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 enhancing T cell Ca2+ load to modulate GVHD and GVL activity in mice. Completion of these experiments will provide novel insights into T cell biology, T cell-mediated inflammatory disorders such as GVHD and autoimmune diseases, and lead to development of novel methods for improving the efficacy of immunotherapy for chronic infections and cancer.
Conditional deletion of either Stromal interaction molecule 1 (Stim1), which functions as crucial Ca2+ sensors and modulators in T cells, or Ezh2, which is a histone methyltransferase, leads to inhibition of alloreactive T cell-mediated graft-versus-host disease (GVHD). Surprisingly, Stim1 deletion rescues Ezh2-null T cells, leading to production of alloreactive effector T cells in mice and severe GVHD. The investigations proposed within focus on: 1) illuminating how Ca2+-driven molecular pathways are integrated with Ezh2-controlled epigenetic effects to control T cell immunity and identifying how Ezh2 deficiency dysregulates Ca2+ entry in T cells, and 2) establishing the beneficial effects of enhancing T cell Ca2+ load to modulate GVHD and GVL activity in mice.