Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties

T cell responses within pediatric brain tumors (PBT) remain poorly understood. We performed single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (TCR-Seq) of patient-derived brain tumor-infiltrating T cells to map T cell molecular profile with TCR repertoire and clonality. We demonstrate marked clonal expansion of intra-tumoral T cells and reveal their differential phenotype, transcriptional state and functional properties within brain tumors. To understand T cell responses within highly immunogenic tumors that respond to checkpoint blockers, we undertook analysis of human non-small cell lung cancer (NSCLC). We performed single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (TCR-Seq) of patient-derived lung tumor-infiltrating T cells to map T cell molecular profile with clonality. PBT: Tumor samples were obtained from pediatric patients with brain tumors. Tumor samples were disintegrated and cells from each donor were labelled with dead cell discriminating dye, fluorophore-tagged FACS antibodies and unique anti-human hashtag antibodies against beta2 microglobulin for donor deconvolution. T cells from each donor were FACS-sorted for scRNA-seq using the 10X genomics platform. Experiments were performed on five separate experimental days (5 batches). Two libraries of pooled, live, singlet-gated, CD45+CD3+ T cells were generated from each of the first 3 experiments. And two libraries of pooled, live, singlet-gated cells (one of CD8+ T cells and another of CD4+ T cells), were generated from each of the last 2 experiments. NSCLC: Tumor samples were obtained from 10 patients with non-small cell lung cancer. Tumor samples were disintegrated and cells from each donor were labelled with dead cell discriminating dye, fluorophore-tagged FACS antibodies and unique anti-human hashtag antibodies against beta2 microglobulin for donor deconvolution. T cells from each donor were FACS-sorted for scRNA-seq using the 10X genomics platform. Two and four libraries of pooled, live, singlet-gated, CD45+CD3+CD4+ T cells and CD45+CD3+CD8+ T cells were generated respectively from the 10 donors.