Background
Immune checkpoint blockade (ICB) provides durable clinical and survival benefits for a fraction of cancer patients.1β3 ICB blocks interactions between inhibitory receptors expressed by exhausted CD8+ T cells and their ligands expressed by tumor cells or antigen-presenting cells, enhancing the functionality and clonal expansion of CD8+ T cells, thereby improving the overall anti-tumor immune response.4 Recent studies have underscored the remarkable heterogeneity within CD8+ T cell exhaustion differentiation states. CD8+ T cells in these states display varying levels of proliferative potential, cytotoxic capacity, inhibitory receptor expression, and responsiveness to ICB.5β10 Numerous mouse and human cancer studies have demonstrated that stem-like progenitor exhausted CD8+ T cells respond the most to ICB.6 11β13 However, the exact anatomic locations that coordinate the differentiation and expansion of CD8+ T cells following ICB, and the precise transcriptional states of exhausted CD8+ T cells that respond best to ICB remain largely unknown.
Methods
We utilized an ICB-responsive KrasG12D P53-/- lung cancer model engineered to express the model antigen SIYRYYGL (SIY). Using paired single-cell RNA and TCR sequencing, we profiled endogenous, tumor-reactive CD8+ T cells isolated from tumors, tumor-draining lymph nodes, and spleens of mice treated with ICB. This allowed us to probe the transcriptional and phenotypic states of SIY-reactive CD8+ T cells that maximally expanded following ICB, as well as the clonal relationships between distinct transcriptional states across anatomic locations.
Results
We demonstrated that SIY-reactive CD8+ T cells expanded the most in the white pulp of the spleen following ICB and transcriptionally, these cells resembled an intermediate-exhausted phenotype. These splenic intermediate-exhausted CD8+ T cells gave rise to the majority of tumor-infiltrating clonotypes. Systemic antigen levels regulated the differentiation and expansion of intermediate-exhausted CD8+ T cells in the spleen following ICB. Increasing systemic antigen enhanced the differentiation of the splenic intermediate-exhausted population towards a blood-resident exhausted_KLR phenotype, thereby reducing numbers of tumor-infiltrating T cells in untreated mice. Conversely, eliminating antigen abrogated the expansion of splenic tumor-reactive CD8+ T cells following ICB. The expansion and differentiation of these splenic intermediate-exhausted T cells in response to ICB was dependent on antigen presentation by cross-presenting dendritic cells within the white pulp of the spleen.
Conclusions
Our findings highlight critical roles for the spleen, systemic antigen levels, and cross-presenting dendritic cells in regulating the expansion and differentiation of intermediate-exhausted CD8+ T cells following ICB, offering valuable insights into the anatomical locations and transcriptional phenotypes that underpin effective anti-tumor immunity.
Acknowledgements
David H. Koch Graduate Fellowship.
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Ethics Approval
All mouse experiments in this study were approved by MITβs Committee on Animal Care (CAC) – DHHS Animal Welfare Assurance # D16-00078.