Morch, A. M. | bioRxiv Science

bioRxiv2023

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

Understanding how cellular decisions by receptor/ligand interactions at cell/cell interface has been challenging because it is difficult to independently vary the surface density of multiple ligands. Here, we exploit the SpyCatcher/SpyTag split-protein system for rapid combinatorial display of native ligands on cells (Combicells). We use this platform to assess T cell antigen sensitivity and the impact of T cell co-stimulation/co-inhibition receptors. The TCR displayed much greater sensitivity to pMHC than CARs and BiTES did to CD19. While TCR sensitivity was greatly enhanced by CD2 ligand, CAR sensitivity to CD19 was primarily but more modestly enhanced by LFA-1 ligand. Lastly, we show that the PD-1/ligand engagement inhibited 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 the study of receptor/ligand interactions at cell/cell interfaces. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/545075v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@d1ec95org.highwire.dtl.DTLVardef@33c0forg.highwire.dtl.DTLVardef@5bfed7org.highwire.dtl.DTLVardef@1b80ee2_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryUsing CombiCells, a platform for the combinatorial display of cell surface ligands, to compare T cell antigen sensitivity mediated by TCRs, CARs, and BiTEs and its dependence on co-stimulation/co-inhibition receptor ligands

immunology↗

bioRxiv2022

The kinase occupancy of T-cell coreceptors reconsidered

The sensitivity of the {beta} T-cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8{beta}, which are expressed primarily by cells of the helper and cytotoxic T-cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor-kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4-Lck interaction was stoichiometric (~100%) and that the CD8{beta}-Lck interaction was also substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor-Lck co-diffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to show that stoichiometric Lck coupling required an amphipathic -helix present in CD4 but not CD8. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 out-competed CD8{beta} for Lck. In T cells, TCR signaling induced CD4-Lck oligomerization but did not affect the high levels of CD4-Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting. Significance statementCD4 and CD8{beta} are archetypal coreceptor proteins that potently enhance T-cell antigen sensitivity but how they function is still debated. A fundamental question that remains incompletely resolved is: what fractions of the coreceptors bind the signal-initiating kinase, Lck? Using in vitro assays and non-invasive fluorescence fluctuation spectroscopy in live cells, we show that most coreceptors are occupied by Lck at the surface of live cells. The structural basis for important differences in the kinase occupancy of CD4 and CD8{beta} is also identified. These results provide important context for refining current models of both TCR antigen recognition and cell fate decisions made during thymopoiesis.

immunology↗