Targeting STAT1 Separates Graft-Versus-Host Disease (GVHD) and Graft-Versus-Leukemia (GVL) Effects in Mice

Abstract 2973

We have recently shown that loss of STAT1 signaling in donor T cells significantly reduces acute GVHD in both MHC-mismatched and mHA-mismatched BMT and is associated with increased expansion of T_reg cells. In this study we further addressed the role of STAT1 in GVL reactions and the underlying mechanisms. We first sought to delineate the role of STAT1 in the generation of CD8⁺ CTL against host-type spleen cells and A20 lymphoma targets using standard CML assays. Whole spleen cells (SPCs) or CD8⁺ selected STAT1^+/+ or STAT1^−/− cells from 129Sv mice (H2^b) were stimulated for 5 days with irradiated BALB/c (H2^d) spleen cells or A20 cells and then assessed for CTL activity against ⁵¹Cr-labelled target cells. STAT1-deficient SPCs appeared to have reduced CTL activity against host SPCs and host type A20 lymphoma cells compared to STAT1^+/+ splenocytes. In contrast, isolated STAT1-deficient CD8⁺ cells had significantly increased CTL activity against both host-type targets and A20 cells. We then assessed the role of STAT1 in mediating GVL effects in vivo using a fully MHC-mismatched (129 [H2^b]→BALB/c [H2^d]) strain combination and the A20 lymphoma model. Lethally irradiated (800rad) BALB/c mice received T cell-depleted (TCD) wildtype 129 bone marrow cells (BMCs, 5×10E6) plus pan-T cells (5×10E5) from either 129.STAT1^−/− or 129.STAT1^+/+ donors. In addition, recipients were injected with 5×10E5 luciferase-expressing A20 lymphoma cells (A20-luc) on day 0. As expected animals receiving STAT1-deficient T cells showed significantly reduced GVHD-induced mortality (mean survival time [MST] not reached versus 11 days, p<0.001). Mice receiving TCD BMCs alone plus A20-luc had a MST of 25 days after BMT with all animals succumbing to death by tumor. Mice receiving TCD BMCs plus wild type pan-T cells and challenged with A20-luc died before day 20 due to severe GVHD (MST 10 days post-BMT). In contrast, mice receiving STAT1^−/− pan-T and A20-luc cells showed significantly prolonged survival (MST 43 days post-BMT, p=0.0027, Log-rank test). Sustained GVL effects of STAT1-deficient splenocytes against A20-luc cells were further confirmed by bioluminescence imaging on days +10 and days +20 post-BMT. As we recently reported that absence of STAT1 in donor lymphocytes impairs Th1 differentiation and enhances T_reg generation in CD4⁺ lymphocytes, we further investigated wether absence of STAT1 would influence differentiation of CD8⁺ cells. Our data revealed that CD8⁺ cells lacking STAT1 failed to be activated into IFN- γ-producing cells upon anti-CD3/CD28 stimulation. However, when cultured under Th1 conditions (i.e. in the presence of IL-12 and neutralizing anti-IL4 antibodies), IFN- γ secretion could be restored. This intact CD8⁺ cell differentiation was also observed in vivo on day +6 post-BMT in recipients of STAT1^−/− BMCs plus splenocytes where we were able to detect comparable levels of IFN- γ-expressing CD8⁺ cells compared to recipients of STAT1^+/+ donor grafts in both MHC- and mHA-mismatched BMT settings. Furthermore, we could demonstrate preserved CTL function of STAT1-deficient CD8⁺ in vivo. For that purpose BALB/c mice were reconstituted with 129 wildtype BMC together with either STAT1^+/+ or STAT1^−/− CD8 T cells. On day+37 post-BMT mice were infused with CFSE-labelled host splenocytes as targets cells. Similar clearance of CFSE-labelled target cells was observed between STAT1-deficient and STAT1 wildtype cells. These results suggest that STAT1 in donor T cells may serve as a potential target to separate GVHD and GVL through promoting T_reg generation from CD4⁺ cells while conserving or even promoting Tc1 differentiation of CD8⁺ cells.