gene exp il21 mm00517640 m1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher gene exp il21 mm00517640 m1
    IL-6-induced <t>IL-4/IL-21</t> production is responsible for the defect of Th1 differentiation. Young naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro . ( a ) Four days after stimulation, expressions of the indicated transcription factors were analysed. ( b , c ) Naive polyclonal CD4 + T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown ( b ). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR ( c ). Results are shown as mean±s.e.m. with n =4–6 per group; * P
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    1) Product Images from "IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age"

    Article Title: IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age

    Journal: Nature Communications

    doi: 10.1038/ncomms7702

    IL-6-induced IL-4/IL-21 production is responsible for the defect of Th1 differentiation. Young naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro . ( a ) Four days after stimulation, expressions of the indicated transcription factors were analysed. ( b , c ) Naive polyclonal CD4 + T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown ( b ). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR ( c ). Results are shown as mean±s.e.m. with n =4–6 per group; * P
    Figure Legend Snippet: IL-6-induced IL-4/IL-21 production is responsible for the defect of Th1 differentiation. Young naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro . ( a ) Four days after stimulation, expressions of the indicated transcription factors were analysed. ( b , c ) Naive polyclonal CD4 + T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown ( b ). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR ( c ). Results are shown as mean±s.e.m. with n =4–6 per group; * P

    Techniques Used: In Vitro, Mutagenesis, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    IL-6-induced IL-4/IL-21/IL-10 production is responsible for the defective CD8 help in aged mice. ( a , b ) OT-II cells were primed in young or aged mice at day 0 as in Fig. 1b , and then were treated with control Ab, anti-IL-4 and anti-IL-21 Abs ( a ) or anti-IL-10 Ab ( b ) on days 3 and 4. Six days after immunization, the frequencies of IFN-γ + cells in the donor OT-II cells were determined. ( b ) Representative plots. ( c ) OT-II transfer, immunization, Ab treatment and MCA-OVA inoculation were performed as in Fig. 1d . Four days after tumour inoculation, IL-10 concentration in serum was determined. ( d , e ) OT-II transfer, immunization and treatments of anti-IL-4 and anti-IL-21 Abs were performed as in a . Five days after immunization, mice were inoculated with MCA-OVA. Anti-IL-10 Ab was injected twice at 2 and 3 days after tumour inoculation. Five days after tumour inoculation, draining LNs were analysed for OVA-specific CD8 + T cells using OVA-Ip tetramer ( d ). OVA-Ip-specific CD8 + T-cell response was also evaluated by the IFN-γ ELISPOT assay ( e ). Data shown are mean±s.e.m. with n =4–5 per group. ( f ) CD62L lo effector OT-II cells primed in young or aged IL-6 +/+ or IL-6 −/− mice were sorted as in Fig. 4a and were co-cultured with CFSE-labelled CD8 + T cells in the presence or absence of anti-IL-10 Ab. After 3 days, CFSE profile and IFN-γ production in CD8 + T cells were determined using a flow cytometer. Representative data from two independent experiments are shown. * P
    Figure Legend Snippet: IL-6-induced IL-4/IL-21/IL-10 production is responsible for the defective CD8 help in aged mice. ( a , b ) OT-II cells were primed in young or aged mice at day 0 as in Fig. 1b , and then were treated with control Ab, anti-IL-4 and anti-IL-21 Abs ( a ) or anti-IL-10 Ab ( b ) on days 3 and 4. Six days after immunization, the frequencies of IFN-γ + cells in the donor OT-II cells were determined. ( b ) Representative plots. ( c ) OT-II transfer, immunization, Ab treatment and MCA-OVA inoculation were performed as in Fig. 1d . Four days after tumour inoculation, IL-10 concentration in serum was determined. ( d , e ) OT-II transfer, immunization and treatments of anti-IL-4 and anti-IL-21 Abs were performed as in a . Five days after immunization, mice were inoculated with MCA-OVA. Anti-IL-10 Ab was injected twice at 2 and 3 days after tumour inoculation. Five days after tumour inoculation, draining LNs were analysed for OVA-specific CD8 + T cells using OVA-Ip tetramer ( d ). OVA-Ip-specific CD8 + T-cell response was also evaluated by the IFN-γ ELISPOT assay ( e ). Data shown are mean±s.e.m. with n =4–5 per group. ( f ) CD62L lo effector OT-II cells primed in young or aged IL-6 +/+ or IL-6 −/− mice were sorted as in Fig. 4a and were co-cultured with CFSE-labelled CD8 + T cells in the presence or absence of anti-IL-10 Ab. After 3 days, CFSE profile and IFN-γ production in CD8 + T cells were determined using a flow cytometer. Representative data from two independent experiments are shown. * P

    Techniques Used: Mouse Assay, Concentration Assay, Injection, Enzyme-linked Immunospot, Cell Culture, Flow Cytometry, Cytometry

    2) Product Images from "IL-21 induces IL-22 production in CD4+ T-cells"

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    Journal: Nature communications

    doi: 10.1038/ncomms4753

    STAT3 controls AhR recruitment to the il22 promoter in IL-21-stimulated CD4+ T cells ( a ) AhR, c-Maf and STAT3 binding sites in the il22 promoter. ( b-c ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21. ChIP analysis of the interaction of AhR ( b ) and STAT3 ( c ) to the AhRand STAT3 binding sites in the il22 promoter, respectively. ( d ) Regulation of the transcriptional activity of the il22 promoter by AhR and STAT3. A reporter construct for the il22 promoter (il22:Luc) was cotransfected in HEK293 cells with vector coding for AhR and/or constitutively activated STAT3 (STAT3c). Firefly luciferase was determined and normalized to renilla luciferase activity. ( e ) ChIP analysis of the recruitment of AhR to the il22 promoter in WT and RORγt-deficient CD4+ cells activated in the presence of IL-21. ( f ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21, and the epigenetic status of the il22 promoter was analyzed by ChIP using antibodies to Acetyl-H3, Acetyl-H4 and H3 trimethylated at Lys4, Lys9 or Lys27. ( g-j ) ChIP analysis of the epigenetic status of the il22 promoter in WT and STAT3-deficient CD4+ T cells activated in the presence of IL-21. STAT3 recruitment (g) , H3 and H4 acetylation in SREs (h) and XREs (i) and AhR recruitment (j) in the il22 promoter. Results are representative of 2-3 independent experiments. * P
    Figure Legend Snippet: STAT3 controls AhR recruitment to the il22 promoter in IL-21-stimulated CD4+ T cells ( a ) AhR, c-Maf and STAT3 binding sites in the il22 promoter. ( b-c ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21. ChIP analysis of the interaction of AhR ( b ) and STAT3 ( c ) to the AhRand STAT3 binding sites in the il22 promoter, respectively. ( d ) Regulation of the transcriptional activity of the il22 promoter by AhR and STAT3. A reporter construct for the il22 promoter (il22:Luc) was cotransfected in HEK293 cells with vector coding for AhR and/or constitutively activated STAT3 (STAT3c). Firefly luciferase was determined and normalized to renilla luciferase activity. ( e ) ChIP analysis of the recruitment of AhR to the il22 promoter in WT and RORγt-deficient CD4+ cells activated in the presence of IL-21. ( f ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21, and the epigenetic status of the il22 promoter was analyzed by ChIP using antibodies to Acetyl-H3, Acetyl-H4 and H3 trimethylated at Lys4, Lys9 or Lys27. ( g-j ) ChIP analysis of the epigenetic status of the il22 promoter in WT and STAT3-deficient CD4+ T cells activated in the presence of IL-21. STAT3 recruitment (g) , H3 and H4 acetylation in SREs (h) and XREs (i) and AhR recruitment (j) in the il22 promoter. Results are representative of 2-3 independent experiments. * P

    Techniques Used: Binding Assay, In Vitro, Chromatin Immunoprecipitation, Activity Assay, Construct, Plasmid Preparation, Luciferase

    Transcriptional profiling of IL-21-stimulated CD4+ T cells Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of IL-21, IL-6 and TGFβ1 or without addition of exogenous cytokines. (a) Heatmap showing genes with at least a 1.5 fold change in expression relative to Th0 as determined by whole-genome microarrays. (b) Principal-component analysis (PCA). (c-d) Heatmaps depicting the expression of key cytokines, chemokines, receptors and transcription factors (c) and transcriptional modules (d) that control the differentiation of Th17 cells as defined in 34 . (e) Quantitative RT-PCR analysis of the expression of genes in Fig. 2a. mRNA expression is shown relative to gapdh . (f) Western blot analysis of the expression of AhR (95kDa) and RORγt (58kDa) on T cells activated in the presence of IL-21 or under Th17-polarizing conditions (left panel). Quantification of western blot reactivity (right panel). (g) Ingenuity Pathways Analysis of the transcriptional response of naïve CD4+ T cells to IL-21. * P
    Figure Legend Snippet: Transcriptional profiling of IL-21-stimulated CD4+ T cells Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of IL-21, IL-6 and TGFβ1 or without addition of exogenous cytokines. (a) Heatmap showing genes with at least a 1.5 fold change in expression relative to Th0 as determined by whole-genome microarrays. (b) Principal-component analysis (PCA). (c-d) Heatmaps depicting the expression of key cytokines, chemokines, receptors and transcription factors (c) and transcriptional modules (d) that control the differentiation of Th17 cells as defined in 34 . (e) Quantitative RT-PCR analysis of the expression of genes in Fig. 2a. mRNA expression is shown relative to gapdh . (f) Western blot analysis of the expression of AhR (95kDa) and RORγt (58kDa) on T cells activated in the presence of IL-21 or under Th17-polarizing conditions (left panel). Quantification of western blot reactivity (right panel). (g) Ingenuity Pathways Analysis of the transcriptional response of naïve CD4+ T cells to IL-21. * P

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Western Blot

    STAT3 controls the production of IL-22 by CD4+ T cells stimulated with IL-21 ( a-b ) NaïveWT CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) FACS analysis of phosphorylated STAT3. Mean fluorescence intensity (MFI) of phosphorylated STAT3 normalized to total STAT3. ( b ) qPCR analysis of stat3 expression. ( c-f ) Naïve WT and STAT3-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( c ) Effect of STAT3 deficiency on IL-22 and IL-17 production. ( d ) Effect of STAT3 deficiency on il21 expression. ( e-f ) Effect of STAT3 deficiency on ahr and rorc expression ( e ) and the receptors il1r , il23r ( f ). ( g ) Induction of stat3 by IL-1β, IL-21 IL-23, IL-1β and IL-21 or IL-21 and IL-23. mRNA expression is shown relative to gapdh . Results are representative of 2-3 independent experiments. * P
    Figure Legend Snippet: STAT3 controls the production of IL-22 by CD4+ T cells stimulated with IL-21 ( a-b ) NaïveWT CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) FACS analysis of phosphorylated STAT3. Mean fluorescence intensity (MFI) of phosphorylated STAT3 normalized to total STAT3. ( b ) qPCR analysis of stat3 expression. ( c-f ) Naïve WT and STAT3-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( c ) Effect of STAT3 deficiency on IL-22 and IL-17 production. ( d ) Effect of STAT3 deficiency on il21 expression. ( e-f ) Effect of STAT3 deficiency on ahr and rorc expression ( e ) and the receptors il1r , il23r ( f ). ( g ) Induction of stat3 by IL-1β, IL-21 IL-23, IL-1β and IL-21 or IL-21 and IL-23. mRNA expression is shown relative to gapdh . Results are representative of 2-3 independent experiments. * P

    Techniques Used: FACS, Fluorescence, Real-time Polymerase Chain Reaction, Expressing

    RORγt and AhR control IL-22 production by CD4+ T cells stimulated with IL-21 (a-c) Naïve WT and RORγt-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) Effect of RORγt deficiency on IL-22 and IL-17 production. ( b ) Effect of RORγt deficiency on il23r and il1r1 expression. ( c ) Effect of RORγt deficiency on the synergism between IL-21 and IL-23. ( d ) Effect of IL-1β and IL-23 in rorc expression. ( e ) Effect of the modulation of AhR activity on IL-22 and IL-17 production. ( f ) IL-22 production by WT and AhR-d CD4+ T cells activated in the presence of IL-21. ( g ) Modulation of ahr expression by IL-1β and IL-23. ( h ) Effect of AhR modulation on STAT3 phosphorylation and stat3 expression. MFI of phosphorylated STAT3 normalized to total STAT3. mRNA expression is shown relative to gapdh . Representative results of 2-3 independent experiments. * P
    Figure Legend Snippet: RORγt and AhR control IL-22 production by CD4+ T cells stimulated with IL-21 (a-c) Naïve WT and RORγt-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) Effect of RORγt deficiency on IL-22 and IL-17 production. ( b ) Effect of RORγt deficiency on il23r and il1r1 expression. ( c ) Effect of RORγt deficiency on the synergism between IL-21 and IL-23. ( d ) Effect of IL-1β and IL-23 in rorc expression. ( e ) Effect of the modulation of AhR activity on IL-22 and IL-17 production. ( f ) IL-22 production by WT and AhR-d CD4+ T cells activated in the presence of IL-21. ( g ) Modulation of ahr expression by IL-1β and IL-23. ( h ) Effect of AhR modulation on STAT3 phosphorylation and stat3 expression. MFI of phosphorylated STAT3 normalized to total STAT3. mRNA expression is shown relative to gapdh . Representative results of 2-3 independent experiments. * P

    Techniques Used: Expressing, Activity Assay

    IL-21 and AhR control IL-22 + CD4+ T cells in vivo and limit mucosal inflammation ( a ) RAG2/IL2RG mice transferred with WT, AhR-d or IL-21R deficient CD4+ T cells were given 3 % DSS ad libitum in their drinking water for 7 days. Body weight was assessed daily, and the percent mass change from day 0 was calculated. Mean ± s.e.m is shown, n =5 mice per group. Effect of genotype on disease course P
    Figure Legend Snippet: IL-21 and AhR control IL-22 + CD4+ T cells in vivo and limit mucosal inflammation ( a ) RAG2/IL2RG mice transferred with WT, AhR-d or IL-21R deficient CD4+ T cells were given 3 % DSS ad libitum in their drinking water for 7 days. Body weight was assessed daily, and the percent mass change from day 0 was calculated. Mean ± s.e.m is shown, n =5 mice per group. Effect of genotype on disease course P

