Dual Functions of Mitochondrial Calcium Uniporter in T Cell Alloimmunity
Authors: WANG Ying , Li Alicia Huaze , sanders-Braggs Ruqayyah , Zhou Yan , Chen Mimi , Bui Tien , Zhu Hongfa , Soboloff Jonathan , Elrod John , Hexner Elizabeth O , Zhang Yi 
Published: December 2023

Despite pharmacological prophylaxis using calcineurin inhibitors, graft-versus-host disease (GVHD) still occurs in patients undergoing allogeneic hematopoietic stem cell transplantation. However, the mechanism that underlies the breakthrough of alloreactive T cell responses following calcium signaling inhibition remains poorly defined. The mitochondrial calcium uniporter (MCU), which is located in the inner mitochondrial membrane, controls Ca2+ uptake into the mitochondria matrix. Past dogma suggested that lack of mitochondria calcium uptake would compromise T cell immune response due to suboptimal NFAT activation upon TCR ligation. However, the exact role of MCU in the regulation of alloreactive T cell responses has not been previously determined. Here, we demonstrate dual functions of MCU in alloreactive T cell response and GVHD maintenance, highlighting MCU as a potential novel target to manipulate T cell alloreactivity. Using T cell-specific Mcu conditional knockout C57/BL6 (B6) mice (Mcu-cKO), we found that compared to WT T cells, B6 Mcu-cKO T cells induced less severe GVHD in Balb/c mice, with prolonged survival and significantly lower number of donor effector cells in the spleen and liver early after transplantation. Surprisingly, these Mcu-cKO T cell recipients developed severe liver GVHD by 60 days after transplantation, as evidenced by extensive lymphocytic infiltration in the liver, while to a lesser extent in the skin and intestine in these GVHD mice. Flow cytometric analysis of donor T cells, which were harvested between 5 and 8 weeks after transplantation, showed that Mcu-cKO T cells retained the IFN-g- and GM-CSF-producing capacity, but acquired stronger capacity of producing the cytotoxic molecule granzyme B. To comprehensively delineate the role of MCU in allogeneic T cell response, we used MHC-identical but minor histocompatibility antigen (miHA)-mismatched B6-to-Balb/b GVHD model, in which alloreactive CD8+ T cells can be identified with H60 peptide/MHC class-I dimer staining. Similarly, loss of Mcu in T cells caused less severe GVHD, attributed to the decreased number of H60+CD8+ effector T cells that showed impaired proliferation capacity. RNA-seq analysis of liver-infiltrating H60+CD8+ T cells revealed that Mcu ablation resulted in decreased expression of cell proliferation genes (Pclaf, Cdc45, Top2a, Cdca8, etc), increased expression of genes (Cd7, Bcl2, CD96, Eomes, Ezh1, etc) associated with memory T cells. These results demonstrated that Mcu is required for the expansion of alloreactive T cells, but restricts their memory-associated transcriptional programs during GVHD maintenance. Further characterization showed these alloreactive Mcu-cKO T cells produced higher levels of tissue migration molecules (CCR9, CCR5, etc) and lower death molecule (Lpin1) than their WT counterparts. Using gain-of-function strategy of retroviral Mcu overexpression in T cells (McuOE), we found McuOE CD8+ T cells displayed enhanced effector differentiation represented by higher expression of effector molecules (Ifng), decreased expression of stemness and memory associated molecules (Tcf7, Id3), and reduced expression of migration molecules (CCR9, CXCR3, etc). The cultured McuOE CD8+ T cells contracted earlier than WT. These results were further validated in vivo using the B6-to-Balb/c GVHD model. These data demonstrate a negative regulatory role of MCU in the tissue migration and persistence of alloreactive T cells. Notably, human T cells from patients with severe GVHD produced lower levels of MCU protein compared to that of non-GVHD patients. Our findings identified for the first time the dual functions of MCU in the regulation of T cell alloimmunity. MCU-mediated mitochondria calcium uptake promotes early expansion of alloreactive T cells but restricts the expression of memory-associated gene programs and tissue migration molecules. Strategies of enhancing MCU functionality in alloreactive T cells could potentially reduce their memory potential and tissue infiltration capacity, thereby reducing GVHD.