During graft-versus-host disease (GVHD), donor T cells require the histone methyltransferase Ezh2 for producing and sustaining effector T cells that mediate host tissue injury. We recently established that Ezh2 serves as a molecular gatekeeper for the generation of CD8 memory T cell precursors in GVHD, critical for the production of effector T cells in response to persistent antigen (Nat Commun 2017). However, our efforts to develop novel approaches to selectively target alloreactive effector T cells has been limited by the lack of understanding of why Ezh2 loss causes cell death of antigen-activated T cells. Stromal interaction molecule (Stim) proteins, Stim1 and Stim2, are crucial dynamic endoplasmic reticulum (ER) Ca2+ sensors and modulators of Ca2+ signals. Upon T cell receptor (TCR) ligation, Stim1 activation causes its translocation towards the plasma membrane, where it activates the Ca2+ channel Orai1, facilitating Ca2+ entry and driving T cell activation. Conditional Stim1 deletion inhibits GVHD in mice due to impaired effector differentiation. Remarkably, Stim1 deletion rescues antigen-activated Ezh2-null T cells, leading to restored production of alloreactive effector T cells in mice and severe GVHD. Therefore, we hypothesize that: A) Ezh2 and Stim1 operate coordinately to regulate the viability and function of antigen-driven T cells; and B) Ezh2/Stim1- regulated molecular pathway(s) are crucial for controlling alloreactive T cell-mediated GVHD. We further establish that the role of Stim1 in Ezh2-mediated cell death is to drive mitochondrial Ca2+ (mitoCa2+) overload since conditional deletion of the mitochondrial calcium uniporter (MCU), leads to rescue of antigen-activated Ezh2-null T cells. To establish the therapeutic potential of these findings, we performed a preliminary screen with an 800 compound library, finding 36 compounds that block T cell proliferation. Amongst them was artesunate (ART), a water-soluble derivative of artemisinin clinically approved for the treatment of malaria and known to target the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA), which pumps Ca2+ from the cytosol to the ER lumen. SERCA inhibition leads to Stim1/Orai1 activation and mitoCa2+ uptake. Preliminary investigations show that ART treatment reduces GVHD in BALB/C mice receiving allogeneic C57BL/6 T cells. Considered collectively, these findings suggest that Ezh2 regulates antigen-specific effector T cell survival through modulation cytosolic Ca2+ entry, thereby limiting mitochondrial Ca2+ loading and protecting against cell death. This hypothesis will be tested through three specific aims. In Aim-1, we will define the mechanisms that regulate the survival and differentiation of antigen-driven Ezh2/Stim1-null T cells. Aim-2 will determine the molecular mechanisms by which Ezh2 deficiency dysregulates cytosolic and mitochondria Ca2+ uptake in activated T cells. Finally, Aim-3 will examine the beneficial effect of en...