    Techniques Used: In Vivo, Mouse Assay

    IL-21 promotes the differentiation of CD4+ T cells that produce IL-22 but not IL-17 Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of different cytokines as indicated. ( a-b ) Effect of IL-21 in the production of IL-22 and IL-17 measured by ELISA in culture supernatants ( a ) and qPCR ( b ). ( c ) Effect of IL-21 on the expression of il21 . ( e ) Effect of IL-21 on the expression of il6r, il21r, il23r . ( d ) Relationship between cell division and IL-22 expression in CD4+ T cells. CFSE-labeled naïve CD4+ T cells were activated in the presence of IL-21 and the production of IL-22 was analyzed by intracellular staining. The data are shown as the Mean fluorescence intensity (MFI) for IL-22 for each division cycle. ( e ) Effects of IL-21 on the expression of il6r, il21r and il23r . ( f-h ) Synergistic effects of IL-21 and IL-23 on IL-22 (f) and IL-17 (g) cytokine and il21 expression ( h ). (i) Effect of IL-21 on il1r expression. ( j-l ) Synergistic effects of IL-21 and IL-1β on IL-22 (j) , Il17a (k) and il21 (l) expression. mRNA expression is shown relative to gapdh . Results are representative of 3-5 independent experiments.* P
    Figure Legend Snippet: IL-21 promotes the differentiation of CD4+ T cells that produce IL-22 but not IL-17 Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of different cytokines as indicated. ( a-b ) Effect of IL-21 in the production of IL-22 and IL-17 measured by ELISA in culture supernatants ( a ) and qPCR ( b ). ( c ) Effect of IL-21 on the expression of il21 . ( e ) Effect of IL-21 on the expression of il6r, il21r, il23r . ( d ) Relationship between cell division and IL-22 expression in CD4+ T cells. CFSE-labeled naïve CD4+ T cells were activated in the presence of IL-21 and the production of IL-22 was analyzed by intracellular staining. The data are shown as the Mean fluorescence intensity (MFI) for IL-22 for each division cycle. ( e ) Effects of IL-21 on the expression of il6r, il21r and il23r . ( f-h ) Synergistic effects of IL-21 and IL-23 on IL-22 (f) and IL-17 (g) cytokine and il21 expression ( h ). (i) Effect of IL-21 on il1r expression. ( j-l ) Synergistic effects of IL-21 and IL-1β on IL-22 (j) , Il17a (k) and il21 (l) expression. mRNA expression is shown relative to gapdh . Results are representative of 3-5 independent experiments.* P

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Labeling, Staining, Fluorescence

    3) Product Images from "CCR7 Deficiency Exacerbates Injury in Acute Nephritis Due to Aberrant Localization of Regulatory T Cells"

    Article Title: CCR7 Deficiency Exacerbates Injury in Acute Nephritis Due to Aberrant Localization of Regulatory T Cells

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2009020133

    The cytokine expression pattern in kidneys and lymph nodes varies between CCR7KO and WT mice. (A and B) The expression of IL-10, TGF-β, IL-6, TNF-α, IFN-γ, IL-17, and IL-21 mRNA was evaluated by real-time PCR on total RNA from kidneys (A) or lymph nodes (B) of CCR7KO ( ; n = 21) or WT (■; n = 18) mice 7 d after anti-GBM injection. The data are shown as fold increase in comparison with healthy tissue of WT mice. The IL-17 expression was evaluated only in lymph nodes, because healthy controls do not express IL-17 in kidneys. CCR7KO mice express significantly more mRNA of IL-6, IL-10, and IL-21 in kidneys, whereas mRNA of IL-6, IFN-γ, and IL-17 was significantly increased in the lymph nodes of CCR7KO mice as compared with WT controls. * P
    Figure Legend Snippet: The cytokine expression pattern in kidneys and lymph nodes varies between CCR7KO and WT mice. (A and B) The expression of IL-10, TGF-β, IL-6, TNF-α, IFN-γ, IL-17, and IL-21 mRNA was evaluated by real-time PCR on total RNA from kidneys (A) or lymph nodes (B) of CCR7KO ( ; n = 21) or WT (■; n = 18) mice 7 d after anti-GBM injection. The data are shown as fold increase in comparison with healthy tissue of WT mice. The IL-17 expression was evaluated only in lymph nodes, because healthy controls do not express IL-17 in kidneys. CCR7KO mice express significantly more mRNA of IL-6, IL-10, and IL-21 in kidneys, whereas mRNA of IL-6, IFN-γ, and IL-17 was significantly increased in the lymph nodes of CCR7KO mice as compared with WT controls. * P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Injection

    4) Product Images from "Germinal Center B Cell Depletion Diminishes CD4+ Follicular T Helper Cells in Autoimmune Mice"

    Article Title: Germinal Center B Cell Depletion Diminishes CD4+ Follicular T Helper Cells in Autoimmune Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102791

    Depletion of B cells in SRBC immunized mice with anti-CD20 plus -CD40L-TM treatment leads to accelerated loss of mature Tfh cells. (A) Diagrammatic representation of experimental protocol. BALB/c mice were immunized with SRBC and treated with either anti-CD20 (0.25 mg mg/mouse) + anti-CD40L-TM (0.40 mg mg/mouse) or control antibodies at day 9 post treatment. Mice were sacrificed on days 5 and 8 post antibody treatment (14 and 17 days post immunization). (B) Relative expression of IL-21 and Bcl-6 in FACS sorted CD4 + CD44 high CD62L low cells from mice treated with either anti-CD20+anti-CD40L-TM or control antibodies. Data are normalized with expression of housekeeping gene 18S. (C) Graphs show number of total B cells (B220 + CD19 + ), GC B cells (PNA + FAS high IgD low ), Tfh cells (CXCR5 + PD1 high and CXCR5 + Bcl-6 + cells) gated on CD4 + CD44 high T cells. ***P
    Figure Legend Snippet: Depletion of B cells in SRBC immunized mice with anti-CD20 plus -CD40L-TM treatment leads to accelerated loss of mature Tfh cells. (A) Diagrammatic representation of experimental protocol. BALB/c mice were immunized with SRBC and treated with either anti-CD20 (0.25 mg mg/mouse) + anti-CD40L-TM (0.40 mg mg/mouse) or control antibodies at day 9 post treatment. Mice were sacrificed on days 5 and 8 post antibody treatment (14 and 17 days post immunization). (B) Relative expression of IL-21 and Bcl-6 in FACS sorted CD4 + CD44 high CD62L low cells from mice treated with either anti-CD20+anti-CD40L-TM or control antibodies. Data are normalized with expression of housekeeping gene 18S. (C) Graphs show number of total B cells (B220 + CD19 + ), GC B cells (PNA + FAS high IgD low ), Tfh cells (CXCR5 + PD1 high and CXCR5 + Bcl-6 + cells) gated on CD4 + CD44 high T cells. ***P

    Techniques Used: Mouse Assay, Expressing, FACS

    5) Product Images from "Early Th1 Cell Differentiation Is Marked by A Tfh-like Cell Transition"

    Article Title: Early Th1 Cell Differentiation Is Marked by A Tfh-like Cell Transition

    Journal: Immunity

    doi: 10.1016/j.immuni.2011.11.012

    Effect of Modulating the Levels of IFN-γand IL-21 during the Course of In Vitro Th1 Differentiation
    Figure Legend Snippet: Effect of Modulating the Levels of IFN-γand IL-21 during the Course of In Vitro Th1 Differentiation

    Techniques Used: In Vitro

    6) Product Images from "Interleukin-21 (IL-21) Downregulates Dendritic Cell Cytokine Responses to Helicobacter pylori and Modulates T Lymphocyte IL-17A Expression in Peyer’s Patches during Infection"

    Article Title: Interleukin-21 (IL-21) Downregulates Dendritic Cell Cytokine Responses to Helicobacter pylori and Modulates T Lymphocyte IL-17A Expression in Peyer’s Patches during Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00237-19

    IL-21 reduces the proinflammatory gene expression profile of H. pylori (Hp)-activated BMDCs. BMDCs were cultured with or without recombinant murine IL-21 and with or without H. pylori (MOI, 25) for 6 h. (A) Real time RT-PCR with the TaqMan technology was used to quantify cytokine transcription. Relative units were calculated using GAPDH as the endogenous control, and untreated BMDCs were used as the calibrator sample. Experimental conditions were set up in triplicate, and the data are representative of those from 4 experiments. ANOVA followed by Dunnett’s correction for multiple comparisons was used. *, P
    Figure Legend Snippet: IL-21 reduces the proinflammatory gene expression profile of H. pylori (Hp)-activated BMDCs. BMDCs were cultured with or without recombinant murine IL-21 and with or without H. pylori (MOI, 25) for 6 h. (A) Real time RT-PCR with the TaqMan technology was used to quantify cytokine transcription. Relative units were calculated using GAPDH as the endogenous control, and untreated BMDCs were used as the calibrator sample. Experimental conditions were set up in triplicate, and the data are representative of those from 4 experiments. ANOVA followed by Dunnett’s correction for multiple comparisons was used. *, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Quantitative RT-PCR

    IL-17 responses are significantly elevated in the MLN and PPs of H. pylori -infected IL-21 −/− mice. Real-time RT-PCR was used to measure Il-21 (A) and Il-17a (B) expression levels in H. pylori -infected mice. (C) Intracellular cytokine staining was used to measure IFN-γ and IL-17A production by CD4 + T cells and γδ-positive T cells from the Peyer’s patches of H. pylori -infected mice (with PMA restimulation [stim] and without PMA restimulation [null]). Four to 7 mice were used for each experiment, and the data presented are representative of those from 3 experiments. (A, B) An unpaired t test was performed to test for statistical significance. (C) ANOVA followed by Dunnett’s correction for multiple comparisons was used. ns, not significant; *, P
    Figure Legend Snippet: IL-17 responses are significantly elevated in the MLN and PPs of H. pylori -infected IL-21 −/− mice. Real-time RT-PCR was used to measure Il-21 (A) and Il-17a (B) expression levels in H. pylori -infected mice. (C) Intracellular cytokine staining was used to measure IFN-γ and IL-17A production by CD4 + T cells and γδ-positive T cells from the Peyer’s patches of H. pylori -infected mice (with PMA restimulation [stim] and without PMA restimulation [null]). Four to 7 mice were used for each experiment, and the data presented are representative of those from 3 experiments. (A, B) An unpaired t test was performed to test for statistical significance. (C) ANOVA followed by Dunnett’s correction for multiple comparisons was used. ns, not significant; *, P

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

    IL-21 does not regulate H. pylori -induced DC maturation based on surface marker expression. BMDCs were cultured with or without recombinant murine IL-21 and with or without H. pylori (MOI, 25) for 6 h. Flow cytometry was used to calculate the level of expression of CD80, CD86, and MHC class II (A) and the percentage of BMDCs expressing CD40 (B). Experimental conditions were set up in triplicate, and the data are representative of those from 4 experiments. ANOVA followed by Dunnett’s correction for multiple comparisons was used. *, P
    Figure Legend Snippet: IL-21 does not regulate H. pylori -induced DC maturation based on surface marker expression. BMDCs were cultured with or without recombinant murine IL-21 and with or without H. pylori (MOI, 25) for 6 h. Flow cytometry was used to calculate the level of expression of CD80, CD86, and MHC class II (A) and the percentage of BMDCs expressing CD40 (B). Experimental conditions were set up in triplicate, and the data are representative of those from 4 experiments. ANOVA followed by Dunnett’s correction for multiple comparisons was used. *, P

    Techniques Used: Marker, Expressing, Cell Culture, Recombinant, Flow Cytometry

    7) Product Images from "Dual Role of Interleukin-10 in Murine NZB/W F1 Lupus"

    Article Title: Dual Role of Interleukin-10 in Murine NZB/W F1 Lupus

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22031347

    Characterization of IL-10-expressing CD4 T cells. ( a ) Splenocytes from NZB/W F1 animals ( n = 4 mice) were examined at the age of 10 weeks, 22 weeks, 28 weeks and with established glomerulonephritis (GN) for IL-10 expression in CD4 T cells by flow cytometry. ( b – e ) Expression of IL-10 was examined in ( b ) FoxP3 + T reg and FoxP3 − effector CD4 T cells; ( c ) ICOS + versus ICOS − CD4 T cells; ( d ) CXCR5 hi PD1 hi T FH , CXCR5 − PD1 hi and CXCR5 − PD1 lo CD4 T cells; and ( e ) in relation to IFN-γ expression in CD4 T cells from splenocytes of 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis by flow cytometry ( n = 5–6 mice). Results are expressed as scatter blots with mean ± SEM, and representative FACS blots are depicted; each data point ( a ) or data symbol ( b – e ) represents an individual mouse. The p value was calculated using a Wilcoxon test ( b , c , e ) or Friedman test for paired analysis ( d ). ( f ) IL-21 and cMAF mRNA expression was determined in IL-10 + and IL-10 - CD4 T cells after purification from splenocytes of 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis ( n = 4–6 mice). Each symbol represents results obtained from different donors depicted as fold difference between IL-10 + and IL-10 − CD4 T cells. Results are expressed as scatter blots with mean ± SEM. The p value was calculated using a one-sample t test. ( g ) IL-27 mRNA expression was examined in spleens of healthy, 10 week old ( n = 4 mice) and sick, 28 week old ( n = 4 mice) NZB/W F1 animals. ( h ) Expression of pSTAT3 on CD4 T cells was determined in splenocytes of healthy, 10 week old ( n = 4 mice) and sick, 28 week old (sick; n = 6 mice) NZB/WF1 animals by flow cytometry. ( g , h ) Results are expressed as scatter blots with mean ± SEM; each data point represents an individual mouse. The p value was calculated using a Mann–Whitney test.
    Figure Legend Snippet: Characterization of IL-10-expressing CD4 T cells. ( a ) Splenocytes from NZB/W F1 animals ( n = 4 mice) were examined at the age of 10 weeks, 22 weeks, 28 weeks and with established glomerulonephritis (GN) for IL-10 expression in CD4 T cells by flow cytometry. ( b – e ) Expression of IL-10 was examined in ( b ) FoxP3 + T reg and FoxP3 − effector CD4 T cells; ( c ) ICOS + versus ICOS − CD4 T cells; ( d ) CXCR5 hi PD1 hi T FH , CXCR5 − PD1 hi and CXCR5 − PD1 lo CD4 T cells; and ( e ) in relation to IFN-γ expression in CD4 T cells from splenocytes of 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis by flow cytometry ( n = 5–6 mice). Results are expressed as scatter blots with mean ± SEM, and representative FACS blots are depicted; each data point ( a ) or data symbol ( b – e ) represents an individual mouse. The p value was calculated using a Wilcoxon test ( b , c , e ) or Friedman test for paired analysis ( d ). ( f ) IL-21 and cMAF mRNA expression was determined in IL-10 + and IL-10 - CD4 T cells after purification from splenocytes of 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis ( n = 4–6 mice). Each symbol represents results obtained from different donors depicted as fold difference between IL-10 + and IL-10 − CD4 T cells. Results are expressed as scatter blots with mean ± SEM. The p value was calculated using a one-sample t test. ( g ) IL-27 mRNA expression was examined in spleens of healthy, 10 week old ( n = 4 mice) and sick, 28 week old ( n = 4 mice) NZB/W F1 animals. ( h ) Expression of pSTAT3 on CD4 T cells was determined in splenocytes of healthy, 10 week old ( n = 4 mice) and sick, 28 week old (sick; n = 6 mice) NZB/WF1 animals by flow cytometry. ( g , h ) Results are expressed as scatter blots with mean ± SEM; each data point represents an individual mouse. The p value was calculated using a Mann–Whitney test.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, FACS, Purification, MANN-WHITNEY

