Figure 4: ITK is required for Tr1 cell differentiation in a kinase dependent manner.

(a,b) ITK kinase activity is required for Tr1 cell differentiation in mouse: WT, Itk^−/− and Itk_as naive CD4⁺ T cells (carrying IL-10^GFP/Foxp3^RFP reporters) were cultured under Tr1 polarizing conditions. (a) Representative FACS plots showing IL-10 and Foxp3 expression and summary of IL-10⁺Foxp3⁻ cell percentage, density (initial density: 0.5 × 10⁶ per ml) and IL-10 relative (Rel) mRNA levels (normalized to Gapdh first, then WT average level set as 1). (b) Representative plots of CD4⁺ T cell viability (first panel), expression of Ki67 (second panel) and LAG3/CD49b (third panel) in viable CD4⁺ T cells; along with summary of percentage of viable CD4⁺, Ki67⁺ CD4⁺ and LAG3⁺CD49b⁺ CD4⁺ T cells. n=6. Data represent results of more than five experiments. (c,d) ITK kinase activity is required for human Tr1 cell differentiation: naive CD4⁺ T cells isolated from human peripheral blood mononuclear cells were cultured under Tr1 polarizing conditions; cells were stimulated and subjected to intracellular staining. (c) Representative FACS plots showing IL-10 and FOXP3 expression and summary of IL-10⁺FOXP3⁻ cell percentage, IL-10 Rel MFI levels (Non-treated group average level set as 1) and IL-10 Rel mRNA levels (normalized to GAPDH first, then WT average level set as 1). (d) Representative plots of CD4⁺ T cell viability (first panel), expression of Ki67 (second panel) and LAG3/CD49b (third panel) in viable CD4⁺ T cells; along with summary of percentage of viable CD4⁺, Ki67⁺ CD4⁺ and LAG3⁺CD49b⁺ CD4⁺ T cells. n=4. Data represent results of two experiments. *P≤0.05, **P≤0.01, ***P≤0.001, by non-parametric Mann–Whitney test. Data presented as mean±s.e.m.