Loss of LDH-a Accelerated CD8⁺ T Memory Cell Differentiation but Impairs Anti-Tumor Responses

Lactate Dehydrogenase A (LDH-A) catalyzes the conversion of pyruvate into lactate in the last step of glycolysis, and its heightened expression is associated with the enhanced glycolytic metabolism in solid and hematopoietic cancers. LDH-A plays an essential role in the survival and pluripotency of hematopoietic stem cells (HSCs) via sustained glycolysis. Loss of LDH-A reduced HSCs mitochondrial fitness, maintenance, and repopulation ability. Activated T cells rely on glycolysis to engage in effector functions however the role for LDH-A in CD8⁺ T cell differentiation is unclear. We generated mice with conditional targeting of the LDH-A allele (LDH-A^f/f) and crossed them with CD4-Cre to selectively delete LDH-A in T cells (LDH-A^f/fCD4-Cre). To generate LDH-A deficient antigen-specific CD8⁺ T cells, we backcrossed LDH-A^f/fCD4-Cre mice with OTI-transgenic mice bearing a TCR recognizing Ovalbumin peptide (Ova_257-264) presented by H-2Kb, and generated OTI/ LDH-A^f/fCD4-Cre (OTI/LDH-A^-/-) mice. Antigen-specific T cells from OTI or OTI/LDH-A^-/- mice were adoptively transferred into syngeneic recipients followed by immunization with Ova-expressing Listeria monocytogenes (Lm-OVA). During the effector phase, 8 days after infection, OTI/LDH-A^-/- T cells had diminished expansion compared to OTI cells and displayed a T memory (T_MEM)-like signature with expansion of memory precursors, higher expression of CD127 and lower expression of KLRG1. Gene expression analysis showed that LDH-A deficient T cells were enriched for genes encoding factors known to identify T_MEM CD8⁺ cells, including Bcl6, Tcf7 and its targets Eomes and Id3, and the Klf2 targets CCR7 and CD62L. In contrast, transcripts encoding regulators of T effector (T_EFF) differentiation such as Prdm1, T-bet, KLRG1, IFNγ were enriched in control OTI cells. The expression of key enzymes involved in lipolysis and FAO were also significantly different. LDH-A deficient cells displayed increased expression of Cpt1a, the rate-limiting enzyme involved in transport of fatty acids into the mitochondria for β-oxidation. These cells also had elevated transcripts encoding regulators of lipolysis, Lal and Atgl, and the glycerol transporter aquaporin 9 (Aqp9), involved in T_MEM development. Metabolomics analysis showed high levels of metabolic intermediates of glycolysis and TCA cycle. During the memory phase, 60 days after infection with Lm-Ova, there were no numerical differences in T_MEM subsets in recipients of OTI and OTI/LDH-A^-/- T cells. We sorted T_MEM cells and adoptively transferred them into new hosts, which were subsequently rechallenged with Lm-OVA. LDH-A^-/- deficient T_MEM cells were functionally superior compared to their OTI counterparts as determined by reduced CFUs per spleen in hosts receiving OTI/LDH-A^-/- T cells. Rechallenged OTI/LDH-A^-/- T_MEM cells also produced higher amounts of IFNg, and contained a greater proportion of stem-like memory T cells (T_SCM). Given these findings, we sought to determine if this superior T_MEM phenotype would confer greater anti-tumor immunity. We injected syngeneic hosts with B16-F10 melanoma expressing OVA (B16-OVA), then transferred OTI or OTI/LDH-A^-/- T cells and monitored tumor growth. Strikingly, we found significantly larger tumors, a higher CD4/8 T cell ratio, and less CD8⁺ T cell infiltration in recipients of OTI/LDH-A^-/- T cells. OTI/LDH-A^-/- T cells within the tumor had higher expression of the immunosuppressive protein ICOS, and hallmark features of T cell exhaustion including higher expression of TIM-3, and lower levels of Slamf6 (Ly108) and CD62L. These results indicate that selective ablation of LDH-A in CD8⁺ T cells imprints a T_MEM-like phenotype but impairs T_EFF differentiation and function within the tumor microenvironment compromising anti-tumor immunity. Our findings uncover a new role of LDH-A in CD8⁺ T_EFF vs. T_MEM responses and indicate that LDH-A has a decisive role in regulating T cell differentiation and functionality.

Vlachos:Harvard Stem Cell Institute: Research Funding; NIDDK: Research Funding; Guidepoint Global: Consultancy; NCI: Research Funding; NHLBI: Research Funding; Mosaic: Consultancy. Seth:Janssen Research & Development: Ended employment in the past 24 months.