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: Type I IFN resistant CAR T cells provide enhanced therapy with VSVmIFNß in lymphodepleted mice.a CAR T cells were genetically modified using CRISPR Cas9 one day after transduction by nucleofection of an RNP complex consisting of Cas9 duplexed with tracrRNA and two IFNAR1 specific or two negative control crRNAs. 48 h following modification, expression of the CAR (Thy1.1) and the IFNAR1 is shown. b Two days after modification, CAR T cells were cultured in IL2 (50 U/mL) in the absence or presence of additional recombinant mouse IFNß. CAR expression is shown for representative CD8 CAR T cells (left) and quantified in three replicates in CD8 and CD4 CAR T cells (right). c The percent of CRISPR IFNAR1 KO or control CD8 and CD4 CAR T cells expressing Fas is shown. d Inhibitory receptor expression (PD1, LAG3, TIM3) quantified on CRISPR IFNAR1 KO or control CD8 CAR T cells cultured in IL2 in the absence or presence of additional IFNß. Data shown are representative of two independent experiments. Technical replicates are shown ± SD (n = 3 (b) n = 4 (c)). e Mice bearing B16EGFRvIII tumors were treated with 5 × 107 pfu VSVmIFNß or PBS 6 h prior to administration of 1 × 107 IFNAR1 KO EGFRvIII CAR T cells on day 7. Select groups received two additional doses of 5 × 107 pfu VSVmIFNß on days 10 and 13. Select groups received a lymphodepleting dose of radiation (5 Gy TBI) on day 6. n = 6/group. f Tumor growth is shown in the left panel and overall survival is shown in right panel. g Mice bearing B16EGFRvIII tumors received a lymphodepleting dose of radiation (5 Gy TBI) on day 6 and were treated with 5 × 107 pfu VSVmIFNß or PBS 6 h prior to administration of 1 × 107 WT or IFNAR1 KO EGFRvIII CAR T cells on day 7. Select groups received six additional doses of 5 × 107 pfu VSVmIFNß on days 10,13,17,20,23, and 27. n = 6/group. Experiments in (e) and (g) were performed once. P-values were determined using the Log-rank Mantel-Cox test (f, h) and a two-way ANOVA with a Tukey multiple comparisons post-test (b, c). Statistical significance set at p < 0.05, ns > 0.05. Source data are provided in the Source Data File.

Fig 4: Recombinant IFNß promotes apoptosis, activation, inhibitory receptor expression, and dysregulated expression of the CAR.a Annexin V expression on CD8 CAR T cells or untransduced (UTD) T cells cultured in IL2 (50 U/mL) in the presence or absence of recombinant murine IFNß and 1µM Ruxolitinib (JAK1/2 inhibitor) for 48 h. b Annexin V expression on CD8 CAR T cells cocultured with B16EGFRvIII cells at an E:T ratio of 1:5 in the presence or absence of recombinant murine IFNß and/or 1µM Ruxolitinib. Fas (c) or cleaved caspase (d) expression on CD8 CAR T cells or UTD cells cultured in IL2 with IFNß. e Median fluorescence intensity (MFI) of CAR expression measured using Biotin-Protein L and streptavidin- PE (Protein L + SA PE) compared to Annexin V on CAR T cells cultured in IL2 with or without IFNß (10 ng/mL). CAR expression on CD8 CAR T cells cultured in IFNß for 48 h in the absence (f) or presence of target tumor cells (g). Representative (h) and mean (i) expression of CD25 and CD69 for CAR T cells cultured in IL2 with IFNß. Statistical comparisons are indicated for CD25- CD69- populations in (i). j Co-expression of the CAR and activation markers on CD8 CAR T cells. k Mean expression of PD1, LAG3, TIM3 on CD8 CAR T or UTD cells cultured in IL2 or in the presence of B16EGFRvIII cells (E:T 1:5). l Fold change in inhibitory receptor and CAR expression is shown for cells grown in IL2 with additional recombinant IFNß relative to culture in IL2 alone. m WT or transgenic IFNAR1 KO CAR T cells were cocultured with B16EGFRvIII (E:T ratio of 1:5) which were mock infected or infected with VSVmIFNß 6 h prior to coculture. Surface CAR expression is represented as fold change relative to coculture with mock infected tumor cells. n Inhibitory receptor expression was quantified on WT or IFNAR1 KO CD8 CAR T cultured as in (m). Data are representative of two independent experiments (panels a–l). Experiments in panels (m, n) were performed once. Means ± SD of n = 4 (panel a) or n = 3 (panels b–d, f, g, i, k, m, n) technical replicates are shown. P-values were determined using a two-way ANOVA with a Tukey multiple comparisons post-test (a–d, f, g, i). Statistical significance set at p < 0.05, ns > 0.05. Source data are provided in the Source Data File.

