Ezh2 and Intracellular Ca2+ Signals Interdependently Coordinate Gvhd and CAR T Cell Responses

Despite significant advances in graft-versus-host disease (GVHD) prevention and treatment, calcineurin inhibitor (CNI)-based standard GVHD prophylaxis in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has limited efficacy in controlling acute and chronic GVHD. Thus, inhibition of calcium (Ca2+) signaling is insufficient to suppress the generation and maintenance of alloreactive T cells that mediate host tissue injury. Recent studies suggested that CNI-dependent alloreactive T cells possess great ability to persist and mediate chronic-like GVHD in mice. The molecular events by which these T cells breakthrough CNI inhibition have not been previously defined. Ezh2, a chromatin-modifying epigenetic regulator, silences expression of gene programs critical for multiple cellular processes. Ablating Ezh2 in T cells inhibits GVHD and anti-tumor activity, largely due to massive antigen-activated T cell death. Increased Ca2+ signals in activated T cells are known to induce their cell death and dysfunction. However, the relationship between Ezh2 and intracellular Ca2+ response generation in GVHD has never been previously examined. We report here that Ezh2 and Ca2+-mediated signals operate coordinately to regulate the viability and effector function of GVHD T cells. Blockade of Ca2+ signal by conditional deletion of Stim1, an endoplasmic reticulum (ER) Ca2+ sensor required for Ca2+ entry in T cells, rescued non-viable Ezh2-null alloreactive T cells, as well as restored their capacity to mediate GVHD in mice after allo-HSCT. Moreover, while STIM1-null T cells typically exhibitdecreased effector differentiation and function of GVHD T cells, this was restored by deletion of Ezh2 in Stim1-null T cells. These data identify the interdependent roles of Ezh2 and Ca2+ signals in activation, effector differentiation and survival of alloreactive T cells. To understand how Ezh2 acts as 'brake“ for Ca2+ signals in T cells, we performed bulk-RNA-sequencing analysis on Ezh2/STIM1 dual knockout T cells. Ezh2 directly repressedexpression Itpr2, which encodes the ER Ca2+ release channel 1,4,5-trisphosphate receptor (IP3R2), thereby interfering with ER Ca2+ release and subsequent cytosolic Ca2+ entry. Combined deletion of Ezh2 and Itpr2 genes restored the inability of allogeneic Ezh2-null T cells to induce lethal GVHD. Furthermore, the co-dependence of Ezh2 and Iptr2 were similarly observed in CD19-directed CAR-T mediated elimination of CD19-expressing C1498 acute myeloid leukemia in mice. Itpr2 loss in Ezh2-null CAR-T cells led to their improved survival, expansion, and production of IFN-g-producing effector CAR-T cells. Collectively, our findings identify that Ezh2 suppresses the expression of Iptr2 to prevent excessive Ca2+ signal generation and antigen-driven T cell death and T cell dysfunction. These observations reveal a potential therapeutic window for the treatment of GVHD focused on increasing intracellular Ca2+ signals to eliminate alloreactive T cells, which is opposing to the current concept of CNI treatment. Furthermore, targeting this Ezh2-Itpr2 axis may have broad implications in the regulation of other types of antigen-driven T cell responses, such as anti-tumor immunity, autoimmunity and graft rejection of solid organ transplantation.