Fig 2: Expression of T-cell activation/exhaustion markers on the CD8+ TIL. a Expression of Nrp-1, PD-1, LAG-3, CTLA-4 and Tim-3 on CD8+ T cells from B16F10 TIL isolated at day 15. Right: percentages of Nrp-1 among CD8+ T cells expressing or not PD-1 (n = 26), Tim-3 (n = 19), LAG-3 (n = 19) or CTLA-4 (n = 16). b Expression of Nrp-1 on CD8+ T cells correlated with that of PD-1 (n = 38). c Expression of Nrp-1 on CD8+ TIL is restricted to a PD-1hi T-cell population. Left, Expression of Nrp-1 and PD-1 on CD8+ TIL; identification of three T-cell subsets: Nrp-1+PD-1hi, Nrp-1−PD-1+and Nrp-1−PD-1−. Right: expression of exhaustion/activation markers, functional proteins and transcription factors in Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− CD8+ T-cell subsets. Heat maps including percentages of cells positive for expression of PD-1, Nrp-1, Tim-3, CTLA-4, LAG-3, granzyme B (GrzB) and Ki-67, and gMFI for NFATc1, IRF-4, Helios, Blimp-1 and T-bet (n = 5). d gMFI of PD-1 on Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− TIL subsets (n = 16). Results are representative of three to five independent experiments. Means ± SEM two-tailed Student’s unpaired t test a or one-way ANOVA test with Bonferroni correction d. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file c

Fig 3: NK1R-signaling Promotes Activation of NFAT1, NFAT2, and NF?b-p65, and T Cell Survival Dependent on IL-2 Secretion(A) Calcineurin activity in WT and NK1RKO T cells incubated with SarSP or CD3 Ab, measured 15 min after treatment. Means ± 1 SD of duplicated results of 1 representative experiment of 2.(B) Western blot analysis of NFAT1, NFAT2, NF?ß-p65, cFos, and cJun, in nuclear extracts of WT and NK1RKO T cells, untreated or after incubation with CD3 and CD28 Ab. Controls were stimulated with ionomycin (Iono). One representative experiment of 3.(C) Concentrations of IL-2 by ELISA in supernatants of WT and NK1RKO T cells untreated or after 24-h stimulation with CD3 and CD28 Ab. Means ± 1 SD, 1 representative of 3 experiments.(D) Surface IL-2Ra expression by FACS in WT and NK1RKO T cells untreated or after 24-h stimulation with CD3 and CD28 Ab. One representative of 3 experiments.(E) Comparison by FACS of T cell proliferation (CFSE dilution), cell death (FVD incorporation), and activation (CD44High) between WT and NK1RKO T cells after 4-day stimulation with CD3 and CD28 Ab. Numbers are cell percentages per quadrant. One representative out of 6 experiments.(F) Comparison by FACS of cycles of cell division (histograms) and percentages of cell proliferation (bar diagram) between WT and NK1RKO T cells. Numbers in histograms are cell percentages. Each dot in the bar diagrams represents a mouse. Means ± 1 SD.(G) Percentages of cell death (by FVD incorporation) of WT and NK1RKO T cells stimulated for 4 days with CD3 and CD28 Ab, alone or plus IL-2. Means ± 1 SD, 6 mice per condition.Results were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (A, C, F, and G). ***p < 0.001, ****p < 0.0001, NS: not significant.