Longitudinal single-cell analysis of a patient receiving adoptive cell therapy reveals potential mechanisms of treatment failure

Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) is being studied in multiple tumor types. However, little is known about clonal cell expansion in vitro and persistence of the ACT product in vivo. We performed single-cell RNA and T-Cell Receptor (TCR) sequencing on serial blood and tumor samples from a patient undergoing ACT, who did not respond. We found that clonal expansion varied during preparation of the ACT product, and only one expanded clone was preserved in the ACT product. The TCR of the preserved clone which persisted and remained activated for five months was previously reported as specific for cytomegalovirus and had upregulation of granzyme family genes and genes associated with effector functions (HLA-DQB1, LAT, HLA-DQA1, and KLRD1). Clones that contracted during TIL preparation had features of exhaustion and apoptosis. At disease progression, all previously detected clonotypes were detected. New clonotypes appearing in blood or tumor at disease progression were enriched for genes associated with cytotoxicity or stemness (FGFBP2, GNLY, GZMH, GZMK, IL7R, SELL and KLF2), and these might be harnessed for alternative cellular therapy or cytokine therapy. In-depth single-cell analyses of serial samples from additional ACT-treated patients is warranted, and viral- versus tumor-specificity should be carefully analyzed.