UCSF

Regulatory T cell (Treg) therapy for type 1 diabetes is a novel approach to restore immune tolerance and halt the autoimmune destruction of the pancreatic beta cells. However, a majority of infused Tregs do not persist. Low-dose IL2 can enhance Treg survival, but off-Treg effects on NK and CD8+ T cells raises concerns of exacerbating autoimmune pathology. In this study, we investigated if orthogonal IL2 could induce selective IL2 signaling in Tregs engineered to express a corresponding orthogonal IL2 receptor (ortho-IL2R), thus enhance Treg persistence and function a NOD mouse model of type 1 diabetes (NOD CD28KO). To investigate this, we utilized in vitro and in vivo assays, flow cytometry, and type 1 diabetes mouse models. We compared two orthogonal IL2 variants, 1G12 or 3A10, to determine which provided greater selectivity and potency. 3A10 showed no reactivity to wildtype (WT) IL2R, thus was a true orthogonal ligand. However, a high concentration of 3A10, 100,000IU/ml, in vitro was required to induce STAT5 phosphorylation and proliferation, likely due to its relatively low affinity for the ortho-IL2R (KD= 0.2nM) (28). Consequently, a high dose of MSA-3A10 in vivo, 30ug once or twice a week, was needed to promote proliferation and lineage stability of ortho-IL2R expressing Tregs in NSG mice. Furthermore, in NOD CD28KO mice, high doses of MSA-3A10, 30ug once or twice a week, did not induce proliferation of ortho-IL2R+ Tregs and subsequently failed to aid 2,000 islet antigen-specific-ortho-IL2R+ Tregs in preventing of diabetes in NOD CD28KO mice. In lieu of these findings, we explored another ortho-IL2, 1G12, which has a high affinity for ortho-IL2R (KD=0.04nM). However, at high concentrations has the potential to induce IL2 signaling in Tregs expressing WT-IL2R (28). In vitro at low concentrations, below 1,000IU/ml, 1G12 selectively induced IL2 signaling in ortho-IL2R+ Tregs and in vivo a low dose of MSA-1G12 (7.6ug) induced proliferation and upregulation of Treg markers in ortho-IL2R+ Tregs. Moreover, in vivo a low dose of MSA-1G12, 7.6ug once a week, enhanced the potency of 2,000 islet antigen-specific-ortho-IL2R+ Tregs resulting in 100% diabetes prevention in NOD CD28KO mice. To investigate further the mechanism of prevention of T1D, we exogenously provided 7.6ug of MSA-1G12 to NOD CD28KO mice twice a week for two weeks and found that endogenous Tregs upregulated Treg markers in response to MSA-1G12. However, we are still exploring the dynamic nature of the main immune cell responder to ortho-IL2 during the progression of T1D. Collectively, these results show both ortho-IL2 ligands could preferentially enhance ortho-IL2R+ Tregs, however, their efficacy in enhancing infused Tregs was limited by their relative affinities for the orthogonal IL2R. Finally, we explored strategies to improve the orthogonal IL2R/IL2 system by creating an autocrine system that eliminates the need for low or high dosing approaches. In turn, we also investigated the consequences of constitutive IL2 signaling. We tethered the orthogonal IL2R with orthogonal IL2 (3A10 or 1G12) with a peptide linker (a truncated 26 amino acid portion of the extracellular domain of IL2RA). We report that selective IL2 signaling can be induced through the 3A10/oIL2R and 1G12/oIL2R tethers. However, IL2 signaling is induced at different degrees which results in differential proliferation rates. The 3A10/oIL2R tether induces greater proliferation and upregulation of Treg markers and pro-survival molecules such as Bcl-2 in vitro and in vivo. While in vivo, 1G12/oIL2R tether potentially induced survival. Efforts are underway to determine if islet-antigen specific Tregs with either tethers can prevent T1D and the mechanisms occurring.