Lineage tracking reveals dynamic relationships of T cells in colorectal cancer

T cells are key elements of cancer immunotherapy(1) but certain fundamental properties, such as the development and migration of T cells within tumours, remain unknown. The enormous T cell receptor (TCR) repertoire, which is required for the recognition of foreign and self-antigens(2), could serve as lineage tags to track these T cells in tumours(3). Here we obtained transcriptomes of 11,138 single T cells from 12 patients with colorectal cancer, and developed single T cell analysis by RNA sequencing and TCR tracking (STARTRAC) indices to quantitatively analyse the dynamic relationships among 20 identified T cell subsets with distinct functions and clonalities. Although both CD8(+) effector and 'exhausted' T cells exhibited high clonal expansion, they were independently connected with tumour-resident CD8(+) effector memory cells, implicating a TCR-based fate decision. Of the CD4(+) T cells, most tumour-infiltrating T regulatory (T-reg) cells showed clonal exclusivity, whereas certain T-reg cell clones were developmentally linked to several T helper (T-H) cell clones. Notably, we identified two IFNG(+) T(H)1-like cell clusters in tumours that were associated with distinct IFN gamma-regulating transcription factors-the GZMK(+) effector memory T cells, which were associated with EOMES and RUNX3, and CXCL13(+) BHLHE40(+) T(H)1-like cell clusters, which were associated with BHLHE40. Only CXCL13(+) BHLHE40(+) T(H)1-like cells were preferentially enriched in patients with microsatellite-instable tumours, and this might explain their favourable responses to immune-checkpoint blockade. Furthermore, IGFLR1 was highly expressed in both CXCL13(+) BHLHE40(+) T(H)1-like cells and CD8(+) exhausted T cells and possessed co-stimulatory functions. Our integrated STARTRAC analyses provide a powerful approach to dissect the T cell properties in colorectal cancer comprehensively, and could provide insights into the dynamic relationships of T cells in other cancers.