Fig 2: 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).