Extended Data Fig. 2

a, Imaging flow cytometry analyzing the physical interaction between HKCA with DCs or with Aire⁺ ILC3s. CD11c⁺ DCs or Lineage negative cells were isolated from mouse pLNs using MACS-beads depletion and then incubated with CPD-stained HKCA for 90 minutes. Samples were stained for Aire, MHCII, CD11c, Lamp1 (lysosomal marker) and DAPI and analyzed by Imaging flow cytometry. In both, Aire⁺ ILC3 cells (Aire⁺) and DCs (CD11c⁺) HKCA associate with lysosomes (Lamp1). Representative images out of five independent repetitions of the experiment are shown. b, Comparison of the capacity of different immune subsets (B cells, Ly6C⁺ MNPs, CD11b⁺ MNPs, CD11c⁺ MNPs and Aire⁺ ILC3s) to endocytose CPD-labeled HKCA in an in vitro endocytosis assay. Subsets were FACS-sorted and incubated with HKCA-CPD for three hours. The frequency of cells that have successfully internalized HKCA-CPD was then assessed by flow cytometry (mean ± SD; n = 3). c,d, Comparison of the capacity of either wild-type or Galectin-3/Dectin-1-double knockout (dKO) Aire⁺ ILC3s (c) or DCs (d) to endocytose CPD-labeled HKCA at 37 °C or at 4 °C. Corresponding populations were isolated from WT or dKO mice and kept either at 37 °C or at 4 °C. The internalization of HKCA-CPD by the cells was measured by FACS (mean ± SD; n = 3). e, Representative gating strategy of APC populations related to Fig. 3c–g. f, FACS analysis of in vitro assay related to Fig. 3c. g, Graphical summary of an experimental setting relevant for data shown in h. Wild-type mice were intravenously stimulated by HKCA in specified timepoints and APC population were isolated using gating strategy depicted in e. h, FACS analysis of in vitro assay related to Fig. 3d–f assessing the antigen presentation of the corresponding cell subsets.