Figure 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 NRP1^high or NRP1^low 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 NRP1^high or NRP1^low 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.