    IL-10 in vitro effects on adaptive immune cells. ( a , d ) Splenocytes or ( b , e ) purified CD4, CD8 T cells or CD19 + B cells from 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis ( n = 4–9 mice) were incubated for 4 days with α-CD3/CD28 (T cell stimulation) or CpG (B cell stimulation), ( a , d ) in the presence or absence of neutralizing α-IL-10 or ( b , e ) in the presence or absence of recombinant mouse IL-10, TNF-α or IL-21. ( a , b ) Proliferation of CD4 T, CD8 T and B cells was examined using CFDA-SE. Depicted is one representative FACS blot showing the proliferation of CD8 T cells and gating strategy to quantify cells with 0–1, 2–4 or ≥5 cycles of proliferation. The mean percentages ± SEM of cells with 0–1, 2–4 or ≥5 cycles of proliferation from 6 ( a ) and 4–9 ( b ) independent donors are expressed as stacked bars. The p value was calculated using a Wilcoxon test with paired data analysis to determine the difference between α-IL-10-treated and untreated cells ( a ) or the difference between untreated cells and those treated with IL-10, IL-21 or TNF-α ( b ) (* p
    Figure Legend Snippet: IL-10 in vitro effects on adaptive immune cells. ( a , d ) Splenocytes or ( b , e ) purified CD4, CD8 T cells or CD19 + B cells from 28 week old NZB/W F1 animals with established autoantibodies and beginning nephritis ( n = 4–9 mice) were incubated for 4 days with α-CD3/CD28 (T cell stimulation) or CpG (B cell stimulation), ( a , d ) in the presence or absence of neutralizing α-IL-10 or ( b , e ) in the presence or absence of recombinant mouse IL-10, TNF-α or IL-21. ( a , b ) Proliferation of CD4 T, CD8 T and B cells was examined using CFDA-SE. Depicted is one representative FACS blot showing the proliferation of CD8 T cells and gating strategy to quantify cells with 0–1, 2–4 or ≥5 cycles of proliferation. The mean percentages ± SEM of cells with 0–1, 2–4 or ≥5 cycles of proliferation from 6 ( a ) and 4–9 ( b ) independent donors are expressed as stacked bars. The p value was calculated using a Wilcoxon test with paired data analysis to determine the difference between α-IL-10-treated and untreated cells ( a ) or the difference between untreated cells and those treated with IL-10, IL-21 or TNF-α ( b ) (* p

    Techniques Used: In Vitro, Purification, Mouse Assay, Incubation, Cell Stimulation, Recombinant, FACS

    8) Product Images from "IL-27 induces the transcription factor c-Maf, cytokine IL-21 and costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells 1"

    Article Title: IL-27 induces the transcription factor c-Maf, cytokine IL-21 and costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells 1

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

    doi: 10.4049/jimmunol.0901233

    IL-21 is necessary for IL-10 production in Tr1 cells
    Figure Legend Snippet: IL-21 is necessary for IL-10 production in Tr1 cells

    Techniques Used:

    IL-27 induces c-Maf, IL-21, IL-21R and ICOS
    Figure Legend Snippet: IL-27 induces c-Maf, IL-21, IL-21R and ICOS

    Techniques Used:

    IL-27 induces c-Maf, which transactivates IL-21
    Figure Legend Snippet: IL-27 induces c-Maf, which transactivates IL-21

    Techniques Used:

    9) Product Images from "Expression of Murine CD80 by Herpes Simplex Virus 1 in Place of Latency-Associated Transcript (LAT) Can Compensate for Latency Reactivation and Anti-apoptotic Functions of LAT"

    Article Title: Expression of Murine CD80 by Herpes Simplex Virus 1 in Place of Latency-Associated Transcript (LAT) Can Compensate for Latency Reactivation and Anti-apoptotic Functions of LAT

    Journal: Journal of Virology

    doi: 10.1128/JVI.01798-19

    Effect of CD80 expression on T cell exhaustion in the TG of latently infected mice. TG from latently infected HSV-CD80 [LAT(−)] and McKrae [LAT(+)] mice were individually isolated on day 28 p.i., and qRT-PCR was performed using total RNA as described in Materials and Methods. CD4, CD8, PD-1, Tim-3, IL-2, IL-21, TNF-α, and IFN-γ expression in naive mice was used as a baseline to estimate the relative expression of each transcript in TG of latently infected mice. GAPDH expression was used to normalize the relative expression of each transcript. Each point represents the mean ± SEM from 10 TG.
    Figure Legend Snippet: Effect of CD80 expression on T cell exhaustion in the TG of latently infected mice. TG from latently infected HSV-CD80 [LAT(−)] and McKrae [LAT(+)] mice were individually isolated on day 28 p.i., and qRT-PCR was performed using total RNA as described in Materials and Methods. CD4, CD8, PD-1, Tim-3, IL-2, IL-21, TNF-α, and IFN-γ expression in naive mice was used as a baseline to estimate the relative expression of each transcript in TG of latently infected mice. GAPDH expression was used to normalize the relative expression of each transcript. Each point represents the mean ± SEM from 10 TG.

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

    10) Product Images from "IL4, IL21, and IFNγ interact to govern TBET and CD11c expression in TLR-activated B cells"

    Article Title: IL4, IL21, and IFNγ interact to govern TBET and CD11c expression in TLR-activated B cells

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

    doi: 10.4049/jimmunol.1600522

    TBET + CD11c + cells delineate a B MEM cell subset and accumulate in Il21 tg mice (A–B) GC B and B MEM cells were analyzed for TBET and CD11c expression by FACS. GC B and B MEM . All panels are representative of 3 independent experiments with ≥ 3 mice per strain. ( A ) TBET staining on GC B cells from C57BL/6 (B6, n=14) or BALB/c (n=23) mice with frequency enumeration. ( B ) TBET and CD11c staining on B MEM cells from B6 mice. ( C ) TBET and CD11c staining on splenic B-2 cells from WT and Il21 tg mice. ( D ) Serum IgG 1 or IgG 2a/c (IgG 2a + IgG 2c ) levels in WT and Il21 tg mice were determined by ELISA. Values are means ± S.E.M. from 5 WT and 7 Il21 tg mice.
    Figure Legend Snippet: TBET + CD11c + cells delineate a B MEM cell subset and accumulate in Il21 tg mice (A–B) GC B and B MEM cells were analyzed for TBET and CD11c expression by FACS. GC B and B MEM . All panels are representative of 3 independent experiments with ≥ 3 mice per strain. ( A ) TBET staining on GC B cells from C57BL/6 (B6, n=14) or BALB/c (n=23) mice with frequency enumeration. ( B ) TBET and CD11c staining on B MEM cells from B6 mice. ( C ) TBET and CD11c staining on splenic B-2 cells from WT and Il21 tg mice. ( D ) Serum IgG 1 or IgG 2a/c (IgG 2a + IgG 2c ) levels in WT and Il21 tg mice were determined by ELISA. Values are means ± S.E.M. from 5 WT and 7 Il21 tg mice.

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

    IL4 and IL21 act in a cell intrinsic manner to regulate TBET expression in vitro Magnetically enriched CD23 + splenic B cells were cultured in vitro ), and IFNγ (γ). Mouse data are representative of 3 independent experiments. ( A ) WT or Cd19 cre/+ Tbx21 f/f B cells treated for 48hrs and probed for TBET (ΔMFI=WT-mutant). ( B ) Tbx21 mRNA levels in WT cells treated for 20hrs. ( C ) WT, Il21r −/− , or Stat6 −/− B cells were labeled with either CFSE (green plots) or Violet Cell Trace (VCT, purple plots), treated with ODN1826 and indicated cytokines for 48h, then stained for CD11c and TBET. ( D ) Magnetically enriched CD27 − CD19 + human B cells were labeled with CFSE, treated for 108h, and probed for TBET on live, CFSE − cells. ( E ) Frequency of TBET + B cells from each treatment across 6 healthy, adult donors.
    Figure Legend Snippet: IL4 and IL21 act in a cell intrinsic manner to regulate TBET expression in vitro Magnetically enriched CD23 + splenic B cells were cultured in vitro ), and IFNγ (γ). Mouse data are representative of 3 independent experiments. ( A ) WT or Cd19 cre/+ Tbx21 f/f B cells treated for 48hrs and probed for TBET (ΔMFI=WT-mutant). ( B ) Tbx21 mRNA levels in WT cells treated for 20hrs. ( C ) WT, Il21r −/− , or Stat6 −/− B cells were labeled with either CFSE (green plots) or Violet Cell Trace (VCT, purple plots), treated with ODN1826 and indicated cytokines for 48h, then stained for CD11c and TBET. ( D ) Magnetically enriched CD27 − CD19 + human B cells were labeled with CFSE, treated for 108h, and probed for TBET on live, CFSE − cells. ( E ) Frequency of TBET + B cells from each treatment across 6 healthy, adult donors.

    Techniques Used: Activated Clotting Time Assay, Expressing, In Vitro, Cell Culture, Mutagenesis, Labeling, Staining

    Influenza virus infection drives TBET + CD11c + B MEM cell formation in the absence of both IFNγ and IL4 Splenocytes were harvested from non-infected (−) or day 10 post i.n. 30 TCID50 PR8 infection (+) WT (n=21, black bars), Ifng −/− (n=10, white bars), or Il4 −/− Ifng −/− (n=13, gray bars) mice across 3–7 experiments with ≥3 mice per group. GC B, B MEM , and T FH . ( A ) Enumeration of GC B cells. ( B ) TBET staining on GC B cells. ( C ) Il4 and ( D ) Il21 mRNA levels from sorted naïve CD62L + CD4 T (T N , n=9) or T FH cells. ( E ) Proportions and ( F ) numbers of TBET + CD11c + B MEM cells.
    Figure Legend Snippet: Influenza virus infection drives TBET + CD11c + B MEM cell formation in the absence of both IFNγ and IL4 Splenocytes were harvested from non-infected (−) or day 10 post i.n. 30 TCID50 PR8 infection (+) WT (n=21, black bars), Ifng −/− (n=10, white bars), or Il4 −/− Ifng −/− (n=13, gray bars) mice across 3–7 experiments with ≥3 mice per group. GC B, B MEM , and T FH . ( A ) Enumeration of GC B cells. ( B ) TBET staining on GC B cells. ( C ) Il4 and ( D ) Il21 mRNA levels from sorted naïve CD62L + CD4 T (T N , n=9) or T FH cells. ( E ) Proportions and ( F ) numbers of TBET + CD11c + B MEM cells.

    Techniques Used: Infection, Mouse Assay, Staining

    Activated B cells express T-BET independent of IFNγ in IL4 limiting conditions Splenocytes and sera were harvested from non-infected (−) or day 14 post oral gavage (+) of 200 H. polygyrus in WT (n=20, black bars), Il4 −/− (n=24, white bars), or Il4 −/− Ifng −/− (n=11, gray bars) mice across 3–6 experiments with ≥3 mice per group. GC B, B MEM , and T FH . ( A ) Enumeration of GC B cells. ( B ) TBET staining on GC B cells. ( C ) Serum concentrations of IgG 1 and IgG 2c + IgG 2b . ( D ) Il21 mRNA levels from sorted T FH cells. ( E ) Proportions and ( F ) numbers of TBET + CD11c + B MEM cells.
    Figure Legend Snippet: Activated B cells express T-BET independent of IFNγ in IL4 limiting conditions Splenocytes and sera were harvested from non-infected (−) or day 14 post oral gavage (+) of 200 H. polygyrus in WT (n=20, black bars), Il4 −/− (n=24, white bars), or Il4 −/− Ifng −/− (n=11, gray bars) mice across 3–6 experiments with ≥3 mice per group. GC B, B MEM , and T FH . ( A ) Enumeration of GC B cells. ( B ) TBET staining on GC B cells. ( C ) Serum concentrations of IgG 1 and IgG 2c + IgG 2b . ( D ) Il21 mRNA levels from sorted T FH cells. ( E ) Proportions and ( F ) numbers of TBET + CD11c + B MEM cells.

    Techniques Used: Infection, Mouse Assay, Staining

    11) Product Images from "IL-6 inhibits upregulation of membrane-bound TGF-beta 1 on CD4+ T cells and blocking IL-6 enhances oral tolerance"

    Article Title: IL-6 inhibits upregulation of membrane-bound TGF-beta 1 on CD4+ T cells and blocking IL-6 enhances oral tolerance

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

    doi: 10.4049/jimmunol.1600921

    IL-6R signaling inhibits llrc32 expression via STAT3 A-D. Expression levels of Lrrc32 (A), Tgfb1 (B), Il21 (C) and Il17a (D) normalized to Gapdh after stimulation of naïve CD4+ T cells with coated anti-CD3 mAb, anti-CD28 mAb and IL-2 in the absence (black circles) presence of IL-6 (red squares) for 2, 5 or 16 hours. Graphs show average values ± SEM for three to five independent experiments. E-F. Percentage of CD4+LAP+ T cells after stimulation of naïve CD4+ T cells from CD4 Cre STAT3 flox/flox (STAT3 −/− ; E), STAT1 −/− (F) mice as compared to wild-type (WT) mice with coated anti-CD3 mAb, anti-CD28 mAb and IL-2 in the absence (black circles) or presence of IL-6 (red squares) for 72 hours. Graphs show average values ± SEM for one representative experiment with three to four samples each and a representative histogram. Statistically significant values are labeled; ***p
    Figure Legend Snippet: IL-6R signaling inhibits llrc32 expression via STAT3 A-D. Expression levels of Lrrc32 (A), Tgfb1 (B), Il21 (C) and Il17a (D) normalized to Gapdh after stimulation of naïve CD4+ T cells with coated anti-CD3 mAb, anti-CD28 mAb and IL-2 in the absence (black circles) presence of IL-6 (red squares) for 2, 5 or 16 hours. Graphs show average values ± SEM for three to five independent experiments. E-F. Percentage of CD4+LAP+ T cells after stimulation of naïve CD4+ T cells from CD4 Cre STAT3 flox/flox (STAT3 −/− ; E), STAT1 −/− (F) mice as compared to wild-type (WT) mice with coated anti-CD3 mAb, anti-CD28 mAb and IL-2 in the absence (black circles) or presence of IL-6 (red squares) for 72 hours. Graphs show average values ± SEM for one representative experiment with three to four samples each and a representative histogram. Statistically significant values are labeled; ***p

    Techniques Used: Expressing, Mouse Assay, Labeling

    12) Product Images from "IL-23 and IL-17A, but Not IL-12 and IL-22, Are Required for Optimal Skin Host Defense against Candida albicans"

    Article Title: IL-23 and IL-17A, but Not IL-12 and IL-22, Are Required for Optimal Skin Host Defense against Candida albicans

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

    doi: 10.4049/jimmunol.1001153

    IL-22 expression is decreased in IL-17A −/− mice, whereas IL-17A expression is normal in IL-22 −/− mice after cutaneous C. albicans infection. IL-17A, IL-17F, IL-22, IL-21, IFN -γ, and TNF -α mRNA expression
    Figure Legend Snippet: IL-22 expression is decreased in IL-17A −/− mice, whereas IL-17A expression is normal in IL-22 −/− mice after cutaneous C. albicans infection. IL-17A, IL-17F, IL-22, IL-21, IFN -γ, and TNF -α mRNA expression

    Techniques Used: Expressing, Mouse Assay, Infection

    Impaired production of IL-17A, IL-17F , and IL-22 in IL-23–deficient mice. IL-17A, IL-17F, IL-22, IL-21, IFN -γ, and TNF -α mRNA expression 48 h after C. albicans skin infection in WT and the various knockout mice as measured by qPCR.
    Figure Legend Snippet: Impaired production of IL-17A, IL-17F , and IL-22 in IL-23–deficient mice. IL-17A, IL-17F, IL-22, IL-21, IFN -γ, and TNF -α mRNA expression 48 h after C. albicans skin infection in WT and the various knockout mice as measured by qPCR.

