CAR+ and CAR- T cells differentiate into an NK-like subset that is associated with increased inflammatory cytokines following infusion

Chimeric antigen receptor (CAR) T cells have demonstrable efficacy in treating B-cell malignancies. Factors such as product composition, lymphodepletion and immune reconstitution are known to influence functional persistence of CAR + T cells. However, little is known about the determinants of differentiation and phenotypic plasticity of CAR + T and immune cells early post-infusion. We report single cell multi-omics analysis of molecular, clonal, and phenotypic profiles of CAR + T and other immune cells circulating in patients receiving donor-derived products. We used these data to reconstruct a differentiation trajectory, which explained the observed phenotypic plasticity and identified cell fate of CAR + and CAR - T cells. Following lympho-depletion, endogenous CAR - CD8 + and γ□ T cells, clonally expand, and differentiate across heterogenous phenotypes, from a dominant resting or proliferating state into precursor of exhausted T cells, and notably into a terminal NK-like phenotype. In parallel, following infusion, CAR + T cells undergo a similar differentiation trajectory, showing increased proliferation, metabolic activity and exhaustion when compared to circulating CAR - T cells. The subset of NK-like CAR + T cells was associated with increasing levels of circulating proinflammatory cytokines, including innate-like IL-12 and IL-18. These results demonstrate that differentiation and phenotype of CAR + T cells are determined by non-CAR induced signals that are shared with endogenous T cells, and condition the patients’ immune-recovery. One Sentence Summary CAR + and CAR - CD8+ T cells share a differentiation trajectory terminating in an NK-like phenotype that is associated with increased inflammatory cytokines levels. Graphical Abstract