CD8 Tregs Promote Gvhd Prevention and Restore Impaired GVL Effect Mediated By CD4 Tregs in Mice

Adoptive regulatory T-cell (Treg) therapy has enhanced the outcome of patients suffering from graft-versus-host (GVH) disease following allogeneic hematopoietic stem cell transplantation (allo-HCT); however, fear of broad immune suppression and subsequent dampening of the beneficial graft-versus-leukemic (GVL) responses remains a challenge. In order to subvert broad immune suppression, we generated alloantigen-specific induced Tregs (iTregs) from resting CD4 or CD8 T cells and tested the ability of iTregs to suppress GVH and maintain GVL responses. We utilized a clinically relevant murine model of haploidentical-HCT with the addition of host-original leukemia cell line to evaluate the effects of CD4 and CD8 iTregs in GVH and GVL responses.  While alloantigen-specific CD4 iTregs were effective in preventing GVHD (Fig. 1 A and C), they completely abrogated the GVL effect against aggressive leukemia resulting in 100% tumor mortality (Fig. 1 B and D). Mechanistically, these CD4 iTregs were found to potently suppress the expansion of effector T cells (Teffs) and their ability to secrete IFNγ and granzyme B in the recipient spleen and liver, which may contribute to the impaired GVL activity. Using similar approach, we generated alloantigen-specific CD8 iTregs and found they express higher levels of granzyme B and CTLA-4 compared to nTreg and CD4 iTregs. In vivo studies showed these CD8 iTregs moderately attenuated GVHD (Fig. 1 A and C) while completely sparing the GVL effect (Fig. 1 B and D). We thus further reasoned that the combination of CD4 and CD8 iTregs could achieve the optimal goal of allo-HCT: GVHD suppression with GVL preservation. Indeed, the combination therapy potently suppressed GVHD resulting in increased survival and decreased pathological injury to target organs than either CD4 or CD8 iTreg singular therapy (Fig. 1 A and C). More importantly, the combination therapy maintained potent GVL responses reflected by significantly decreased tumor mortality and load (Fig. 1 B and D).  Mechanistically, we observed addition of CD8 iTregs maintained the suppression of Teff expansion but restored the ability of Teffs in producing inflammatory cytokines (e.g. IFNγ and TFNα) and cytolytic effector molecules (e.g. granzyme B and TRAIL).  To our knowledge the current findings are the first to support the use of combinational iTreg therapy to achieve optimal suppression of GVHD while maintaining GVL responses.

This work was supported by NIH grants: R01 CA118116 and R01 CA169116