Fig. 4: SHP-2 bridges two PD-1 molecules via PD-1 ITSM pY248.

a The NanoBiT proximity assay: PD-1-LgBiT and PD-1-SmBiT induce luciferase activity only if two PD-1 molecules stably interact resulting in the formation of an active luciferase enzyme. b HEK-293 cells were co-transfected as indicated and luciferase activity was assessed. Comparisons were performed by two-way ANOVA and Tukey’s multiple comparisons test (****P < 0.0001, PD1sm + PD1Lg + SHP2WT + Fyn(+) vs. all other samples, **P < 0.005, PD1sm + PD1Lg + SHP2R32A + Fyn(+) vs. PD1sm + PD1Lg + SHP2DM + Fyn(+), n = 6 experiments). c HEK-293 cells were co-transfected with the indicated constructs together with either kinase active (+) or inactive (-) Fyn and luciferase activity was assessed (****P < 0.0001, n = 3 experiments). d HEK-293 cells co-transfected with PD1SmBiT, PD1LgBiT and SHP-2 WT, together with either kinase active (+) or inactive (−) Fyn, were cultured in the presence of hPD-L1-Ig (dimer), hPD-L1 monomer or control IgG and luciferase activity was assessed (*P < 0.05, n = 3 experiments). e HEK-293 cells co-transfected with PD1SmBiT, PD1LgBiT, SHP-2 WT, and kinase active Fyn were cultured in the presence of increasing amounts of the allosteric SHP-2 inhibitor SHP099 or vehicle control (0) and luciferase activity was assessed (arrow indicates baseline luciferase activity). f The BiFC assay is based on structural complementation of two non-fluorescent N- and C-terminal fragments of Venus YFP-fluorescent protein fused to a pair of interacting proteins. g HEK-293 cells transfected with indicated constructs were analyzed by confocal microscopy. Representative fluorescent images of each indicated transfection condition. Polymerized actin was visualized with rhodamine-conjugated phalloidin. YFP signal from PD1-VN + PD1-VC complementation was visualized in the green channel. Intensity of yellow color in the merged images is indicative of co-localization of PD-1 dimer with the actin cytoskeleton. h Quantitative analysis of images from g and comparison of total corrected cellular YFP fluorescence (TCCF-YFP) in the indicated conditions after subtraction of TCCF-YFP from control transfection PD1-VN + PD1-VC + 2xVector, **P < 0.005, n = 50 from one of two independent experiments. i Primary human T cells co-transfected with PD1SmBiT, PD1LgBiT and the indicated SHP-2 constructs were cultured with Raji-PD-L1 with or without prior loading with SEE and luciferase activity was assessed 6 h later (****P < 0.0001, ***P < 0.0005, **P < 0.005, *P < 0.05, n = 3 experiments).