    Techniques Used: Mouse Assay, Expressing, Infection, Knock-Out, Real-time Polymerase Chain Reaction

    13) Product Images from "T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice"

    Article Title: T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131377

    IL-21 promotes autophagy expression in neuronal cells after hypoxia/ischemia. (a) Il21r mRNA expression relative to GAPDH expression levels is shown in normoxic and hypoxic primary mouse neurons after OGD or control treatment. (b) Viability of Neuro2A cells treated with the indicated doses of IL-21 after OGD. (c) Il21r mRNA expression relative to GAPDH in neuronal (Neura2A), astrocytic, and endothelial cell lines (MB114) expressed relative to BMDC expression. (d) ATG6 expression in primary neurons treated with PBS, etoposide, or 32–256 ng/ml rIL-21 for 4 h after 1–2 h oxygen glucose deprivation as measured by RT-PCR. Cells treated in triplicate. (e) Number of ATG6 + cells per field in the same regions of WT and IL-21 KO mouse brains after tMCAO as assessed by immune staining ( n = 3 mice per group). Arrows indicate ATG-6 + cells in periinfarcted brain tissue of WT and IL-21 KO mice. Bars, 100 µm. Data are representative of two independent experiments. *, P
    Figure Legend Snippet: IL-21 promotes autophagy expression in neuronal cells after hypoxia/ischemia. (a) Il21r mRNA expression relative to GAPDH expression levels is shown in normoxic and hypoxic primary mouse neurons after OGD or control treatment. (b) Viability of Neuro2A cells treated with the indicated doses of IL-21 after OGD. (c) Il21r mRNA expression relative to GAPDH in neuronal (Neura2A), astrocytic, and endothelial cell lines (MB114) expressed relative to BMDC expression. (d) ATG6 expression in primary neurons treated with PBS, etoposide, or 32–256 ng/ml rIL-21 for 4 h after 1–2 h oxygen glucose deprivation as measured by RT-PCR. Cells treated in triplicate. (e) Number of ATG6 + cells per field in the same regions of WT and IL-21 KO mouse brains after tMCAO as assessed by immune staining ( n = 3 mice per group). Arrows indicate ATG-6 + cells in periinfarcted brain tissue of WT and IL-21 KO mice. Bars, 100 µm. Data are representative of two independent experiments. *, P

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

    Blockade of IL-21 signaling before or after tMCAO reduces infarct size in WT mice. (a) Infarct volumes 24 h after tMCAO in WT mice treated with 500 µg recombinant mIL-21R.Fc or PBS 1 h before (pretreatment) or 2 h after (posttreatment) surgery. Representative TTC-stained brain slices shown on left ( n = 3–4 mice per group). (b) Still image from Video 1 depicting behavioral differences between WT mice posttreated with IL-21R.Fc or PBS. (c) IL-21R.Fc protein levels in the indicated organs 20–24 h after tMCAO in WT mice injected with 500 µg IL-21R.Fc 2 h after start of reperfusion ( n = 2–4 mice per group). N.D., not detected. Data are representative of two independent experiments. **, P
    Figure Legend Snippet: Blockade of IL-21 signaling before or after tMCAO reduces infarct size in WT mice. (a) Infarct volumes 24 h after tMCAO in WT mice treated with 500 µg recombinant mIL-21R.Fc or PBS 1 h before (pretreatment) or 2 h after (posttreatment) surgery. Representative TTC-stained brain slices shown on left ( n = 3–4 mice per group). (b) Still image from Video 1 depicting behavioral differences between WT mice posttreated with IL-21R.Fc or PBS. (c) IL-21R.Fc protein levels in the indicated organs 20–24 h after tMCAO in WT mice injected with 500 µg IL-21R.Fc 2 h after start of reperfusion ( n = 2–4 mice per group). N.D., not detected. Data are representative of two independent experiments. **, P

    Techniques Used: Mouse Assay, Recombinant, Staining, Injection

    IL-21 is primarily produced by brain-infiltrating CD4 + T cells. (a) Intracellular cytokine staining of lymphocytes isolated from n = 5 pooled healthy WT, ischemic IL-21 −/− , or ischemic WT mouse brains 24 h after tMCAO or sham procedure showing IL-21 versus CD8α expression. Histograms show CD4, NK1.1, and TCRγδ expression on IL-21 + cells from ischemic WT brain. (b) CD45, CD4, and LFA-1 expression by negative fractions purified from WT and IL-21 −/− lymph node cells by CD4 + negative selection using magnetic cell separation before transfer into RAG2 −/− recipients. (c) Infarct volume in WT mice ( n = 4), RAG2 −/− mice ( n = 4), RAG2 −/− mice + WT CD4 T cells ( n = 10), and RAG2 −/− mice + IL-21 −/− CD4 T cells ( n = 10) 24 h after tMCAO. Representative TTC-stained 2-mm mouse brain slices shown on top. Data are representative of two independent experiments. **, P
    Figure Legend Snippet: IL-21 is primarily produced by brain-infiltrating CD4 + T cells. (a) Intracellular cytokine staining of lymphocytes isolated from n = 5 pooled healthy WT, ischemic IL-21 −/− , or ischemic WT mouse brains 24 h after tMCAO or sham procedure showing IL-21 versus CD8α expression. Histograms show CD4, NK1.1, and TCRγδ expression on IL-21 + cells from ischemic WT brain. (b) CD45, CD4, and LFA-1 expression by negative fractions purified from WT and IL-21 −/− lymph node cells by CD4 + negative selection using magnetic cell separation before transfer into RAG2 −/− recipients. (c) Infarct volume in WT mice ( n = 4), RAG2 −/− mice ( n = 4), RAG2 −/− mice + WT CD4 T cells ( n = 10), and RAG2 −/− mice + IL-21 −/− CD4 T cells ( n = 10) 24 h after tMCAO. Representative TTC-stained 2-mm mouse brain slices shown on top. Data are representative of two independent experiments. **, P

    Techniques Used: Produced, Staining, Isolation, Expressing, Purification, Selection, Magnetic Cell Separation, Mouse Assay

    IL-21 is up-regulated early in mouse brain, and IL-21–deficient mice are protected after tMCAO. GeArray S Series Mouse Autoimmune and Inflammatory Response gene array of transcripts expressed in pooled brain tissues 24 h after tMCAO or sham procedure ( n = 3–6 mice per group). (a) Bar graphs show PCR array spot intensity of genes with a greater than sixfold difference in gene expression in tMCAO compared with sham, normalized to the interquartile mean spot intensity. (b) IL-21 mRNA expression level in ipsilateral hemisphere relative to contralateral hemisphere 24 h after tMCAO ( n = 3 per group). Mann-Whitney rank sum test *, P
    Figure Legend Snippet: IL-21 is up-regulated early in mouse brain, and IL-21–deficient mice are protected after tMCAO. GeArray S Series Mouse Autoimmune and Inflammatory Response gene array of transcripts expressed in pooled brain tissues 24 h after tMCAO or sham procedure ( n = 3–6 mice per group). (a) Bar graphs show PCR array spot intensity of genes with a greater than sixfold difference in gene expression in tMCAO compared with sham, normalized to the interquartile mean spot intensity. (b) IL-21 mRNA expression level in ipsilateral hemisphere relative to contralateral hemisphere 24 h after tMCAO ( n = 3 per group). Mann-Whitney rank sum test *, P

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, MANN-WHITNEY

    Lymphocyte recruitment to brain is diminished in IL-21 deficient mice. (a) Gating strategy for leukocytes isolated from brain after MCAO. (b) WT and IL-21 KO spleen cells 24 h after tMCAO or sham procedure ( n = 3 mice per group). (c) Relative change in spleen weight of WT and IL-21 KO mice after tMCAO ( n = 3–7 mice per group). (d) Percentage of blood and spleen CD4 + T cells expressing IL-21 after 5-h ex vivo stimulation with PMA (10 ng/ml) and Ionomycin (1 µg/ml) 4 d after MCAO or control treatment. (e and f) Leukocyte accumulation in the brain of WT mice compared with IL-21 KO mice 1, 4, and 7 d after tMCAO ( n = 3–6 mice per group). (g–k) In vitro cytokine expression by WT and IL-21 KO CD4 + and CD8α + T cells after 5-h stimulation under indicated conditions with or without recombinant mouse IL-21 (100 ng/ml). (l) TNF production by CD11b + myeloid cells stimulated with LPS (500 ng/ml) for 5 h with or without recombinant mouse IL-21 (100 ng/ml). Cells isolated from n = 3 mice per group. Data are representative of two to four independent experiments. *, P
    Figure Legend Snippet: Lymphocyte recruitment to brain is diminished in IL-21 deficient mice. (a) Gating strategy for leukocytes isolated from brain after MCAO. (b) WT and IL-21 KO spleen cells 24 h after tMCAO or sham procedure ( n = 3 mice per group). (c) Relative change in spleen weight of WT and IL-21 KO mice after tMCAO ( n = 3–7 mice per group). (d) Percentage of blood and spleen CD4 + T cells expressing IL-21 after 5-h ex vivo stimulation with PMA (10 ng/ml) and Ionomycin (1 µg/ml) 4 d after MCAO or control treatment. (e and f) Leukocyte accumulation in the brain of WT mice compared with IL-21 KO mice 1, 4, and 7 d after tMCAO ( n = 3–6 mice per group). (g–k) In vitro cytokine expression by WT and IL-21 KO CD4 + and CD8α + T cells after 5-h stimulation under indicated conditions with or without recombinant mouse IL-21 (100 ng/ml). (l) TNF production by CD11b + myeloid cells stimulated with LPS (500 ng/ml) for 5 h with or without recombinant mouse IL-21 (100 ng/ml). Cells isolated from n = 3 mice per group. Data are representative of two to four independent experiments. *, P

    Techniques Used: Mouse Assay, Isolation, Expressing, Ex Vivo, In Vitro, Recombinant

    14) Product Images from "Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation"

    Article Title: Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation

    Journal: Nature immunology

    doi: 10.1038/ni.3683

    The effects of IRF1 and BATF deficiency on Tr1 function (a) Irf1 mRNA expression in Stat1 −/− (left) and Stat3 −/− (right) cells primed in the presence of IL-27. (b) Batf mRNA expression in Stat1 −/− (left) and Stat3 −/− (right) cells primed in the presence of IL-27. (c) Effects of IRF1 and BATF retroviral overexpression on Il10 , Il21 , Maf and AhR expression in cells treated in T H 0 or Tr1 conditions; mRNA levels were quantified using qPCR. Data are pooled from 3 independent experiments (a ( Stat1 −/− ), b, c; n=3 samples, dots represent biological replicates) or representative of 4 independent experiments (a ( Stat3 −/− ); n=3 samples, dots represent technical replicates). NS, not significant ( P > 0.05); * P
    Figure Legend Snippet: The effects of IRF1 and BATF deficiency on Tr1 function (a) Irf1 mRNA expression in Stat1 −/− (left) and Stat3 −/− (right) cells primed in the presence of IL-27. (b) Batf mRNA expression in Stat1 −/− (left) and Stat3 −/− (right) cells primed in the presence of IL-27. (c) Effects of IRF1 and BATF retroviral overexpression on Il10 , Il21 , Maf and AhR expression in cells treated in T H 0 or Tr1 conditions; mRNA levels were quantified using qPCR. Data are pooled from 3 independent experiments (a ( Stat1 −/− ), b, c; n=3 samples, dots represent biological replicates) or representative of 4 independent experiments (a ( Stat3 −/− ); n=3 samples, dots represent technical replicates). NS, not significant ( P > 0.05); * P

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction

    15) Product Images from "The Peptide Specificity of the Endogenous T Follicular Helper Cell Repertoire Generated after Protein Immunization"

    Article Title: The Peptide Specificity of the Endogenous T Follicular Helper Cell Repertoire Generated after Protein Immunization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046952

    The peptide-specific hierarchies of the Tfh and non-Tfh population are remarkably similar, Independent of the cytokine assayed. The peptide specificities of Tfh and non-Tfh cells were evaluated with IL-2, IL-21, IL-4, and IFNγ ELISPOT assays on day 8 or 9 post-immunization. The percent of the total response for each peptide tested (peptide-specific/sum of all peptide responses measured) generated after immunization with MalE encoding the S1 epitope (A), HEL (B), or OVA (C) are shown for both Tfh (open bars) and non-Tfh (shaded bars). Data are presented as the mean of two experiments with error bars representing the range.
    Figure Legend Snippet: The peptide-specific hierarchies of the Tfh and non-Tfh population are remarkably similar, Independent of the cytokine assayed. The peptide specificities of Tfh and non-Tfh cells were evaluated with IL-2, IL-21, IL-4, and IFNγ ELISPOT assays on day 8 or 9 post-immunization. The percent of the total response for each peptide tested (peptide-specific/sum of all peptide responses measured) generated after immunization with MalE encoding the S1 epitope (A), HEL (B), or OVA (C) are shown for both Tfh (open bars) and non-Tfh (shaded bars). Data are presented as the mean of two experiments with error bars representing the range.

    Techniques Used: Enzyme-linked Immunospot, Generated

    Peptide-specific Tfh responses can be measured with ELISPOT assays. On day 8 or 9 days post-immunization with MalE containing the HA-derived S1 epitope (A), HEL (B), or OVA (C), Tfh and non-Tfh cells were separated and peptide-specific responses were measured with IL-21, IL-4, IL-2 and IFNγ ELISPOT assays. The frequency of peptide-specific cytokine-secreting Tfh and non-Tfh cells are shown as cytokine-specific spots per 1,000,000 cells. Open and shaded bars show Tfh and non-Tfh cells respectively. The means of two experiments are shown, with error bars indicating the range.
    Figure Legend Snippet: Peptide-specific Tfh responses can be measured with ELISPOT assays. On day 8 or 9 days post-immunization with MalE containing the HA-derived S1 epitope (A), HEL (B), or OVA (C), Tfh and non-Tfh cells were separated and peptide-specific responses were measured with IL-21, IL-4, IL-2 and IFNγ ELISPOT assays. The frequency of peptide-specific cytokine-secreting Tfh and non-Tfh cells are shown as cytokine-specific spots per 1,000,000 cells. Open and shaded bars show Tfh and non-Tfh cells respectively. The means of two experiments are shown, with error bars indicating the range.

