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Background The efficacy of immunotherapy depends on the presence and persistence of functional immune cells within the tumor. While tumor-specific T cells can be activated and infiltrate the tumor microenvironment, they are progressively rendered dysfunctional by the combination of chronic antigen stimulation and metabolic stress, resulting in an altered differentiation state, termed exhaustion. Indeed, exhaustion remains a significant hurdle for immunotherapeutic success. Previously, we have shown that the tumor microenvironment represses mitochondrial metabolism and T cell metabolic fitness directly impacts effector function. However, how persistent immunologic signals directly cross talk with mitochondria remains unclear. We have shown that exhausted T cells accumulate stored carbon outside the mitochondria in the form of lipid droplets and protein hyperacetylation. We hypothesize that excess carbon is exported from dysfunctional mitochondria to the cytosol, however; it remains unclear whether this accumulation of stored carbon contributes to exhausted T cell dysfunction or represents an untapped source of fuel that may be key to their reinvigoration.Methods We have evaluated the effect of blocking mitochondrial carbon export via the citrate carrier (CIC, encoded by Slc25a1) both in vitro and in vivo. Using CRISPR-Cas9, we deleted Slc25a1 in primary murine OT-I T cells and assessed their metabolic capacity. We also adoptively transferred these cells into ovalbumin-expressing B16OVA tumor bearing mice to evaluate the effect of this gene deletion on antigen-specific T cell function in the tumor microenvironment. We immunophenotyped the cells for markers of terminal exhaustion, cytokine production, lipid accumulation, and protein acetylation.Results Exhausted T cells, both in vitro and ex vivo, accumulate stored carbon due to heightened export of citrate from the mitochondria. Inhibition of mitochondrial citrate export via genetic deletion or pharmacologic inhibition of Slc25a1 increased both oxidative and glycolytic metabolism and also reduced the accumulation of lipid and protein acetylation in exhausted T cells. Additionally, deletion of Slc25a1 reduces exhaustion and improves tumor control in adoptively transferred, tumor-specific T cells in tumor-bearing hosts.Conclusions Our results support a model in which mitochondrial citrate export results in accumulation of stored carbon and progression to exhaustion in T cells. As exhausted T cells experience mitochondrial stress, they shuttle TCA-generated citrate to the cytosol, where it fuels lipid accumulation and protein acetylation. Our study provides new insight into the metabolic mechanisms of T cell exhaustion and may inform future immunotherapeutic development, as these pathways may be leveraged to both delay exhaustion or alter the functional status of pre-existing exhausted T cells.Ethics Approval Approval Date: 7/25/2023 To Whom It May Concern: The University of Pittsburgh’s Institutional Animal Care and Use Committee has reviewed and approved the research proposal referenced above. The committee finds that the protocol meets the standards for humane animal care and use as set by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals. Sincerely, Deborah L. Chapman, PhD Institutional Animal Care and Use Committee The three year term of this protocol will expire on 7/25/2026. A full de novo rewrite and review must be completed and approved before this date to continue the project after this protocol expires.