SIIN when compared with controls cells transfected with relevant parental EBN1-GFP plasmids that lack the SIIN epitope. ( B ) FLR-specific T cells were incubated overnight in the presence of brefeldin A at a responder to stimulator ratio of 5:1 with fixed EBV-negative SVMR6 cells transfected with selected EBNA1-GFP expression vectors. IFN-γ production by CD3 + CD8 + cells was determined by intracellular cytokine staining. ( C ) HPVGEADYFEY-specific T cells were incubated in duplicate in the presence of brefeldin A for 6 h at a responder-to-stimulator ratio of 5:1 with fixed EBV-negative DG75 cells transfected with selected EBNA1-GFP expression vectors. Cells were incubated with a HPV-specific pentamer followed by anti-CD8 and incubated with anti-IFN-γ and analyzed on a FACSCanto. The top right hand corner of each panel represents the percentage of HPV-specific CD8 + lymphocytes producing IFN-γ. Data shown in C and D are for one representative experiment of three separate experiments.
Figure Legend Snippet: A codon-modified GAr sequence enhances the presentation of CD8 + T cell epitopes. ( A ) Expression of surface H-2K b –SIIN complexes was assessed by flow cytometry on 293KbC2 cells transfected with EBNA1-SIIN-GFP constructs encoding native or codon-modified GAr sequences (as shown). Data are the percent of cells positive for H-2K b –SIIN when compared with controls cells transfected with relevant parental EBN1-GFP plasmids that lack the SIIN epitope. ( B ) FLR-specific T cells were incubated overnight in the presence of brefeldin A at a responder to stimulator ratio of 5:1 with fixed EBV-negative SVMR6 cells transfected with selected EBNA1-GFP expression vectors. IFN-γ production by CD3 + CD8 + cells was determined by intracellular cytokine staining. ( C ) HPVGEADYFEY-specific T cells were incubated in duplicate in the presence of brefeldin A for 6 h at a responder-to-stimulator ratio of 5:1 with fixed EBV-negative DG75 cells transfected with selected EBNA1-GFP expression vectors. Cells were incubated with a HPV-specific pentamer followed by anti-CD8 and incubated with anti-IFN-γ and analyzed on a FACSCanto. The top right hand corner of each panel represents the percentage of HPV-specific CD8 + lymphocytes producing IFN-γ. Data shown in C and D are for one representative experiment of three separate experiments.

Techniques Used: Modification, Sequencing, Expressing, Flow Cytometry, Cytometry, Transfection, Construct, Incubation, Staining

23) Product Images from "Reprogramming the adjuvant properties of aluminum oxyhydroxide with nanoparticle technology"

Article Title: Reprogramming the adjuvant properties of aluminum oxyhydroxide with nanoparticle technology

Journal: NPJ Vaccines

doi: 10.1038/s41541-018-0094-0

Augmentation of TH1 immunity is not a general property of nanoalum formulations or free PAA. a . b – d C57Bl/6 mice (five per group) were immunized twice with 0.5 µg of ID93 alone or adjuvanted with Alhydrogel, PAA, PAA:nanoalum, or PEG:nanoalum. b One week after the second immunization, splenocytes were isolated and either unstimulated or stimulated with the ID93 protein in the presence of brefeldin A for 8 h at 37 °C. Cells were then stained for surface expression of CD4 and CD44, as well as intracellular expression of IFN-ƴ, TNF and/or CD154. c Splenocytes from immunized mice were stimulated with the ID93 protein and assessed for secretion of IFN-ƴ. d Serum was collected from immunized animals 3 weeks after the first immunization and assessed for ID93 binding antibody titers by ELISA for IgG1 and IgG2 subclasses. ***( P
Figure Legend Snippet: Augmentation of TH1 immunity is not a general property of nanoalum formulations or free PAA. a . b – d C57Bl/6 mice (five per group) were immunized twice with 0.5 µg of ID93 alone or adjuvanted with Alhydrogel, PAA, PAA:nanoalum, or PEG:nanoalum. b One week after the second immunization, splenocytes were isolated and either unstimulated or stimulated with the ID93 protein in the presence of brefeldin A for 8 h at 37 °C. Cells were then stained for surface expression of CD4 and CD44, as well as intracellular expression of IFN-ƴ, TNF and/or CD154. c Splenocytes from immunized mice were stimulated with the ID93 protein and assessed for secretion of IFN-ƴ. d Serum was collected from immunized animals 3 weeks after the first immunization and assessed for ID93 binding antibody titers by ELISA for IgG1 and IgG2 subclasses. ***( P

Techniques Used: Mouse Assay, Isolation, Staining, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay

24) Product Images from "DNA-Containing Immunocomplexes Promote Inflammasome Assembly and Release of Pyrogenic Cytokines by CD14+ CD16+ CD64high CD32low Inflammatory Monocytes from Malaria Patients"

Article Title: DNA-Containing Immunocomplexes Promote Inflammasome Assembly and Release of Pyrogenic Cytokines by CD14+ CD16+ CD64high CD32low Inflammatory Monocytes from Malaria Patients

Journal: mBio

doi: 10.1128/mBio.01605-15

Purified ICs induce high levels of proinflammatory cytokines by CD14 + CD16 + monocytes from malaria patients. (A) PBMCs from P. vivax malaria patients ( n = 6) before and 30 to 45 days after treatment were isolated and stimulated with 60 µg/ml of ICs for 24 h. The levels of TNF-α, IL-1β, and IL-10 were measured in supernatants by CBA. The P values were determined by a paired t test. (B) Flow cytometric analysis shows an increased frequency of CD14 + CD16 + cells in PBMCs from two P. vivax malaria patients. The frequency of CD14 + CD16 + cells decreased to levels similar to those for healthy donors at 30 to 40 days after treatment and parasitological cure. Results of three additional patients and healthy donors are shown in Fig. S4A in the supplemental material . (C) PBMCs from two different P. vivax malaria patients were isolated and stimulated with 60 µg/ml of ICs from three different patients for 10 h in culture containing brefeldin A and submitted to flow cytometric analysis to measure the expression of intracellular TNF-α and IL-1β in CD14 + CD16 − as well as CD14 + CD16 + monocytes. The results are representative of two out of five patients. (D) Top panels show the gating strategy to identify the monocyte subsets: CD14 + CD16 − , CD14 + CD16 + , and CD14 lo CD16 + . The middle two panels are representative histograms of CD64 and CD32 expression in P. vivax -infected patients and healthy donors (HD). Bottom panels show the mean fluorescence intensity (MFI) ratios of CD16/CD32 (left) and CD64/CD32 (right) of the three monocyte subsets from P. vivax -infected patients ( n = 6) and in healthy donors (HD, n = 4). *, P
Figure Legend Snippet: Purified ICs induce high levels of proinflammatory cytokines by CD14 + CD16 + monocytes from malaria patients. (A) PBMCs from P. vivax malaria patients ( n = 6) before and 30 to 45 days after treatment were isolated and stimulated with 60 µg/ml of ICs for 24 h. The levels of TNF-α, IL-1β, and IL-10 were measured in supernatants by CBA. The P values were determined by a paired t test. (B) Flow cytometric analysis shows an increased frequency of CD14 + CD16 + cells in PBMCs from two P. vivax malaria patients. The frequency of CD14 + CD16 + cells decreased to levels similar to those for healthy donors at 30 to 40 days after treatment and parasitological cure. Results of three additional patients and healthy donors are shown in Fig. S4A in the supplemental material . (C) PBMCs from two different P. vivax malaria patients were isolated and stimulated with 60 µg/ml of ICs from three different patients for 10 h in culture containing brefeldin A and submitted to flow cytometric analysis to measure the expression of intracellular TNF-α and IL-1β in CD14 + CD16 − as well as CD14 + CD16 + monocytes. The results are representative of two out of five patients. (D) Top panels show the gating strategy to identify the monocyte subsets: CD14 + CD16 − , CD14 + CD16 + , and CD14 lo CD16 + . The middle two panels are representative histograms of CD64 and CD32 expression in P. vivax -infected patients and healthy donors (HD). Bottom panels show the mean fluorescence intensity (MFI) ratios of CD16/CD32 (left) and CD64/CD32 (right) of the three monocyte subsets from P. vivax -infected patients ( n = 6) and in healthy donors (HD, n = 4). *, P

Techniques Used: Purification, Isolation, Crocin Bleaching Assay, Flow Cytometry, Expressing, Infection, Fluorescence

25) Product Images from "Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis"

Article Title: Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis

Journal: Scientific Reports

doi: 10.1038/s41598-017-11450-3

Chol-HCQ liposomes suppress bleomycin-induced pulmonary fibrosis through anti-inflammatory effects and by inhibiting the CTGF/ERK signalling pathways. ( a , up) Specific esterase staining of neutrophils in rat lung sections on day 7 of the experiment. The rats were treated with bleomycin before administration (400×). ( b ) Neutrophils were counted in 5 random 200 s fields. ( a , below, and c )Immunohistochemistry analysis of CTGF levels in the lung tissues from BLM-induced pulmonary fibrosis rats. Original magnification is 200 s. Positive cells were countered in 5 random fields. ( d and e ) TNF-α and TGF-β1 contents in plasma from rats on day 7 were determined by ELISA kits. (f) Isolated alveolar macrophage cells were stimulated with bleomycin (25 μg/ml) and treated with Chol-HCQ (10 μM) or HCQ (10 μM) and etanercept (5 μg/ml) in the presence of brefeldin A in 24-well plate at 37 °C for 24 h. Flow cytometry was conducted to detect macrophage intracellular TNF-α expression. ( g – j ) On day 14, rats were sacrificed and lung tissues lysate were made for Western blot analysis. Chol-HCQ inhibits ERK1/2 (Thy202/Thr204) and NF-κB phosphorylation in vivo . Data are representative of three separate experiments. *p
Figure Legend Snippet: Chol-HCQ liposomes suppress bleomycin-induced pulmonary fibrosis through anti-inflammatory effects and by inhibiting the CTGF/ERK signalling pathways. ( a , up) Specific esterase staining of neutrophils in rat lung sections on day 7 of the experiment. The rats were treated with bleomycin before administration (400×). ( b ) Neutrophils were counted in 5 random 200 s fields. ( a , below, and c )Immunohistochemistry analysis of CTGF levels in the lung tissues from BLM-induced pulmonary fibrosis rats. Original magnification is 200 s. Positive cells were countered in 5 random fields. ( d and e ) TNF-α and TGF-β1 contents in plasma from rats on day 7 were determined by ELISA kits. (f) Isolated alveolar macrophage cells were stimulated with bleomycin (25 μg/ml) and treated with Chol-HCQ (10 μM) or HCQ (10 μM) and etanercept (5 μg/ml) in the presence of brefeldin A in 24-well plate at 37 °C for 24 h. Flow cytometry was conducted to detect macrophage intracellular TNF-α expression. ( g – j ) On day 14, rats were sacrificed and lung tissues lysate were made for Western blot analysis. Chol-HCQ inhibits ERK1/2 (Thy202/Thr204) and NF-κB phosphorylation in vivo . Data are representative of three separate experiments. *p

