In Vitro expanded STAT1^−/− Regulatory T Cells Prevent Acute Graft Versus Host Disease (GVHD) and Promote Donor Cell Engraftment In Allogeneic Fully MHC-Mismatched Bone Marrow Transplant

Abstract 732

T_reg cells have been recognized as critical regulators of the immune response and shown to prevent the development of GVHD. However, little is known about of the role of STAT1 signaling in T_reg cells during the development of GVHD. In this study, we tried to investigate how STAT1 signaling controls donor T_reg development and function in the setting of GVHD. For this purpose we studied the role of STAT1 in natural and inducible T_reg (nT_reg and iT_reg, respectively). To better understand the influence of STAT1-deficiency on the proliferation of nT_reg cells, purified splenic STAT1^−/− or STAT1^+/+ CD4⁺CD25⁺ cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and cultured on anti-CD3 coated plates in the presence of anti-CD28 and IL-2 for 3 days and analyzed for proliferation and viability. After 72h of in vitro culture 50% of the STAT1^+/+ starting population were no longer viable compared to only 10% of STAT1^−/− cells. Furthermore, we noted a significantly increased expansion of STAT1-deficient CD4⁺CD25⁺Foxp3⁺ T_reg cells compared to STAT1^+/+ T_reg cells (p<0.001). In line with these findings, STAT1-deficiency resulted in a significantly higher proportion of CFSE^lo cells indicating vigorous proliferation (85% Foxp3⁺CFSE^lo in STAT1^−/− compared to only 65% Foxp3⁺CFSE^lo in STAT1^+/+ T_reg cells. Furthermore, at the end of the culture 30% of the STAT1^+/+ CD4⁺CD25⁺ population were Foxp3-negative compared to only 10% of the STAT1^−/− cells. We next determined the impact of STAT1 on the generation of iT_reg cells in vitro. For this purpose CD4⁺CD25⁻ cells from STAT1^−/− or STAT1^+/+ mice were cultured for 3 days on anti-CD3 coated plates in the presence of anti-CD28 antibodies, hTGF-β, mIL-2, anti-IFN-γ and anti-IL-4 for 3 days. Compared to STAT1^+/+, we observed significantly enhanced generation of iT_regs from STAT1^−/− splenocytes (19.9%±3.0% vs. 10.6%±1.3%, p=0.008). We then performed studies to assess the in vivo generation of iT_reg. For that purpose BALB/c mice were reconstituted with T Cell Depleted (TCD) 129.STAT1^+/+Bone Marrow Cells (BMC) following lethal irradiation and recipients were co-injected with CD4⁺CD25⁻ cells purified from either 129.STAT1^+/+ or 129.STAT1^−/− splenocytes. We again noted a significantly higher proportion of CD4⁺CD25⁺ Foxp3⁺ cells in recipients of CD4⁺CD25⁻STAT1^−/− cells compared to recipients of STAT1^+/+ T cells indicating a significantly increased conversion of CD4+CD25- cells into T_reg cells. To confirm the in vitro results we tested the functional ability of in vitro expanded (using anti-CD3, anti-CD28, IL-2 and TGF-β) STAT1^+/+ or STAT1^−/− T_reg cells to block induction of GVHD. GVHD was induced in BALB/c mice following lethal irradiation (800rad) and fully MHC-mismatched BMT using 129.STAT1^+/+ bone marrow cells plus 129.STAT^+/+ conventional T cells (T_con). Animals were co-injected with expanded T_reg cells from either 129.STAT1^+/+ or 129.STAT1^−/− donors at a ratio of 1:1 or 1:4 (T_reg:T_con). STAT1^−/− or STAT1^+/+ T_reg cells were equipotent in completely preventing GVHD mortality. However, compared to recipients of STAT1^+/+ T_reg recipients of STAT1^−/− T_reg showed reduced signs of GVHD morbidity as determined by a significantly improved weight development. Furthermore, recipients of STAT1^−/− T_reg showed significantly increased donor cell engraftment compared to recipients of STAT1^+/+T_reg (donor CD4⁺ [87% vs. 60%, p=0.03], CD8⁺[99% vs. 96%, p=0.04], Mac1⁺[96% vs. 77%, p=0.02] and B220⁺[100% vs. 96%, p=0.007]) cells in the recipient spleen. These observations clearly demonstrate that STAT1 is a critical regulator of T_reg cell development and expansion and that targeting STAT1 in CD4⁺ T cells may facilitate in vitro and in vivo generation/expansion of T_reg cells for therapeutic use in GVHD while also promoting donor cell engraftment.