Integrative single-cell multi-omics of CD19-CAR ^pos and CAR ^neg T cells suggest drivers of immunotherapy response in B-cell neoplasias

How phenotypic, clonal, and functional heterogeneity of CAR-T-cells impact clinical outcomes remain understudied. Here, we integrated clonal kinetics with transcriptomic heterogeneity resolved by single-cell omics to explore cellular dynamics response of both non-transduced (CAR ^neg ) and transduced (CAR ^pos )T-cells. CAR ^neg and CAR ^pos T-cells were longitudinally interrogated in the manufactured infusion product (IP) and in-vivo at CAR-T cell expansion peak in five B-ALL patients treated with CD19CAR-T-cells (varni-cel). Significant differences were found in the cellular dynamics between CAR ^pos and CAR ^neg T-cells in response to therapy. CAR ^pos T-cells in the IP exhibited a significant higher CD4:CD8 ratio than CAR ^neg T-cells, and the CD4:CD8 CAR ^pos T-cell composition impacted therapy outcome as confirmed in a larger cohort of 24 varni-cel-treated B-ALL patients. Conversely, an inverted trend in the CD4:CD8 CAR ^pos T-cell ratio was consistently observed at the expansion peak, with clonally expanding CD8 ⁺ effector memory and cytotoxic T-cells being the most abundant populations. Expanded cytotoxic CAR ^pos γδT cells emerged at the expansion peak, and the extent of their in-vivo expansion positively correlated with treatment efficacy, which was validated in a large cohort of B-ALL patients (n=18) treated with varni-cell and B-cell lymphoma patients (n=58) treated with either lisa-cel or axi-cel. Our data provide insights into the complexity and diversity of T-cell responses following CAR-T cell therapy and suggest drivers of immunotherapy response.