Non-canonical IL-2 Signalling in Treg Differentiation and Suppression 2260578

Abstract

Abstract Introduction Interleukin-2 (IL-2) is indispensable for regulatory T cell (Treg) differentiation and function, signaling through the high-affinity IL-2 receptor αβÏ’ to phosphorylate STAT5. We previously identified a non-canonical pathway, termed Matrix-directed Alternative Cytokine Signaling (MACS), in which CD25—IL-2 engages CCR7 to drive integrin activation in Tregs. Enforcement of MACS by the antibody PC61 enhances Treg suppressive function and ameliorates disease in experimental autoimmune encephalomyelitis. Methods We generated a mouse carrying a point mutation (IL-2 E66K) that disrupts MACS while preserving canonical STAT5 signaling. Results These mice exhibit age-associated immune dysregulation in the gut, characterized by reduced frequencies of Foxp3+ cells and a selective loss of peripheral (Nrp1⁻) Tregs. Single-cell RNA-seq of CD4+ T cells from mesenteric lymph nodes (mLNs) of WT and IL-2 E66K/E66K mice revealed that within the suppressive Treg subset, IL-2 E66K/E66K mice exhibited reduced IL-10 expression, suggesting impaired regulatory function. Consistent with this, suppression assays demonstrated that IL-2 E66K/E66K Tregs displayed impaired suppressive function in vitro. Additionally, analysis revealed broad downregulation of AP-1 family genes (Fos, Fosb, Jun, Junb) across CD4+ T cells, raising the possibility that disruption of this transcriptional program contributes to impaired Treg biology. In vitro, PC61 treatment enhanced Treg differentiation from IL-2WT/WT CD4+ naïve T cells but failed to do so in IL-2E66K/E66K cultures. Conclusion Together, these findings establish that loss of MACS compromises Treg suppressive capacity and differentiation and point to AP-1—driven transcriptional programs as a potential mechanistic link. Funding Source na Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)