PD1 triggering decreases IS size through inhibition of PKCθ recruitment. (A) Assessment of the effect of PD1 triggering on the size (maximal diameter of the contact zone) of the IS. Jurkat-PD1-NFAT cells (labeled with CMTMR) were incubated with Raji Ctrl and Raji PDL2 cells (labeled with CFSE in presence of 6 ng/mL SEE), at a 1:1 cell ratio for 30 minutes at 37°C to facilitate IS formation. Image data from representative experiment, highlighting quantified IS by white arrowheads. Representative images of 2 independent experiments are shown. (B-C) Quantification of the size of the IS formed as described in panel A, using Jurkat-PD1-WT or DM cells (B) or Jurkat-PD1-WT cells, at different time points of incubation with Raji Ctrl and Raji PDL2 cells (C). Representative results of 2 and 3 independent experiments, respectively, are shown. Each dot represents a value from 1 Jurkat cell, (B) 51, 26, 50 and 33 cells; (C)16, 25, 16 and 27 cells were quantified, respectively. (D) Assessment of the effect of PD1 triggering on the size (maximal diameter of the contact zone) of the IS. Jurkat-PD1-WT and Jurkat-PD1-DM cells (labeled with CMTMR) were incubated with Raji PDL2 cells (labeled with CFSE in presence of 6 ng/mL SEE), at a 1:1 cell ratio for 30 minutes at 37°C to facilitate IS formation. Image data from representative experiments, highlighting quantified IS by white arrowheads. Representative images of 3 independent experiments are shown. Scale bar, 10 μm. Graphs in panels B-C show mean value ± SEM. Statistical analysis was performed using 1-way ANOVA with Šídák’s multiple comparisons test. ∗∗∗∗P < .0001; ∗∗P < .01; ns, not significant.