Techniques Used: Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Cytometry, Expressing, Western Blot, In Vivo

26) Product Images from "4-1BB and OX40 stimulation enhance CD8 and CD4 T-cell responses to a DNA prime, poxvirus boost vaccine"

Article Title: 4-1BB and OX40 stimulation enhance CD8 and CD4 T-cell responses to a DNA prime, poxvirus boost vaccine

Journal: Immunology

doi: 10.1111/j.1365-2567.2004.01917.x

4-1BB enhances CD8 T-cell activation and memory. BALB/c mice were primed with 100 µg of plasmid pTH.HM twice, 1 week apart. Two weeks later, mice were boosted with 1 × 10 6  pfu of MVA.HM and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. with the boost. At the indicated number of days later, unfractionated spleen cells were cultured directly  ex vivo  for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml RGPGRAFVTI peptide, then stained for CD8 and intracellular IFN-γ. One representative is shown from each group (a). Each point represents the average percentage (b) and number (c) of antigen-specific CD8 T cells ± SEM (day 6,  n  = 3 mice per group; day 22,  n  = 4 mice per group). A second experiment gave similar results.
Figure Legend Snippet: 4-1BB enhances CD8 T-cell activation and memory. BALB/c mice were primed with 100 µg of plasmid pTH.HM twice, 1 week apart. Two weeks later, mice were boosted with 1 × 10 6 pfu of MVA.HM and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. with the boost. At the indicated number of days later, unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml RGPGRAFVTI peptide, then stained for CD8 and intracellular IFN-γ. One representative is shown from each group (a). Each point represents the average percentage (b) and number (c) of antigen-specific CD8 T cells ± SEM (day 6, n = 3 mice per group; day 22, n = 4 mice per group). A second experiment gave similar results.

Techniques Used: Activation Assay, Mouse Assay, Plasmid Preparation, Purification, Cell Culture, Ex Vivo, Staining

4-1BB and OX40 cooperate to enhance CD8 T and CD4 T cells. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, 2 weeks apart. Thirty days later, mice were vaccinated with 2 × 10 5  pfu of rVV-OVA i.p. At the time of the rVV boost, mice were divided into four groups and received either 200 µg of anti-4-1BB mAb + 200 µg rat IgG, 200 µg of anti-OX40 mAb + 200 µg rat IgG, 200 µg anti-4-1BB mAb + 200 µg anti-OX40 mAb, or 400 µg of rat IgG. Five weeks later, spleen cells were examined for antigen-specific CD4 and CD8 T-cell responses. (a) Unfractionated spleen cells were cultured directly  ex vivo  in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. OVA-specific CD4 T cells are shown as both a percentage of CD4 +  T cells and as the number per spleen. (b) Unfractionated spleen cells were cultured directly  ex vivo  for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ. SIINFEKL-specific CD8 T cells are shown as both a percentage of CD8 +  T cells and total number per spleen. Data shown are the average ± SEM ( n  = 10 mice per group). A second experiment gave similar results.
Figure Legend Snippet: 4-1BB and OX40 cooperate to enhance CD8 T and CD4 T cells. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, 2 weeks apart. Thirty days later, mice were vaccinated with 2 × 10 5 pfu of rVV-OVA i.p. At the time of the rVV boost, mice were divided into four groups and received either 200 µg of anti-4-1BB mAb + 200 µg rat IgG, 200 µg of anti-OX40 mAb + 200 µg rat IgG, 200 µg anti-4-1BB mAb + 200 µg anti-OX40 mAb, or 400 µg of rat IgG. Five weeks later, spleen cells were examined for antigen-specific CD4 and CD8 T-cell responses. (a) Unfractionated spleen cells were cultured directly ex vivo in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. OVA-specific CD4 T cells are shown as both a percentage of CD4 + T cells and as the number per spleen. (b) Unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ. SIINFEKL-specific CD8 T cells are shown as both a percentage of CD8 + T cells and total number per spleen. Data shown are the average ± SEM ( n = 10 mice per group). A second experiment gave similar results.

Techniques Used: Mouse Assay, Plasmid Preparation, Cell Culture, Ex Vivo, Staining

4-1BB improves long-term CD8 T-cell memory. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, two weeks apart. Thirty days later, mice were vaccinated with 2 × 10 5  pfu of rVV-OVA and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. (a) At the indicated number of days later, unfractionated spleen cells were cultured directly  ex vivo  for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ or (b) TNF-α. (c) The percentage of total spleen cells that were CD8 + . (d) OVA-specific CD4 T cells were quantified in parallel to SIINFEKL-specific CD8 T cells. Unfractionated spleen cells were cultured directly  ex vivo  in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. Data shown are the average ± SEM (day 6,  n  = 6 mice per group; day 60,  n  = 10 mice per group). A second experiment gave similar results.
Figure Legend Snippet: 4-1BB improves long-term CD8 T-cell memory. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, two weeks apart. Thirty days later, mice were vaccinated with 2 × 10 5 pfu of rVV-OVA and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. (a) At the indicated number of days later, unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ or (b) TNF-α. (c) The percentage of total spleen cells that were CD8 + . (d) OVA-specific CD4 T cells were quantified in parallel to SIINFEKL-specific CD8 T cells. Unfractionated spleen cells were cultured directly ex vivo in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. Data shown are the average ± SEM (day 6, n = 6 mice per group; day 60, n = 10 mice per group). A second experiment gave similar results.

Techniques Used: Mouse Assay, Plasmid Preparation, Purification, Cell Culture, Ex Vivo, Staining

27) Product Images from "Human T‐cell lymphotropic/leukemia virus type 1 ( HTLV‐1) Tax‐specific T‐cell exhaustion in HTLV‐1‐infected individuals, et al. Human T‐cell lymphotropic/leukemia virus type 1 (HTLV‐1) Tax‐specific T‐cell exhaustion in HTLV‐1‐infected individuals"

Article Title: Human T‐cell lymphotropic/leukemia virus type 1 ( HTLV‐1) Tax‐specific T‐cell exhaustion in HTLV‐1‐infected individuals, et al. Human T‐cell lymphotropic/leukemia virus type 1 (HTLV‐1) Tax‐specific T‐cell exhaustion in HTLV‐1‐infected individuals

Journal: Cancer Science

doi: 10.1111/cas.13654

Relationships between programmed cell death protein 1 ( PD ‐1) positivity of Human T‐cell lymphotropic/leukemia virus type 1 ( HTLV ‐1) Tax‐specific cytotoxic T cells (Tax‐ CTL ) and the production of interferon ( IFN )‐γ and tumor necrosis factor ( TNF )‐α ex vivo in response to cognate peptide in HTLV ‐1 asymptomatic carriers ( AC ). A, Correlation between HTLV ‐1 provirus load in PBMC and the percentage of PD ‐1‐positive Tax‐ CTL in HTLV ‐1 AC . There was a significant positive correlation between these two factors ( R s = 0.574, P = .013). B, Correlation between the percentage of PD ‐1‐positive Tax‐ CTL and the percentage of both IFN ‐γ‐ and TNF ‐α‐producing Tax‐ CTL ex vivo in response to cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.542, P = .020). C, Correlation between the HTLV ‐1 provirus load in PBMC and the percentage of both IFN ‐γ‐ and TNF ‐α‐producing Tax‐ CTL ex vivo in response to cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.494, P = .037). D, Lymphocyte populations from HTLV ‐1 AC donors 1, 2, 3, and 4 showing CD 8 and HTLV ‐1 Tax tetramer positivity. CD 8 and HTLV ‐1 Tax tetramer‐positive cells are gated as shown by the red squares (left panels), and plotted to show T‐cell immunoglobulin and mucin domain‐containing protein‐3 ( TIM ‐3) and PD ‐1 positivity. Percentages of PD ‐1‐positive but TIM ‐3‐negative, and of PD ‐1‐ and TIM ‐3‐double‐positive cells are indicated in each panel (second left). CD 8 and HTLV ‐1 Tax tetramer‐positive cells are plotted to show lymphocyte‐activation gene 3 ( LAG ‐3) and cytotoxic T‐lymphocyte‐associated antigen 4 ( CTLA ‐4) positivity. Percentage of LAG ‐3‐positive but CTLA ‐4‐negative cells is indicated (second right). PBMC obtained from HTLV ‐1 AC 1, 2, 3, and 4 were cocultured with or without cognate peptide (final concentration 100 nmol/L) at 37°C in 5% CO 2 for 3 h, after which brefeldin A was added. The cells were then incubated for an additional 2 h. Subsequently, IFN ‐γ and TNF ‐α production from Tax‐ CTL was evaluated. Percentage of Tax‐ CTL producing both IFN ‐γ and TNF ‐α in response to cognate peptide is indicated in each panel (right‐hand side). Plots labeled 1, 2, 3, and 4 in (A,B,C), correspond to 1, 2, 3, and 4, in (D), respectively. Plots labeled 2, 3, and 4 in (A,B,C) are colored blue, green, and brown, respectively
Figure Legend Snippet: Relationships between programmed cell death protein 1 ( PD ‐1) positivity of Human T‐cell lymphotropic/leukemia virus type 1 ( HTLV ‐1) Tax‐specific cytotoxic T cells (Tax‐ CTL ) and the production of interferon ( IFN )‐γ and tumor necrosis factor ( TNF )‐α ex vivo in response to cognate peptide in HTLV ‐1 asymptomatic carriers ( AC ). A, Correlation between HTLV ‐1 provirus load in PBMC and the percentage of PD ‐1‐positive Tax‐ CTL in HTLV ‐1 AC . There was a significant positive correlation between these two factors ( R s = 0.574, P = .013). B, Correlation between the percentage of PD ‐1‐positive Tax‐ CTL and the percentage of both IFN ‐γ‐ and TNF ‐α‐producing Tax‐ CTL ex vivo in response to cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.542, P = .020). C, Correlation between the HTLV ‐1 provirus load in PBMC and the percentage of both IFN ‐γ‐ and TNF ‐α‐producing Tax‐ CTL ex vivo in response to cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.494, P = .037). D, Lymphocyte populations from HTLV ‐1 AC donors 1, 2, 3, and 4 showing CD 8 and HTLV ‐1 Tax tetramer positivity. CD 8 and HTLV ‐1 Tax tetramer‐positive cells are gated as shown by the red squares (left panels), and plotted to show T‐cell immunoglobulin and mucin domain‐containing protein‐3 ( TIM ‐3) and PD ‐1 positivity. Percentages of PD ‐1‐positive but TIM ‐3‐negative, and of PD ‐1‐ and TIM ‐3‐double‐positive cells are indicated in each panel (second left). CD 8 and HTLV ‐1 Tax tetramer‐positive cells are plotted to show lymphocyte‐activation gene 3 ( LAG ‐3) and cytotoxic T‐lymphocyte‐associated antigen 4 ( CTLA ‐4) positivity. Percentage of LAG ‐3‐positive but CTLA ‐4‐negative cells is indicated (second right). PBMC obtained from HTLV ‐1 AC 1, 2, 3, and 4 were cocultured with or without cognate peptide (final concentration 100 nmol/L) at 37°C in 5% CO 2 for 3 h, after which brefeldin A was added. The cells were then incubated for an additional 2 h. Subsequently, IFN ‐γ and TNF ‐α production from Tax‐ CTL was evaluated. Percentage of Tax‐ CTL producing both IFN ‐γ and TNF ‐α in response to cognate peptide is indicated in each panel (right‐hand side). Plots labeled 1, 2, 3, and 4 in (A,B,C), correspond to 1, 2, 3, and 4, in (D), respectively. Plots labeled 2, 3, and 4 in (A,B,C) are colored blue, green, and brown, respectively

