![]() To better understand changes in the composition of CAR-T cells after infusion, we studied a cohort of patients ( n = 10) who received CD19-specific CAR-T cells manufactured from bulk CD4 + T cells and CD8 + central memory-enriched (T CM) cells, which were infused in a 1:1 ratio of CD4 +:CD8 + CAR-T cells. Using single-cell RNA sequencing (scRNA-seq), we identify transcriptionally distinct clusters of infused CD8 + CAR-T cells that differ in their contribution to the CAR-T cell repertoire in blood after infusion.Ĭlonal diversity of CAR-T cells decreases after infusion We find distinct patterns of clonal behavior that contribute to the CAR-T cell population in the recipient after infusion. Thus, we examine the T cell receptor beta (TCRB) repertoire and lentiviral integration sites of CD8 + CAR-T cells isolated from the IP and from blood of patients treated with CD19-targeted CAR-T cell immunotherapy. These highly disparate patterns suggest variability in the clonal composition of infused CAR-T cells and potential differences in the ability of individual CAR-T cell clones to expand after adoptive transfer. Another group reported one patient in whom a single CD8 + CAR-T cell clone, in which the transgene had integrated into the TET2 locus, dominated at the peak of in vivo expansion 19. We previously reported in a small subset of patients with acute lymphoblastic leukemia (ALL) that CAR-T cells were polyclonal, both in the IP and at the peak of expansion in the recipient 4. Despite the importance of in vivo CAR-T cell expansion to clinical response and toxicities of CAR-T cell therapy, little is known about the clonal composition of CAR-T cells in the infusion product (IP), how clonal composition of CAR-T cells changes in the recipient after adoptive transfer, and how distinct transcriptional signatures in the CAR-T IP might affect cell fate in vivo. In vivo CAR-T cell proliferation after infusion is important for anti-tumor efficacy and may depend on several factors including the design of the CAR construct, the quality of T cells for CAR T manufacturing, the manufacturing process, the lymphodepletion regimen, infused cell phenotype, and the tumor burden and tumor microenvironment 3, 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. Lymphodepletion chemotherapy followed by infusion of CD19-specific chimeric antigen receptor modified-T (CAR-T) cells has produced durable responses in a subset of patients with relapsed and refractory B-cell malignancies 1, 2, 3, 4, 5, 6, 7, 8. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion. Although integration site does not appear to be a key driver of clonal kinetics, scRNA-seq demonstrates that clones that expand after infusion mainly originate from infused clusters with higher expression of cytotoxicity and proliferation genes. ![]() We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool after infusion. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. Here we perform TCRB sequencing, integration site analysis, and single-cell RNA sequencing (scRNA-seq) to profile CD8 + CAR-T cells from infusion products (IPs) and blood of patients undergoing CD19 CAR-T immunotherapy. However, clonal kinetics and transcriptional programs that regulate the fate of CAR-T cells after infusion remain poorly understood. Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. ![]()
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