292 - Regulation of Foxp3 expression in TGF-β induced regulatory T cells derived from human umbilical cord blood vs adult blood CD4 T cells

Abstract

Natural T regulatory cells (nTreg) have shown the potential to safely and effectively induce immune tolerance in several in vivo applications. However, the limited proliferative capacity of nTreg remains a challenge to broader clinical applications. Inducible Treg (iTreg) can restore immune tolerance in settings where nTregs are decreased or defective. However, the instability of expression of the transcription factor Fork head box P3 (Foxp3) poses a significant barrier to successful immune therapy. Further understanding of Foxp3 binding partners and their function is emerging. The transcription factor BACH2 has been shown to repress effector programs to stabilize Treg-mediated immune homeostasis in murine KO studies. However, the potential of BACH2 to stabilize Foxp3 expression in human CD4 T-cells has not been explored. In the current studies, we compared the influence of BACH2 on Foxp3 expression in UCB vs. AB naive CD4 T-cells. UCB naïve CD4 T-cells highly express BACH2, 21-fold higher compared to AB naive CD4 T-cells, at the protein and mRNA levels (p < 0.01). Expression of Foxp3 is 2.5-fold higher in iTreg derived from UCB vs. AB naïve CD4 T-cells (p < 0.001). Also, the absolute number of Foxp3+ iTreg generated from UCB vs. AB naïve CD4 T-cells is 4-fold higher. Of particular interest, the suppressive function of UCB Foxp3+ iTreg is more potent in mixed lymphocyte cultures compared to AB Foxp3+ iTreg (p < 0.001). UCB iTreg also exhibit higher surface CD25 and CTLA-4 expression as measured by flow cytometry (p < 0.001). Transient BACH2 shRNA knockdown in UCB CD4 T-cells results in ~ 42% loss of Foxp3 expression (Empty shRNA: 78.8% vs. Bach2 shRNA: 46.2%, p < 0.001) and reduced expression of molecules down-stream of Foxp3, including CTLA-4, as measured by qPCR (p < 0.01) . Chromatin immunoprecipitation (ChIP) analysis revealed that BACH2 binds to the Foxp3 promoter in UCB iTreg but not AB iTreg (p < 0.01). Furthermore, Foxp3 luciferase reporter assays demonstrated that BACH2 is transcriptionally active at the Foxp3 promoter. Overall, our results suggest that BACH2 is an important transcription factor partner that stabilizes Foxp3 expression in UCB naïve CD4 T-cells.