    Techniques Used: Enzyme-linked Immunospot, Derivative Assay

    The immunodominance hierarchies established within the Tfh and non-Tfh populations during the peak of the immune response are maintained late in the immune response. As previously described, peptide-specific Tfh and non-Tfh cells responses were measured with IL-21 ELISPOT assays on day 9, 15 and 26 after MalE-LACK(I > A) protein immunization (A). On day 26 after protein immunization, peptide-specific Tfh and non-Tfh responses were measured with IL-21, IL-4, IL-2, and IFNγ ELISPOT assays. The percent of peptide responses are shown in B. Data shown represent a single experiment from cells pooled from 40–80 mice per time point.
    Figure Legend Snippet: The immunodominance hierarchies established within the Tfh and non-Tfh populations during the peak of the immune response are maintained late in the immune response. As previously described, peptide-specific Tfh and non-Tfh cells responses were measured with IL-21 ELISPOT assays on day 9, 15 and 26 after MalE-LACK(I > A) protein immunization (A). On day 26 after protein immunization, peptide-specific Tfh and non-Tfh responses were measured with IL-21, IL-4, IL-2, and IFNγ ELISPOT assays. The percent of peptide responses are shown in B. Data shown represent a single experiment from cells pooled from 40–80 mice per time point.

    Techniques Used: Enzyme-linked Immunospot, Mouse Assay

    The kinetic stability of peptide∶MHC class II complexes determines the immunodominance of both Tfh and non-Tfh responses. Mice were immunized with MalE protein expressing either a high or low kinetic stability peptide variant. The persistence of peptide variants with MHC class II molecules are shown in Table 2 . Two pairs (LACK and HA) of kinetic stability variants were evaluated and 20–25 mice were used per group. LACK responses are shown in the top row (A, B, and C) and HA responses are shown in the bottom row (D, E, and F). The left column (A and D) shows the percent of the IL-21 secreting Tfh (open bars) or non-Tfh (shaded bars) recalled after immunizing mice with the MalE protein bearing the low stability variant (LACK(WT) or HA(T > V)), while the center column (B and E) shows the percent of response recalled after immunizing mice with the MalE protein bearing the high stability variant (LACK(I > A) or HA(T > G)). The right column (C and F) depicts the fractional response of peptide variant (white) and sum of endogenous MalE peptides (gray) of either the Tfh or non-Tfh response. Data are shown as the mean of two experiments with error bars representing the range.
    Figure Legend Snippet: The kinetic stability of peptide∶MHC class II complexes determines the immunodominance of both Tfh and non-Tfh responses. Mice were immunized with MalE protein expressing either a high or low kinetic stability peptide variant. The persistence of peptide variants with MHC class II molecules are shown in Table 2 . Two pairs (LACK and HA) of kinetic stability variants were evaluated and 20–25 mice were used per group. LACK responses are shown in the top row (A, B, and C) and HA responses are shown in the bottom row (D, E, and F). The left column (A and D) shows the percent of the IL-21 secreting Tfh (open bars) or non-Tfh (shaded bars) recalled after immunizing mice with the MalE protein bearing the low stability variant (LACK(WT) or HA(T > V)), while the center column (B and E) shows the percent of response recalled after immunizing mice with the MalE protein bearing the high stability variant (LACK(I > A) or HA(T > G)). The right column (C and F) depicts the fractional response of peptide variant (white) and sum of endogenous MalE peptides (gray) of either the Tfh or non-Tfh response. Data are shown as the mean of two experiments with error bars representing the range.

    Techniques Used: Mouse Assay, Expressing, Variant Assay

    16) Product Images from "The Aryl hydrocarbon Receptor (AhR) interacts with c-Maf to promote the differentiation of IL-27-induced regulatory type 1 (TR1) cells"

    Article Title: The Aryl hydrocarbon Receptor (AhR) interacts with c-Maf to promote the differentiation of IL-27-induced regulatory type 1 (TR1) cells

    Journal: Nature immunology

    doi: 10.1038/ni.1912

    AhR signaling dictates IL-21 secretion in T R 1 cells Naïve T cells were differentiated into T R 1 cells without (Ctrl) or with FICZ (100nM) and a) IL-21 cytokine production was assessed by cytokine bead array analysis after 72 hours of culture; b) The transcription factor Maf was quantified by RT-PCR at 48 hours c) and d) Naïve T cells from wild type and Il21r −/− mice were differentiated into T R 1 cells and IL-21 and IL-10 production were analyzed by cytokine bead array analysis after 48 hours of culture e) mRNA for Maf , Ahr and Tbx21 in the cells described in c) was quantified by RT-PCR relative to the expression of mRNA encoding β-actin . Data are from one of three experiments with similar results. (*p
    Figure Legend Snippet: AhR signaling dictates IL-21 secretion in T R 1 cells Naïve T cells were differentiated into T R 1 cells without (Ctrl) or with FICZ (100nM) and a) IL-21 cytokine production was assessed by cytokine bead array analysis after 72 hours of culture; b) The transcription factor Maf was quantified by RT-PCR at 48 hours c) and d) Naïve T cells from wild type and Il21r −/− mice were differentiated into T R 1 cells and IL-21 and IL-10 production were analyzed by cytokine bead array analysis after 48 hours of culture e) mRNA for Maf , Ahr and Tbx21 in the cells described in c) was quantified by RT-PCR relative to the expression of mRNA encoding β-actin . Data are from one of three experiments with similar results. (*p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Expressing

    AhR and c-Maf transactivate the Il10 and Il21 promoters in T R 1 cells a) AhR and c-Maf binding sites in the Il10 and the Il21 promoters. Schematic representation of the Il10 and the Il21 promoters, AhR binding sites (XRE) are depicted as open boxes and c-Maf binding sites (MARE) are depicted as filled boxes. b) ChIP analysis of the interaction of AhR or isotype control antibody (IgG) to the XRE in the Il10 and c) the Il21 promoter in in vitro differentiated T R 1 or control T H 0 cells. (*p
    Figure Legend Snippet: AhR and c-Maf transactivate the Il10 and Il21 promoters in T R 1 cells a) AhR and c-Maf binding sites in the Il10 and the Il21 promoters. Schematic representation of the Il10 and the Il21 promoters, AhR binding sites (XRE) are depicted as open boxes and c-Maf binding sites (MARE) are depicted as filled boxes. b) ChIP analysis of the interaction of AhR or isotype control antibody (IgG) to the XRE in the Il10 and c) the Il21 promoter in in vitro differentiated T R 1 or control T H 0 cells. (*p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, In Vitro

    17) Product Images from "Blimp-1 is essential for Th2 cell development and allergic asthma"

    Article Title: Blimp-1 is essential for Th2 cell development and allergic asthma

    Journal: bioRxiv

    doi: 10.1101/766246

    IL-10 and IL-21 do not cooperatively regulate Blimp-1 expression. A) Percent and Geo MFI of IL-10 expressing cells isolated from the lung gated on Live, CD4 + TCR β + FoxP3 - CD44 + . B) Percent of Th1 (IFN γγ + T-bet + ) and Th2 (IL-13 + GATA3 + ) cells in the lung isolated from Blimp-1 YFP animals treated as Control (IgG) or treated with anti-IL-10R, anti-IL21R, or both anti-IL-10R and anti-IL21R for the duration of the HDM-induced allergic lung inflammation model. C) Percent of Blimp-1 YFP + Th1 and Th2 cells in B. Data are pooled from 2-3 experiments with 6-10 total mice per group. Kruskal-Wallis One-way ANOVA. *p
    Figure Legend Snippet: IL-10 and IL-21 do not cooperatively regulate Blimp-1 expression. A) Percent and Geo MFI of IL-10 expressing cells isolated from the lung gated on Live, CD4 + TCR β + FoxP3 - CD44 + . B) Percent of Th1 (IFN γγ + T-bet + ) and Th2 (IL-13 + GATA3 + ) cells in the lung isolated from Blimp-1 YFP animals treated as Control (IgG) or treated with anti-IL-10R, anti-IL21R, or both anti-IL-10R and anti-IL21R for the duration of the HDM-induced allergic lung inflammation model. C) Percent of Blimp-1 YFP + Th1 and Th2 cells in B. Data are pooled from 2-3 experiments with 6-10 total mice per group. Kruskal-Wallis One-way ANOVA. *p

    Techniques Used: Expressing, Isolation, Mouse Assay

    IL-6 and IL-21 are not required for Blimp-1 mediated Th2 cell differentiation. A) Percent of T cell subsets (gated on Live, CD4 + TCR β + FoxP3 - (non-Treg) or FoxP3 + (Treg)) or B) Percent of Blimp-1 + cells (gated on Live, CD4 + TCR β + FoxP3 - CD44 + ) isolated from lungs of control (IL-6R α f/f CD4Cre - ) or IL6R α CD4Cre (IL-6R α f/f CD4Cre + ) animals i.n. immunized with HDM. C) Percent of eosinophils in the BAL, D) Th1 (IFN γγ + , T-bet + ) and Th2 (IL-13 + GATA3 + ) cells (gated on Live, CD4 + TCR β + FoxP3 - ) and E) percent of Blimp-1 + or Bcl6 + (gated on Live, CD4 + TCR β + FoxP3 - ) T cells in lung isolated from IL-21R-intact or IL-21R-deficient animals immunized with HDM. Data are pooled from 2-3 experiments with 9-10 total mice per group, mean ± SD. F) Percent of Th1 (IFN γγ + T-bet + ) and Th2 (IL-13 + GATA3 + ) cells (gated on Live, CD4 + TCR β + FoxP3 - ) and G) Blimp-1 YFP + cells within Th1 and Th2 cells isolated from the lung from control (IgG) or anti-IL-21R treated animals immunized with HDM. Data are pooled from 2 experiments with 6-7 mice total per group, mean ± SD, Mann-Whitney t-test. *p
    Figure Legend Snippet: IL-6 and IL-21 are not required for Blimp-1 mediated Th2 cell differentiation. A) Percent of T cell subsets (gated on Live, CD4 + TCR β + FoxP3 - (non-Treg) or FoxP3 + (Treg)) or B) Percent of Blimp-1 + cells (gated on Live, CD4 + TCR β + FoxP3 - CD44 + ) isolated from lungs of control (IL-6R α f/f CD4Cre - ) or IL6R α CD4Cre (IL-6R α f/f CD4Cre + ) animals i.n. immunized with HDM. C) Percent of eosinophils in the BAL, D) Th1 (IFN γγ + , T-bet + ) and Th2 (IL-13 + GATA3 + ) cells (gated on Live, CD4 + TCR β + FoxP3 - ) and E) percent of Blimp-1 + or Bcl6 + (gated on Live, CD4 + TCR β + FoxP3 - ) T cells in lung isolated from IL-21R-intact or IL-21R-deficient animals immunized with HDM. Data are pooled from 2-3 experiments with 9-10 total mice per group, mean ± SD. F) Percent of Th1 (IFN γγ + T-bet + ) and Th2 (IL-13 + GATA3 + ) cells (gated on Live, CD4 + TCR β + FoxP3 - ) and G) Blimp-1 YFP + cells within Th1 and Th2 cells isolated from the lung from control (IgG) or anti-IL-21R treated animals immunized with HDM. Data are pooled from 2 experiments with 6-7 mice total per group, mean ± SD, Mann-Whitney t-test. *p

    Techniques Used: Cell Differentiation, Isolation, Mouse Assay, MANN-WHITNEY

    18) Product Images from "Antigen-presenting ILC3 regulate T cell–dependent IgA responses to colonic mucosal bacteria"

    Article Title: Antigen-presenting ILC3 regulate T cell–dependent IgA responses to colonic mucosal bacteria

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20180871

    ILC3 suppress TfH-dependent B cell class switching in an MHCII and IL-4–dependent manner. B cells were sort-purified and cultured in vitro alone ( n = 12), with TfH ( n = 12) or with TfH in combination with either (CD3 + CD4 + /CXCR5 + PD1 + )CD25 + GITR + TfR ( n = 11), (CD3 + CD4 + /CXCR5 − PD1 − )CD25 + GITR + T reg cells ( n = 6), wild-type ILC3 ( n = 8), or ILC3 from MHCII ΔILC3 mice ( n = 6). Statistical comparisons performed by one-way ANOVA; data pooled from three independent experiments. (A and B) Representative flow cytometry plots (A) and frequencies (B) of (CD3 − MHCII + /B220 + CD19 + )GL7 + IgG1 + class-switched B cells. (C–F) Expression of Cd40l (C) , Tgfb, Ifng, and Il17a (D) , Il21 (E), and Il4 (F) in sort-purified B cells, T cells, or TfH assessed by qPCR ( n = 3 per group), representative of two independent experiments. Statistical comparisons performed by Student’s t test. (G) Concentration of IL-4 protein in supernatants from cocultures containing B cells and TfH cultured with wild-type of MHCII-deficient ILC3, as in A and B. Statistical comparisons performed by Student’s t test. (H and I) Cell numbers of mLN (CD3 − MHCII + /B220 + CD19 + )GL7 + Fas + GC B cells (H) and (CD3 − MHCII + )B220 − IgA + plasma cells in the cLPL of anti–IL-4 ( n = 5) or isotype ( n = 5) control–treated MHCII ΔILC3 and H2-Ab1 fl/fl ( n = 6) mice (I). (J and K) Relative abundance of Helicobacter spp. (J) and H. typhlonius (K) in total colonic mucosal bacteria preparations from anti–IL-4 or isotype control–treated MHCII ΔILC3 and H2-Ab1 fl/fl mice. (H–K) Statistical comparisons performed by one-way ANOVA; data representative of two independent experiments. All data shown as mean ± SEM; * P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.001.
    Figure Legend Snippet: ILC3 suppress TfH-dependent B cell class switching in an MHCII and IL-4–dependent manner. B cells were sort-purified and cultured in vitro alone ( n = 12), with TfH ( n = 12) or with TfH in combination with either (CD3 + CD4 + /CXCR5 + PD1 + )CD25 + GITR + TfR ( n = 11), (CD3 + CD4 + /CXCR5 − PD1 − )CD25 + GITR + T reg cells ( n = 6), wild-type ILC3 ( n = 8), or ILC3 from MHCII ΔILC3 mice ( n = 6). Statistical comparisons performed by one-way ANOVA; data pooled from three independent experiments. (A and B) Representative flow cytometry plots (A) and frequencies (B) of (CD3 − MHCII + /B220 + CD19 + )GL7 + IgG1 + class-switched B cells. (C–F) Expression of Cd40l (C) , Tgfb, Ifng, and Il17a (D) , Il21 (E), and Il4 (F) in sort-purified B cells, T cells, or TfH assessed by qPCR ( n = 3 per group), representative of two independent experiments. Statistical comparisons performed by Student’s t test. (G) Concentration of IL-4 protein in supernatants from cocultures containing B cells and TfH cultured with wild-type of MHCII-deficient ILC3, as in A and B. Statistical comparisons performed by Student’s t test. (H and I) Cell numbers of mLN (CD3 − MHCII + /B220 + CD19 + )GL7 + Fas + GC B cells (H) and (CD3 − MHCII + )B220 − IgA + plasma cells in the cLPL of anti–IL-4 ( n = 5) or isotype ( n = 5) control–treated MHCII ΔILC3 and H2-Ab1 fl/fl ( n = 6) mice (I). (J and K) Relative abundance of Helicobacter spp. (J) and H. typhlonius (K) in total colonic mucosal bacteria preparations from anti–IL-4 or isotype control–treated MHCII ΔILC3 and H2-Ab1 fl/fl mice. (H–K) Statistical comparisons performed by one-way ANOVA; data representative of two independent experiments. All data shown as mean ± SEM; * P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.001.

