Abstract There is growing appreciation for the emergence of CAR neg bystander T cells after CAR-T cell infusion. However, their phenotypic and transcriptomic hallmarks and mechanisms of activation remain uncertain. We performed single-cell RNA-Seq (scRNA-Seq) on non-human primate (NHP) and patient-derived T cells to interrogate CAR neg T cells following B cell targeted CAR-T cell therapy. In a 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 NK-cell markers ( KIR3DL2, CD160, KLRD1 ), chemokines and chemokine receptors ( CCL5, XCL1, CCR9 ), and downregulation of naive T cell-associated genes ( SELL, CD28 ). A transcriptionally similar population was identified in patients following Tisangelecleucel infusion. Mechanistic studies revealed that IL-2 and IL-15 exposure induced bystander-like CD8+ T cells. These T cells efficiently killed leukemic cells through a TCR-independent mechanism. Together, these data identify bystander CD8+ T cells as a novel mechanism by which CAR-T cell infusion can induce further anti-leukemic activity, measurable in both NHP and in patients. Statement of Significance We have deeply interrogated CAR neg bystander CD8+ T cells during CAR-T cell expansion in non­human primates and patients receiving Tisangelecleucel to identify the unique transcriptomic signature defining these cells, and to determine that IL-2-and IL-15-induced cytotoxic bystander T cells are capable of killing in a TCR-independent manner. These data highlight the potential of bystander T cells for leukemia control and provide a critical foundation for their future analysis.

B cell directed CAR-T cell therapy results in activation of CD8+ cytotoxic CAR-negative bystander T cells in both non-human primates and patients