Fig 2: aVhigh tumours promote TIL exclusion and decreased T cell proliferation and activation.a Left, B16F10E and B16F10E-KO tumour growth kinetics (p = 0.800). Right, tumour weights of B16F10E and B16F10E-KO tumours recovered at day 14 after engraftment (p = 0.846). Tumour volumes and tumour weights are given as means ± SEM of eight mice/group. Data represent one independent experiment out of three. b The ratio of active versus total TGF-ß from B16F10E and B16F10E-KO tumours (n = 8) was measured ex vivo on day 8 by ELISA (*p = 0.038). c Absolute cell counts of CD3+ (*p = 0.040) and CD8+ T cells (*p = 0.034) infiltrating B16F10E and B16F10E-KO tumours (n = 8). Data are from one independent experiment out of three. d Representative flow cytometry profiles (bi-exponential scale) of the expression of CD62L and CD44 in CD8+ T cells from B16F10E and B16F10E-KO tumours (n = 8). Right, percentage of CD44+CD62Lneg cells among CD8+ T cells in B16F10E and B16F10E-KO (***p = 0.0006). e KLRG1 (n = 18, *p = 0.014), CD69 (n = 13, *p = 0.025) and CD103 (n = 17, ***p = 0.0003) expression in CD8+ T cells infiltrating B16F10E and B16F10E-KO tumours. Data are from two independent experiments out of three. f Representative flow cytometry profiles of PD-1 expression on CD8+ TIL from B16F10E and B16F10E-KO. Right, expression of PD-1 on CD8+ T cells infiltrating B16F10E and B16F10E-KO tumours (n = 12, *p = 0.016). g Representative profiles of Ki-67 expression in CD8+ T cells from B16F10E and B16F10E-KO. Right, expression of Ki-67 in CD8+ T cells from B16F10E and B16F10E-KO tumours (n = 11, *p = 0.028). Data are from two independent experiments out of three. Each symbol represents an individual tumour (a-g.). Horizontal lines correspond to mean ± SEM (a, right-g.). Data were calculated with unpaired Student t-tests (a, right-g.) and two-way ANOVA (a, left). Source data are provided as a Source Data file.

Fig 3: Murine UPS tumour growth is unaffected by lymphocyte deficiency and resistant to immunological checkpoint blockade therapy.(A-B) Survival and growth kinetics of UPS cells engrafted into lymphocyte deficient Rag2 KO mice were no different from control C57Bl/6 mice. (C) Despite 45–55% of CD4 and CD8 TILs expressing PD1, UPS bearing mice were resistant to anti-PD1 and anti-CTLA-4 therapy, whereas survival was 66% in the inflamed M3-9-M model similarly treated with anti-PD1 (D-F).

Fig 4: CaV1.4 deficiency results in T cell activation/potential exhaustion. Splenocytes of WT (n = 15 or n = 22) and KO (n = 18 or n = 26) mice were stained with different antibodies and analyzed by flow cytometry. (A) The PD-1+ frequency and (B) CTLA-4 mean fluorescent intensity (MFI) are shown in boxplots for different populations, which are classified as CD62L+ IL-7R+ CD44- (naïve), CD62L+ IL-7R+ CD44+ (TCM), CD62L- IL-7R+ (TEM), CD62L- IL-7R- (end-stage TEff). Boxplots show four pooled experiments. Histograms show one representative sample of each genotype. The dashed lines show FMO controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, MFI = mean fluorescent intensity.

Fig 5: Expression of Sema-3A and Nrp-1 in human lung tumour cell lines and CTL clone. a Sema-3A expression in human lung tumour cell lines. Total protein extracts were analysed by western blot using anti-Sema-3A mAb. The 16HBE cell line was included as a control. Full length and proteolytically processed proteins are indicated. b Co-expression of Nrp-1 and CD25, and Nrp-1 and PD-1 on P62 T cells stimulated with immobilised anti-CD3. c The P62 clone was unstimulated or stimulated with anti-CD3, pre-incubated with Sema-3A-Fc, and then labelled with anti-human IgG-Fc secondary mAb. Results are gMFI mean ± SEM of triplicate samples. d Sema-3A-Fc inhibits CTL migration toward a CXCL12 gradient. The T-cell clone was stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwell plates and exposed to a gradient of CXCL12 loaded in the lower chambers. The number of T cells that had migrated into the lower chambers was determined. Results are mean chemotaxis index ± SEM of triplicate samples. e Sema-3A-Fc inhibits CD8+ TIL migration toward CXCL12. CD8+ TIL isolated from three human NSCLC tumours were stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwells and exposed to a CXCL12 gradient. Results are mean chemotaxis index ± SEM of triplicates. f Cytotoxicity of the CTL clone toward autologous tumour cells. The P62 clone was stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward the cognate IGR-Pub cell line was determined. g Cytotoxic activity of freshly isolated TIL toward autologous tumour cells. TIL, freshly isolated from a NSCLC tumour, were stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward freshly isolated autologous tumour cells was determined. Data shown for cytotoxic assay correspond to one of three independent experiments. Means ± SEM two-tailed Student’s paired t test c, one-way ANOVA test with Bonferroni correction d, e or two-way ANOVA test with Bonferroni correction f, g. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file a