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

Program: Oral and Poster Abstracts
Type: Oral
Session: 203. Lymphocytes and Acquired or Congenital Immunodeficiency Disorders: Know Thy(mus) Self, Know Thy Enemy: From Lymphocyte Genetic Variation to Disease
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Saturday, December 7, 2024: 12:30 PM

Rushil Shah, BS1*, Konstantinos Aliazis, PhD2*, Leda Xanthi Katopodi, MS3*, Anthos A Christofides, MD, PhD1*, Halil-Ibrahim Aksoylar, PhD4*, Dimitra Karagkouni, PhD5,6*, Rinku Pal, BS7*, Lequn Li, MD, PhD8*, Nikolaos Patsoukis, PhD7*, Ioannis S Vlachos, PhD3,6,9*, Pankaj Seth, PhD10* and Vassiliki A Boussiotis, MD, PhD11

1Beth Israel Deaconess Medical Center, Harvard Medical School, BOSTON, MA
2Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Brookline, MA
3Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA
4Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, BOSTON, MA
5Department of Pathology, Cancer Research Institute, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA
6Broad Institute of Harvard and MIT, Cambridge, MA
7Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
8Beth Isreal Deaconess Medical Center, Boston, MA
9Beth Israel Deaconess Medical Center, Boston, MA
10Janssen Research & Development, Spring House, PA
11Hematology-Oncology, Beth Israel Deaconess Medical Center, Boston, MA

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-Af/f) and crossed them with CD4-Cre to selectively delete LDH-A in T cells (LDH-Af/fCD4-Cre). To generate LDH-A deficient antigen-specific CD8+ T cells, we backcrossed LDH-Af/fCD4-Cre mice with OTI-transgenic mice bearing a TCR recognizing Ovalbumin peptide (Ova257-264) presented by H-2Kb, and generated OTI/ LDH-Af/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 (TMEM)-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 TMEM 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 (TEFF) 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 TMEM 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 TMEM subsets in recipients of OTI and OTI/LDH-A-/- T cells. We sorted TMEM cells and adoptively transferred them into new hosts, which were subsequently rechallenged with Lm-OVA. LDH-A-/- deficient TMEM 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-/- TMEM cells also produced higher amounts of IFNg, and contained a greater proportion of stem-like memory T cells (TSCM). Given these findings, we sought to determine if this superior TMEM 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 TMEM-like phenotype but impairs TEFF differentiation and function within the tumor microenvironment compromising anti-tumor immunity. Our findings uncover a new role of LDH-A in CD8+ TEFF vs. TMEM responses and indicate that LDH-A has a decisive role in regulating T cell differentiation and functionality.

Disclosures: 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.

*signifies non-member of ASH