B-cell–directed CAR T-cell therapy activates CD8⁺ cytotoxic CAR^neg bystander T cells in patients and nonhuman primates

• Transcriptomic analysis of NHP and patient-derived T cells following CAR T-cell infusion identified activated CAR⁻ bystander CD8⁺ T cells.

• Cytokine exposure–induced bystander-like CD8⁺ T cells capable of T-cell receptor–independent cytotoxic killing of leukemia cells.

Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell–derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CAR^neg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CAR^neg T cells following B-cell–targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8⁺ CAR^neg T cells emerging during CAR T-cell expansion. These bystander CD8⁺ CAR^neg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8⁺ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor–independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CAR^neg bystander CD8⁺ T cells following B-cell–targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.