Fig. 6.

Anti-Nrp-1 mAb combined with anti-PD-1 improve tumour progression control. a Mice were engrafted with B16F10 and then treated i.p. with anti-PD-1, i.t. with anti-Nrp-1, or a combination of both mAb injected in parallel at each site at days 6, 8, 10, 12 and 14 after tumour inoculation. Tumour volumes are given as means ( ± SEM) of nine mice/group. Mice were killed at day 17. Data are means of two independent experiments out of three. b Weight of tumours from mice untreated and treated with blocking mAb in a. at day 17. c C57BL/6 mice were engrafted with MC-38 tumour cells and then treated i.p. with anti-PD-1, i.t. or i.p. with anti-Nrp-1, or a combination of anti-PD-1 (i.p.) plus anti-Nrp-1 (i.t. or i.p.) injected at days 4, 7, 10 and 16 after tumour inoculation. Tumour volumes are given as means ( ± SEM) of 4–5 mice/group. d Weight of tumours from mice untreated and treated with blocking mAb in c. recovered at day 21. e Numbers of CD8⁺ T cells per milligram of B16F10 tumour were determined at day 17. f Ratio of CD8⁺/CD4⁺ Treg cells in B16F10 tumours from mice treated with blocking mAb or an isotype control. g Numbers of KLRG1⁺, Ki-67⁺ and granzyme B⁺ CD8⁺ T-cell per milligram of B16F10 tumour from mice treated with blocking mAb or an isotype control. h Numbers of CD8⁺ T cells per milligram of MC-38 tumour were determined at day 21. i Cytotoxic activity of freshly isolated CD8⁺ TIL toward MC-38 tumour cells. CD8⁺ TIL were isolated at day 21 in c. and then cytotoxicity toward autologous tumour cells was determined. j Absolute cell counts of granzyme B⁺ CD8⁺ T cells from MC-38 TIL. Numbers of T-cell subsets per milligram of tumour from mice treated i.p. with anti-PD-1, i.t. or i.p. with anti-Nrp-1, or a combination of anti-PD-1 (i.p.) plus anti-Nrp-1 (i.t. or i.p.) mAb. Means ± SEM two-way ANOVA test with Bonferroni correction a, c, i or one-way ANOVA test with Bonferroni correction b, d, e, f, g, j. *P < 0.05; **P < 0.01; *** P < 0.001