Fig. 5
From: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1
The Nrp-1+PD-1hi TIL subset is enriched with activated antigen-specific CD8+ T cells. a Staining of CD8+ TIL with dextramers. C57BL/6 mice were engrafted with B16F10 and then vaccinated with Trp2 and gp100 peptides. On day 15, TIL were isolated from tumours. Right: Percentages of Trp2 (n = 8) and gp100 (n = 5) dextramer-positive T cells among Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− CD8+ TIL. b Expression of IFNγ (n=20) and TNF (n=18) in Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− CD8+ T cells. TIL were stimulated for 4 h with autologous tumour cells. c Degranulation of CD8+ TIL. TIL were stimulated with autologous tumour cells; then, T-cell subsets were analysed for expression of CD107a (n = 18). d Cytotoxicity of freshly isolated CD8+ TIL. CD8+ TIL were pre-incubated in medium or with anti-Nrp-1, anti-PD-1 or a combination of both mAb; then, cytotoxicity toward autologous tumour cells was determined. e Increase in MHC-I and PD-L1 expression on tumour cells co-cultured with autologous CD8+ TIL. Kinetic studies of H-2-Kb/-Db and PD-L1 expression on B16F10 co-cultured with CD8+ TIL. Expression profiles (left), percentages of positive cells (middle) and gMFI (right) of MHC-I (upper panels) and PD-L1 (lower panels) are shown. f Expression of perforin in CD8+ T cells. TIL were stimulated with autologous tumour cells in the absence or presence of neutralising anti-Nrp-1, anti-PD-1 or anti-Nrp-1 plus anti-PD-1 then, T-cell subsets were analysed for expression of perforin (n = 5). g Anti-Nrp-1 re-establishes migration of Nrp-1+PD-1hi T cells toward B16F10. TIL were pre-incubated in medium or with neutralising anti-Nrp-1, -PD-1, a combination of both mAb or an isotype control. Cells were seeded in the upper chambers of transwells and then exposed to a gradient of B16F10 supernatant, enriched in Sema-3B, loaded in the lower chambers. The numbers of Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− T cells that had migrated were determined by flow cytometry. Results are representative of three independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction a–g. *P < 0.05; **P < 0.01; ***P < 0.001
