αil 4  (Bio X Cell)

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

Article Title: Intrinsic STAT4 expression controls effector CD4 T cell migration and Th17 pathogenicity

doi: 10.4049/jimmunol.2200606

Figure Lengend Snippet: (A) Stat4 mRNA expression from isolated WT, STAT4−/−, or STAT4ΔdLck CD4 T cells. (B-G) Naïve CD4 T cells were isolated from the spleens of WT, STAT4−/−, or STAT4ΔdLck mice and polarized under Th1 cell conditions. Cells were analyzed on day 3 of differentiation. (B) Representative plots of pSTAT4 staining. (C) Frequency of pSTAT4+ cells. (D) Relative expression of Ifng mRNA. (E) Representative plots of IFNγ staining. (F) Frequency of IFNγ+ cells. (G) ELISA of IFNγ production. (H-I) Naïve CD4 T cells were isolated from the spleens of WT, STAT4−/−, or STAT4ΔdLck mice and polarized under Th17 cell conditions. Cells were analyzed on day 3 of differentiation. (H) Representative plots of IL-17A staining. (I) Frequency of IL-17+ cells. All representative plots are gated on live CD4+ cells. Data represent 3–5 independent experiments. Unpaired T test; ns = not significant, *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: For Th1 conditions, cultures were supplemented with IL-12 (10ng/mL; Peprotech) and αIL-4 (10 mg/ml; Bio X Cell).

Techniques: Expressing, Isolation, Staining, Enzyme-linked Immunosorbent Assay

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

Article Title: Intrinsic STAT4 expression controls effector CD4 T cell migration and Th17 pathogenicity

doi: 10.4049/jimmunol.2200606

Figure Lengend Snippet: (A) Splenocytes were isolated from previously immunized mice and cultured under Th17 conditions for three days. CD4 T cells were isolated and transferred into CD45.1 WT recipient mice. Disease symptoms were monitored daily (n=30–36, 10 independent experiments). (B) Representative flow diagram of CD45.2 donor cells in the brain and spinal cord (gated on CD4+ T cells). (C) Frequency and (D) number of CD45.2 donor cells in the spleen, brain, and spinal cord (gated on CD4+ T cells) (n=18–20, 3 independent experiments). (E) Representative flow diagram of donor cell cytokine profile. (F-H) Frequency (top) and number (bottom) of (F) IL-17A+, (G) IFNγ+ (H) IL-17A+IFNγ+ T cells Gated on (B-D) CD4+ and (E-H) CD4+CD45.2+ cells (n=18–20, 3 independent experiments). (A) Two- way ANOVA and (C, D, F-H). Unpaired T test; ns = not significant, *=p<0.05. **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: For Th1 conditions, cultures were supplemented with IL-12 (10ng/mL; Peprotech) and αIL-4 (10 mg/ml; Bio X Cell).

Techniques: Isolation, Cell Culture

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

Article Title: Intrinsic STAT4 expression controls effector CD4 T cell migration and Th17 pathogenicity

doi: 10.4049/jimmunol.2200606

Figure Lengend Snippet: (A) STAT4ΔdLCK and littermate WT controls were immunized for EAE. Disease symptoms were monitored daily (n=9–14, 5 independent experiments). (B) Frequency and (C) number of CD4 T cells infiltrating to the CNS at the peak of disease (n=7–11, 3 independent experiments). (D) Representative flow plot of the expression of IL-17A+ and IFN γ+ cells (gated on CD4+ T cells). (E-G) Frequency (top) and number (bottom) of CD4+ T cells in the CNS producing (E) IFNγ (F) IL-17A and (G) IFNγ and IL17A. (n=7–11, 3 independent experiments). (A) Two- way ANOVA and (E, F, G) unpaired T test; ns = not significant, *=p<0.05. **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: For Th1 conditions, cultures were supplemented with IL-12 (10ng/mL; Peprotech) and αIL-4 (10 mg/ml; Bio X Cell).