Fig 5: The CGRP–RAMP1 axis promotes intratumoral CD8+ T cell exhaustion.(a–e) Orthotopic B16F10-mCherry-OVA (5x105 cells; i.d.) cells were injected to nociceptor intact (Nav1.8WT::DTAfl/WT) and ablated mice (Nav1.8cre::DTAfl/WT). As measured fifteen days post inoculation, NaV1.8+ nociceptor-ablated mice had lower proportion of PD-1+LAG3+TIM3+ (a) CD8+ T cells, but increased levels of IFN?+ (b), TNF+ (c), IL-2+ (d) CD8+ T cells. B16F10-mCherry-OVA (5x105 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1+LAG3+TIM3+ CD8+ T cells (e). (f) Orthotopic B16F10-mCherry-OVA cells (5x105 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8+ T cells exhaustion (PD-1+LAG3+TIM3+). (g) Orthotopic B16F10-mCherry-OVA cells (5x105 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). (h–m) Orthotopic B16F10-mCherry-OVA cells (5x105 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm3 (defined as therapeutic). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8+ T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume (h) and weight (i) but increased the relative proportion of IFN?+ (k), TNF+ (l), and IL-2+ (m) CD8+ T cells. BIBN4096 had no effect on the number of intratumoral CD8+ T cells (j). When administered as therapeutic, BIBN4096 reduced tumour volume (h) and weight (i) but had limited effect on CD8+ T cells’ cytotoxicity (j–m). (n) Orthotopic B16F10-mCherry-OVA cells (5x105 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact (Trpv1WT::DTAfl/WT) and ablated (Trpv1cre::DTAfl/WT) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. (o–s) Splenocytes-isolated CD8+ T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions (ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 µM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival (o) of cultured cytotoxic CD8+ T cells, nor their relative expression of PD-1+LAG3+TIM3+ (p), IFN?+ (q), TNF+ (r), and IL-2+ (s). (t) B16F10 cells (1x105 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 µM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. (u-w) Naive splenocyte CD8+ T cells were FACS purified from Ramp1WT (CD45.1+) or Ramp1-/- (CD45.2+) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. 8-week-old female Rag1-/- mice were transplanted (i.v., 2.5x106 cells) with either Ramp1-/- or Ramp1WT CD8+ T cells or 1:1 mix of Ramp1-/- and Ramp1WT CD8+ T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x105 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8+ T cells across the three tested groups (u). The relative proportion of intra-tumour PD-1+LAG3+TIM3+ CD8+ T cells was lower in Ramp1-/- transplanted mice (v). Within the same tumour, intratumoral CD8+ T cell exhaustion was immunophenotyped by flow cytometry (representative panel shown inw) and showed that the relative proportion of PD-1+LAG3+TIM3+ CD8+ T cells was ~3-fold lower in Ramp1-/- CD8+ T cells than in Ramp1WT CD8+ T cells (w). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25th to 75th percentile and the middle line indicates the median), for which individual data points are given (a–d, f, h–m, o–v), linear regression (e), Mantel–Cox regression (g), mean ± S.E.M (n), or as FACS plot (w). N are as follows a–e: Nav1.8WT::DTAfl/WT (n = 18), Nav1.8cre::DTAfl/WT (n = 10), f: Trpv1WT::DTAfl/WT (n = 16), Trpv1cre::DTAfl/WT +CGRP (n = 11), g: vehicle (n = 89), BIBN4096 (n = 16), h–m: Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n: Trpv1WT::DTAfl/WT + vehicle (n = 8), Trpv1WT::DTAfl/WT + BIBN4096 (n = 9), Trpv1cre::DTAfl/WT + vehicle (n = 7), Trpv1cre::DTAfl/WT + BIBN4096 (n = 7), o: vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s: n = 5/groups, t: n = 4/groups, u–w: n = 5/groups. Experiments were independently repeated twice (a–f, n–w) or four (g–m) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test (a–d, f,v), simple linear regression analysis (e), Mantel–Cox regression (g), by one-way ANOVA posthoc Bonferroni (h–m; o–u), or two-way ANOVA post-hoc Bonferroni (n). Source Data