Monocarboxylate transporter 1 deficient donor T cells rewire metabolism and ameliorate aGVHD lethality

Abstract

Abstract Introduction Allogeneic hematopoietic stem cell transplantation is limited by complications, including acute graft-versus-host disease (aGVHD). Donor graft naive T cells that are alloreactive differentiate into effector T cells, as key drivers of aGVHD, and produce proinflammatory cytokines and cytotoxic molecules. Alloreactive effector T cells rapidly proliferate and predominantly rely on aerobic glycolysis to generate ATP and NADH. The end product of aerobic glycolysis in effector T cells is lactate, which regenerates NAD+. Lactate is co-exported with H+ ion to the extracellular space via monocarboxylate transporter 1 (MCT1), thereby maintaining intracellular pH and lactate balance. Whereas effector T cells rely on aerobic glycolysis, regulatory T cells (Tregs) that inhibit effector T cells and central memory T cells that have a reduced aGVHD capacity primarily depend on mitochondrial respiration. We hypothesized that MCT1 deletion in donor T cells would render alloreactive effector T cells acidotic, rewire metabolism, and reduce proliferation. Therefore, we investigated the metabolic and functional effects of MCT1 deletion in T cells and its impact on aGVHD progression. Methods To study the role of MCT1 in aGVHD progression, we used donor T cell-specific MCT1 knockout (KO) mice (MCT1fl/fl× CD4-Cre+) and lethally irradiated fully MHC-mismatched recipients that received bone marrow plus MCT1 KO or wild-type (WT) donor T cells. Outcomes included survival, weight loss, T cell phenotype, and infiltration into aGVHD target organs (spleen, mesenteric lymph node, liver, small intestine, and large intestine). In vitro metabolic profiling of MCT1 KO T cells stimulated via anti-CD3/CD28 was performed using flow cytometry, Seahorse analyzer, and isotope-labeled glucose tracing by mass spectrometry. Results As compared to WT T cells, MCT1 KO T cells showed up to 30 percent reduced proliferation, diminished extracellular acidification rate, and increased oxygen consumption rate. [U-13C6]glucose tracing that showed an increase in M+2 citrate and malate enrichment and a decrease in extracellular lactate in MCT1 KO T cells. Moreover, a two-fold increase in the lactate dehydrogenase (LDH)B to LDHA ratio, and a 3.9-fold increase in the M+3 pyruvate to M+3 lactate ratio from isotope-labeled glucose tracing were observed. In vitro, MCT1 KO and WT T cells differentiated into Tregs to a similar extent, but MCT1 KO Tregs had elevated expression of Treg-associated antigens, including Foxp3, LAG3, CD39, ICOS, and PD1, likely due to increased oxidative metabolism and high intracellular lactate that can be used as a Treg fuel source in contrast to effector T cells. Irradiated recipients receiving MCT1 KO vs WT donor T cells had reduced aGVHD lethality (2 pooled experiments, n = 17/group; 3-month survival of 70 percent in KO vs 0 percent in WT; p<0.0001) along with improved clinical scores. In recipients of MCT1 KO vs WT T cells assessed on day 14, aGVHD organs had an increased frequency of central memory T cell, known to have a lower aGVHD capacity, and up to a 10-fold increase in Treg (CD25+Foxp3+):T cell ratio. In recipients of MCT1 KO T cells, a lower T cell frequency and over 70 percent reduction in number was seen in liver, small intestine, and large intestine. Splenic and mesenteric lymphoid MCT1 KO T cells downregulated non-lymphoid homing markers CCR9 and ɑ4β7 and upregulated lymphoid homing markers CCR7 and CD62L, findings that may contribute to fewer lymphoid T cells migrating to GVHD organs. Purified MCT1 KO donor T cells in the spleen, liver, small intestines, and large intestine had attenuated expression of cytotoxic molecules, including Granzyme A, Granzyme B, and Perforin. Despite fewer effector T cells, lower cytolytic molecule expression, and a higher Treg frequency, under aGVHD conditions, MCT1 KO donor T cells effectively cleared A20-luciferase+ lymphoma cells for 30 days with a 75 percent survival rate, in contrast to uniform aGVHD lethality in WT mice that died without evidence of tumor cells. Conclusions These findings underscore an obligate role of MCT1in alloreactive T cell responses and its divergent effects on alloreactive effector versus central memory and Treg subsets. Targeting MCT1 with inhibitors including AZD3965 may represent a novel therapeutic strategy to mitigate aGVHD without abrogation of graft-versus-tumor effects.