Fig. 3: Loss of Ch25h in LECs dampens immunotherapy efficacy in lymphangiogenic melanoma.

A–E LEC^ΔCh25h and LEC^WT mice were inoculated with B16F10-OVA VEGF-C (B16-OVA-VC) cells and vaccinated with OVA protein and CpG-B at day 5 (A) or at day 4 and day 8 (B–E). A Ch25h expression (GFP MFI) was measured by flow cytometry on day 11. n = 5 mice/ group. B Tumor growth was followed and compared to unvaccinated B16F10-OVA VEGF-C tumor-bearing WT mice. Representative of four independent experiments, n = 5–6 mice/group each. C–E Tumor were harvested on day 24 and analyzed by flow cytometry. C CD45⁺/tumor cell ratio and tumor cell proliferation (Ki67⁺) and CD45⁺ cell frequency in living cells. D Myeloid cells (CD11b⁺CD68⁺) were separated into monocytes (Ly6C⁺F4/80^-) and macrophages (Ly6C^-F4/80⁺) and were analyzed for their expression of indicated markers. E CD4⁺ and CD8⁺ T cells were analyzed for their expression of indicated markers. C–E Representative of two independent experiments, n = 5–8 mice/group each. Data were presented as mean values ± SD (F, G) LEC^ΔCh25h and LEC^WT mice were inoculated with B16F10-OVA VEGF-C cells and adoptively transferred 8 days later with OT-1 effector T cells. Tumor growth was followed and normalized to the size of WT at day 6 (F), and OT-1 cells were analyzed in tumors 2 days after transfer for indicated markers (G). Data were pooled from two independent experiments, n = 2–5 mice/group each, and are presented as mean values ± SEM (F) or as mean values ± SD (G). B, F Two-way ANOVA, ^*P < 0. 05. (A, C, D, E, G) Two-tailed unpaired t-test. ^*P < 0.05; ^**P < 0.01; ^***P < 0.0001.