ITK signalling via the Ras/IRF4 pathway regulates the development and function of Tr1 cells

Abstract

Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging the T cell receptor (TCR), express high levels of the immunosuppressive cytokine IL-10, but not Foxp3, and can suppress inflammation and promote immune tolerance. Here we show that ITK, an important modulator of TCR signalling, is required for the TCR-induced development of Tr1 cells in various organs, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell differentiation. Tr1 cell development and suppressive function of Itk deficient cells can be restored by the expression of the transcription factor interferon regulatory factor 4 (IRF4). Downstream of ITK, Ras activity is responsible for Tr1 cell induction, as expression of constitutively active HRas rescues IRF4 expression and Tr1 cell differentiation in Itk^-/- cells. We conclude that TCR/ITK signalling through the Ras/IRF4 pathway is required for functional development of Tr1 cells.

Figure 1. ITK is required for Tr1 cell development in vivo.

WT and Itk^−/− IL-10^GFP/Foxp3^RFP dual reporter mice were treated with αCD3ε antibody or control, and live cells from the indicated organs were analysed. (a) Representative FACS plots showing IL-10^GFP expression by Foxp3^RFP− CD4⁺ T cells and summary of IL-10⁺ cell percentage over Foxp3⁻CD4⁺ T cells and number in blood (number per ml), spleen, lung, small intestine and fat (number per gram). (b) Representative FACS plots showing the LAG3/CD49b expression by IL-10^GFP+ Foxp3^RFP− CD4⁺ T cells, summary of LAG3⁺CD49b⁺ cell percentage over IL-10⁺Foxp3⁻CD4⁺ T cells and number of LAG3⁺CD49b⁺ IL-10⁺Foxp3⁻ Tr1 cells from samples shown in (a). ^§P≤0.05, ^§§P≤0.01, ^§§§P≤0.001, compared with the levels in PBS-treated group; *P≤0.05, **P≤0.01, ***P≤0.001, comparing groups connected, by non-parametric Mann–Whitney test. Data were pooled from three experiments, ‘n’ indicates number of replicates in each group/point. Data presented as mean±s.e.m.

Figure 2. ITK is required for parasitic/viral infection-induced Tr1 cell development in vivo.

(a,b) ITK is required for Tr1 cell development during parasitic infection: WT and Itk^−/− mice were infected with 500 L3 N. brasiliensis (N.B.) and lungs analyzed 7 days post infection (d.p.i.). (a) Representative FACS plots showing IL-10^GFP and Foxp3^RFP expression by pulmonary CD4⁺ T cells and summary of IL-10⁺Foxp3⁻ T cell percentage, number and IL-10 expression levels (WT average level set as 1). (b) Representative FACS plots showing the LAG3 and CD49b expression by IL-10^GFP+ Foxp3^RFP− CD4⁺ T cells, summary of LAG3⁺CD49b⁺ IL-10⁺Foxp3⁻CD4⁺ Tr1 cell percentage and number from samples shown in a. ^§P≤0.05, ^§§P≤0.01, ^§§§P≤0.001, compared with PBS-treated group; *P≤0.05, **P≤0.01, ***P≤0.001, comparing groups connected, by non-parametric Mann-Whitney test. (c,d) ITK is required for Tr1 cell development during viral infection: WT and Itk^−/− mice were infected with 10⁴ PFU Influenza A/WSN/1933 (WSN) and lungs analyzed 3 and 7 d.p.i. (c) Representative FACS plots showing IL-10^GFP and Foxp3^RFP expression by pulmonary CD4⁺ T cells and summary of IL-10⁺Foxp3⁻ T cell percentage, number and IL-10 expression levels (WT average level set as 1). (d) Representative FACS plots showing the LAG3/CD49b expression by IL-10^GFP+ Foxp3^RFP− CD4⁺ T cells, summary of LAG3⁺CD49b⁺ IL-10⁺Foxp3⁻CD4⁺ Tr1 cell percentage and number from samples shown in c. P values on plots were calculated by two-way ANOVA. Data were pooled from three experiments, ‘n’ indicates number of replicates in each group/point. Data presented as mean±s.e.m.

Figure 3. ITK is required for Tr1 cell differentiation in vitro.

