Using CombiCells, a platform enabling titration and combinatorial display of cell surface ligands, to investigate the sensitivity and costimulatory requirements of TCRs and CARs

Receptor/ligand interactions at cellular interfaces are ubiquitous and the integrated signals from multiple interactions determines cellular decision-making. However, our understanding of cell-cell recognition is hampered by the inherent difficulty in precisely controlling the density of cell-surface ligands. Here, we adapt the protein Spycatcher, which forms a spontaneous covalent bond with Spytag, to enable the presentation of native ligands on cells. By expressing Spycatcher on the surface of cells using a short membrane-anchoring hinge, we show that addition of different concentrations/combinations of purified Spytag-ligands allows for the combinatorial display of ligands on cells (CombiCells) within minutes. We demonstrate the utility of the platform by quantifying T cell antigen sensitivity and the contribution of the CD2, LFA-1, CD28, and PD-1 accessory receptors. We show that T cell recognition of pMHC antigens is enhanced mostly by CD2/ligand engagement whereas recognition of the antigen CD19 by CAR-T cells is enhanced mostly by LFA-1/ligand engagement. Moreover, we show that PD-1/ligand engagement can inhibit T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate our understanding of how receptor/ligand interactions at cellular interfaces control cellular decisions. Graphical abstract

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