Clonal replacement of tumor-specific T cells following PD-1 blockade [bulk RNA]

Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, the clonal origin of tumor-specific T cells following checkpoint blockade in patients remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)- sequencing on site-matched tumors from patients with basal cell carcinoma (BCC) pre- and post-anti-PD-1 therapy. Tracking TCR clonotypes and transcriptome phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the response to treatment was accompanied by a clonal expansion of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this response was not accompanied by an expansion of pre-existing tumor-specific T cell clonotypes; rather, expanded T cell clones post-therapy comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in tumor-specific exhausted CD8+ T cells, compared to other distinct T cell phenotypes, and was more evident in patients who exhibited a clinical response to treatment. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that the chronic activation of pre-existing tumor-infiltrating T cells may limit their re-invigoration following checkpoint blockade, and that a successful response relies on the expansion of a distinct repertoire of tumor-specific T cell clones. Overall design: CD4+ T helper cells were sorted as naive T cells (CD4+CD25-CD45RA+), Treg (CD4+CD25+IL7Rlo), Th1 (CD4+CD25-IL7RhiCD45RA-CXCR3+CCR6-), Th2 (CD4+CD25-IL7RhiCD45RA-CXCR3-CCR6-), Th17 (CD4+CD25-IL7RhiCD45RA-CXCR3-CCR6+), Th1-17 (CD4+CD25-IL7RhiCD45RA-CXCR3+CCR6+), and Tfh subsets (CXCR5+ counterparts of each). RNA-seq cDNA library construction was performed using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) with 2?ng of input RNA. Sequencing libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina).

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Yost KE, Satpathy AT, Wells DK, Qi Y, Wang C, Kageyama R, McNamara KL, Granja JM, Sarin KY, Brown RA, Gupta RK, Curtis C, Bucktrout SL, Davis MM, Chang ALS, Chang HY