Mechanisms by Which Low-Dose IL-2/CD25 Fusion Protein Limits Diabetes in NOD Mice

Suppression of autoreactive T cells by regulatory T cells (Tregs) is essential for immune tolerance and homeostasis. Interleukin-2 (IL-2) is critical for Treg development, function, and maintenance. Tregs constitutively express the high-affinity IL-2 receptor (IL-2R), enabling them to respond to much lower IL-2 levels compared to T effector (Teff) cells. Many autoimmune and inflammatory diseases exhibit quantitative and qualitative defects in Tregs and IL-2/IL-2R signaling. Clinical trials in autoimmune patients receiving low-dose (LD) recombinant (r) IL-2 have shown increases in Tregs and improvement in clinical outcomes. However, achieving robust control of autoimmunity is unlikely due to poor pharmacodynamics of rIL-2 which cannot sustain a constant elevation of highly functional Tregs. Here we utilize the NOD mouse model of type 1 diabetes and a novel, long-lasting IL-2-based biologic, where mouse IL-2 is covalently linked to mouse IL-2R CD25 subunit, to investigate the cellular mechanisms underlying the tolerance-enhancing effects of LD IL-2. By varying the administration of IL-2/CD25 fusion protein, we show that a sustained, but not a periodic increase in Tregs is highly effective in inhibiting autoimmunity. Analyses of pancreatic islets using flow cytometry and single-cell RNA sequencing revealed that IL-2/CD25 rapidly changes the islet immune microenvironment. IL-2/CD25 induces a marked increase in activated effector (e) Tregs, particularly ST2⁺ eTregs, type 2 innate lymphoid cells (ILC2s), and cytokine-producing regulatory natural killer (NK) cells, while islet antigen-reactive CD4⁺ and CD8⁺ T cells decrease. T cell receptor repertoire analysis revealed a reduction in multiple dominant clonotypes associated with effector CD4⁺ and CD8⁺ T cells. These effects were amplified upon extended treatment with IL-2/CD25. Thus, sustained suppression of self-reactive CD4⁺ and CD8⁺ Teff cells and robust control of autoimmunity requires a constant elevation of Tregs and suggests a potential beneficial role for ILC2s and cytokine-producing NK cells.