Dissecting Systemic T Cell Responses after Stereotactic Ablative Radiotherapy in NSCLC by Single-Cell RNA and T Cell Receptor Sequencing

Abstract

There is accumulating evidence that stereotactic ablative radiotherapy (SABR) modulates immune responses to cancer; combining SABR and immunotherapy could promote the abscopal effect, but the precise effects of SABR on patients' systemic T cells is unclear. Here, we investigated SABR-induced systemic T cell response in early-stage non-small cell lung cancer (NSCLC) by single-cell RNA and T cell receptor sequencing. We performed single-cell RNA and T cell receptor sequencing on 29,439 T cells from four pairs of peripheral blood before and after SABR in early-stage NSCLC patients. Cell clustering and dimensionality reduction, SingleR, feature genes score, and TCR profiling analyses were used to investigate the heterogeneity of T cells and their changes following SABR. We identified fourteen T cell subtypes using unsupervised graph-based clustering of uniform manifold approximation and projection. By comparing the gene set scores of CD8_TE and CD8_EM pre- and post-SABR, we found both cytotoxic and inhibitory scores were significantly elevated in CD8_TE (both P < 0.001), while cytotoxic score was significantly increased in CD8_EM (P < 0.001) after SABR. We also found that CD4_TE showed increased cytotoxic scores and decreased Treg scores (P < 0.001 and < 0.05, respectively), while Treg cells showed decreased inhibitory and Treg scores (P < 0.001 and <0.01, respectively) after SABR. The proportion of large TCR clones was higher after SABR, which was accompanied by a decrease in proportion of single clones. When we compared the transcriptomes of CD8_TE cells between the single, small and large clones post-SABR, we found high expression of GZMB and KLRC3 in cells with large clones, and GZMK, IL7R, and SELL in small and single clones. This suggested that T cells after SABR with large clones may have higher cytotoxicity than those with small and single clones. Our study identified systemic T cell activation after SABR at single-cell resolution, providing unprecedented insight into the immune-modulatory role of SABR in early-stage NSCLC.