Fig. 2: Induction of CD137L enhanced cancer immune surveillance both in immune-competent mice and melanoma cells.

a Representative images of the endpoint tumors in BALB/c nude mice. b B16F10 cells with CD137L stable expression and vector cells were injected into BALB/c nude mice on day 0, tumor volume was measured on the indicated time points. c Representative images of the endpoint tumors in C57BL/6 mice. d B16F10 cells with CD137L stable expression and vector cells were injected into C57BL/6 mice on day 0, tumor volume was measured on the indicated time points. e Kaplan–Meier survival curves for the survival of mice bearing CD137L overexpression or vector tumors. f Correlations with immune cells for CD137L. Spearman’s correlation test was performed. g–j CD137L⁺ cells (g), CD3⁺ T cells (h), CD8⁺ T cells (i), and GZMB ⁺ CD8⁺ T cells (j) were analyzed in mouse tumors by flow cytometry. k, l Immunostaining of CD3⁺ (Cyan), CD4⁺ (Magenta), CD8⁺ (Red), GZMB⁺ (Green) in the B16F10 tumor mass. Scale bar, 100 μm. m A375 shNC and shCD137L knockdown cells cocultured with activated T cells for 24 h were subjected to crystal violet staining. Tumor cell to T-cell ratio, 1:2.5 or 1:5, crystal staining (left panel) and frequency (right panel), Student’s t-test. n Vector and overexpression of CD137L A375 cells cocultured with activated T cells for 48 h were subjected to crystal violet staining. Tumor cell to T-cell ratio, 1:2.5 or 1:5 crystal staining (left panel) and frequency (right panel). Data: mean ± SD (a, c, g–j, l–n), mean ± SEM (b, d). Significance was determined by the log-rank test (e), two-sided unpaired Student’s t-test (a–d, g–j, l, n) and one-way ANOVA followed by Tukey’s multiple comparisons test (m). n = 3 biologically independent samples per group, representative of three independent experiments with similar results in (k–n). n  =  6 mice per group in (a–d, g–j). n  =   5 mice per group in (e). Consistent in vivo results were observed in at least two independent experiments, with at least five mice per condition.