    Techniques Used: Purification, Cell Culture, In Vitro, Mouse Assay, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay

    19) Product Images from "Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses"

    Article Title: Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171450

    Potent Tfh cell responses are elicited by a single immunization with m1Ψ-mRNA-LNPs in mice. Mice were immunized once i.d. with 30 µg of Luc or HA m1Ψ-mRNA-LNPs, a single i.m. injection with 1,000 HAU of inactivated PR8 virus, MF59-adjuvanted recombinant PR8 HA protein, or intranasally infected with 25 TCID 50 of live PR8 influenza virus, and immune responses were examined 10 d after immunization (A and B). (A) Total numbers of splenic Tfh cells were determined by staining for TCR + CD19 − CD4 + CD62L − CXCR5 + PD-1 + T cells. (B) IFN-γ, IL-4, and IL-21 transcript levels in sorted Tfh cells from PR8 HA m1Ψ-mRNA-LNP–immunized mice were determined by quantitative real-time RT-PCR. Fold induction of cytokines compared with total universal RNA is shown. (C and D) Mice were immunized with a single i.d. injection of 30 µg of HA or Env m1Ψ-mRNA-LNPs, and immune responses were examined 12 d after immunization. Percentage of IFN-γ producing CD4 + Bcl-6 + Tfh-like cells was measured by flow cytometry after HA (C) or Env (D) peptide stimulation. (E) Mice were immunized with a single i.d. injection of 30 µg of PR8 HA m1Ψ-mRNA-LNPs, and rates of binding to PR8 HA were examined 2, 4, and 8 wk later by biolayer interferometry. The apparent nanomolar affinity of anti–HA antibodies, derived from the mean rates of HA-binding in polyclonal sera, is plotted for each serum sample, with lower values corresponding to higher apparent affinity. n = 5–8 mice, and each symbol represents values for one animal. Experiments were repeated at least two times to achieve sufficient numbers of values for mice in each group. Error bars are SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P
    Figure Legend Snippet: Potent Tfh cell responses are elicited by a single immunization with m1Ψ-mRNA-LNPs in mice. Mice were immunized once i.d. with 30 µg of Luc or HA m1Ψ-mRNA-LNPs, a single i.m. injection with 1,000 HAU of inactivated PR8 virus, MF59-adjuvanted recombinant PR8 HA protein, or intranasally infected with 25 TCID 50 of live PR8 influenza virus, and immune responses were examined 10 d after immunization (A and B). (A) Total numbers of splenic Tfh cells were determined by staining for TCR + CD19 − CD4 + CD62L − CXCR5 + PD-1 + T cells. (B) IFN-γ, IL-4, and IL-21 transcript levels in sorted Tfh cells from PR8 HA m1Ψ-mRNA-LNP–immunized mice were determined by quantitative real-time RT-PCR. Fold induction of cytokines compared with total universal RNA is shown. (C and D) Mice were immunized with a single i.d. injection of 30 µg of HA or Env m1Ψ-mRNA-LNPs, and immune responses were examined 12 d after immunization. Percentage of IFN-γ producing CD4 + Bcl-6 + Tfh-like cells was measured by flow cytometry after HA (C) or Env (D) peptide stimulation. (E) Mice were immunized with a single i.d. injection of 30 µg of PR8 HA m1Ψ-mRNA-LNPs, and rates of binding to PR8 HA were examined 2, 4, and 8 wk later by biolayer interferometry. The apparent nanomolar affinity of anti–HA antibodies, derived from the mean rates of HA-binding in polyclonal sera, is plotted for each serum sample, with lower values corresponding to higher apparent affinity. n = 5–8 mice, and each symbol represents values for one animal. Experiments were repeated at least two times to achieve sufficient numbers of values for mice in each group. Error bars are SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P

    Techniques Used: Mouse Assay, Injection, Recombinant, Infection, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Binding Assay, Derivative Assay

    20) Product Images from "Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses"

    Article Title: Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171450

    Potent Tfh cell responses are elicited by a single immunization with m1Ψ-mRNA-LNPs in mice. Mice were immunized once i.d. with 30 µg of Luc or HA m1Ψ-mRNA-LNPs, a single i.m. injection with 1,000 HAU of inactivated PR8 virus, MF59-adjuvanted recombinant PR8 HA protein, or intranasally infected with 25 TCID 50 of live PR8 influenza virus, and immune responses were examined 10 d after immunization (A and B). (A) Total numbers of splenic Tfh cells were determined by staining for TCR + CD19 − CD4 + CD62L − CXCR5 + PD-1 + T cells. (B) IFN-γ, IL-4, and IL-21 transcript levels in sorted Tfh cells from PR8 HA m1Ψ-mRNA-LNP–immunized mice were determined by quantitative real-time RT-PCR. Fold induction of cytokines compared with total universal RNA is shown. (C and D) Mice were immunized with a single i.d. injection of 30 µg of HA or Env m1Ψ-mRNA-LNPs, and immune responses were examined 12 d after immunization. Percentage of IFN-γ producing CD4 + Bcl-6 + Tfh-like cells was measured by flow cytometry after HA (C) or Env (D) peptide stimulation. (E) Mice were immunized with a single i.d. injection of 30 µg of PR8 HA m1Ψ-mRNA-LNPs, and rates of binding to PR8 HA were examined 2, 4, and 8 wk later by biolayer interferometry. The apparent nanomolar affinity of anti–HA antibodies, derived from the mean rates of HA-binding in polyclonal sera, is plotted for each serum sample, with lower values corresponding to higher apparent affinity. n = 5–8 mice, and each symbol represents values for one animal. Experiments were repeated at least two times to achieve sufficient numbers of values for mice in each group. Error bars are SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P
    Figure Legend Snippet: Potent Tfh cell responses are elicited by a single immunization with m1Ψ-mRNA-LNPs in mice. Mice were immunized once i.d. with 30 µg of Luc or HA m1Ψ-mRNA-LNPs, a single i.m. injection with 1,000 HAU of inactivated PR8 virus, MF59-adjuvanted recombinant PR8 HA protein, or intranasally infected with 25 TCID 50 of live PR8 influenza virus, and immune responses were examined 10 d after immunization (A and B). (A) Total numbers of splenic Tfh cells were determined by staining for TCR + CD19 − CD4 + CD62L − CXCR5 + PD-1 + T cells. (B) IFN-γ, IL-4, and IL-21 transcript levels in sorted Tfh cells from PR8 HA m1Ψ-mRNA-LNP–immunized mice were determined by quantitative real-time RT-PCR. Fold induction of cytokines compared with total universal RNA is shown. (C and D) Mice were immunized with a single i.d. injection of 30 µg of HA or Env m1Ψ-mRNA-LNPs, and immune responses were examined 12 d after immunization. Percentage of IFN-γ producing CD4 + Bcl-6 + Tfh-like cells was measured by flow cytometry after HA (C) or Env (D) peptide stimulation. (E) Mice were immunized with a single i.d. injection of 30 µg of PR8 HA m1Ψ-mRNA-LNPs, and rates of binding to PR8 HA were examined 2, 4, and 8 wk later by biolayer interferometry. The apparent nanomolar affinity of anti–HA antibodies, derived from the mean rates of HA-binding in polyclonal sera, is plotted for each serum sample, with lower values corresponding to higher apparent affinity. n = 5–8 mice, and each symbol represents values for one animal. Experiments were repeated at least two times to achieve sufficient numbers of values for mice in each group. Error bars are SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P

    Techniques Used: Mouse Assay, Injection, Recombinant, Infection, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Binding Assay, Derivative Assay

    21) Product Images from "Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13"

    Article Title: Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13

    Journal: Mucosal immunology

    doi: 10.1038/mi.2010.82

    Gene expression analysis of isolated NK populations. Relative fold expression in natural killer (NK)/natural killer T (NKT)-positive and -negative populations. mRNA transcript levels of interleukin 13 (IL-13), interferon-γ (IFN-γ), IL-17A, IL-17F, IL-22, IL-21, and IL-4 in freshly isolated NK positive (NK +) and negative (NK −) cells isolated cells from nonstressed (NS) and desiccating-stressed (DS) ocular surface (OS) for 5 (DS5) or 10 days (DS10) and normal spleen. *** P
    Figure Legend Snippet: Gene expression analysis of isolated NK populations. Relative fold expression in natural killer (NK)/natural killer T (NKT)-positive and -negative populations. mRNA transcript levels of interleukin 13 (IL-13), interferon-γ (IFN-γ), IL-17A, IL-17F, IL-22, IL-21, and IL-4 in freshly isolated NK positive (NK +) and negative (NK −) cells isolated cells from nonstressed (NS) and desiccating-stressed (DS) ocular surface (OS) for 5 (DS5) or 10 days (DS10) and normal spleen. *** P

    Techniques Used: Expressing, Isolation

    Effects of CsA treatment on NK and NKT cells. ( a ) Mean±s.d. of conjunctival goblet cell (GC) density in nonstressed (NS) C57BL/6 control mice and mice subjected to desiccating stress for 5 or 10 days (DS5 and DS10, respectively) or DS5 treated with Cyclosporine A (DS5 + CsA) or DS5 treated with CsA vehicle (DS5 + Veh). ( b ) mRNA transcript levels of MUC5AC in conjunctiva of NS C57BL/6 control mice and mice subjected to DS5 and DS10, respectively. ( c ) Mean±.d. of protein concentrations of MUC5AC in conjunctiva of NS C57BL/6 control mice and mice subjected to DS5 and DS10, respectively. ( d, e ) Representative flow cytometry analysis of freshly isolated ocular surface cells stained with pan-NK marker DX5 –fluorescein isothiocyanate (FITC)-conjugated antibody in NS C57BL/6 controls mice and mice subjected to DS5 and DS10, respectively ( d ) or DS5 treated with CsA (DS5 + CsA) or DS5 treated with CsA vehicle (DS5 + Veh) ( e ). Lymphocytes were gated based on characteristic light-scatter properties, and single lymphocytes were gated based on forward scatter height vs. forward scatter area (FSC-A). Numbers in the quadrants indicate the percentage of cells. ( f ) Mean±s.d. of the percentage of DX5+ cells evaluated by flow cytometry in three independent experiments. ( g ) Percentage change in the level of expression of interferon-γ (IFN-γ), IL-13, IL-17A, IL-21, and IL-22 mRNA transcripts in CsA-treated NK + and NK − cell populations, compared with vehicle. *** P
    Figure Legend Snippet: Effects of CsA treatment on NK and NKT cells. ( a ) Mean±s.d. of conjunctival goblet cell (GC) density in nonstressed (NS) C57BL/6 control mice and mice subjected to desiccating stress for 5 or 10 days (DS5 and DS10, respectively) or DS5 treated with Cyclosporine A (DS5 + CsA) or DS5 treated with CsA vehicle (DS5 + Veh). ( b ) mRNA transcript levels of MUC5AC in conjunctiva of NS C57BL/6 control mice and mice subjected to DS5 and DS10, respectively. ( c ) Mean±.d. of protein concentrations of MUC5AC in conjunctiva of NS C57BL/6 control mice and mice subjected to DS5 and DS10, respectively. ( d, e ) Representative flow cytometry analysis of freshly isolated ocular surface cells stained with pan-NK marker DX5 –fluorescein isothiocyanate (FITC)-conjugated antibody in NS C57BL/6 controls mice and mice subjected to DS5 and DS10, respectively ( d ) or DS5 treated with CsA (DS5 + CsA) or DS5 treated with CsA vehicle (DS5 + Veh) ( e ). Lymphocytes were gated based on characteristic light-scatter properties, and single lymphocytes were gated based on forward scatter height vs. forward scatter area (FSC-A). Numbers in the quadrants indicate the percentage of cells. ( f ) Mean±s.d. of the percentage of DX5+ cells evaluated by flow cytometry in three independent experiments. ( g ) Percentage change in the level of expression of interferon-γ (IFN-γ), IL-13, IL-17A, IL-21, and IL-22 mRNA transcripts in CsA-treated NK + and NK − cell populations, compared with vehicle. *** P

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

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    Article Title: CCR7 Deficiency Exacerbates Injury in Acute Nephritis Due to Aberrant Localization of Regulatory T Cells
    Article Snippet: For linear amplification of TNF-α, FoxP3, and β-actin (reference gene), SYBR Green Master Mix (Invitrogen) and the primers shown in were used. .. For quantification of IFN-γ, IL-10, IL-6, IL-17, and IL-21, TaqMan Mastermix (Applied Biosystems) and the gene expression assays Mm00801778_m1, Mm00439616_m1, Mm00446190_m1, Mm00439619_m1, and Mm00517640_m1 (Applied Biosystems) were used. .. Kidney samples were homogenized and lysed on ice by using Triton lysis buffer (37.6 mM KCl, 24.8 mM Tris base, and 1% Triton X-100) supplemented with 1% protease inhibitor cocktail (Sigma).

    Article Title: Interleukin-21 (IL-21) Downregulates Dendritic Cell Cytokine Responses to Helicobacter pylori and Modulates T Lymphocyte IL-17A Expression in Peyer’s Patches during Infection
    Article Snippet: All real-time RT-PCRs were analyzed using an Applied Biosystems StepOne Plus real-time PCR instrument. .. The following primers and probes were purchased as TaqMan Gene Expression Assays from Thermo Fisher: Il1b (catalog no. Mm00434228_m1), Il6 (catalog no. Mm00446190_m1), Il10 (catalog no. Mm01288386_m1), Il12 (catalog no. Mm00434165_m1), Il17a (catalog no. Mm00439619_m1), Il21 (catalog no. Mm00517640_m1), Ifng (catalog no. Mm99999071_m1), and Il23a and Gapdh (catalog no. Mm99999915_g1). .. T cell isolation.

    other:

    Article Title: Dual Role of Interleukin-10 in Murine NZB/W F1 Lupus
    Article Snippet: Probes with the following Applied Biosystems assay identification numbers were used: Mm99999915_g1 (GAPDH ), Mm01288386_m1 (IL-10 ), Mm00434151_m1 (IL-10R ), Mm00517640_m1 (IL-21 ), Mm02581355_s1 (cMAF ), Mm00461162_m1 (IL-27 ).

