The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 + helper T (T H ) cells and CD8 + cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T H 1 and T H 2 signature cytokines, e.g. , interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3 . However, rather than conforming to stringent T H 1 or T H 2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T H 1/T H 2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T H 1/T H 2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19 4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 + 'helper' and CD8 + cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T H 1 and T H 2 signature cytokines (e.g., IFNγ, TNFα, IL5, and IL13), as confirmed by the expression of master transcription factors TBX21 (T-bet) and GATA3. However, rather than conforming to stringent T H 1 or T H 2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T H 1/T H 2 function in the same cell and the regulatory T cell (T reg ) activity, although observed in a small fraction of activated cells, emerges from this hybrid T H 1/T H 2 population. GM-CSF is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status although all major differentiation subsets such as naïve, central memory, effector memory and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GMCSF, supporting the notion that 'polyfunctional' CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights to the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.