Extended Data Fig. 3.

(a) Dynamic association of LAG3, TCR and CD8 on Lag3^+/+ CD8⁺ T cells stimulated with TCRβ Ab using sensitized emission FRETc with quantification shown (Scale bar = 5μm). (b) The average number of LAG3 and CD4 molecules that are within 25 nm × 25 nm × 50 nm area around the TCR in Lag3^+/+ CD4⁺ T cells, isolated as above, following stimulation with TCRβ Ab as determined by the STORM-derived coordinates. Data are representative of at least 3–5 experiments. (c) Diagram depicting a fluid planar lipid bilayer system for analysis of protein-protein interactions. In the system, fluorescently tagged (TAMRA) CD4^CT or CD8^CT are anchored to the lipid bilayer via his-binding phospholipids resulting in fluid, randomly distributed TAMRA molecules visualized using confocal microscopy. Protein interactions are observed following addition of a peptide (LAG3^CT) resulting in phase separation and redistribution of molecules into supramolecular clusters. (d, e) Molecular distribution of CD4 (d), CD8 (e) and Lck within the IS of Lag3^+/+ and Lag3^−/− CD4⁺ or CD8⁺ T cells, isolated as above, as determined by STORM with quantification of the Co-localization index (Scale bar = 0.5μm). STORM imaging data are representative of 15 data sets derived from 3 separate experiments. Statistics determined by unpaired Student’s two sided t test (a, b) and by Wilcoxon matched pairs signed rank test (d, f). P values are noted in figures.