Techniques: Expressing

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

Article Title: Intrinsic STAT4 expression controls effector CD4 T cell migration and Th17 pathogenicity

doi: 10.4049/jimmunol.2200606

Figure Lengend Snippet: (A) STAT4ΔIL17A and littermate WT controls were immunized for EAE, and disease was scored daily (n=14–20, 6 independent experiments). (B) Number of CD4 T cells infiltrating to the CNS at the peak of disease. (C) Representative flow diagram YFP+ cells (gated on CD4+). (D) Frequency and (E) number of YFP+ cells (gated on CD4+) (n=7–9, 6 independent experiments). (F) Representative flow diagram of IL17A+ and IFNγ+ cells (gated on CD4+). (G-I) Frequency (top) and number (bottom) of (G) IL-17A+ (H) IFNγ+ and (I) IFNγ+IL17A+ cells (gated on CD4+) (n=10, 5 independent experiments). (A) Two- way ANOVA and (B, D, E, G-I) unpaired T test; ns = not significant, *=p<0.05. **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: For Th1 conditions, cultures were supplemented with IL-12 (10ng/mL; Peprotech) and αIL-4 (10 mg/ml; Bio X Cell).

Techniques:

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

Article Title: Intrinsic STAT4 expression controls effector CD4 T cell migration and Th17 pathogenicity

doi: 10.4049/jimmunol.2200606

Figure Lengend Snippet: (A, B) WT:WT and WT:STAT4−/− mixed bone marrow chimeric mice were immunized for EAE. (A) Representative plots show CD45.1 and CD45.2 staining gated on CD4 T cells from the spleen, brain, and spinal cord on day 17 post immunization. (B) Normalized accumulation frequencies of CD45.2+ WT and STAT4−/− CD4 T cells in the brain and spinal cord were determined on day 11 and day 17 of disease. Normalization frequency = (%CD45.2+CD4+ brain or spinal cord ÷ %CD45.2+CD4+ spleen) x 100. (C-D) Ki67 staining in CNS infiltrating CD45.2+ WT or STAT4−/− CD4 T cells from mixed bone marrow chimeric mice on day 17 post immunization. (C) Representative plots are gated on CD45.2+ CD4 T cells. (D) Cumulative frequencies of Ki67+ CD45.2+ WT or STAT4−/− CD4 T cells in the CNS. (E-F) Phosphorylated STAT3 (pSTAT3) staining in CD45.2+ WT or STAT4−/− CD4 T cells following media or IL-23 stimulation of pooled CNS cells. (E) Representative plots gated on CD45.2+ CD4 T cells illustrate media (grey filled) and IL-23 (black line) induced pSTAT3. (F) Cumulative frequencies of IL-23 induced pSTAT3+ CD45.2+ WT or STAT4−/− CD4 T cells from pooled CNS tissue. Data represent 2–6 independent experiments with (A-B) 2–5, (C-D) 3–4, or (E-F) 2–5 mice in each group (mean ± SD). ns=not significant; ***=p<0.001; ****=p<0.0001.

Article Snippet: For Th1 conditions, cultures were supplemented with IL-12 (10ng/mL; Peprotech) and αIL-4 (10 mg/ml; Bio X Cell).

Techniques: Staining

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

Article Title: Intrinsic STAT4 expression controls effector CD4 T cell migration and Th17 pathogenicity

doi: 10.4049/jimmunol.2200606

Figure Lengend Snippet: (A-B) Splenocytes from previously immunized mice were cultured under Th17 conditions. CD4 T cells from these cultures were isolated and RNA sequencing was performed. (A) Volcano plot of differentially expressed genes between WT and STAT4−/− Th17 cells (red) genes significantly elevated in STAT4−/− Th17 cells and (blue) genes significantly elevated in the WT Th17 cells. (B) GSEA plots show enrichment of genes elevated in WT Th17 cells → elevated in STAT4−/− Th17 cells (left → right) when compared with pathogenic and non-pathogenic Th17 cells (GSE43955), (C-H) Splenocytes were isolated from previously immunized WT IL17AGFP+ or STAT4−/− IL17AGFP+ mice and cultured under Th17 conditions for three days. GFP+CD4+ T cells were isolated and transferred into CD45.1 WT recipient mice. Disease symptoms were monitored daily (n=10–11, 3 independent). (D) Number of recovered donor cells in the CNS (n=4–8, 3 independent experiments). (E) Representative flow diagram of IFNγ and IL-17A expression (gated on CD4+CD45.2+). (F-H) Frequency (top) and number (bottom) of (F) IL-17A+ (G) IFNγ and (H) IL-17A+IFNγ+ producing cells (gated on CD4+CD45.2+) (n-4–8, 3 independent experiments). (C) Two- way ANOVA and (D,F-H). Unpaired T test; ns = not significant, *=p<0.05. **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: For Th1 conditions, cultures were supplemented with IL-12 (10ng/mL; Peprotech) and αIL-4 (10 mg/ml; Bio X Cell).

Techniques: Cell Culture, Isolation, RNA Sequencing Assay, Expressing