Techniques Used: CTL Assay, Ex Vivo, Activation Assay, Concentration Assay, Incubation, Labeling

Relationships between programmed cell death protein 1 ( PD ‐1) positivity of Human T‐cell lymphotropic/leukemia virus type 1 ( HTLV ‐1) Tax‐specific cytotoxic T cells (Tax‐ CTL ) and their production of interferon ( IFN )‐γ and tumor necrosis factor ( TNF )‐α ex vivo in response to cognate peptide in adult T‐cell leukemia/lymphoma ( ATL ) patients. A, Correlation between HTLV ‐1 provirus load in PBMC and the percentage of PD ‐1‐positive Tax‐ CTL in ATL patients. There was a significant positive correlation between these 2 factors ( R s = 0.676, P = .006). B, Correlation between the percentage of PD ‐1‐positive Tax‐ CTL and the percentage of Tax‐ CTL producing both IFN ‐γ and TNF ‐α ex vivo in response to the cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.639, P = .010). C, Correlation between HTLV ‐1 provirus load in PBMC and the percentage of Tax‐ CTL producing both IFN ‐γ and TNF ‐α ex vivo in response to cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.774, P = .001). The 4 symbols in gray in the plots (A,B,C) indicate ATL patients after allogeneic hematopoietic stem cell transplantation ( HSCT ). D, Lymphocyte populations of PBMC , ex vivo, from ATL patients 1, 2, 3, and 4 are plotted to show CD 8 and HTLV ‐1 Tax tetramer positivity. CD 8 and HTLV ‐1 Tax tetramer‐positive cells are shown gated by the red squares (left panels), and are plotted to show T‐cell immunoglobulin and mucin domain‐containing protein‐3 ( TIM ‐3) and PD ‐1 positivity. Percentages of PD ‐1‐positive but TIM ‐3‐negative, and of PD ‐1‐, TIM ‐3‐double‐positive cells are indicated in each panel (second left). CD 8 and HTLV ‐1 Tax tetramer‐positive cells are plotted to show lymphocyte‐activation gene 3 ( LAG ‐3) and cytotoxic T‐lymphocyte‐associated antigen 4 ( CTLA ‐4) positivity. Percentage of LAG ‐3‐positive but CTLA ‐4‐negative cells is indicated second right. PBMC obtained from ATL patients 1, 2, 3, and 4 were cocultured with or without cognate peptide (final concentration 100 nmol/L) at 37°C in 5% CO 2 for 3 h, after which brefeldin A was added. The cells were then incubated for an additional 2 h. Subsequently, IFN ‐γ and TNF ‐α production from Tax‐ CTL was evaluated. Percentage of both IFN ‐γ‐ and TNF ‐α‐producing Tax‐ CTL in response to cognate peptide is indicated in each panel (right panels). Plots labeled 1, 2, 3, and 4 in (A,B,C) correspond to 1, 2, 3, and 4, in (D), respectively. Plots labeled 2, 3, and 4 in (A,B,C) are colored blue, green, and brown, respectively
Figure Legend Snippet: Relationships between programmed cell death protein 1 ( PD ‐1) positivity of Human T‐cell lymphotropic/leukemia virus type 1 ( HTLV ‐1) Tax‐specific cytotoxic T cells (Tax‐ CTL ) and their production of interferon ( IFN )‐γ and tumor necrosis factor ( TNF )‐α ex vivo in response to cognate peptide in adult T‐cell leukemia/lymphoma ( ATL ) patients. A, Correlation between HTLV ‐1 provirus load in PBMC and the percentage of PD ‐1‐positive Tax‐ CTL in ATL patients. There was a significant positive correlation between these 2 factors ( R s = 0.676, P = .006). B, Correlation between the percentage of PD ‐1‐positive Tax‐ CTL and the percentage of Tax‐ CTL producing both IFN ‐γ and TNF ‐α ex vivo in response to the cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.639, P = .010). C, Correlation between HTLV ‐1 provirus load in PBMC and the percentage of Tax‐ CTL producing both IFN ‐γ and TNF ‐α ex vivo in response to cognate peptide. There was a significant inverse correlation between these 2 factors ( R s = −0.774, P = .001). The 4 symbols in gray in the plots (A,B,C) indicate ATL patients after allogeneic hematopoietic stem cell transplantation ( HSCT ). D, Lymphocyte populations of PBMC , ex vivo, from ATL patients 1, 2, 3, and 4 are plotted to show CD 8 and HTLV ‐1 Tax tetramer positivity. CD 8 and HTLV ‐1 Tax tetramer‐positive cells are shown gated by the red squares (left panels), and are plotted to show T‐cell immunoglobulin and mucin domain‐containing protein‐3 ( TIM ‐3) and PD ‐1 positivity. Percentages of PD ‐1‐positive but TIM ‐3‐negative, and of PD ‐1‐, TIM ‐3‐double‐positive cells are indicated in each panel (second left). CD 8 and HTLV ‐1 Tax tetramer‐positive cells are plotted to show lymphocyte‐activation gene 3 ( LAG ‐3) and cytotoxic T‐lymphocyte‐associated antigen 4 ( CTLA ‐4) positivity. Percentage of LAG ‐3‐positive but CTLA ‐4‐negative cells is indicated second right. PBMC obtained from ATL patients 1, 2, 3, and 4 were cocultured with or without cognate peptide (final concentration 100 nmol/L) at 37°C in 5% CO 2 for 3 h, after which brefeldin A was added. The cells were then incubated for an additional 2 h. Subsequently, IFN ‐γ and TNF ‐α production from Tax‐ CTL was evaluated. Percentage of both IFN ‐γ‐ and TNF ‐α‐producing Tax‐ CTL in response to cognate peptide is indicated in each panel (right panels). Plots labeled 1, 2, 3, and 4 in (A,B,C) correspond to 1, 2, 3, and 4, in (D), respectively. Plots labeled 2, 3, and 4 in (A,B,C) are colored blue, green, and brown, respectively

Techniques Used: CTL Assay, Ex Vivo, Transplantation Assay, Activation Assay, Concentration Assay, Incubation, Labeling

28) Product Images from "Immunologic Human Renal Allograft Injury Associates with an Altered IL-10/TNF-α Expression Ratio in Regulatory B Cells"

Article Title: Immunologic Human Renal Allograft Injury Associates with an Altered IL-10/TNF-α Expression Ratio in Regulatory B Cells

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013080837

Analysis of IL-10 and TNF- α expression by B subsets. Magnetic bead–enriched CD19 + B cells are stimulated with CpG and CD40L for 48 hours plus phorbol ester, ionomycin, and brefeldin A (last 5 hours). (A and C) Each representative dot plot
Figure Legend Snippet: Analysis of IL-10 and TNF- α expression by B subsets. Magnetic bead–enriched CD19 + B cells are stimulated with CpG and CD40L for 48 hours plus phorbol ester, ionomycin, and brefeldin A (last 5 hours). (A and C) Each representative dot plot

Techniques Used: Expressing

29) Product Images from "Sustained interactions between T cell receptor and antigen promote the differentiation of CD4+ memory T cells"

Article Title: Sustained interactions between T cell receptor and antigen promote the differentiation of CD4+ memory T cells

Journal: Immunity

doi: 10.1016/j.immuni.2013.08.033

Activated CD4 +  T cells in SMα mice undergo physiological expansion and differentiation following infection with LCMV or Lm-gp61. B6 or SMα mice were injected with 5 × 10 6  naïve CD8 +  T cells isolated from the spleens of B6 mice. One day later, mice were infected with LCMV or Lm-gp61. A) Representative plots indicate the frequency of IFN-γ-producing CD4 +  T cells in the spleen at the indicated time points post-infection following  ex vivo  restimulation with GP 61-80  peptide in the presence of Brefeldin A. B-C) Graphs indicate the number of IFN-γ-producing cells in the spleen of B6 or SMα mice in a time course post-infection with either LCMV or Lm-gp61. Error bars indicate SEM (n=4 mice/group). D) Plot displays estimated fold expansion of GP 61-80 -specific CD4 +  T cells during the first 8 days post-infection based on our calculations of naïve precursor frequency in B6 or SMα mice. “NS” indicates “not significant”, as measured by a two-tailed student's T-test ( p > .05). E) Representative flow plots indicate the frequency of IFNγ + TNFγ +  and IFNγ + IL-2 +  CD4 +  double producers in the spleen at the indicated time points post-infection for B6 and SMα mice. Results are representative of three independent experiments.
Figure Legend Snippet: Activated CD4 + T cells in SMα mice undergo physiological expansion and differentiation following infection with LCMV or Lm-gp61. B6 or SMα mice were injected with 5 × 10 6 naïve CD8 + T cells isolated from the spleens of B6 mice. One day later, mice were infected with LCMV or Lm-gp61. A) Representative plots indicate the frequency of IFN-γ-producing CD4 + T cells in the spleen at the indicated time points post-infection following ex vivo restimulation with GP 61-80 peptide in the presence of Brefeldin A. B-C) Graphs indicate the number of IFN-γ-producing cells in the spleen of B6 or SMα mice in a time course post-infection with either LCMV or Lm-gp61. Error bars indicate SEM (n=4 mice/group). D) Plot displays estimated fold expansion of GP 61-80 -specific CD4 + T cells during the first 8 days post-infection based on our calculations of naïve precursor frequency in B6 or SMα mice. “NS” indicates “not significant”, as measured by a two-tailed student's T-test ( p > .05). E) Representative flow plots indicate the frequency of IFNγ + TNFγ + and IFNγ + IL-2 + CD4 + double producers in the spleen at the indicated time points post-infection for B6 and SMα mice. Results are representative of three independent experiments.

