Single-cell integrative analysis of CAR-T cell activation reveals a predominantly T_H1/T_H2 mixed response independent of differentiation

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 TH1 and TH2 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 TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell and the regulatory T cell (Treg) activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 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.