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 quickly 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. We have shown T cell exhaustion can be driven by mitochondrial stress.¹ These features evoke an image of weak, starving T cells that are unable to sufficiently fuel their effector function. However, we and others have observed that CD8 T cells accumulate large lipid stores as they progress towards exhaustion.² What remains unclear is whether lipid accumulation in these cells contributes to their dysfunction or represents an untapped source of fuel that may be the key to their reinvigoration.

Methods Using an in vitro model of T cell exhaustion developed in our lab, we have evaluated the effect of blocking several steps in fatty acid synthesis, including mitochondrial citrate transport through the citrate carrier and malonyl-CoA production by acetyl-CoA carboxylase (ACC).³ We also deleted Slc25a1 (encoding the citrate carrier) in primary murine OT-I T cells and adoptively transferred these cells into ovalbumin-expressing B16^OVA 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, and lipid accumulation.

Results Exhausted T cells, both in vitro and ex vivo, accumulate lipid droplets due to heightened export of citrate from the mitochondria. Inhibition of mitochondrial citrate export via genetic deletion or pharmacologic inhibition of the citrate carrier SLC25A1 resulted in reduced lipid accumulation and improved polyfunctional cytokine production in vitro. Additionally, deletion of Slc25a1 improves cytokine production in adoptively transferred, tumor-specific T cells in tumor-bearing hosts.

Conclusions Taken together, our results suggest a role for mitochondrial citrate export in the accumulation of cytosolic lipids and progression of CD8 T cell exhaustion. We propose that as exhausted T cells experience mitochondrial stress, they shuttle TCA-generated citrate to the cytosol where it fuels de novo fatty acid synthesis and lipid accumulation. This pathway may be targeted to delay exhaustion or reinvigorate exhausted T cells within tumors. These data provide new insight into the metabolic mechanisms of T cell exhaustion and may inform future immunotherapeutic development.

References

1. Scharping NE, Rivadeneira DB, Menk AV, Vignali PDA, Ford BR, Rittenhouse NL, et al. Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion. Nat Immunol. 2021;22:205–15.

2. Manzo T, Prentice BM, Anderson KG, Raman A, Schalck A, Codreanu GS, et al. Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8+ T cells. J Exp Med. 2020;217.

3. Scharping NE, Menk AV, Moreci RS, Whetstone RD, Dadey RE, Watkins SC, et al. The tumor microenvironment represses T cell mitochondrial biogenesis to drive intratumoral T cell metabolic insufficiency and dysfunction. Immunity. 2016;45:374–88.