Techniques Used: Mouse Assay, Infection, Injection, Isolation, Ex Vivo, Two Tailed Test, Flow Cytometry

30) Product Images from "Monocyte glycolysis determines CD8+ T cell functionality in human Chagas disease"

Article Title: Monocyte glycolysis determines CD8+ T cell functionality in human Chagas disease

Journal: JCI Insight

doi: 10.1172/jci.insight.123490

Monocytes from  T.cruzi –infected patients exhibit higher functional activity potential. ( A ) For intracellular cytokine analysis, PBMCs isolated from peripheral blood from control donors and from seropositive patients were cultured with monensin, brefeldin A, and ( B ) LPS (CON,  n  = 5; CHAG,  n  = 5) or ( C ) parasite lysate (CON,  n  = 6; CHAG,  n  = 4) and stained with anti–CD14-PECy5. After staining of surface markers, cells were fixed and made permeable. Then the cells were stained with anti–IL-1β, anti–IL-6, and anti–IL-10 antibodies and analyzed by flow cytometry. PBMCs were gated according to their CD14 +  staining after exclusion of doublets and debris by using FSC-H/FSC-A dot plots. Frequency and MFI of IL-1β–, IL-6–, and IL-10–producing monocytes. Data are presented as mean ± SEM. * P
Figure Legend Snippet: Monocytes from T.cruzi –infected patients exhibit higher functional activity potential. ( A ) For intracellular cytokine analysis, PBMCs isolated from peripheral blood from control donors and from seropositive patients were cultured with monensin, brefeldin A, and ( B ) LPS (CON, n = 5; CHAG, n = 5) or ( C ) parasite lysate (CON, n = 6; CHAG, n = 4) and stained with anti–CD14-PECy5. After staining of surface markers, cells were fixed and made permeable. Then the cells were stained with anti–IL-1β, anti–IL-6, and anti–IL-10 antibodies and analyzed by flow cytometry. PBMCs were gated according to their CD14 + staining after exclusion of doublets and debris by using FSC-H/FSC-A dot plots. Frequency and MFI of IL-1β–, IL-6–, and IL-10–producing monocytes. Data are presented as mean ± SEM. * P

Techniques Used: Infection, Functional Assay, Activity Assay, Isolation, Cell Culture, Staining, Cytometry

31) Product Images from "Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant"

Article Title: Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant

Journal: Leukemia

doi: 10.1038/leu.2013.66

Functional assays in TAAmix-specific CTL lines in two donors. ( a ) Recognition of TAAmix and SAs by IFNγ-ELISpot (mean spot count ± s.d.) and ( b ) cytolytic activity (mean ± s.d.) in a CFSE-based cytotoxicity assay against peptide-pulsed target cells correlating with these results, with no lysis of unpulsed PHAB (dotted line). ( c ) Intracellular cytokine detection of peptide stimulated CTLs in the presence of CD28/CD49d, Brefeldin A and Monensin showing secretion of multiple cytokines, (IFNγ, TNFα, IL2) and CD154 and CD107a-expression as a marker of activation and degranulation, respectively, in both CD4 + and CD8 + T-cell populations. ( d ) Recognition of TAAmix and SAs in IFNγ-ELISpot assay (mean spot count ± s.d.) by TAAmix CTLs from a representative donor. ( e ) Specific lysis (mean ± s.d.) of peptide-pulsed autologous PHAB in a CFSE-based cytotoxicity assay. ( f ) Cytokine secretion by CD4 + and CD8 + T cells.
Figure Legend Snippet: Functional assays in TAAmix-specific CTL lines in two donors. ( a ) Recognition of TAAmix and SAs by IFNγ-ELISpot (mean spot count ± s.d.) and ( b ) cytolytic activity (mean ± s.d.) in a CFSE-based cytotoxicity assay against peptide-pulsed target cells correlating with these results, with no lysis of unpulsed PHAB (dotted line). ( c ) Intracellular cytokine detection of peptide stimulated CTLs in the presence of CD28/CD49d, Brefeldin A and Monensin showing secretion of multiple cytokines, (IFNγ, TNFα, IL2) and CD154 and CD107a-expression as a marker of activation and degranulation, respectively, in both CD4 + and CD8 + T-cell populations. ( d ) Recognition of TAAmix and SAs in IFNγ-ELISpot assay (mean spot count ± s.d.) by TAAmix CTLs from a representative donor. ( e ) Specific lysis (mean ± s.d.) of peptide-pulsed autologous PHAB in a CFSE-based cytotoxicity assay. ( f ) Cytokine secretion by CD4 + and CD8 + T cells.

Techniques Used: Functional Assay, CTL Assay, Enzyme-linked Immunospot, Activity Assay, Cytotoxicity Assay, Lysis, Expressing, Marker, Activation Assay

32) Product Images from "ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling"

Article Title: ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling

Journal: Blood

doi: 10.1182/blood-2009-10-250415

ITAM signaling controls the half-life and surface retention of peptide-MHCII complexes. DCs were pulse-labeled with [ 35 S] for 30 minutes, washed, and chased for the indicated time points. Cells were then lysed, and mature MHCII complexes were immunoprecipitated with Y3P antibody before resolution by PAGE and autoradiography. In WT DCs, stable peptide-MHCII complexes (αβ-peptide), which are SDS-resistant in nonboiled samples, appeared by 3 hours and were slightly reduced after a 9-hour chase. While Vav NULL (A) or DF (B) DCs generated stable peptide-MHCII complexes at 3 hours, they rapidly decayed by the 9-hour time point. (C) Quantification of pMHCII decay from 3-9 hours was achieved by densitometric analysis of pMHCII bands at 9 hours normalized to 3 hours. Data represent the relative mean reduction in pixel intensity ± SD from 4 independent experiments. (D) WT and Vav NULL DCs were treated with Brefeldin A to block transport of newly synthesized MHCII through the Golgi network, and surface MHCII was detected by FACS at the indicated time points to determine the rate at which MHCII was removed from the cell surface. Data represent the mean percentage ± SD of remaining surface MHCII (based on mean fluorescence intensity [MFI] at time 0) from 5 independent experiments; * P
Figure Legend Snippet: ITAM signaling controls the half-life and surface retention of peptide-MHCII complexes. DCs were pulse-labeled with [ 35 S] for 30 minutes, washed, and chased for the indicated time points. Cells were then lysed, and mature MHCII complexes were immunoprecipitated with Y3P antibody before resolution by PAGE and autoradiography. In WT DCs, stable peptide-MHCII complexes (αβ-peptide), which are SDS-resistant in nonboiled samples, appeared by 3 hours and were slightly reduced after a 9-hour chase. While Vav NULL (A) or DF (B) DCs generated stable peptide-MHCII complexes at 3 hours, they rapidly decayed by the 9-hour time point. (C) Quantification of pMHCII decay from 3-9 hours was achieved by densitometric analysis of pMHCII bands at 9 hours normalized to 3 hours. Data represent the relative mean reduction in pixel intensity ± SD from 4 independent experiments. (D) WT and Vav NULL DCs were treated with Brefeldin A to block transport of newly synthesized MHCII through the Golgi network, and surface MHCII was detected by FACS at the indicated time points to determine the rate at which MHCII was removed from the cell surface. Data represent the mean percentage ± SD of remaining surface MHCII (based on mean fluorescence intensity [MFI] at time 0) from 5 independent experiments; * P

Techniques Used: Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Generated, Blocking Assay, Synthesized, FACS, Fluorescence

33) Product Images from "Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies"

Article Title: Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies

Journal: Retrovirology

doi: 10.1186/1742-4690-11-33

Sustained expression of BG505 SOSIP.664 gp140 by stable cell lines. (A)  Intracellular Env expression with continued passage of the 293 T 13# 3–5 and CHO B-D7 stable cell lines (blue curves). The fixed and permeabilized cells were stained with FITC-2G12 (20 μg/ml) after culture for 6 h in the presence of Brefeldin A. MFI values for the Env-expressing clones are recorded in the top right corner of each histogram. Parental cells served as negative controls (red curves show MFI values: ranges 9.12-12.9 for 293 T cells and 7.23-13.2 for CHO cells).  (B)  Production of BG505 SOSIP.664 gp140 trimers by the stable cell lines throughout the culture period, as determined by ELISA using 2G12 and bio-PGT145.  (C)  Viability of stable cell clones during passage. The cells were stained with trypan blue, with the percentage of viable cells (parental  vs.  stable) shown as the passage number increases.
Figure Legend Snippet: Sustained expression of BG505 SOSIP.664 gp140 by stable cell lines. (A) Intracellular Env expression with continued passage of the 293 T 13# 3–5 and CHO B-D7 stable cell lines (blue curves). The fixed and permeabilized cells were stained with FITC-2G12 (20 μg/ml) after culture for 6 h in the presence of Brefeldin A. MFI values for the Env-expressing clones are recorded in the top right corner of each histogram. Parental cells served as negative controls (red curves show MFI values: ranges 9.12-12.9 for 293 T cells and 7.23-13.2 for CHO cells). (B) Production of BG505 SOSIP.664 gp140 trimers by the stable cell lines throughout the culture period, as determined by ELISA using 2G12 and bio-PGT145. (C) Viability of stable cell clones during passage. The cells were stained with trypan blue, with the percentage of viable cells (parental vs. stable) shown as the passage number increases.

