Fig. 6: Lack of UBL3 impairs DC-targeted antigen presentation in vivo.

a Left: spleen cDC1 expression of CLEC9A from wild type (WT) or Ubl3^−/− mice by flow cytometry, n = 11 mice examined over three experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). Right: Spleen cDCs from WT or Ubl3^−/− mice were labeled with FIP-conjugated anti-CLEC9A mAb. After 30 mins at 37°C, quencher (Q) was added and percentage internalization was calculated as described in Methods. Histograms show representative CLEC9A-FIP signal. Right: quantification of internalization from one experiment performed in triplicate, with bars mean ± SD, ns (not significant) p > 0.05, unpaired t test (two-sided). b In vivo antigen presentation assay. Purified CellTrace Violet (CTV)-labeled OT-I and OT-II cells were adoptively transferred into WT and Ubl3^−/− mice. 24 hours later, mice were injected with 0.2 µg anti-CLEC9A mAb-targeted OVA and spleens harvested after 64 hours. Antigen presentation capacity was assessed by flow cytometric analysis of CTV-dilution by OT-I and OT-II proliferation. Data from n = 9 (OT-I) and n = 8 for WT, 7 for Ubl3^−/− (OT-II) mice, two experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). c Quantification of spleen cDC1 and cDC2 from WT or Ubl3^−/− mice. Data from n = 9 mice, three independent experiments, bars mean ± SD, symbols representing individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). d Left: C3 expression on spleen cDCs from WT, Ubl3^−/− or Marchf1^−/− mice by flow cytometry. Data from n = 5 from one experiment, bars mean ± SD, symbols represent individual mice. Two-way ANOVA with Bonferroni’s test. Right: Quantification of trogocytic B cells gated on B220⁺CD19⁺ cells in Nycodenz-enriched splenocytes from WT, Ubl3^−/− or Marchf1^−/− mice. Representative dot plots and data from one experiment with n = 5, bars mean ± SD, and symbols representing individual mice. Significant p values shown above bars, with ****p < 0.0001, ns (not significant) p > 0.05, one-way ANOVA with Bonferroni’s test.