# Synthetic rescue of antigen-driven T cells and alloimmunity

> **NIH NIH R01** · TEMPLE UNIV OF THE COMMONWEALTH · 2021 · $637,613

## Abstract

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...

## Key facts

- **NIH application ID:** 10074532
- **Project number:** 5R01AI143256-03
- **Recipient organization:** TEMPLE UNIV OF THE COMMONWEALTH
- **Principal Investigator:** Jonathan A Soboloff
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $637,613
- **Award type:** 5
- **Project period:** 2019-08-12 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10074532

## Citation

> US National Institutes of Health, RePORTER application 10074532, Synthetic rescue of antigen-driven T cells and alloimmunity (5R01AI143256-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10074532. Licensed CC0.

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