    Article Title: IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age
    Article Snippet: TaqMan probes were as follows: c-maf (Mm02581355_s1), Tbx21 (Mm00450960_m1), Gata3 (Mm00484683_m1), Rorc (Mm01261022_m1), Foxp3 (Mm00475162_m1), Il10 (Mm01288386_m1), Bcl6 (Mm00477633_m1), Il17a (Mm00439618_m1), Il4 (Mm00445259_m1), Ifng (Mm01168134_m1), Il21 (Mm00517640_m1) and Gapdh (Mm99999915_g1).

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells
    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

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    Thermo Fisher gene exp il21 mm00517640 m1
    IL-6-induced <t>IL-4/IL-21</t> production is responsible for the defect of Th1 differentiation. Young naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro . ( a ) Four days after stimulation, expressions of the indicated transcription factors were analysed. ( b , c ) Naive polyclonal CD4 + T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown ( b ). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR ( c ). Results are shown as mean±s.e.m. with n =4–6 per group; * P
    Gene Exp Il21 Mm00517640 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher gene exp ahr mm00478932 m1
    Induction of protein tyrosine phosphatases following T-cell activation limits STAT1 signalling. (a) CD4 + T N and CD4 + T EM cells were pre-treated for 5 min with 5mM sodium orthovanadate (vanadate) prior to IL-6 (20ng/ml) stimulation for 30 min. Changes in pY-STAT1 and pY-STAT3 activity were monitored by intracellular flow cytometry (MFI). A comparable analysis of pS-STAT1 and pS-STAT3 is shown as a control (n=3). (b) Quantitative PCR for <t>Ahr</t> , Il21 , Stat3 and Socs3 after vanadate pre-treatment and 20ng/ml IL-6 stimulation in CD4 + T EM cells (n=3). (c) Heatmap analysis of Affymetrix transcriptomic data identifies the top 20 genes ( P
    Gene Exp Ahr Mm00478932 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IL-6-induced IL-4/IL-21 production is responsible for the defect of Th1 differentiation. Young naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro . ( a ) Four days after stimulation, expressions of the indicated transcription factors were analysed. ( b , c ) Naive polyclonal CD4 + T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown ( b ). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR ( c ). Results are shown as mean±s.e.m. with n =4–6 per group; * P

    Journal: Nature Communications

    Article Title: IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age

    doi: 10.1038/ncomms7702

    Figure Lengend Snippet: IL-6-induced IL-4/IL-21 production is responsible for the defect of Th1 differentiation. Young naive CD4 + T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro . ( a ) Four days after stimulation, expressions of the indicated transcription factors were analysed. ( b , c ) Naive polyclonal CD4 + T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown ( b ). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR ( c ). Results are shown as mean±s.e.m. with n =4–6 per group; * P

    Article Snippet: TaqMan probes were as follows: c-maf (Mm02581355_s1), Tbx21 (Mm00450960_m1), Gata3 (Mm00484683_m1), Rorc (Mm01261022_m1), Foxp3 (Mm00475162_m1), Il10 (Mm01288386_m1), Bcl6 (Mm00477633_m1), Il17a (Mm00439618_m1), Il4 (Mm00445259_m1), Ifng (Mm01168134_m1), Il21 (Mm00517640_m1) and Gapdh (Mm99999915_g1).

    Techniques: In Vitro, Mutagenesis, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    IL-6-induced IL-4/IL-21/IL-10 production is responsible for the defective CD8 help in aged mice. ( a , b ) OT-II cells were primed in young or aged mice at day 0 as in Fig. 1b , and then were treated with control Ab, anti-IL-4 and anti-IL-21 Abs ( a ) or anti-IL-10 Ab ( b ) on days 3 and 4. Six days after immunization, the frequencies of IFN-γ + cells in the donor OT-II cells were determined. ( b ) Representative plots. ( c ) OT-II transfer, immunization, Ab treatment and MCA-OVA inoculation were performed as in Fig. 1d . Four days after tumour inoculation, IL-10 concentration in serum was determined. ( d , e ) OT-II transfer, immunization and treatments of anti-IL-4 and anti-IL-21 Abs were performed as in a . Five days after immunization, mice were inoculated with MCA-OVA. Anti-IL-10 Ab was injected twice at 2 and 3 days after tumour inoculation. Five days after tumour inoculation, draining LNs were analysed for OVA-specific CD8 + T cells using OVA-Ip tetramer ( d ). OVA-Ip-specific CD8 + T-cell response was also evaluated by the IFN-γ ELISPOT assay ( e ). Data shown are mean±s.e.m. with n =4–5 per group. ( f ) CD62L lo effector OT-II cells primed in young or aged IL-6 +/+ or IL-6 −/− mice were sorted as in Fig. 4a and were co-cultured with CFSE-labelled CD8 + T cells in the presence or absence of anti-IL-10 Ab. After 3 days, CFSE profile and IFN-γ production in CD8 + T cells were determined using a flow cytometer. Representative data from two independent experiments are shown. * P

    Journal: Nature Communications

    Article Title: IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age

    doi: 10.1038/ncomms7702

    Figure Lengend Snippet: IL-6-induced IL-4/IL-21/IL-10 production is responsible for the defective CD8 help in aged mice. ( a , b ) OT-II cells were primed in young or aged mice at day 0 as in Fig. 1b , and then were treated with control Ab, anti-IL-4 and anti-IL-21 Abs ( a ) or anti-IL-10 Ab ( b ) on days 3 and 4. Six days after immunization, the frequencies of IFN-γ + cells in the donor OT-II cells were determined. ( b ) Representative plots. ( c ) OT-II transfer, immunization, Ab treatment and MCA-OVA inoculation were performed as in Fig. 1d . Four days after tumour inoculation, IL-10 concentration in serum was determined. ( d , e ) OT-II transfer, immunization and treatments of anti-IL-4 and anti-IL-21 Abs were performed as in a . Five days after immunization, mice were inoculated with MCA-OVA. Anti-IL-10 Ab was injected twice at 2 and 3 days after tumour inoculation. Five days after tumour inoculation, draining LNs were analysed for OVA-specific CD8 + T cells using OVA-Ip tetramer ( d ). OVA-Ip-specific CD8 + T-cell response was also evaluated by the IFN-γ ELISPOT assay ( e ). Data shown are mean±s.e.m. with n =4–5 per group. ( f ) CD62L lo effector OT-II cells primed in young or aged IL-6 +/+ or IL-6 −/− mice were sorted as in Fig. 4a and were co-cultured with CFSE-labelled CD8 + T cells in the presence or absence of anti-IL-10 Ab. After 3 days, CFSE profile and IFN-γ production in CD8 + T cells were determined using a flow cytometer. Representative data from two independent experiments are shown. * P

    Article Snippet: TaqMan probes were as follows: c-maf (Mm02581355_s1), Tbx21 (Mm00450960_m1), Gata3 (Mm00484683_m1), Rorc (Mm01261022_m1), Foxp3 (Mm00475162_m1), Il10 (Mm01288386_m1), Bcl6 (Mm00477633_m1), Il17a (Mm00439618_m1), Il4 (Mm00445259_m1), Ifng (Mm01168134_m1), Il21 (Mm00517640_m1) and Gapdh (Mm99999915_g1).

    Techniques: Mouse Assay, Concentration Assay, Injection, Enzyme-linked Immunospot, Cell Culture, Flow Cytometry, Cytometry

    STAT3 controls AhR recruitment to the il22 promoter in IL-21-stimulated CD4+ T cells ( a ) AhR, c-Maf and STAT3 binding sites in the il22 promoter. ( b-c ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21. ChIP analysis of the interaction of AhR ( b ) and STAT3 ( c ) to the AhRand STAT3 binding sites in the il22 promoter, respectively. ( d ) Regulation of the transcriptional activity of the il22 promoter by AhR and STAT3. A reporter construct for the il22 promoter (il22:Luc) was cotransfected in HEK293 cells with vector coding for AhR and/or constitutively activated STAT3 (STAT3c). Firefly luciferase was determined and normalized to renilla luciferase activity. ( e ) ChIP analysis of the recruitment of AhR to the il22 promoter in WT and RORγt-deficient CD4+ cells activated in the presence of IL-21. ( f ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21, and the epigenetic status of the il22 promoter was analyzed by ChIP using antibodies to Acetyl-H3, Acetyl-H4 and H3 trimethylated at Lys4, Lys9 or Lys27. ( g-j ) ChIP analysis of the epigenetic status of the il22 promoter in WT and STAT3-deficient CD4+ T cells activated in the presence of IL-21. STAT3 recruitment (g) , H3 and H4 acetylation in SREs (h) and XREs (i) and AhR recruitment (j) in the il22 promoter. Results are representative of 2-3 independent experiments. * P

    Journal: Nature communications

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    doi: 10.1038/ncomms4753

    Figure Lengend Snippet: STAT3 controls AhR recruitment to the il22 promoter in IL-21-stimulated CD4+ T cells ( a ) AhR, c-Maf and STAT3 binding sites in the il22 promoter. ( b-c ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21. ChIP analysis of the interaction of AhR ( b ) and STAT3 ( c ) to the AhRand STAT3 binding sites in the il22 promoter, respectively. ( d ) Regulation of the transcriptional activity of the il22 promoter by AhR and STAT3. A reporter construct for the il22 promoter (il22:Luc) was cotransfected in HEK293 cells with vector coding for AhR and/or constitutively activated STAT3 (STAT3c). Firefly luciferase was determined and normalized to renilla luciferase activity. ( e ) ChIP analysis of the recruitment of AhR to the il22 promoter in WT and RORγt-deficient CD4+ cells activated in the presence of IL-21. ( f ) WT naïve CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence or absence of IL-21, and the epigenetic status of the il22 promoter was analyzed by ChIP using antibodies to Acetyl-H3, Acetyl-H4 and H3 trimethylated at Lys4, Lys9 or Lys27. ( g-j ) ChIP analysis of the epigenetic status of the il22 promoter in WT and STAT3-deficient CD4+ T cells activated in the presence of IL-21. STAT3 recruitment (g) , H3 and H4 acetylation in SREs (h) and XREs (i) and AhR recruitment (j) in the il22 promoter. Results are representative of 2-3 independent experiments. * P

    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

    Techniques: Binding Assay, In Vitro, Chromatin Immunoprecipitation, Activity Assay, Construct, Plasmid Preparation, Luciferase

    Transcriptional profiling of IL-21-stimulated CD4+ T cells Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of IL-21, IL-6 and TGFβ1 or without addition of exogenous cytokines. (a) Heatmap showing genes with at least a 1.5 fold change in expression relative to Th0 as determined by whole-genome microarrays. (b) Principal-component analysis (PCA). (c-d) Heatmaps depicting the expression of key cytokines, chemokines, receptors and transcription factors (c) and transcriptional modules (d) that control the differentiation of Th17 cells as defined in 34 . (e) Quantitative RT-PCR analysis of the expression of genes in Fig. 2a. mRNA expression is shown relative to gapdh . (f) Western blot analysis of the expression of AhR (95kDa) and RORγt (58kDa) on T cells activated in the presence of IL-21 or under Th17-polarizing conditions (left panel). Quantification of western blot reactivity (right panel). (g) Ingenuity Pathways Analysis of the transcriptional response of naïve CD4+ T cells to IL-21. * P

    Journal: Nature communications

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    doi: 10.1038/ncomms4753

    Figure Lengend Snippet: Transcriptional profiling of IL-21-stimulated CD4+ T cells Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of IL-21, IL-6 and TGFβ1 or without addition of exogenous cytokines. (a) Heatmap showing genes with at least a 1.5 fold change in expression relative to Th0 as determined by whole-genome microarrays. (b) Principal-component analysis (PCA). (c-d) Heatmaps depicting the expression of key cytokines, chemokines, receptors and transcription factors (c) and transcriptional modules (d) that control the differentiation of Th17 cells as defined in 34 . (e) Quantitative RT-PCR analysis of the expression of genes in Fig. 2a. mRNA expression is shown relative to gapdh . (f) Western blot analysis of the expression of AhR (95kDa) and RORγt (58kDa) on T cells activated in the presence of IL-21 or under Th17-polarizing conditions (left panel). Quantification of western blot reactivity (right panel). (g) Ingenuity Pathways Analysis of the transcriptional response of naïve CD4+ T cells to IL-21. * P

    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

    Techniques: In Vitro, Expressing, Quantitative RT-PCR, Western Blot

    STAT3 controls the production of IL-22 by CD4+ T cells stimulated with IL-21 ( a-b ) NaïveWT CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) FACS analysis of phosphorylated STAT3. Mean fluorescence intensity (MFI) of phosphorylated STAT3 normalized to total STAT3. ( b ) qPCR analysis of stat3 expression. ( c-f ) Naïve WT and STAT3-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( c ) Effect of STAT3 deficiency on IL-22 and IL-17 production. ( d ) Effect of STAT3 deficiency on il21 expression. ( e-f ) Effect of STAT3 deficiency on ahr and rorc expression ( e ) and the receptors il1r , il23r ( f ). ( g ) Induction of stat3 by IL-1β, IL-21 IL-23, IL-1β and IL-21 or IL-21 and IL-23. mRNA expression is shown relative to gapdh . Results are representative of 2-3 independent experiments. * P

    Journal: Nature communications

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    doi: 10.1038/ncomms4753

    Figure Lengend Snippet: STAT3 controls the production of IL-22 by CD4+ T cells stimulated with IL-21 ( a-b ) NaïveWT CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) FACS analysis of phosphorylated STAT3. Mean fluorescence intensity (MFI) of phosphorylated STAT3 normalized to total STAT3. ( b ) qPCR analysis of stat3 expression. ( c-f ) Naïve WT and STAT3-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( c ) Effect of STAT3 deficiency on IL-22 and IL-17 production. ( d ) Effect of STAT3 deficiency on il21 expression. ( e-f ) Effect of STAT3 deficiency on ahr and rorc expression ( e ) and the receptors il1r , il23r ( f ). ( g ) Induction of stat3 by IL-1β, IL-21 IL-23, IL-1β and IL-21 or IL-21 and IL-23. mRNA expression is shown relative to gapdh . Results are representative of 2-3 independent experiments. * P

