Fig. 3. CH25H expression in DCs enables efficient antigen cross-presentation otherwise inhibited by factors of tumor microenvironment.

a Merged MFI expression of SIINFEKL-bound H-2kb in DCs from indicated mice after treatment of OVA protein (200 μg/mL, 18 h). n = 3 biologically independent samples. b Percentage of IFNγ⁺ in OT-I CD8⁺ T cells after 72 h co-culture of naïve OT-I CD8⁺ T cells with splenic CD11c⁺ myeloid cells from indicated mouse pulsed with OVA (10:1) or MC38-OVA lysate (10:4). n = 3 biologically independent samples. c Antigen cross presentation analysis for WT or Ch25h^−/− DCs pre-treated or not with 25HC (50 nM, 4 h before adding soluble sOVA at indicated concentrations). OVA-pulsed DCs were then co-cultured (10:1 for 72 h) with OT-I CD8⁺ T cells labeled with carboxy fluorescein succinimidyl ester (CFSE). Proliferation of these T cells was assessed by CFSE dilution. n = 3 biologically independent samples. d Antigen cross presentation analysis for WT or Ch25h^−/− DCs pre-treated or not with 25HC (50 nM, 4 h before adding beads loaded with OVA at indicated percentage) was carried out as in panel C. n = 3 biologically independent samples. e Antigen cross presentation analysis for Ch25h^−/− DCs transduced with retroviruses for expression of CH25H^WT or catalytically inactive CH25H^H242,243Q mutant. n = 4 biologically independent samples. f Antigen cross presentation analysis for WT DCs pre-treated with or without PGE₂ (10 ng/mL), VEGF (20 ng/mL) or TCM from LLC cells (75%, v/v) for 24 h with or without the treatment of 25HC (50 nM) or DC661 (5 μM) for 4 h as indicated. Then DCs were pulsed with soluble OVA protein (50 or 100 μg/mL, 6 h) and co-cultured with CFSE-labeled OT-I T cells (10:1 for 72 h). n = 3 biologically independent samples. g Antigen cross presentation analysis for WT DCs pre-treated with or without PGE₂ (10 ng/mL), VEGF (20 ng/mL) or TCM from LLC cells (75%, v/v) for 24 h with or without the treatment of 25HC (50 nM) or DC661 (5 μM) for 4 h as indicated. Then DCs were then treated with beads-bound OVA protein (25% or 50%, 1 h) and co-cultured with CFSE-labeled OT-I T cells (10:1 for 72 h). n = 3 biologically independent samples. h Antigen cross presentation analysis for DCs of indicated genotypes. T cell proliferation was assessed by flow cytometry in CFSE-labeled OT-I T cells co-cultured (10:1 for 72 h) with DCs from indicated mice, pretreated with conditioned media from LLC cells (70%, v/v) for 24 h and pulsed with soluble OVA protein (200 μg/mL, 6 h). n = 5 biologically independent samples. h Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Students’ t-test (A, B, C, D, E and H) or 1-way ANOVA with Tukey’s multiple-comparison test (F and G) test. n.s., not significant. Source data are provided as a Source Data file.