Background Expansion of systemic tumor-reactive T cells after immune checkpoint blockade (ICB) has been linked to successful tumor control, but regulators of systemic T cell expansion remain unknown. Using a murine lung cancer model, we previously demonstrated that flank tumors respond to ICB, while lung tumors do not. ICB-mediated control of flank tumors correlated with stronger expansion of systemic anti-tumor T cell responses, and the spleen, not tumor-draining lymph nodes (TdLNs), appeared to be the source of ICB-expanded tumor-reactive T cells.
Methods The murine KP lung cancer cell line was engineered to express the CD8+ T cell antigen SIY (KP.SIY). Mice were inoculated subcutaneously to generate flank tumors or intravenously to generate lung tumors. For single cell sequencing experiments, endogenous SIY-reactive CD8+ T cells were isolated using fluorescence activated cell sorting (FACS) and RNA sequencing was performed using SeqWell. Mice expressing the transgenic 2C T cell receptor, specific for SIY, were used as T cell donors in adoptive transfer experiments.
Results KP.SIY flank tumors generated higher numbers of splenic SIY-reactive CD8+ T cells than KP.SIY lung tumors, and the spleen was the major site of SIY-reactive CD8+ T cell accumulation after ICB. Paired single cell RNA and TCR sequencing revealed extensive clonal expansion of SIY-reactive CD8+ T cells in flank tumor-bearing mice, including multiple recurrent TCR sequences shared between mice, that was significantly reduced in the lung tumor setting. To determine the influence of anatomic site on the response to ICB, we adoptively transferred naive 2C TCR-transgenic T cells to mice bearing KP.SIY flank tumors. In vivo primed 2C T cells were FACS isolated from TdLNs or spleens and transferred into secondary tumor-bearing hosts prior to ICB treatment. Surprisingly, only 2C T cells isolated from spleens of primary tumor-bearing mice expanded in secondary hosts following ICB. Single cell sequencing of endogenous SIY-reactive CD8+ T cells confirmed distinct gene expression profiles between SIY-reactive T cells from TdLNs and spleens, and that tumor-infiltrating clonotypes were more prevalent in spleens of tumor-bearing mice than in TdLNs.
Conclusions The major source of systemic tumor-reactive CD8+ T cells after ICB in mice is the spleen, not the TdLN. The enhanced ability of splenic CD8+ T cells to expand after ICB therapy was driven by T cell-intrinsic differences which govern ICB responsiveness.
Ethics Approval The experiments in this abstract were approved by the Massachusetts Institute of Technology Committee on Animal Care, protocol number 0220-006-23, NIH assurance ID D16-00078.
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