Single-cell molecular pixelation of CAR T-cell surface proteome reveals spatial patterns characteristic of chronic stimulation

Customized immunotherapies using CAR T-cells are already making their way into the clinic and are showing much promise for the treatment of cancers including leukemia. However, stimulating CAR T-cells against the target antigen, such as that presented on a tumor, has the challenge that chronic stimulation leads to deterioration of the treatment potential of the CAR T-cells. Identifying the effects of chronic stimulation is currently challenging, making it difficult to select CAR T-cells that are effective for treatment. We applied a recently developed antibody-based spatial profiling technique for characterizing the surface proteome, named molecular pixelation, to determine characteristic surface-based signatures of chronic CAR T-cell stimulation. This technique uses DNA-conjugated antibodies against 80 markers, 4 controls and 76 target proteins, and two rounds of gap-fill ligation and rolling circle amplification to determine the relative position of the protein molecules on the surface of a cell. We analyzed 8504 CAR T-cells at a single-cell level, collected from 3 donors and stimulated once for acutely stimulated cells and stimulated repeatedly over 14 days for chronically stimulated cells, followed by selection for live cells and a surface-based marker NGFR indicating the expression of the CAR construct. The sequencing counts that correspond to individual antibodies allow us to distinguish acutely and chronically stimulated cells, and the molecular pixelation protocol allows us to identify polarization and colocalization of the markers on the cell surface. A total of 33 of the 76 proteins (43.4%) exhibited significant changes in polarization, and there was an overall increase in polarization after chronic stimulation. Significant colocalization changes were observed for 561 proteins (19.7% of all 2850 combinations); one particular colocalization event that stood out was CD37/CD82, which increased significantly after chronic stimulation (adjusted p-value < 1e-250). Molecular pixelation is a new spatial proteomics technique for the discovery of proteomic polarization and colocalization on the cell surface that can identify new types of biomarker candidates, such as were identified for chronically stimulated T-cells in this study.