Techniques Used: Expressing, Stable Transfection, Staining, Clone Assay, Enzyme-linked Immunosorbent Assay

Vector for constitutive secretion of BG505 SOSIP.664 gp140 in a Flp-In™ based expression system, and stable cell line selection. (A)  Design of the pAM/C construct for expressing BG505 SOSIP.664 gp140. The plasmid map shows the site of the  env  and  furin  gene insertions, the promoters and the Poly A sequences.  (B)  Intracellular Env expression in transfected 293 T and CHO cells. The histograms represent parental cells (red) and stable cell clones (blue); the numbers (top right of each panel) are the mean fluorescence intensity (MFI) values after staining with FITC-2G12.  (C)  Secretion of BG505 SOSIP.664 gp140 trimers by Stable 293 T and CHO cell clones. The trimer concentrations in the culture supernatants were determined by ELISA using 2G12 and bio-PGT145.  (D)  Fluorescent microscopy of stable cell clones. Cells were grown in an 8-well chamber slide, treated with Brefeldin A, fixed, permeabilized and stained for Env (FITC-2G12; green) or nuclear DNA (DAPI; blue). The left panels show parental 293 T and CHO cells, the right, the stable cell clones.
Figure Legend Snippet: Vector for constitutive secretion of BG505 SOSIP.664 gp140 in a Flp-In™ based expression system, and stable cell line selection. (A) Design of the pAM/C construct for expressing BG505 SOSIP.664 gp140. The plasmid map shows the site of the env and furin gene insertions, the promoters and the Poly A sequences. (B) Intracellular Env expression in transfected 293 T and CHO cells. The histograms represent parental cells (red) and stable cell clones (blue); the numbers (top right of each panel) are the mean fluorescence intensity (MFI) values after staining with FITC-2G12. (C) Secretion of BG505 SOSIP.664 gp140 trimers by Stable 293 T and CHO cell clones. The trimer concentrations in the culture supernatants were determined by ELISA using 2G12 and bio-PGT145. (D) Fluorescent microscopy of stable cell clones. Cells were grown in an 8-well chamber slide, treated with Brefeldin A, fixed, permeabilized and stained for Env (FITC-2G12; green) or nuclear DNA (DAPI; blue). The left panels show parental 293 T and CHO cells, the right, the stable cell clones.

Techniques Used: Plasmid Preparation, Expressing, Stable Transfection, Selection, Construct, Transfection, Fluorescence, Staining, Clone Assay, Enzyme-linked Immunosorbent Assay, Microscopy

34) Product Images from "A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials"

Article Title: A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials

Journal: Particle and Fibre Toxicology

doi: 10.1186/1743-8977-8-17

Early induction of TNF-α expression followed by late induction of mannose receptor (MR) in 3D cultures . Cells were exposed to 0.5 μg/ml (0.38 μg/cm 2 ) of particulates. After 1, 3, 7, 10 and 14 days, cells were incubated with Brefeldin A for 6 hrs, fixed in situ and co-immunostained for TNF-α (red), MR (green), and counterstained with DAPI (blue nuclear fluorescence). A three-day exposure to asbestos or MWCNTs resulted in an increase in TNF-α expression. Prolonged exposure to all nanomaterials resulted in an increase in MR expression. Samples were analyzed by confocal microscopy using the appropriate filters. Photographs are representative of Z-stacks of three individual samples. Magnification: 400×.
Figure Legend Snippet: Early induction of TNF-α expression followed by late induction of mannose receptor (MR) in 3D cultures . Cells were exposed to 0.5 μg/ml (0.38 μg/cm 2 ) of particulates. After 1, 3, 7, 10 and 14 days, cells were incubated with Brefeldin A for 6 hrs, fixed in situ and co-immunostained for TNF-α (red), MR (green), and counterstained with DAPI (blue nuclear fluorescence). A three-day exposure to asbestos or MWCNTs resulted in an increase in TNF-α expression. Prolonged exposure to all nanomaterials resulted in an increase in MR expression. Samples were analyzed by confocal microscopy using the appropriate filters. Photographs are representative of Z-stacks of three individual samples. Magnification: 400×.

Techniques Used: Expressing, Incubation, In Situ, Fluorescence, Confocal Microscopy

35) Product Images from "A Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence"

Article Title: A Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence

Journal: Nature immunology

doi: 10.1038/ni.1847

Defect in peripheral T cells in  elektra  homozygotes (a)  Cells from spleen, lymph node (LN), and blood of WT or homozygous  elektra  mice were analyzed by flow cytometry for CD4 and CD8 expression.  (b)  Thymocytes from WT or homozygous  elektra  mice were analyzed by flow cytometry for CD4 and CD8 expression. For  a  and  b , results are representative of 10 mice per genotype.  (c)  WT and homozygous  elektra  mice were i.v. injected with either 200 or 2 × 10 6  PFU of LCMV (Armstrong strain). Splenocytes were isolated 7 days post-injection and restimulated  ex vivo  with either GP33 or NP396, peptides derived from LCMV, in the presence of Brefeldin A. Total numbers of CD8 +  splenocytes were determined by flow cytometry (top). CD8 +  cells were then fixed, permeabilized, and stained for intracellular IFN-γ expression (bottom).  n =3 mice of each genotype per condition. Results are representative of 2 independent experiments. ***  P
Figure Legend Snippet: Defect in peripheral T cells in elektra homozygotes (a) Cells from spleen, lymph node (LN), and blood of WT or homozygous elektra mice were analyzed by flow cytometry for CD4 and CD8 expression. (b) Thymocytes from WT or homozygous elektra mice were analyzed by flow cytometry for CD4 and CD8 expression. For a and b , results are representative of 10 mice per genotype. (c) WT and homozygous elektra mice were i.v. injected with either 200 or 2 × 10 6 PFU of LCMV (Armstrong strain). Splenocytes were isolated 7 days post-injection and restimulated ex vivo with either GP33 or NP396, peptides derived from LCMV, in the presence of Brefeldin A. Total numbers of CD8 + splenocytes were determined by flow cytometry (top). CD8 + cells were then fixed, permeabilized, and stained for intracellular IFN-γ expression (bottom). n =3 mice of each genotype per condition. Results are representative of 2 independent experiments. *** P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Injection, Isolation, Ex Vivo, Derivative Assay, Staining

36) Product Images from "Neither Lys- and DAP-type peptidoglycans stimulate mouse or human innate immune cells via Toll-like receptor 2"

Article Title: Neither Lys- and DAP-type peptidoglycans stimulate mouse or human innate immune cells via Toll-like receptor 2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193207

Mouse macrophage response to B . anthracis - and S . aureus -derived PGN. (A-B) Differentiated BMDMs from WT or TLR2 -/- mice were plated into wells of a 96-well plate (1.5 x 10 5 cells per well) and stimulated with LPS (1 μg/ml), B . anthracis PGN (10 μg/ml), or S . aureus PGN (10 μg/ml) in the presence of Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of 3 independent experiments (solid grey peak is unstimulated sample). Results are mean ± SEM from three independent experiments. The p values were calculated using a two-way ANOVA using Bonferroni post hoc test for multiple comparisons. # p
Figure Legend Snippet: Mouse macrophage response to B . anthracis - and S . aureus -derived PGN. (A-B) Differentiated BMDMs from WT or TLR2 -/- mice were plated into wells of a 96-well plate (1.5 x 10 5 cells per well) and stimulated with LPS (1 μg/ml), B . anthracis PGN (10 μg/ml), or S . aureus PGN (10 μg/ml) in the presence of Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of 3 independent experiments (solid grey peak is unstimulated sample). Results are mean ± SEM from three independent experiments. The p values were calculated using a two-way ANOVA using Bonferroni post hoc test for multiple comparisons. # p

Techniques Used: Derivative Assay, Mouse Assay, Staining, Flow Cytometry, Cytometry

Lipid modification of contaminating peptides in purified S . aureus PGN is important for TLR2 signaling. PGN from Newman S . aureus (A) and Newman S . aureus Δlgt (B) was digested with mutanolysin. After digestion samples were run in triplicate for amino acid analysis as described in Fig 2 . (C-D) Differentiated BMDMs were stimulated with LPS (1 μg/ml), B . anthracis PGN (10 μg/ml), Newman S . aureus PGN (10 μg/ml), or Newman S . aureus Δlgt PGN (10 μg/ml) in the presence of Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of three independent experiments (solid grey peak is unstimulated sample). Results are mean ± SEM from three independent experiments. The p values were calculated using a one-way ANOVA using Bonferroni post hoc test for multiple comparisons. # p
Figure Legend Snippet: Lipid modification of contaminating peptides in purified S . aureus PGN is important for TLR2 signaling. PGN from Newman S . aureus (A) and Newman S . aureus Δlgt (B) was digested with mutanolysin. After digestion samples were run in triplicate for amino acid analysis as described in Fig 2 . (C-D) Differentiated BMDMs were stimulated with LPS (1 μg/ml), B . anthracis PGN (10 μg/ml), Newman S . aureus PGN (10 μg/ml), or Newman S . aureus Δlgt PGN (10 μg/ml) in the presence of Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of three independent experiments (solid grey peak is unstimulated sample). Results are mean ± SEM from three independent experiments. The p values were calculated using a one-way ANOVA using Bonferroni post hoc test for multiple comparisons. # p

Techniques Used: Modification, Purification, Staining, Flow Cytometry, Cytometry

Human monocyte responses to PGN from lipoprotein deficient S . aureus require serum opsonins. (A) PBMCs were plated into wells of a 96-well plated (4–8 x 10 5 cells per well) and stimulated with B . anthracis PGN (10 μg/ml) in presence of FCS (1% v/v) with or without normal human serum (1% v/v) and with Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. (B) PBMCs were plated into wells of a 96-well plated (4–8 x 10 5 cells per well) and stimulated with Newman S . aureus PGN (10 μg/ml), or Newman S . aureus Δlgt PGN (10 μg/ml) in presence of FCS (1% v/v) with or without normal human serum (1% v/v) and with Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of experiments from three donors (solid grey peak is unstimulated sample). Results are mean ± SEM from three donors. The p values were calculated using either a two-way (B) or one-way (D) ANOVA using Bonferroni post hoc test for multiple comparisons. # p ≤ .0001 compared to unstimulated control, ** p = .001, *** p = 0001, **** p
Figure Legend Snippet: Human monocyte responses to PGN from lipoprotein deficient S . aureus require serum opsonins. (A) PBMCs were plated into wells of a 96-well plated (4–8 x 10 5 cells per well) and stimulated with B . anthracis PGN (10 μg/ml) in presence of FCS (1% v/v) with or without normal human serum (1% v/v) and with Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. (B) PBMCs were plated into wells of a 96-well plated (4–8 x 10 5 cells per well) and stimulated with Newman S . aureus PGN (10 μg/ml), or Newman S . aureus Δlgt PGN (10 μg/ml) in presence of FCS (1% v/v) with or without normal human serum (1% v/v) and with Brefeldin A for 6 hours at 37°C. The cells were stained for intracellular TNFα, and then analyzed by flow cytometry. Graphs are representative of experiments from three donors (solid grey peak is unstimulated sample). Results are mean ± SEM from three donors. The p values were calculated using either a two-way (B) or one-way (D) ANOVA using Bonferroni post hoc test for multiple comparisons. # p ≤ .0001 compared to unstimulated control, ** p = .001, *** p = 0001, **** p

