Distinct phenotypic states and spatial distribution of CD8⁺ T cell clonotypes in human brain metastases

Metastatic disease in the brain is difficult to control and predicts poor prognosis. Here, we analyze human brain metastases and demonstrate their robust infiltration by CD8⁺ T cell subsets with distinct antigen specificities, phenotypic states, and spatial localization within the tumor microenvironment. Brain metastases are densely infiltrated by T cells; the majority of infiltrating CD8⁺ T cells express PD-1. Single-cell RNA sequencing shows significant clonal overlap between proliferating and exhausted CD8⁺ T cells, but these subsets have minimal clonal overlap with circulating and other tumor-infiltrating CD8⁺ T cells, including bystander CD8⁺ T cells specific for microbial antigens. Using spatial transcriptomics and spatial T cell receptor (TCR) sequencing, we show these clonally unrelated, phenotypically distinct CD8⁺ T cell populations occupy discrete niches within the brain metastasis tumor microenvironment. Together, our work identifies signaling pathways within CD8⁺ T cells and in their surrounding environment that may be targeted for immunotherapy of brain metastases.