Fig 2: The CQC, GGXXG + CV+C, and IKRLL motifs differentially impact proximal TCR-CD3 signaling.(A) Phosphorylation intensity for CD3? (left), Zap70 (center), and Plc?1 (right) are shown for paired (connecting line) WT and Clasp mutant cell lines. Four independently generated cell lines were tested. (B) Phosphorylation intensity for CD3? (left), Zap70 (center), and Plc?1 (right) are shown for paired (connecting line) WT and TP mutant cell lines. Three independently generated cell lines were tested. (C) Phosphorylation of CD3? (left), Zap70 (center), and Plc?1 (right) are shown for paired (connecting line) WT and LL mutant cell lines. Five independently generated cell lines were tested. For (A–C), filled symbols represents the mean ± SEM of concatenated data for coupled cells from three independent experiments. 10,000 coupled cells were collected per experiment (technical replicates), resulting in the concatenation of 30,000 coupled cells total from the 3 independent biological replicates. For (C), the open symbols represent the mean and ± SEM for one single experiment (10,000 coupled cells analyzed). One-way ANOVA was performed with a Dunnett's posttest when the experiments involved multiple comparisons. Student's t-test were performed for when only WT and mutant pairs were analyzed in an experiment. The derived p values for each independent cell line comparing the mutant CD4 to its paired WT is shown. Next to each symbol the number of cells determined to have responded to stimuli are shown with the percentage of responding cells.

Fig 3: Targeting both NRP1 and PD1 has a synergistic effect in human and mouse CD8+ T cells immune response(A) Flow cytometry analysis of NRP1 expression according to cell trace on human CD8+ T cells 96 h after activation with anti-CD3 and anti-CD28, or on nonactivated cells. Data are representative of 5 independent experiments.(B) Flow cytometry analysis of NRP1 and PD1 expression in human CD8+ T cells 96 h after in vitro activation with anti-CD3 and anti-CD28 or non-activated cells. Data are representative of 3 independent experiments.(C) Flow cytometry analysis of NRP1 and PD1 expression in CD8+ TILs. Data are representative of 3 independent experiments in human endometrial, kidney, and ovarian cancer.(D) Flow cytometry analysis of phospho-ZAP70 in PD1+CD8+ TILs according to NRP1 expression. Data from one experiment in human endometrial cancer.(E) Flow cytometry analysis of percentage of divided CD8+ T cells from a patient bearing an NRP1 haploinsufficiency (patient) or from controls (N = 5), respective to SEB superantigen concentration (0, 1, 10, or 100 ng/mL), in the presence or not of anti-PD1 antibody. Activation was performed during 72 h. Data are presented as the mean ± SEM. Data representative of 1 experiment.(F) Flow cytometry analysis of CD25 expression in CD8+ T cells from a patient bearing an NRP1 haploinsufficiency (patient) or from controls (N = 5), respective to SEB superantigen concentration (0, 1, 10, or 100 ng/mL), in the presence or not of anti-PD1 antibody. Activation was performed during 72 h. Data are presented as the mean % of CD25 expression ± SEM. Data representative of 1 experiment.(G) NRP1/PD-1 complexes detection by Proximity-Ligation-Assay (PLA) technology on CD8+ TILs in human colon cancer. Left panel: Representative area of tumor tissue observed. Acquisition with NDPI view software. Middle panel: Tumor infiltrating NRP1+PD1+CD8+ T cells (pink): Merge of green CD8 staining (FITC) and orange NRP1/PD-1 spots (TRITC). Acquisition with NDPI view software: zoom in x20. Right panel: CD8+ NRP1/PD1 positive cell (Pink) and CD8+ NRP1/PD1 negative cells (green). Acquisition with NDPI view software: zoom in x40. Nuclei are stained with DAPI (blue). Images have been observed by confocal microscopy ×20 oil objective. Data are representative of 10 independent experiments.(H) CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumin and poly-IC and treated or not with anti-PD1 antibody in vivo. B16-OVA tumor volume was assessed until 35 days after immunization. Data are presented as the mean ± SEM and as Kaplan Meyer curve. p values were determined by two-way ANOVA test ∗∗∗p < 0.001 ∗∗p < 0.01. Data are representative of 5 experiments.(I) CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumin and poly-IC and treated or not with anti-PD1 antibody in vivo. Overall survival was assessed until 50 days after immunization. Data are presented as the mean ± SEM and as Kaplan Meyer curve. p values were determined by Log rank test ∗∗∗p < 0.001. Data are representative of 5 experiments.(J) Analysis of overall survival of patients with metastatic melanoma treated with anti-PD1, according to RNA NRP1 expression (low or high expression: groups have been determined according to ROC curve analysis) assessed in the tumor before anti-PD1 treatment. Data from transcriptomics analysis of metastatic melanoma tumors were available from Hugo et al. (Hugo et al., 2016) Data are presented as Kaplan Meyer curve. p value (p = 0.040) was determined by Log-rank test (n = 25 patients).(K) Analysis of relapse free survival of patients with metastatic melanoma treated with anti-PD1 and reached at least a partial response, according to NRP1 expression (NRP1-/low compared with NRP1+/high) in CD8+ TILs assessed by immunohistochemistry before starting therapy. Blind analysis has been performed to assess NRP1 expression. Data are presented as the Kaplan Meyer curve. p value (p = 0.042) was determined by Log-rank test (n = 15 patients).