965 Deficiency of metabolic regulator PKM2 activates the pentose phosphate pathway to generate TCF1+ progenitor CD8 T cells to improve efficacy of PD-1 checkpoint blockade

BackgroundTCF1high progenitor CD8+ T cells have been shown to mediate the efficacy of PD-1 checkpoint blockade.1-3 However, the mechanisms that govern generation of TCF1high cells are poorly understood.MethodsWe sequenced bulk RNA from tumor-infiltrating lymphocytes to identify differentially expressed genes based on tumor progression. We utilized in vitro co-cultures of tumor-specific T cells tumor cells to examine differentiation, effector function, and metabolism of T cells with different genetic and pharmacologic manipulations by flow cytometry, metabolic flux analyses, and metabolomic profiling. We performed in vivo adoptive transfers of control and genetically manipulated tumor-specific T cells into tumor-bearing mice from both a non-small cell lung cancer and a melanoma model to examine effects of genetic manipulation on differentiation and effector function, as well as determine tumor burden and overall mouse survival both in the treatment-naïve and anti-PD-1 treated contexts.ResultsRNA Sequencing demonstrated a metabolically active response in tumor-infiltrating CD8+ T cells isolated from large and late-stage tumors. Using a genetic screen targeting glycolytic enzymes up-regulated in tumor-infiltrating CD8+ T cells, we demonstrate that PKM2 deficiency (PKM2KO) enriched for TCF1high progenitor cells (figure 1). Antigen-specific PKM2KO CD8+ T cells from draining lymph nodes and tumors exhibited a central memory-like phenotype (figure 2) with reduced effector cytokine production, increased CD44/CD62L expression, and increased TCF1 and Eomes in non-small cell lung cancer and melanoma. Adoptive transfer of PKM2KO CD8+ T cells in combination with PD-1 blockade impaired tumor growth and improved survival (figure 3). PKM2KO CD8+ T cells showed reduced glycolytic flux and accumulation of glycolytic intermediates with concomitant increases in pentose phosphate pathway (PPP) metabolites. Importantly, small molecule agonism of PPP was sufficient to skew activated CD8+ T cells towards the TCF1high population (figure 4).ConclusionsHere we show that targeting glycolytic flux by deletion of pyruvate kinase muscle 2 (PKM2) results in elevated pentose phosphate pathway activity, leading to generation of an altered differentiation state responsive to PD-1 blockade. Our study demonstrates a novel metabolic reprogramming that contributes to a memory-like T cell state amenable to checkpoint blockade.AcknowledgementsThis work was supported by NIH grants T32 CA203702 and KL2-TR-002385 to GJM, and funds from the Neuberger Berman Foundation Lung Cancer Research Center to NKA and VM.ReferencesEscobar G, Mangani D & Anderson AC. T cell factor 1: A master regulator of the T cell response in disease. Sci Immunol 2020;5. Siddiqui I, et al. I Intratumoral Tcf1 + PD-1 + CD8 + T Cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 2019;50:195–211.e110. Kurtulus S, et al. Checkpoint blockade immunotherapy induces dynamic changes in PD-1-CD8+ Tumor-Infiltrating T Cells. Immunity 2019;50:181–194.e186. Luckey CJ, et al. Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells. Proc Natl Acad Sci USA Feb 2006;28:103(9):3304–9.Ethics ApprovalAll animal work was performed in accordance with an animal protocol approved by the institutional Animal Care and Use Committee at Weill Cornell Medical College (Protocol number 0806-762A).