All experiments were performed with cells carrying the IL-10^GFP/Foxp3^RFP dual reporter system for live cell analysis. Naive WT and Itk^−/− CD4⁺ T cells were cultured under Tr1 polarizing condition. (a) Representative FACS plots showing the IL-10^GFP and Foxp3^RFP expression by naive CD4⁺ T cells cultured under Tr1 differentiation conditions for the indicated time. (b) Summary of IL-10⁺Foxp3⁻ cell percentage and number (number per ml, initial naive CD4⁺ T cell density: 0.5 × 10⁶ per ml). p values calculated by two-way ANOVA. (c) Representative plots of CD25 and CD69 expression by naive CD4⁺ T cells cultured under Tr1 cell-differentiation conditions for 48 h. (d) Representative FACS plots showing the dilution of cell proliferation dye eF450 and expression of IL-10 by CD4⁺ T cells cultured under Tr1 differentiation conditions for 48 or 72 h. Mean±s.e.m. of six replicates were indicated on the flow cytometric plots. Representative plots and summary of percentages of double positive of (e) LAG3/CD49b and (f) ICOS/PD-1 expression by the total and IL-10⁺Foxp3⁻ CD4⁺ T cells 72 h post culture. Naive CD4⁺ T cells were used as controls in c–f. n=6. Data represent results of more than three experiments. ***P≤0.001, by non-parametric Mann–Whitney test. Data presented as mean±s.e.m.

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.

Figure 5. The kinase activity of ITK is required for Tr1 cell suppressive function.

Differentiated and sort purified WT and Itk_as Tr1 cells (CD45.2⁺, carrying IL-10^GFP/Foxp3^RFP reporters) were co-cultured with CFSE stained naive CD4⁺ T cell responders (CD45.1⁺). 3MBPP1 was added to specifically inhibit ITK_as activity. (a) Representative FACS plots of WT and Itk_as Tr1 cells and CFSE dilution of the CD45.1⁺ responders with or without stimulation in the presence or absence of ITK-inhibited Tr1 cells or control cells (responder cell density was fixed and Tr1 cells were added at the indicated ratios). (b) Summary of percentage of suppression of the responder proliferation by the ITK-inhibited Tr1 cells or control cells. P value calculated by two-way ANOVA. (c) Relative (Rel) IL-10 MFI in CD45.2⁺ Tr1 cells in the co-culture system. WT average level was set as 1. *P≤0.05, **P≤0.01, ***P≤0.001, by non-parametric Mann–Whitney test. n=6. Data represents results of three experiments. Data presented as mean±s.e.m.

Figure 6. ITK regulates IRF4 expression during mouse and human Tr1 cell development.

(a) Representative FACS plots and summary of Rel MFI (naive cells level set as 1) of AHR expression in mouse cells cultured under Tr1 polarizing conditions. (b) Naive WT and Itk^−/− IL-10^GFP/Foxp3^RFP reporter CD4⁺ T cells were cultured under Tr1 polarizing conditions; AHR agonist TCDD was added to activate AHR, and antagonist GNF was added to inhibit AHR activity. Representative FACS plots of IL-10^GFP and Foxp3^RFP expression, with summary of percentage of IL-10⁺Foxp3⁻ cells are shown. (c,d) Representative FACS plots and summary of Rel MFI (naive cells level set as 1) of cMAF expression in (c) mouse and (d) human CD4⁺ T cells cultured under Tr1 polarizing conditions. (e,f) Representative FACS plots and summary of Rel MFI (naive cells level set as 1) of IRF4 expression in (e) mouse and (f) human CD4⁺ T cells cultured under Tr1 polarizing conditions. n=6; data represent results of three experiments in mouse. n=4; data represent results of two experiments in human. Naive CD4⁺ T cells without stimulation were used as control. ^§P≤0.05, ^§§§P≤0.001, compared with the control level; ***P≤0.001; NS, no significance, comparing groups connected, by Non-parametric Mann–Whitney test. Data presented as mean±s.e.m.

Figure 7. Re-expression of IRF4 rescues Tr1 cell differentiation and function in Itk^−/− cells.

