Fig. 6. Elucidating intercellular surfaceome signaling domains in functional immunosynapses.

a Schematic of coupling immunogenic peptide gp33 to the singlet oxygen generator (SOG) thiorhodamine. The crystal structure of gp33 (dark gray) presented by MHC class I H-2D^B alpha chain (red) in complex with beta-2 microglobulin (dark red) is shown (PDB identifier: 1FG2), b Schematic of the SILAC-DIA based LUX-MS workflow. Isotopically labeled (heavy) mouse dendritic cells presenting gp33-SOG2 establish synchronized immunosynapses with isolated (light), gp33-specific CD8⁺ T cells enabling light-activated in situ labeling and molecular analysis of intercellular surfaceome signaling interactions within synapses using data-independent acquisition (DIA) mass spectrometry. c Histogram plot showing distribution of heavy-to-light abundance ratios of LUX-MS identified proteins. Ratio-boundaries for cell type assignment and representative proteins are shown. d Volcano plot showing relative abundance changes of LUX-MS quantified proteins from the two-cell systems with and without illumination for 15 min, tested using a two-sided Student’s t test. Dots and crosses represent cell surface and otherwise annotated proteins, respectively. Red and blue dots represent dendritic cell (DC) and T-cell (TC)-derived proteins, respectively. Known immunosynaptic constituents are highlighted with direct gp33 interactors shown in boxes. e Schematic of the LUX-MS identified immunosynaptic surfaceome interaction network. f Schematic highlighting the interconnectivity of the immunological synapse with endolysosomal pathways in T cells and showing proteins reported at the cell-to-cell interface by LUX-MS. Source data are provided as a Source Data file and interactive volcano plots (Supplementary Data 6 and 7).