Enforced Expression of Mitochondrial Calcium Uniporter in Donor T Cells Abolishes Gvhd Progression

During the early/intermediate phases of the immune response, calcium (Ca²⁺) signals are crucial for T cell activation, proliferation and effector differentiation. Yet, inhibiting Ca²⁺ signaling-activated master transcription factor NFAT has achieved limited success in the long-term restriction of graft-versus-host disease (GVHD), while high remission and infection rates remain major issues. This underscores the need to better understand how different calcium channels coordinate to regulate T cell alloimmunity. Mitochondria is one of the two intracellular calcium stores that buffers and modulates cytosolic Ca²⁺ response. Mitochondria calcium uniporter (MCU) complex is the sole channel through which Ca²⁺ enters the mitochondria. The role of MCU in T cell-mediated GVHD remains undetermined.

In this study, we report that enforced Mcu expression in donor T cells abolished their capacity to induce lethal GVHD. Using retroviral gene delivery system to induce or constitutively overexpress Mcu in T cells, we discovered that Mcu ectopic expression impaired donor T cell survival. RNA-seq analysis identified the activation of gene programs mediating death of activated T cells. This was induced by persistent alloantigen exposure (restimulation-induced cell death, RICD), leading to massive cell death in the liver, a GVHD target organ. Pharmacological enhancement of MCU function with a natural compound phenocopied this finding while preserving anti-leukemia potency. These data indicate that boosting MCU function in donor T cells could be a novel and effective strategy to mitigate GVHD after allogeneic hematopoietic stem cell transplantation.

Interestingly, we discovered T cells naturally downregulated MCU function during differentiation, indicating intrinsic repressive mechanisms that protect T cells from RICD. This led us to interrogate whether MCU is required for T cell alloimmunity. We generated T cell-specific Mcu conditional knockout C57/BL6 mice (Mcu-cKO). Balb/c recipients infused with Mcu-KO B6 mouse T cells developed severe liver GVHD, manifested by the dramatically reduced liver size, bile duct lesion, portal and lobular inflammation associated with massive lymphocyte infiltration. However, these Mcu-KO T cell recipients survived longer with 69% compared to 100% mortality in WT T cell recipients. Mechanistic studies showed Mcu-KO recipients had significantly fewer donor T cells in the spleen and liver, attributed to reduced proliferation capacity; and decreased IFN-g-producing cells. Meanwhile, Mcu-KO donor T cells retained GMCSF- and granzyme B-producing capacity. These data demonstrate MCU promotes T cell alloresponse, distinguishing its dispensable role in autoimmune disease and anti-viral infection models.

To delineate the molecular mechanisms through which MCU regulates T cell alloresponse, we performed transcriptome profiling on sort-purified alloreactive CD8 T cells recognizing the alloantigen H60 in balb/b mice. Mcu-KO alloreactive CD8 T cells were characterized with enhanced effector programming, loss of memory potential, positive enrichment of exhaustion feature through gene set enrichment and DEG analysis. In depth mechanistic analysis unveiled STAT5 as the master upstream regulator through which MCU modulated transcriptome. Elevated STAT5 and the expression of its target genes (Bcl2, Tox, Lef1, Gzma, etc) were observed in Mcu-KO CD8 T cells. STAT5 is a predominant IL2 signaling molecule. Mcu deficiency increased the level of CD25, which is required for the formation of high affinity IL2 receptor in CD8 T cells. IL2a (CD25) is transcriptionally activated by NFkb, the elevated activity of which was identified in Mcu-KO CD8 T cells. These observations suggest that Mcu ablation led to activation of IL2-CD25-STAT5 axis, sustaining the persistence of activated T cells while reinforcing effector programs.

Findings from this study elucidate the intricate regulatory network through which MCU regulates T cell alloimmunity, highlighting the fundamental translational potential of enhancing MCU functionality in alloreactive T cells through gene editing or pharmacological approach to reduce GVHD.