WT and Itk^−/− naive IL-10^GFP/Foxp3^RFP reporter CD4⁺ cells were cultured under Tr1 differentiation conditions, and retrovirally transduced with control-YFP or IRF4-YFP. YFP⁺ (IRF4⁺) or YFP^- (IRF4^-) Foxp3^RFP− CD4⁺ T cells were gated for analysis: representative FACS plots of (a) IL-10^GFP expression in Foxp3⁻CD4⁺ T cells and summary of percentage of IL-10⁺ cells over live Foxp3⁻CD4⁺ T cells; (b) LAG3 and CD49b expression and summary of LAG3⁺CD49b⁺ Tr1 cells over live Foxp3⁻CD4⁺ T cells. n=6. Data represent results of three experiments. (c) WT and Itk^−/− Tr1 cells (CD45.2⁺, carrying IL-10^GFP/Foxp3^RFP reporters) with or without IRF4-RV were flow sorted and co-cultured with CFSE stained naive CD4⁺ T cell responders (CD45.1⁺). Representative FACS plots of CFSE dilution of the responders with or without stimulation in the presence or absence of WT or Itk^−/− Tr1 cells that are IRF4-RV⁻ or IRF4-RV⁺ (Tr1: responder cell ratio=1:2); and summary of percentage of suppression of the responder proliferation by the indicated Tr1 cells. n=6. *P≤0.05, ***P≤0.001, by one-way ANOVA with Tukey’s post-hoc test. Data presented as mean±s.e.m.

Figure 8. ITK regulates but does not depend on the expression of Blimp-1 during Tr1 cell differentiation.

(a,b) Representative FACS plots and summary of Rel MFI (WT naive cell level set as 1) of Blimp-1 expression in WT and Itk^−/− naive CD4⁺ T cells or CD4⁺ T cells cultured under Tr1 cell-polarizing condition. n=6. Data represent results of two experiments. ^§§§P≤0.001, compared with the level in naive cells; ***P≤0.001, comparing groups connected, by Non-parametric Mann–Whitney test. (c–e) WT and Itk^−/− naive IL-10^GFP/Foxp3^RFP reporter CD4⁺ cells were cultured under Tr1 polarizing condition, retrovirally transduced with control-hCD2 or Blimp-1-hCD2, and analyzed. (c) Representative FACS dot plots for the expression of Blimp-1 in RV transduced cells. (d) Histogram showing Blimp-1 signal intensity (left) and summary (right) of Rel Blimp-1 MFI. Naive CD4⁺ T cell population in grey was used as control. n=6. Data represent results of two experiments. ***P≤0.001, comparing groups connected, by Non-parametric Mann–Whitney test. (e) Representative FACS plots of (top) IL-10^GFP expression in Foxp3⁻CD4⁺ T cells and summary of percentage of IL-10⁺ cells over live Foxp3⁻CD4⁺ T cells; (bottom) LAG3 and CD49b expression and summary of LAG3⁺CD49b⁺ Tr1 cells over live Foxp3⁻CD4⁺ T cells. n=4. Data represent results of three experiments. ***P≤0.001, by one-way ANOVA with Tukey’s post-hoc test. Data presented as mean±s.e.m.

Figure 9. Ras/MAPK activity is required for Tr1 cell development and IRF4 expression.

(a,b) WT naive IL-10^GFP/Foxp3^RFP reporter CD4⁺ cells were cultured under Tr1 differentiation conditions, in the presence or absence of ERK1/2 inhibitor (PD098059), JNK inhibitor (SP600125), p38 inhibitor (SB203580), or Ras inhibitor (Kobe0065). Viable CD4⁺ T cells were analyzed. (a) Representative FACS plots of IL-10^GFP/Foxp3^RFP, CD69/CD25 and IRF4 expression, and (b) summary of percentage of IL-10⁺Foxp3⁻ and CD25⁺CD69⁺ cells over viable CD4⁺ T cells, and relative IRF4 MFI (naive cell level set as 1). n=6. Data represent results of two experiments. *P≤0.05, **P≤0.01, ***P≤0.001, by one-way ANOVA with Tukey’s post-hoc test. Data presented as mean±s.e.m.

Figure 10. HRas activity rescues Tr1 cell development and IRF4 expression in Itk^−/− cells.

WT and Itk^−/− naive IL-10^GFP/Foxp3^RFP reporter CD4⁺ cells were cultured under Tr1 differentiation conditions, retrovirally transduced with control-YFP, WT HRas-YFP or its constitutively active mutant HRas^G12V-YFP. (a) Representative FACS plots of IL-10^GFP+ T cells. (b) Summary of percentage of IL-10^GFP cells over viable CD4⁺Foxp3⁻ T cells and relative IL-10 MFI in IL-10⁺Foxp3⁻ cells (WT control level set as 1). (c) Representative FACS plots of IRF4 expression in RV-transduced cells. Naive CD4⁺ T cells were used as control in grey. n=6. Data represent results of three experiments. *P≤0.05, ***P≤0.001; NS, no significance, by one-way ANOVA with Tukey’s post-hoc test. Data presented as mean±s.e.m.