Molecular pixelation of the CAR T cell surface proteome

Abstract Immunotherapies using CAR T cells are revolutionizing B-cell acute lymphoblastic leukemia treatments. However, the majority of patients remain unresponsive, and chronic stimulation of T cells is a common contributor that reduces effector function and persistence. We apply Molecular Pixelation, a recently developed single-cell technology for characterizing cellular surface proteomes, to determine characteristic topological surface-based proteomic signatures of CAR T cell exhaustion. We analyze 76 surface proteins on 8504 CAR T cells at a single-cell level, collected from three donors and either stimulated once or repeatedly, six times over two weeks. The abundances, polarizations, and colocalizations of surface proteins can each distinguish CAR T cells that were stimulated acutely or chronically, and all but one marker with polarization changes increased in polarization. These data also reveal disrupted adhesion signatures of protein colocalization in the peripheral supramolecular activation complex (pSMAC) and increased CD37/CD82 colocalization after chronic stimulation. These Molecular Pixelation results convey new spatial signatures for proteomic polarization and colocalization on the cell surface that represent new cell-state axes for immunology and systems biology. Competing Interest Statement T.L.R. is a founder of Arsenal Biosciences. A.T.S. is a founder of Immunai, Cartography Biosciences, Santa Ana Bio, and Arpelos Biosciences, an advisor to Wing Venture Capital, and receives research funding from Merck Research Laboratories, Allogene Therapeutics, and Astellas Pharma. E.L. is a founder of Genbio.ai and an advisor for Element Biosciences, Cartography Biosciences, Pfizer, Moleculent AB, and Pixelgen Technologies AB. The remaining authors declare no competing interests.