Determining FoxP3’s role in hCD8+ iTreg generation, stability, phenotype and function 3731

Abstract Description FoxP3 is a critical transcription factor for CD4 regulatory T cell (Treg) and CD4 induced Treg (iTreg) development and function and has been assumed to play a similar role in CD8 Treg. However, we now find that FoxP3 may be dispensable for human TGFb/RAPA induced CD8 Tregs (hCD8 iTreg) suppressive function. Using spectral flow cytometry and single cell RNA-sequencing of hCD8 iTreg induced from peripheral blood (PB) hCD8 T cells, we find hCD8 iTreg express many canonical CD4 Treg markers including CD39, CD25, CTLA-4, GITR, PD-1 and FoxP3. Additionally, GSEA analysis revealed significant CD4 iTreg and CD8 Treg gene-set enrichment (p < 0.0001). While hCD8 and CD4 iTreg were similarly suppressive in vitro, hCD8 iTreg maintained their suppressive potential even after FoxP3 loss. When exposed in vitro to destabilizing conditions, hCD8 iTregs, unlike CD4 iTreg, maintained their suppressive capacity, despite a significant reduction in FoxP3 expression (>80% to < 20%) at 72h. To more definitively assess whether FoxP3 is required for hCD8 iTreg generation and/or suppressor function, we tested 3 distinct methodologies that were applied to mature PB CD8 + 25- T cells during hCD8 iTreg induction: deleting FoxP3 gene; 2) knocking in a strong promoter to the endogenous FoxP3 locus; and 3) insertion of a FoxP3 over expressing construct. Results of ongoing studies ascertaining the role of FoxP3 expression in the generation, stability, phenotype and function of hCD8 iTreg will be presented. Funding Sources R01 HL11879; P01 CA065493; Hematology Research Training Program T32 HL007062; Children’s Cancer Research Fund Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)