Summary

Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. However, clonal kinetics and transcriptional programs that regulate the fate of CAR-T cells after infusion remain poorly understood. 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. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool 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. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion.

Overall design

We performed scRNA-seq on CD8+ CAR-T cells isolated from the IP and blood at the early, late, and very late time points from four patients who received CD19-specific CAR-T cells for refractory B cell malignancies. Single CD8+ CAR-T cells were sorted as CD8+/CD4-/EGFRt+ events and scRNA-seq was performed using the Chromium Single Cell 5’ library and V(D)J enrichment kit (10x Genomics). Libraries were sequenced on HiSeq2500.

Contributors

Alyssa Sheih 1, Valentin Voillet 2, Laïla-Aïcha Hanafi 1, Hannah A DeBerg 3, Masanao Yajima 4, Reed Hawkins 1, Vivian Gersuk 3, Stanley R Riddell 1 5 6, David G Maloney 1 5 6, Martin E Wohlfahrt 1, Dnyanada Pande 1, Mark R Enstrom 1, Hans-Peter Kiem 1 5 7, Jennifer E Adair 1 5 6, Raphaël Gottardo 2 5 6, Peter S Linsley 3, Cameron J Turtle 8 9 10

Contact

cturtle@fredhutch.org.(Cameron J Turtle)