Techniques Used: Staining, Flow Cytometry, Cytometry

37) Product Images from "Fasciola hepatica Immune Regulates CD11c+ Cells by Interacting with the Macrophage Gal/GalNAc Lectin"

Article Title: Fasciola hepatica Immune Regulates CD11c+ Cells by Interacting with the Macrophage Gal/GalNAc Lectin

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00264

FhTE induces Th2 polarization by reducing IFNγ and increasing IL-4 production . moDCs were incubated with coated FhTE in the presence or absence of LPS (10 μg/ml) for 48 h and the expression of costimulatory molecules was evaluated by flow cytometry (A) . To evaluate the capacity of the stimulated moDCs to polarize naïve CD4 T cells, cells were washed and incubated with CD4 + CD45RA + T cells (ratio 1:10) in the presence of Staphylococcal Enterotoxin B (10 pg/ml). After 5 days, supernatants were harvested for the evaluation of IFNγ (B,C) , and replaced with 100 U of IL-2. After 5 days, Th1/Th2 polarization of T cells was evaluated by intracellular staining of IFNγ and IL-4 after stimulation with PMA and Ionomycin in the presence of Brefeldin A (D–F) . When indicated (C,F) , moDCs were preincubated with an anti-hMGL antibody or an isotype control, before stimulation. IL-4/IFNγ ratio was evaluated relative to the control, based in single positive cells. Concentration of FhTE used: 200, 100, and 50 μg/ml. A representative result of one out of four donors is shown (±SEM, indicated by error bars). Asterisks indicate statistically significant differences (* p
Figure Legend Snippet: FhTE induces Th2 polarization by reducing IFNγ and increasing IL-4 production . moDCs were incubated with coated FhTE in the presence or absence of LPS (10 μg/ml) for 48 h and the expression of costimulatory molecules was evaluated by flow cytometry (A) . To evaluate the capacity of the stimulated moDCs to polarize naïve CD4 T cells, cells were washed and incubated with CD4 + CD45RA + T cells (ratio 1:10) in the presence of Staphylococcal Enterotoxin B (10 pg/ml). After 5 days, supernatants were harvested for the evaluation of IFNγ (B,C) , and replaced with 100 U of IL-2. After 5 days, Th1/Th2 polarization of T cells was evaluated by intracellular staining of IFNγ and IL-4 after stimulation with PMA and Ionomycin in the presence of Brefeldin A (D–F) . When indicated (C,F) , moDCs were preincubated with an anti-hMGL antibody or an isotype control, before stimulation. IL-4/IFNγ ratio was evaluated relative to the control, based in single positive cells. Concentration of FhTE used: 200, 100, and 50 μg/ml. A representative result of one out of four donors is shown (±SEM, indicated by error bars). Asterisks indicate statistically significant differences (* p

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

38) Product Images from "Role of Specific Innate Immune Responses in Herpes Simplex Virus Infection of the Central Nervous System"

Article Title: Role of Specific Innate Immune Responses in Herpes Simplex Virus Infection of the Central Nervous System

Journal: Journal of Virology

doi: 10.1128/JVI.06010-11

Murine astrocytes and microglial cells produce inflammatory cytokines predominantly through TLR2 following infection with HSV-1. Primary brain mixed glial cultures, which are a mixed population of astrocytes and microglial cells, were obtained from wild-type and TLR2-, TLR9-, TLR2/TLR9-, and UNC93B1-deficient mice and cultured with TLR ligands, virus, or medium alone for 1 h and then with brefeldin A for 18 h. (A) Flow cytometry shows that populations of primary brain mixed glial cells produce cytokines in response to stimulation with HSV-1 (MOI of 3). Cells were fixed, permeabilized, stained for a surface marker (CD11b) and intracellular cytokines (TNF [upper panels] and MCP-1 [lower panels]), and analyzed by flow cytometry. Data shown are percentages of cells expressing cytokines. Microglial cells (CD11b + ) make TNF (upper right panel), while astrocytes (CD11b − ) make MCP-1 (lower right panel) when infected with HSV. APC, allophycocyanin; PE, phycoerythrin. (B) Astrocytes (CD11b − ) produce MCP-1 in a TLR2-dependent but TLR9-independent manner following challenge with HSV (MOI of 10 and 3) or Pam 2 CSK 4 (100 ng/ml). Astrocytes respond to CpG 1826 (10 μM) in a TLR9- and UNC93B1-dependent manner. Astrocytes respond to poly(IC) (25 μg/ml) in a UNC93B1-dependent manner. LPS is a TLR2-, TLR9-, and UNC93B1-independent control. (C) Microglial cells (CD11b + ) produce TNF in a TLR2-dependent but TLR9-independent manner following challenge with HSV (MOI of 10 and 3) or Pam 2 CSK 4 (100 ng/ml). Microglial cells respond to CpG 1826 (10 μM) in a TLR9-dependent manner. Microglial cells produce minimal TNF in response to poly(IC) (25 μg/ml). LPS serves as a TLR2-, TLR9-, and UNC93B1-independent control.
Figure Legend Snippet: Murine astrocytes and microglial cells produce inflammatory cytokines predominantly through TLR2 following infection with HSV-1. Primary brain mixed glial cultures, which are a mixed population of astrocytes and microglial cells, were obtained from wild-type and TLR2-, TLR9-, TLR2/TLR9-, and UNC93B1-deficient mice and cultured with TLR ligands, virus, or medium alone for 1 h and then with brefeldin A for 18 h. (A) Flow cytometry shows that populations of primary brain mixed glial cells produce cytokines in response to stimulation with HSV-1 (MOI of 3). Cells were fixed, permeabilized, stained for a surface marker (CD11b) and intracellular cytokines (TNF [upper panels] and MCP-1 [lower panels]), and analyzed by flow cytometry. Data shown are percentages of cells expressing cytokines. Microglial cells (CD11b + ) make TNF (upper right panel), while astrocytes (CD11b − ) make MCP-1 (lower right panel) when infected with HSV. APC, allophycocyanin; PE, phycoerythrin. (B) Astrocytes (CD11b − ) produce MCP-1 in a TLR2-dependent but TLR9-independent manner following challenge with HSV (MOI of 10 and 3) or Pam 2 CSK 4 (100 ng/ml). Astrocytes respond to CpG 1826 (10 μM) in a TLR9- and UNC93B1-dependent manner. Astrocytes respond to poly(IC) (25 μg/ml) in a UNC93B1-dependent manner. LPS is a TLR2-, TLR9-, and UNC93B1-independent control. (C) Microglial cells (CD11b + ) produce TNF in a TLR2-dependent but TLR9-independent manner following challenge with HSV (MOI of 10 and 3) or Pam 2 CSK 4 (100 ng/ml). Microglial cells respond to CpG 1826 (10 μM) in a TLR9-dependent manner. Microglial cells produce minimal TNF in response to poly(IC) (25 μg/ml). LPS serves as a TLR2-, TLR9-, and UNC93B1-independent control.

Techniques Used: Infection, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Staining, Marker, Expressing

39) Product Images from "Gradual Increase of FcγRIIIa/CD16a Expression and Shift toward IFN-γ Secretion during Differentiation of CD56dim Natural Killer Cells"

Article Title: Gradual Increase of FcγRIIIa/CD16a Expression and Shift toward IFN-γ Secretion during Differentiation of CD56dim Natural Killer Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.01556

Degranulation and IFN-γ synthesis by CD56 dim  NK cells in response to FcγRIIIa engagement by plate-bound anti-CD16 monoclonal antibody (mAb). Culture plates were sensitized overnight with a saturating concentration 5 μg/mL  (A,C)  or increasing concentrations  (B)  of anti-CD16 3G8 mAb. Freshly isolated natural killer (NK) cells were then incubated for 4 h  (A,B)  or for the times indicated  (C)  on coated plates, in the presence of anti-CD107a mAb and brefeldin A. Cells were then stained with anti-CD56 mAb. Fixed and permeabilized NK cells were stained for intracellular IFN-γ expression and analyzed by FCM. Results are from one representative of three independent experiments (obtained with NK cells from three donors).
Figure Legend Snippet: Degranulation and IFN-γ synthesis by CD56 dim NK cells in response to FcγRIIIa engagement by plate-bound anti-CD16 monoclonal antibody (mAb). Culture plates were sensitized overnight with a saturating concentration 5 μg/mL (A,C) or increasing concentrations (B) of anti-CD16 3G8 mAb. Freshly isolated natural killer (NK) cells were then incubated for 4 h (A,B) or for the times indicated (C) on coated plates, in the presence of anti-CD107a mAb and brefeldin A. Cells were then stained with anti-CD56 mAb. Fixed and permeabilized NK cells were stained for intracellular IFN-γ expression and analyzed by FCM. Results are from one representative of three independent experiments (obtained with NK cells from three donors).

