IL-10 Receptor Signaling Is Essential for TR1 Cell Function In Vivo

Abstract

IL-10 is essential to maintain intestinal homeostasis. CD4⁺ T regulatory type 1 (T_R1) cells produce large amounts of this cytokine and are therefore currently being examined in clinical trials as T cell therapy in patients with inflammatory bowel disease. However, factors and molecular signals sustaining T_R1 cell regulatory activity still need to be identified to optimize the efficiency and ensure the safety of these trials. We investigated the role of IL-10 signaling in mature T_R1 cells in vivo. Double IL-10^eGFP Foxp3^mRFP reporter mice and transgenic mice with impairment in IL-10 receptor signaling were used to test the activity of T_R1 cells in a murine inflammatory bowel disease model, a model that resembles the trials performed in humans. The molecular signaling was elucidated in vitro. Finally, we used human T_R1 cells, currently employed for cell therapy, to confirm our results. We found that murine T_R1 cells expressed functional IL-10Rα. T_R1 cells with impaired IL-10 receptor signaling lost their regulatory activity in vivo. T_R1 cells required IL-10 receptor signaling to activate p38 MAPK, thereby sustaining IL-10 production, which ultimately mediated their suppressive activity. Finally, we confirmed these data using human T_R1 cells. In conclusion, T_R1 cell regulatory activity is dependent on IL-10 receptor signaling. These data suggest that to optimize T_R1 cell-based therapy, IL-10 receptor expression has to be taken into consideration.

Fig. 1. In vivo differentiated T_R1 cells can respond to IL-10

(A) IL-10Rα expression and MFI of indicated cell populations. Black area represents the isotype control. Four independent experiments were performed. (B) Immunofluorescence staining (two independent experiments) of IL-10Rα on wild type T_R1 cells. (C–D) ΔMFI (compared to unstimulated cells) of pSTAT3 levels. (C) Naïve T cells, Foxp3⁺ T cells and IL-10Rα^WT or IL-10Rα^Impaired T_R1 cells were stimulated with IL-10 (100 ng/ml) for indicated time points. Black area represents the unstimulated control. (D) IL-10Rα^WT or IL-10Rα^Impaired T_R1 cells were stimulated for 20 min with the indicated concentrations of IL-10 or IL-6. Data are representative of three independent experiments.

Fig. 2. IL-10 signaling in T cells is not essential for the differentiation of T_R1 cells

IL-10Rα^WT or IL-10Rα^Impaired Foxp3^RFP IL-10^eGFP double reporter mice were treated with anti-CD3. (A) Frequency of T_R1 cells in the small intestine and CD49b and LAG-3 expression by T_R1 cells are shown (IL-10Rα^WT n=4; IL-10Rα^Impaired n=5) (B) Gene expression analysis, comparing IL-10Rα^WT T_R1 cells and IL-10Rα^Impaired T_R1 cells. Fold change expression of T_R1 cell signature genes of IL-10Rα^Impaired T_R1 cells compared to IL-10Rα^WT T_R1 cells. (C) Maf, Ahr, Prdm1, Tgfb1, Ctla4 and Gzmb mRNA expression normalized to Hprt (data pooled of three independent experiments). (D) T_R1-mediated in vitro suppression. Data are representative of five independent experiments.

Fig. 3. IL-10 signaling in T_R1 cells is essential for their in vivo function

In vivo induced IL-10Rα^WT or IL-10Rα^Impaired T_R1 were injected alone or together with in vivo differentiated effector (e)T_H17 cells. (A) Mass loss, endoscopic and histological colitis score 5 weeks upon transfer (eT_H17 n=7; eT_H17+ IL-10Rα^WT T_R1 n=7; eT_H17+ IL-10Rα^Impaired T_R1 n=10; IL-10Rα^WT T_R1 n=8; IL-10Rα^Impaired T_R1 n=8; lines indicate mean ± SEM). Representative endoscopic pictures (B) and histology (scale bars, 200 µm) (C) are shown. Results are cumulative of two independent experiments. One-way ANOVA (post-test Tukey) was used to calculate significance (** p < 0.01; *** p<0.001).

Fig. 4. IL-10 signaling in T_R1 cells sustains IL-10 expression

(A) In vivo induced IL-10Rα^WT or IL-10Rα^Impaired T_R1 cells were injected into Rag1^−/− mice. Cells were isolated 5 weeks after transfer. Representative dot plots of IL-10^eGFP expression of 4 pooled mice per group are shown. Data are representative of three independent experiments. (B) Cytokine production of T_R1 cells was quantified using Cytometric Bead array. Mean ± SEM from three independent experiments are shown. Mann-Whitney U test was used to calculate significance. (C) Tgfb1, Ctla4 and Gzmb mRNA expression normalized to Hprt. Data are cumulative of three independent experiments.

Fig. 5. IL-10 signaling is dispensable for the in vitro differentiation of T_R1 cells with IL-27 and IL-10 sustains IL-10 production in in vitro differentiated T_R1 cells

(A) In vitro differentiation of IL-10Rα^WT and IL-10Rα^Impaired T_R1 cells. Five independent experiments were performed. (B) IL-10^eGFP ΔMFI of re-stimulated IL-10Rα^Impaired T_R1 cells and IL-10Rα^WT T_R1 cells + αIL-10R antibody compared to IL-10Rα^WT T_R1 cells are shown. Results are cumulative of three independent experiments.

Fig. 6. IL-10 signaling sustains IL-10 production in T_R1 cells via activation of p38 MAPK

(A) pSTAT3 and pp38 ΔMFI of re-stimulated IL-10Rα^Impaired T_R1 cells compared to IL-10Rα^WT T_R1 cells are shown. Results are cumulative of three independent experiments. Paired t test was used to test significance. (B) Frequency of IL-10^eGFP of re-stimulated T_R1 cells in the presence of indicated inhibitors are shown (mean ± SEM of three independent experiments). One-way ANOVA (post-test Tukey) was used to calculate significance (* P<0,05). (C) Tgfb1, Ctla4 and Gzmb mRNA expression normalized to Hprt. Results are cumulative of three independent experiments.

Fig. 7. IL-10 signaling sustains IL-10 production in human T_R1 cell

(A–C) Circulating human T_R1 cells (CD4⁺CD45RA^lowCD49b⁺LAG-3⁺) were FACS-sorted from PBMCs of healthy donors (n=5) (A). T_R1 cells were re-stimulated with anti-CD3 and anti-CD28 for 96 hours with either 50 µg/ml human IL-10Rα or isotype control antibody and the indicted cytokines were quantified. A paired t tested was used to calculate significance. (B). TGFB1, CTLA4 and GZMB mRNA expression normalized to HPRT (C). (D) In vitro differentiated T_R1 and non-T_R1 cells were re-stimulated with anti-CD3/TPA or allogeneic mDC for 48 hours with 50 µg/ml human IL-10Rα blocking antibody or isotype control. A dual IFNγ/IL-10 ELISPOT was performed. Data are cumulative of two independent experiments.