We present a protocol to localize T cell receptor clones using the Visium spatial transcriptomics platform. This approach permits simultaneous localization of both gene expression and T cell clonotypes in situ within tissue sections. T cell receptor sequences identified by this protocol are readily recapitulated by single-cell sequencing. This technique enables detailed studies of the spatial organization of the human T cell repertoire, such as the localization of infiltrating T cell clones within the tumor microenvironment. For complete details on the use and execution of this protocol, please refer to Sudmeier et al. (2022).
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.