Fig 1: 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).
Fig 2: NRP1 modulates PD1 activity at the synapse between CD8+ T cells and tumor cells(A) Illustrative image of phalloidin (yellow), CD8 (pink), CFP from EL4 (purple), and NRP1 (red) labeling in the synapse model between activated OT1 CD8+ T cells and EL4-CFP tumor cells bearing OVA257 (SIINFEKL), observed by ImageStream. The synapse is the high phalloidin labeling zone. Bright field image is in white (scale bar = 7µm). Data are representative of 4 independent experiments from 2 synapse models.(B) Quantification by ImageStream of NRP1 expression (mean pixel intensity/MPI) in an allogeneic synapse model between activated CD8+ T cells and cell tracer violet labeled A20 cells. NRP1 expression was analyzed in activated CD8+ T cells at the synapse junction (high phalloidin labeling zone). Data are presented as the mean MPI ± SEM. p value (p < 0.0001) was determined by Wilcoxon matched pairs test. Data are representative of 4 independent experiments from 2 synapse models.(C) Illustrative image of phalloidin (yellow), phospho-ZAP70 (green), CD8 (red), cell tracer violet labeled A20 tumor cells (purple), and NRP1 (white) between activated NRP1high or NRP1low CD8+ T cells and cell tracer violet labeled A20 tumor cells observed by ImageStream. The synapse is the high phalloidin labeling zone. Bright field image is in white (scale bar = 7µm). Data are representative of 2 independent experiments.(D) Quantification by Imagestream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labeling zone) between activated NRP1high or NRP1low CD8+ T cells and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.(E) Left panel: CD8 (green) NRP1 (red), PD1 (blue), and NRP1/PD1 merge (purple) expression observed by confocal microscopy in CD8+ TILs from control mice (WT) at day 21 post-activation (x63 oil objective, scale bar = 10 µm). Data are representative of 3 tumors. Right panel: Colocalization of NRP1 and PD1 was assessed by the calculation of Pearson coefficient. Data from 10 CD8+ TILs analyzed are presented as mean ± SEM.(F) Phospho-ZAP70 signal according to its localization within the synapse between the CD8+ T cells and the tumor cells. Illustrative image of phalloidin (yellow), phospho-ZAP70 (green), CD8 (red), and Cell Tracer (A20 cells, purple) labeling in the synapse model between activated CD8+ T cells from CD8Nrp1KO mice (KO) or controls (WT) and allogeneic A20 tumor cells, by ImageStream. Bright field image is in white (scale bar = 7µm). Data are representative of 2 independent experiments.(G) Phospho-ZAP70 signal according to its localization within the synapse between the CD8+ T cells and the tumor cells. Quantification by Image stream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labeling zone) between activated CD8+ T cells from CD8Nrp1KO mice (KO) or control mice (WT), and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.(H) Proximity of NRP1 and PD1 proteins demonstrated by Duolink assay on in vitro activated CD8+ T cells from C57BL/6J mice. Upper panel: Left: Negative control experiments performed using anti-IRAP and anti-NRP1 antibodies (PLA-Duolink). Right: NRP1/PD1 complexes (anti-NRP1 and anti-PD1 antibodies with PLA-Duolink). The red spots indicate less than 40nm proximity between cellular-bound antibodies. Nuclei are stained with DAPI (blue). Images have been observed by confocal microscopy (x63 oil objective, scale bar = 10µm). Data are representative of 5 independent experiments. Lower panel: Comparison of number of PLA plots per cell. Data are presented as mean ± SEM.(I) NRP1 and PD1 interaction was demonstrated by CoIP experiments performed in splenocytes from C57BL/6J mice activated with anti-CD3 and anti-CD28 antibodies. NRP1 and PD1 immunoblot (IB) detection is shown in total lysate (TL) as control, in eluate from IgG Control (ctl) IP, and from NRP1 IP (N = 1 experiment). NRP1/PD1 Co-IP was also observed after PD1 IP (N = 2 experiment). Data are representative of 3 independent experiments.(J) Quantification by Imagestream of PD1 expression (MPI) in the synapse junction (high phalloidin labeling zone) between activated CD8+ T cells from CD8Nrp1KO mice (KO) or controls (WT), and allogeneic A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.
Fig 3: PD-1 and PD-L1 protein expressions in the tissue of the examined patients with CRSwNP and HNC (A). Representative Western blots of the PD-1 and PD-L1 protein expressions (B): control subjects (I); CRSwNP patients (II); HNC patients (III). ** p < 0.01 versus the control group.
Fig 4: mRNA expression level of the PD-1 and PD-L1 genes in the examined patients with CRSwNP and HNC. *** p < 0.001 versus control group.
Fig 5: Correlation between the mRNA expression levels of the PD-1 (A,C) and PD-L1 (B,D) genes and the extent of the diseases calculated using the Kennedy score for the CRSwNP patients (A,B) and TNM staging system for the HNC patients (C,D).
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