Integration of IL-2 and IL-4 Signals Coordinates Divergent Regulatory T cell Responses and Drives Therapeutic Efficacy

Abstract

19Cells exist within complex milieus of communicating factors, such as cytokines, that combine to 20 generate context-specific responses, yet nearly all knowledge about the function of each cytokine and 21 the signaling propagated downstream of their recognition is based upon the response to individual 22cytokines. Here, we found that regulatory T cells (Tregs) integrate concurrent signaling initiated by IL-2 23and IL-4 to generate a response divergent from the sum of the two pathways in isolation. IL-4 24 stimulation of STAT6 phosphorylation was blocked by IL-2, while IL-2 and IL-4 synergized to enhance 25 STAT5 phosphorylation, IL-10 production, and the selective proliferation of IL-10-producing Tregs, 26leading to increased inhibition of conventional T cell activation and the reversal of asthma and multiple 27 sclerosis in mice. These data define a mechanism of combinatorial cytokine signaling and lay the 28 foundation upon which to better understand the origins of cytokine pleiotropy while informing 29improved the clinical use of cytokines. 30 picture of cytokine pleiotropy and potentially leading to enhancements to the use of cytokines in the 77 clinical setting. 78 79 Results 80 IL-2 and IL-4 synergistically promote IL-10 production by Tregs 81Our previous findings demonstrated that IL-4 impacts IL-10 production in FoxP3 + Tregs (29), but the 82 nature of the functional interaction between IL-4 and IL-2, a key survival factor for Tregs in vitro and in 83 vivo, remained obscure. Thus, we created a novel dual reporter mouse by crossing FoxP3 RFP (30) and IL-84 10 GFP (31) mice, thereby enabling live sorting of FoxP3 + cells and analysis of IL-10 production on a per-cell 85 basis. CD4 + FoxP3 + Tregs isolated from the spleens of naïve dual reporter mice (Figure 1 -figure 86 supplement 1A-B) by magnetic bead and sterile fluorescence-activated cell sorting (FACS) were cultured 87 with T cell receptor (TCR) activation using αCD3ε antibody and all combinations of IL-2 and IL-4 for 3 88 days. We found that Tregs cultured with combinatorial cytokine stimulation resulted in synergistically 89higher numbers of IL-10 expressing cells (Figures 1A-1C) and IL-10 secretion ( Figure 1D) compared to 90 single cytokine stimulation. However, analysis of IL-10 + cells revealed that IL-10 expression was no 91 different than IL-2-stimulation in isolation ( Figure 1E), suggesting that the cytokines in combination do 92 not elicit a synergistic increase in IL-10 production on a per-cell basis. The sex-independent ( Figure 1F) 93and TCR-stimulation dependent synergy ( Figures 1A-1D) was present in FoxP3 + Tregs but not FoxP3 -94Tconv ( Figure 1A), and no loss of FoxP3 expression was observed ( Figure 1G), suggesting that the 95 machinery required for this effect was unique to FoxP3 + Tregs. 96In order to determine whether the cytokines must be present at the same time, we isolated Tregs 97 and stimulated them with cytokines in series over a 3-day culture and compared the response to 98 simultaneous stimulation. We discovered that b...