    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

    Techniques: FACS, Fluorescence, Real-time Polymerase Chain Reaction, Expressing

    RORγt and AhR control IL-22 production by CD4+ T cells stimulated with IL-21 (a-c) Naïve WT and RORγt-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) Effect of RORγt deficiency on IL-22 and IL-17 production. ( b ) Effect of RORγt deficiency on il23r and il1r1 expression. ( c ) Effect of RORγt deficiency on the synergism between IL-21 and IL-23. ( d ) Effect of IL-1β and IL-23 in rorc expression. ( e ) Effect of the modulation of AhR activity on IL-22 and IL-17 production. ( f ) IL-22 production by WT and AhR-d CD4+ T cells activated in the presence of IL-21. ( g ) Modulation of ahr expression by IL-1β and IL-23. ( h ) Effect of AhR modulation on STAT3 phosphorylation and stat3 expression. MFI of phosphorylated STAT3 normalized to total STAT3. mRNA expression is shown relative to gapdh . Representative results of 2-3 independent experiments. * P

    Journal: Nature communications

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    doi: 10.1038/ncomms4753

    Figure Lengend Snippet: RORγt and AhR control IL-22 production by CD4+ T cells stimulated with IL-21 (a-c) Naïve WT and RORγt-deficient CD4+ T cells were activated in the presence of IL-21 or IL-6 and TGFβ1. ( a ) Effect of RORγt deficiency on IL-22 and IL-17 production. ( b ) Effect of RORγt deficiency on il23r and il1r1 expression. ( c ) Effect of RORγt deficiency on the synergism between IL-21 and IL-23. ( d ) Effect of IL-1β and IL-23 in rorc expression. ( e ) Effect of the modulation of AhR activity on IL-22 and IL-17 production. ( f ) IL-22 production by WT and AhR-d CD4+ T cells activated in the presence of IL-21. ( g ) Modulation of ahr expression by IL-1β and IL-23. ( h ) Effect of AhR modulation on STAT3 phosphorylation and stat3 expression. MFI of phosphorylated STAT3 normalized to total STAT3. mRNA expression is shown relative to gapdh . Representative results of 2-3 independent experiments. * P

    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

    Techniques: Expressing, Activity Assay

    IL-21 and AhR control IL-22 + CD4+ T cells in vivo and limit mucosal inflammation ( a ) RAG2/IL2RG mice transferred with WT, AhR-d or IL-21R deficient CD4+ T cells were given 3 % DSS ad libitum in their drinking water for 7 days. Body weight was assessed daily, and the percent mass change from day 0 was calculated. Mean ± s.e.m is shown, n =5 mice per group. Effect of genotype on disease course P

    Journal: Nature communications

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    doi: 10.1038/ncomms4753

    Figure Lengend Snippet: IL-21 and AhR control IL-22 + CD4+ T cells in vivo and limit mucosal inflammation ( a ) RAG2/IL2RG mice transferred with WT, AhR-d or IL-21R deficient CD4+ T cells were given 3 % DSS ad libitum in their drinking water for 7 days. Body weight was assessed daily, and the percent mass change from day 0 was calculated. Mean ± s.e.m is shown, n =5 mice per group. Effect of genotype on disease course P

    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

    Techniques: In Vivo, Mouse Assay

    IL-21 promotes the differentiation of CD4+ T cells that produce IL-22 but not IL-17 Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of different cytokines as indicated. ( a-b ) Effect of IL-21 in the production of IL-22 and IL-17 measured by ELISA in culture supernatants ( a ) and qPCR ( b ). ( c ) Effect of IL-21 on the expression of il21 . ( e ) Effect of IL-21 on the expression of il6r, il21r, il23r . ( d ) Relationship between cell division and IL-22 expression in CD4+ T cells. CFSE-labeled naïve CD4+ T cells were activated in the presence of IL-21 and the production of IL-22 was analyzed by intracellular staining. The data are shown as the Mean fluorescence intensity (MFI) for IL-22 for each division cycle. ( e ) Effects of IL-21 on the expression of il6r, il21r and il23r . ( f-h ) Synergistic effects of IL-21 and IL-23 on IL-22 (f) and IL-17 (g) cytokine and il21 expression ( h ). (i) Effect of IL-21 on il1r expression. ( j-l ) Synergistic effects of IL-21 and IL-1β on IL-22 (j) , Il17a (k) and il21 (l) expression. mRNA expression is shown relative to gapdh . Results are representative of 3-5 independent experiments.* P

    Journal: Nature communications

    Article Title: IL-21 induces IL-22 production in CD4+ T-cells

    doi: 10.1038/ncomms4753

    Figure Lengend Snippet: IL-21 promotes the differentiation of CD4+ T cells that produce IL-22 but not IL-17 Naïve WT CD4+ T cells were stimulated in vitro with antibodies to CD3 and CD28 in the presence of different cytokines as indicated. ( a-b ) Effect of IL-21 in the production of IL-22 and IL-17 measured by ELISA in culture supernatants ( a ) and qPCR ( b ). ( c ) Effect of IL-21 on the expression of il21 . ( e ) Effect of IL-21 on the expression of il6r, il21r, il23r . ( d ) Relationship between cell division and IL-22 expression in CD4+ T cells. CFSE-labeled naïve CD4+ T cells were activated in the presence of IL-21 and the production of IL-22 was analyzed by intracellular staining. The data are shown as the Mean fluorescence intensity (MFI) for IL-22 for each division cycle. ( e ) Effects of IL-21 on the expression of il6r, il21r and il23r . ( f-h ) Synergistic effects of IL-21 and IL-23 on IL-22 (f) and IL-17 (g) cytokine and il21 expression ( h ). (i) Effect of IL-21 on il1r expression. ( j-l ) Synergistic effects of IL-21 and IL-1β on IL-22 (j) , Il17a (k) and il21 (l) expression. mRNA expression is shown relative to gapdh . Results are representative of 3-5 independent experiments.* P

    Article Snippet: The following probe mixtures were used: il1r (Mm01226962_m1), il6r (Mm00439653_m1), il21r (Mm00600319_m1), il23r (Mm00519942_m1), il10 (Mm0043614_m1), il17 (Mm00439619_m1), il21 (Mm00517640_m1), il22 (Mm00444241_m1), ahr (Mm01291777_m1), maf (Mm02581355_s1), rora (Mm01173766_m1), rorc (Mm00441144_g1), stat3 (Mm01219775_m1), foxp3 (Mm00475156_m1), socs3, mt1 (Mm00496660_g1), mt2 (Mm00809556_s1), gzma (Mm01304452_m1), gzmb (Mm00442834_m1), tbx21 (Mm00450960_m1), gapdh (Mm99999915_g1).

    Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Labeling, Staining, Fluorescence

    The cytokine expression pattern in kidneys and lymph nodes varies between CCR7KO and WT mice. (A and B) The expression of IL-10, TGF-β, IL-6, TNF-α, IFN-γ, IL-17, and IL-21 mRNA was evaluated by real-time PCR on total RNA from kidneys (A) or lymph nodes (B) of CCR7KO ( ; n = 21) or WT (■; n = 18) mice 7 d after anti-GBM injection. The data are shown as fold increase in comparison with healthy tissue of WT mice. The IL-17 expression was evaluated only in lymph nodes, because healthy controls do not express IL-17 in kidneys. CCR7KO mice express significantly more mRNA of IL-6, IL-10, and IL-21 in kidneys, whereas mRNA of IL-6, IFN-γ, and IL-17 was significantly increased in the lymph nodes of CCR7KO mice as compared with WT controls. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: CCR7 Deficiency Exacerbates Injury in Acute Nephritis Due to Aberrant Localization of Regulatory T Cells

    doi: 10.1681/ASN.2009020133

    Figure Lengend Snippet: The cytokine expression pattern in kidneys and lymph nodes varies between CCR7KO and WT mice. (A and B) The expression of IL-10, TGF-β, IL-6, TNF-α, IFN-γ, IL-17, and IL-21 mRNA was evaluated by real-time PCR on total RNA from kidneys (A) or lymph nodes (B) of CCR7KO ( ; n = 21) or WT (■; n = 18) mice 7 d after anti-GBM injection. The data are shown as fold increase in comparison with healthy tissue of WT mice. The IL-17 expression was evaluated only in lymph nodes, because healthy controls do not express IL-17 in kidneys. CCR7KO mice express significantly more mRNA of IL-6, IL-10, and IL-21 in kidneys, whereas mRNA of IL-6, IFN-γ, and IL-17 was significantly increased in the lymph nodes of CCR7KO mice as compared with WT controls. * P

    Article Snippet: For quantification of IFN-γ, IL-10, IL-6, IL-17, and IL-21, TaqMan Mastermix (Applied Biosystems) and the gene expression assays Mm00801778_m1, Mm00439616_m1, Mm00446190_m1, Mm00439619_m1, and Mm00517640_m1 (Applied Biosystems) were used.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Injection

    Induction of protein tyrosine phosphatases following T-cell activation limits STAT1 signalling. (a) CD4 + T N and CD4 + T EM cells were pre-treated for 5 min with 5mM sodium orthovanadate (vanadate) prior to IL-6 (20ng/ml) stimulation for 30 min. Changes in pY-STAT1 and pY-STAT3 activity were monitored by intracellular flow cytometry (MFI). A comparable analysis of pS-STAT1 and pS-STAT3 is shown as a control (n=3). (b) Quantitative PCR for Ahr , Il21 , Stat3 and Socs3 after vanadate pre-treatment and 20ng/ml IL-6 stimulation in CD4 + T EM cells (n=3). (c) Heatmap analysis of Affymetrix transcriptomic data identifies the top 20 genes ( P

    Journal: Nature immunology

    Article Title: Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells

    doi: 10.1038/s41590-019-0350-0

    Figure Lengend Snippet: Induction of protein tyrosine phosphatases following T-cell activation limits STAT1 signalling. (a) CD4 + T N and CD4 + T EM cells were pre-treated for 5 min with 5mM sodium orthovanadate (vanadate) prior to IL-6 (20ng/ml) stimulation for 30 min. Changes in pY-STAT1 and pY-STAT3 activity were monitored by intracellular flow cytometry (MFI). A comparable analysis of pS-STAT1 and pS-STAT3 is shown as a control (n=3). (b) Quantitative PCR for Ahr , Il21 , Stat3 and Socs3 after vanadate pre-treatment and 20ng/ml IL-6 stimulation in CD4 + T EM cells (n=3). (c) Heatmap analysis of Affymetrix transcriptomic data identifies the top 20 genes ( P

    Article Snippet: Gene expression was determined by Q-PCR , using the QuantStudio 12K Flex Real-Time PCR system and the following TaqMan probes from Thermofisher: Ahr (Mm00478932_m1), Bcl3 (Mm00504306_m1), Bcl6 (Mm01342164_m1), Il10 (Mm00439614_m1), Il21 (Mm00517640_m1), Irf1 (Mm01288580_m1), Socs3 (Mm00545913_s1), Stat3 (Mm01219775_m1), Pim1 (Mm00435712_m1) and Actb (Mm01205647_g1) as a housekeeping gene.

    Techniques: Activation Assay, Activity Assay, Flow Cytometry, Real-time Polymerase Chain Reaction

    Infiltrating T-cells showed impaired STAT1 activity in response to arthritis induction. (a) Representative H E staining of knee joints at day 10 post disease induction (antigen-induced arthritis, AIA) (bar: 500μm); boxed area shows the location of the immunofluorescence. Representative immunofluorescence with antibodies against CD3 (red), pY-STAT1 or pY-STAT3 (green) is shown together with DAPI counterstaining (blue) (bar: 100μm). Graph shows the proportion of CD3 + T cells displaying either pY STAT1 or pY-STAT3 (n=3). (b) Phosphorylation of STAT1 and STAT3 by flow cytometry of infiltrating synovial CD4 + T cells during AIA after stimulation with 20ng/ml IL-6 compare to CD4 + T N cells. (c) Representative flow cytometry of pY-STAT1 and pY-STAT3 in CD4 + T cells extracted from inguinal lymph nodes of mBSA challenged (n=4) and non-challenged mice (control) (n=3) following stimulation with 20ng/ml IL-6 for 30 min. Graphs show quantification of pY-STAT1 and pY-STAT3 activity in CD4 + T N and CD4 + T EM cells (n=4). (d) Quantitative PCR of Ahr, Ifng, Il17a, Il21, Rorc, Socs3 and Stat3 in CD4 + T N (n=4) and CD4 + T EM cells (n=2) extracted from inguinal lymph nodes of mBSA challenged mice. (e) Intracellular flow cytometry analysis of IL-21 production in CD4 + T N and CD4 + T EM cells extracted from inguinal lymph nodes after 4 hours stimulation with PMA, ionomycin and monensin (n=4). Data are representative of three independent experiments (c,e), two independent experiments (a,b) and one experiment involving biological replicates (d). **** P

    Journal: Nature immunology

    Article Title: Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells

    doi: 10.1038/s41590-019-0350-0

    Figure Lengend Snippet: Infiltrating T-cells showed impaired STAT1 activity in response to arthritis induction. (a) Representative H E staining of knee joints at day 10 post disease induction (antigen-induced arthritis, AIA) (bar: 500μm); boxed area shows the location of the immunofluorescence. Representative immunofluorescence with antibodies against CD3 (red), pY-STAT1 or pY-STAT3 (green) is shown together with DAPI counterstaining (blue) (bar: 100μm). Graph shows the proportion of CD3 + T cells displaying either pY STAT1 or pY-STAT3 (n=3). (b) Phosphorylation of STAT1 and STAT3 by flow cytometry of infiltrating synovial CD4 + T cells during AIA after stimulation with 20ng/ml IL-6 compare to CD4 + T N cells. (c) Representative flow cytometry of pY-STAT1 and pY-STAT3 in CD4 + T cells extracted from inguinal lymph nodes of mBSA challenged (n=4) and non-challenged mice (control) (n=3) following stimulation with 20ng/ml IL-6 for 30 min. Graphs show quantification of pY-STAT1 and pY-STAT3 activity in CD4 + T N and CD4 + T EM cells (n=4). (d) Quantitative PCR of Ahr, Ifng, Il17a, Il21, Rorc, Socs3 and Stat3 in CD4 + T N (n=4) and CD4 + T EM cells (n=2) extracted from inguinal lymph nodes of mBSA challenged mice. (e) Intracellular flow cytometry analysis of IL-21 production in CD4 + T N and CD4 + T EM cells extracted from inguinal lymph nodes after 4 hours stimulation with PMA, ionomycin and monensin (n=4). Data are representative of three independent experiments (c,e), two independent experiments (a,b) and one experiment involving biological replicates (d). **** P

    Article Snippet: Gene expression was determined by Q-PCR , using the QuantStudio 12K Flex Real-Time PCR system and the following TaqMan probes from Thermofisher: Ahr (Mm00478932_m1), Bcl3 (Mm00504306_m1), Bcl6 (Mm01342164_m1), Il10 (Mm00439614_m1), Il21 (Mm00517640_m1), Irf1 (Mm01288580_m1), Socs3 (Mm00545913_s1), Stat3 (Mm01219775_m1), Pim1 (Mm00435712_m1) and Actb (Mm01205647_g1) as a housekeeping gene.

    Techniques: Activity Assay, Staining, Immunofluorescence, Flow Cytometry, Mouse Assay, Real-time Polymerase Chain Reaction