Techniques Used: Concentration Assay, Isolation, Incubation, Staining, Expressing

40) Product Images from "Polymicrobial Sepsis Diminishes Dendritic Cell Numbers and Function Directly Contributing to Impaired Primary CD8 T-Cell Responses in vivo"

Article Title: Polymicrobial Sepsis Diminishes Dendritic Cell Numbers and Function Directly Contributing to Impaired Primary CD8 T-Cell Responses in vivo

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

doi: 10.4049/jimmunol.1601463

Sepsis diminishes DCs capacity to respond to TLR stimulation in vitro (A) Experimental Design. Enriched CD11c +  DCs from the spleens of sham and CLP mice were incubated in the presence or absence of TLR agonists (LPS and CpG) for 7 h at 37° C in the presence of Brefeldin A. (B) Representative histograms show the frequency of CD11c hi  cells producing IL-12p40 after in vitro incubation. (C) The percentage of CD11c hi  cells producing IL-12p40 and TNF upon TLR stimulation. Data are presented as mean ± SEM of 4 mice per group in pooled samples of two spleens. Data are representative of three similar and independent experiments.  *p  ≤ 0.05,  **p  ≤ 0.01 as determined by student T-test.
Figure Legend Snippet: Sepsis diminishes DCs capacity to respond to TLR stimulation in vitro (A) Experimental Design. Enriched CD11c + DCs from the spleens of sham and CLP mice were incubated in the presence or absence of TLR agonists (LPS and CpG) for 7 h at 37° C in the presence of Brefeldin A. (B) Representative histograms show the frequency of CD11c hi cells producing IL-12p40 after in vitro incubation. (C) The percentage of CD11c hi cells producing IL-12p40 and TNF upon TLR stimulation. Data are presented as mean ± SEM of 4 mice per group in pooled samples of two spleens. Data are representative of three similar and independent experiments. *p ≤ 0.05, **p ≤ 0.01 as determined by student T-test.

Techniques Used: In Vitro, Mouse Assay, Incubation

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Activation Assay:

Article Title: Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)
Article Snippet: .. To be able to use the same cells to assess cytokines both in the culture supernatants as well as intracellularly, the culture supernatant was collected first (after 12 hr or 5 days of culture) for Luminex analysis and then cultured cells were re-supplemented for the last 4 hours of incubation with IMDM +10% FCS and PMA/Ionomycin in presence of brefeldin-A (Leukocyte Activation Cocktail [BD]). .. The stimulated cells were then stained for both surface and intracellular antigens and analyzed and the culture supernatants kept at −800 until analysis of soluble cytokines by multiplex bead array.

Incubation:

Article Title: Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)
Article Snippet: .. To be able to use the same cells to assess cytokines both in the culture supernatants as well as intracellularly, the culture supernatant was collected first (after 12 hr or 5 days of culture) for Luminex analysis and then cultured cells were re-supplemented for the last 4 hours of incubation with IMDM +10% FCS and PMA/Ionomycin in presence of brefeldin-A (Leukocyte Activation Cocktail [BD]). .. The stimulated cells were then stained for both surface and intracellular antigens and analyzed and the culture supernatants kept at −800 until analysis of soluble cytokines by multiplex bead array.

Article Title: iNKT Cell Production of GM-CSF Controls Mycobacterium tuberculosis
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Article Title: Mononuclear Phagocyte-Derived Interleukin-10 Suppresses the Innate Pulmonary Granuloma Cytokine Response in Aged Mice
Article Snippet: .. For intracellular cytokine staining, single-cell suspensions were incubated at 37°C in a humidified 5% CO2 atmosphere for 3 hours in complete medium in the presence of brefeldin A (1 μg/ml) (BD PharMingen) with or without PPD (5 μg/ml). .. Peripheral blood leukocytes from young and aged mice were isolated from heparinized blood after red cell lysis using a mouse erythrocyte lysing kit (R & D Systems, Minneapolis, MN) followed by three washes by centrifugation with final suspension in complete medium for culture.

Cell Culture:

Article Title: Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)
Article Snippet: .. To be able to use the same cells to assess cytokines both in the culture supernatants as well as intracellularly, the culture supernatant was collected first (after 12 hr or 5 days of culture) for Luminex analysis and then cultured cells were re-supplemented for the last 4 hours of incubation with IMDM +10% FCS and PMA/Ionomycin in presence of brefeldin-A (Leukocyte Activation Cocktail [BD]). .. The stimulated cells were then stained for both surface and intracellular antigens and analyzed and the culture supernatants kept at −800 until analysis of soluble cytokines by multiplex bead array.

Article Title: Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome
Article Snippet: .. Cells were stained directly after thawing, as described below, or first cultured for 5 hours in the presence of 10 ng/mL phorbol acetate myristate (PMA), 1 μg/mL ionomycin (both from Sigma-Aldrich), and Brefeldin-A (BD Biosciences) in RPMI 1640 supplemented with 10% FCS. .. PBMCs were washed in phosphate-buffered saline (PBS) (Invitrogen) and then incubated with ViViD (Invitrogen) for dead cell exclusion.

Article Title: Costimulation through TLR2 drives polyfunctional CD8+ T cell responses
Article Snippet: .. For intracellular cytokine staining, cells were cultured in the presence of 1μg/ml brefeldin A (BD Biosciences) as indicated. ..

Expressing:

Article Title: Cutting Edge: A Single MHC Class Ia Is Sufficient for CD8 Memory T Cell Differentiation 1
Article Snippet: .. In duplicate wells, Brefeldin A (GolgiPlug, Cytofix/Cytoperm kit; BD Pharmingen) was added for the final 3 h of stimulation, and cells were subsequently stained and analyzed for intracellular IFN- γ expression. .. Cells were stained with the following Abs: CD8-FITC, CD8-PE, IL-2-PE, IL-7R α -PE, CD25-allophycocyanin, IFN- γ -allophycocyanin, H-2Dd -FITC, H-2Kb -PE, CD4-PerCP, CD8-allophycocyanin (BD Pharmingen).

Staining:

Article Title: Mononuclear Phagocyte-Derived Interleukin-10 Suppresses the Innate Pulmonary Granuloma Cytokine Response in Aged Mice
Article Snippet: .. For intracellular cytokine staining, single-cell suspensions were incubated at 37°C in a humidified 5% CO2 atmosphere for 3 hours in complete medium in the presence of brefeldin A (1 μg/ml) (BD PharMingen) with or without PPD (5 μg/ml). .. Peripheral blood leukocytes from young and aged mice were isolated from heparinized blood after red cell lysis using a mouse erythrocyte lysing kit (R & D Systems, Minneapolis, MN) followed by three washes by centrifugation with final suspension in complete medium for culture.

Article Title: Cutting Edge: A Single MHC Class Ia Is Sufficient for CD8 Memory T Cell Differentiation 1
Article Snippet: .. In duplicate wells, Brefeldin A (GolgiPlug, Cytofix/Cytoperm kit; BD Pharmingen) was added for the final 3 h of stimulation, and cells were subsequently stained and analyzed for intracellular IFN- γ expression. .. Cells were stained with the following Abs: CD8-FITC, CD8-PE, IL-2-PE, IL-7R α -PE, CD25-allophycocyanin, IFN- γ -allophycocyanin, H-2Dd -FITC, H-2Kb -PE, CD4-PerCP, CD8-allophycocyanin (BD Pharmingen).

Article Title: Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome
Article Snippet: .. Cells were stained directly after thawing, as described below, or first cultured for 5 hours in the presence of 10 ng/mL phorbol acetate myristate (PMA), 1 μg/mL ionomycin (both from Sigma-Aldrich), and Brefeldin-A (BD Biosciences) in RPMI 1640 supplemented with 10% FCS. .. PBMCs were washed in phosphate-buffered saline (PBS) (Invitrogen) and then incubated with ViViD (Invitrogen) for dead cell exclusion.

Article Title: RNA binding protein PCBP1 is an intracellular immune checkpoint for shaping T cell responses in cancer immunity
Article Snippet: .. For intracellular cytokine staining, cells were stimulated for 4 hours with PMA (phorbol 12-myristate-13-acetate; 50 ng/ml) and ionomycin (1 μg/ml; Sigma-Aldrich) in the presence of brefeldin A (5 μg/ml; BD Biosciences). .. Intracellular staining, including FoxP3 (FJK-16) was performed with the eBioscience Foxp3 staining buffer set (00-5523-00, eBioscience).

Article Title: Costimulation through TLR2 drives polyfunctional CD8+ T cell responses
Article Snippet: .. For intracellular cytokine staining, cells were cultured in the presence of 1μg/ml brefeldin A (BD Biosciences) as indicated. ..

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    Becton Dickinson brefeldin a
    A.  Stimulation of sorted CD8+CD146+ T cells and CD8+CD146− T cells from healthy donors for 5 days in vitro with CD3/CD28 without the addition of any exogenous polarizing cytokines. PMA, ionomycin ,  and brefeldin A was added for the last 4 hours
    Brefeldin A, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brefeldin a/product/Becton Dickinson
    Average 94 stars, based on 355 article reviews
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    88
    Becton Dickinson protein transport inhibitors brefeldin a
    HLA-A2-specific binding affinity and stability of XBP1 US, XBP1 SP, CD138, and CS1 multipeptide Figure 1a. HLA-A2 binding capacity of multipeptide (MP) cocktail T2 cells were pulsed overnight with a cocktail of heteroclitic XBP1 US 184–192 (YISPWILAV), heteroclitic XBP1 SP 367–375 (YLFPQLISV), native CD138 260–268 (GLVGLIFAV) and native CS1 239–247 (SLFVLGLFL) peptides in serum-fee AIM-V media at total peptide concentrations ranging from 0 μg/ml to 50 μg/ml. Influenza virus matrix protein 58–66 (IVMP 58–66; GILGFVFTL) was used as an HLA-A2-specific positive control peptide. Following overnight peptide pulsing, T2 cells were harvested, washed, and stained with HLA-A2-FITC mAb for flow cytometric analyses. HLA-A2-specificity of the MP cocktail is shown as an increase in HLA-A2 mean fluorescence intensity (MFI) on T2 cells. The HLA-A2 binding was dose-dependent with the MFI plateau observed at the concentration of 25 μg/ml. The values represent the mean MFI ± SE of three separate experiments. Figure 1b. HLA-A2 stability of MP cocktail The MP cocktail (25 μg/ml; 6.25 μg/peptide) pulsed T2 cells were washed and incubated with <t>Brefeldin</t> A (BFA) to block the protein transport of newly synthesized HLA-A2 molecules. The binding stability of MP was measured on T2 cells at 0, 2, 4, 6 and 14 hrs post-BFA treatment and analyzed for HLA-A2 MFI by flow cytometry. An increase in the HLA-A2 MFI was observed at each time point on T2 cells pulsed with the MP from T2 cells alone. The binding of MP was highly stable for up to 6 hours post-BFA treatment. At 14 hrs post-BFA treatment, the stability of MP cocktail was greater than the control IVMP 58–66 peptide. The values represent the mean MFI ± SE of three separate experiments.
    Protein Transport Inhibitors Brefeldin A, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein transport inhibitors brefeldin a/product/Becton Dickinson
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    A.  Stimulation of sorted CD8+CD146+ T cells and CD8+CD146− T cells from healthy donors for 5 days in vitro with CD3/CD28 without the addition of any exogenous polarizing cytokines. PMA, ionomycin ,  and brefeldin A was added for the last 4 hours

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)

    doi: 10.1016/j.clim.2014.01.009

    Figure Lengend Snippet: A. Stimulation of sorted CD8+CD146+ T cells and CD8+CD146− T cells from healthy donors for 5 days in vitro with CD3/CD28 without the addition of any exogenous polarizing cytokines. PMA, ionomycin , and brefeldin A was added for the last 4 hours

    Article Snippet: To be able to use the same cells to assess cytokines both in the culture supernatants as well as intracellularly, the culture supernatant was collected first (after 12 hr or 5 days of culture) for Luminex analysis and then cultured cells were re-supplemented for the last 4 hours of incubation with IMDM +10% FCS and PMA/Ionomycin in presence of brefeldin-A (Leukocyte Activation Cocktail [BD]).

