Combining mass cytometry with analytical approaches, Fisher et al. performed a detailed, quantitative, single-cell analysis of signaling interactions in CD28-CD3ζ CAR T cell activation. Cell expansion boosted response to stimulation, but also increased tonic signaling via CD3ζ, which increased CAR T cell exhaustion. Transduction of γδT cells with a chimeric costimulatory receptor (CCR) that contained a targeting domain and the DAP10 signaling domain set up an AND logic gate that required both CCR and γδ-TCR (which provided CD3ζ signaling) ligation for activation, thus reducing tonic signaling and off-tumor toxicity and enhancing the cytotoxic response.
Learn more about the use of γδ cells in CAR T cell therapy at ReFigure.
Despite the benefits of chimeric antigen receptor (CAR)-T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3zeta, we performed similar analyses on gammadeltaT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3zeta but containing DAP10 stimulatory domains. These CCR-gammadeltaT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-gammadeltaT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies.
Author Info: (1) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial
Author Info: (1) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. (2) Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Department of Applied Physics and Applied Mathematics, Columbia University, New York, NY 10027, USA. (3) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. (4) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. (5) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. (6) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. (7) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. (8) UCL Cancer Institute, 72 Huntley St., Fitzrovia, London WC1E 6AG, UK. (9) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. (10) Department of Haematology and Oncology, Great Ormond Street Hospital, London WC1N 3JH, UK. (11) UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK. peerster@gmail.com j.anderson@ucl.ac.uk. UCL Cancer Institute, 72 Huntley St., Fitzrovia, London WC1E 6AG, UK. (12) Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. peerster@gmail.com j.anderson@ucl.ac.uk. Parker Institute for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
