B-cell-directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates.

ABSTRACT: 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 CARneg 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 CARneg 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+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg 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 CARneg 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.

Identification and Tracking of Alloreactive T Cell Clones in Rhesus Macaques Through the RM-scTCR-Seq Platform.

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.