Fig. 6: TMEM175 knockout augments the cross-presentation of tumor antigens.

a–g Wild type C57BL/6 J mice were subcutaneously injected with \(1\times {10}^{6}\) B16-F10-OVA cells. On day 10, the splenic lymphocytes from tumor bearing mice were harvested, CFSE-labeled, and cultured with WT or Tmem175^−/− BMDMs in the presence of IL-2 (20 U/mL) for 48 h before FCM and ELISA analysis (n = 6 biologically independent samples). During the co-culture, B16-F10-OVA cell debris (2.5 × 10⁵/mL), IL-1β (500 pg/mL), and IL-18 (500 pg/mL) were added in the desired group. b, c Splenic lymphocytes were labeled by CFSE, thus the CFSE^low cells represented the successive generations of cells. Tmem175^−/− BMDMs induced more robust proliferation of CD8⁺ T cells (CFSE^low cells gated from CD3⁺ CD8⁺ cells). IL-1β and IL-18 supplementation further promoted the CD8⁺ T cell proliferation in Tmem175^−/− group. d Tmem175^−/− BMDMs induced more CD8⁺ OVA-tetramer⁺ T cells (gated from CD3⁺ cells). IL-18 significantly promoted the CD8⁺ OVA-tetramer⁺ T cell expansion. e Tmem175^−/− BMDMs induced more robust CD8⁺ CD69⁺ activated T cells (gated from CD3⁺ cells). IL-1β and IL-18 supplementation further promoted the CD8⁺ T cell activation in Tmem175^−/− group. f Tmem175^−/− BMDMs induced more IFN-γ secretion. IL-1β supplementation promoted IFN-γ secretion in WT and Tmem175^−/− group. IL-18 supplementation promoted the IFN-γ secretion in WT group. g Tmem175^−/− BMDMs induced more TNF secretion. IL-18 supplementation promoted the TNF secretion in WT group. h Brief illustration of the engulfment and digestion capacity examination. i Fluorescence intensities of intracellular p15E in Tmem175^−/− BMDMs (gated from CD45⁺ CD11b⁺ F4/80⁺ cells) were significantly higher than in WT BMDMs (n = 3 biologically independent samples). j Fluorescence intensities of FITC-dextran in Tmem175^−/− BMDMs (gated from CD45⁺ CD11b⁺ F4/80⁺ cells) were comparable with those in WT BMDMs (n = 3 biologically independent samples). k, l Cells in the co-culture system were harvested for RNA-seq to investigate the alterations in the gene expression, GO terms, and KEGG pathways (n = 3 biologically independent samples). Up-regulated GO terms by TMEM175 knockout without (k) or with (l) B16-F10-OVA cell debris treatment. m, n CD8⁺ T cells in the co-culture system were harvested and adopted to Cd8a^−/− mice 1 day before and on day 7th after B16-F10-OVA implantation (n = 5 mice). Tumor bearing mice were euthanized on day 14th after B16-F10-OVA implantation to weigh the tumors. Representative results from two independent experiments are presented as mean ± SEM, ns denotes not significant. Statistical significances in (c–g, i, j) were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Statistical significances in (n) were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Source data are provided as a Source data file. a–m Created in BioRender. Wang, Y. (2026) https://BioRender.com/yaf354h. h Created in BioRender. Wang, Y. (2026) https://BioRender.com/69fpsu9.