    Techniques: In Vitro

    Production of intracellular IFN-γ and IL-17A in CD8+ T cells in relationship to expression of CD146 in a representative sample of fresh peripheral blood from a healthy donor stimulated for 6 hours with PMA, ionomycin, and brefeldin A.

    Journal: Clinical immunology (Orlando, Fla.)

    Article Title: Secretion of Interleukin-17 by CD8+ T Cells Expressing CD146 (MCAM)

    doi: 10.1016/j.clim.2014.01.009

    Figure Lengend Snippet: Production of intracellular IFN-γ and IL-17A in CD8+ T cells in relationship to expression of CD146 in a representative sample of fresh peripheral blood from a healthy donor stimulated for 6 hours with PMA, ionomycin, and brefeldin A.

    Article Snippet: To be able to use the same cells to assess cytokines both in the culture supernatants as well as intracellularly, the culture supernatant was collected first (after 12 hr or 5 days of culture) for Luminex analysis and then cultured cells were re-supplemented for the last 4 hours of incubation with IMDM +10% FCS and PMA/Ionomycin in presence of brefeldin-A (Leukocyte Activation Cocktail [BD]).

    Techniques: Expressing

    CpG motifs and in a U-rich context determine the ssRNA capacity to induce TNF-α production by pDCs. Pure pDCs were stimulated with the indicated CpG/GpC RNA sequences for 18 h in the presence of brefeldin A. Intracellular detection of TNF-α was carried out using specific antibodies for the cytokine in previously gated pDCs (CD123 +  cells). (A) Cytometry panels show TNF-α production in stimulated pDCs from one donor after CpG/GpC RNA stimulation. (B) TNF-α-induced production comparing GpC RNA and CpG RNA sequences is shown. The value for CpG RNA-induced production was set at 100%. The MFI for TNF-α is represented ( n  = 4, in duplicate). Statistical analysis was done using unpaired Student's  t  test (2-tailed), comparing in each sequence CpG RNA- and A RNA- versus GpC-induced cytokine production. *,  P

    Journal: Journal of Virology

    Article Title: Oligonucleotide Motifs That Disappear during the Evolution of Influenza Virus in Humans Increase Alpha Interferon Secretion by Plasmacytoid Dendritic Cells ▿

    doi: 10.1128/JVI.01908-10

    Figure Lengend Snippet: CpG motifs and in a U-rich context determine the ssRNA capacity to induce TNF-α production by pDCs. Pure pDCs were stimulated with the indicated CpG/GpC RNA sequences for 18 h in the presence of brefeldin A. Intracellular detection of TNF-α was carried out using specific antibodies for the cytokine in previously gated pDCs (CD123 + cells). (A) Cytometry panels show TNF-α production in stimulated pDCs from one donor after CpG/GpC RNA stimulation. (B) TNF-α-induced production comparing GpC RNA and CpG RNA sequences is shown. The value for CpG RNA-induced production was set at 100%. The MFI for TNF-α is represented ( n = 4, in duplicate). Statistical analysis was done using unpaired Student's t test (2-tailed), comparing in each sequence CpG RNA- and A RNA- versus GpC-induced cytokine production. *, P

    Article Snippet: pDCs stimulated with CpG/GpC RNA for 18 h in the presence of brefeldin A (added 4 h after stimulation) were first stained with PE-CD123 monoclonal antibody (MAb) (BD), fixed with 4% paraformaldehyde, and treated with permeabilizing (0.1% saponin solution) and blocking solution.

    Techniques: Gel Permeation Chromatography, Cytometry, Sequencing

    HDAC inhibitors impair IFN-γ production from PMA/ionomycin stimulated CD4 +  and CD8 +  T-cells. A, B . PBMC from subject OM292 were exposed to HDACis for 4 hours. Cells were then washed and cultured in fresh medium with PMA/ionomycin for an additional 5 hours in the presence of brefeldin A.  A . IFN-γ production in CD4 +  and CD8 +  T-cells was measured by intracellular cytokine staining flow cytometry. Shown are mean ± SEM values. P values were calculated by two-way ANOVA with Dunnett's multiple comparison test (comparing to the no drug control) * p

    Journal: PLoS Pathogens

    Article Title: Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes

    doi: 10.1371/journal.ppat.1004287

    Figure Lengend Snippet: HDAC inhibitors impair IFN-γ production from PMA/ionomycin stimulated CD4 + and CD8 + T-cells. A, B . PBMC from subject OM292 were exposed to HDACis for 4 hours. Cells were then washed and cultured in fresh medium with PMA/ionomycin for an additional 5 hours in the presence of brefeldin A. A . IFN-γ production in CD4 + and CD8 + T-cells was measured by intracellular cytokine staining flow cytometry. Shown are mean ± SEM values. P values were calculated by two-way ANOVA with Dunnett's multiple comparison test (comparing to the no drug control) * p

    Article Snippet: Following 4 hours of exposure, cells were washed with 2×250 µl medium and then cultured with 200 µl fresh R10 medium+1/500 dilution of the 500× PMA/ionomycin cell stimulation cocktail (eBioscience) in the presence of 1 µg/ml Brefeldin A (BD).

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

    HLA-A2-specific binding affinity and stability of XBP1 US, XBP1 SP, CD138, and CS1 multipeptide Figure 1a. HLA-A2 binding capacity of multipeptide (MP) cocktail T2 cells were pulsed overnight with a cocktail of heteroclitic XBP1 US 184–192 (YISPWILAV), heteroclitic XBP1 SP 367–375 (YLFPQLISV), native CD138 260–268 (GLVGLIFAV) and native CS1 239–247 (SLFVLGLFL) peptides in serum-fee AIM-V media at total peptide concentrations ranging from 0 μg/ml to 50 μg/ml. Influenza virus matrix protein 58–66 (IVMP 58–66; GILGFVFTL) was used as an HLA-A2-specific positive control peptide. Following overnight peptide pulsing, T2 cells were harvested, washed, and stained with HLA-A2-FITC mAb for flow cytometric analyses. HLA-A2-specificity of the MP cocktail is shown as an increase in HLA-A2 mean fluorescence intensity (MFI) on T2 cells. The HLA-A2 binding was dose-dependent with the MFI plateau observed at the concentration of 25 μg/ml. The values represent the mean MFI ± SE of three separate experiments. Figure 1b. HLA-A2 stability of MP cocktail The MP cocktail (25 μg/ml; 6.25 μg/peptide) pulsed T2 cells were washed and incubated with Brefeldin A (BFA) to block the protein transport of newly synthesized HLA-A2 molecules. The binding stability of MP was measured on T2 cells at 0, 2, 4, 6 and 14 hrs post-BFA treatment and analyzed for HLA-A2 MFI by flow cytometry. An increase in the HLA-A2 MFI was observed at each time point on T2 cells pulsed with the MP from T2 cells alone. The binding of MP was highly stable for up to 6 hours post-BFA treatment. At 14 hrs post-BFA treatment, the stability of MP cocktail was greater than the control IVMP 58–66 peptide. The values represent the mean MFI ± SE of three separate experiments.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Myeloma-specific multiple peptides able to generate cytotoxic T lymphocytes: A potential therapeutic application in multiple myeloma and other plasma cell disorders

    doi: 10.1158/1078-0432.CCR-11-2776

    Figure Lengend Snippet: HLA-A2-specific binding affinity and stability of XBP1 US, XBP1 SP, CD138, and CS1 multipeptide Figure 1a. HLA-A2 binding capacity of multipeptide (MP) cocktail T2 cells were pulsed overnight with a cocktail of heteroclitic XBP1 US 184–192 (YISPWILAV), heteroclitic XBP1 SP 367–375 (YLFPQLISV), native CD138 260–268 (GLVGLIFAV) and native CS1 239–247 (SLFVLGLFL) peptides in serum-fee AIM-V media at total peptide concentrations ranging from 0 μg/ml to 50 μg/ml. Influenza virus matrix protein 58–66 (IVMP 58–66; GILGFVFTL) was used as an HLA-A2-specific positive control peptide. Following overnight peptide pulsing, T2 cells were harvested, washed, and stained with HLA-A2-FITC mAb for flow cytometric analyses. HLA-A2-specificity of the MP cocktail is shown as an increase in HLA-A2 mean fluorescence intensity (MFI) on T2 cells. The HLA-A2 binding was dose-dependent with the MFI plateau observed at the concentration of 25 μg/ml. The values represent the mean MFI ± SE of three separate experiments. Figure 1b. HLA-A2 stability of MP cocktail The MP cocktail (25 μg/ml; 6.25 μg/peptide) pulsed T2 cells were washed and incubated with Brefeldin A (BFA) to block the protein transport of newly synthesized HLA-A2 molecules. The binding stability of MP was measured on T2 cells at 0, 2, 4, 6 and 14 hrs post-BFA treatment and analyzed for HLA-A2 MFI by flow cytometry. An increase in the HLA-A2 MFI was observed at each time point on T2 cells pulsed with the MP from T2 cells alone. The binding of MP was highly stable for up to 6 hours post-BFA treatment. At 14 hrs post-BFA treatment, the stability of MP cocktail was greater than the control IVMP 58–66 peptide. The values represent the mean MFI ± SE of three separate experiments.

    Article Snippet: After 1 hour incubation, CD28/CD49d mAb (BD), as well as protein transport inhibitors Brefeldin A (BD) and Monensin (BD), were added to the cultures and incubated for an additional 5 hours.

    Techniques: Binding Assay, Positive Control, Staining, Flow Cytometry, Fluorescence, Concentration Assay, Incubation, Blocking Assay, Synthesized, Cytometry