Fig 2: Uptake of B16F10-derived EVs by LN LECs is partly dependent on VCAM-1. (A, B) Representative histogram (left panels) and quantification (right panels) of a4 integrin (ITGA4, A, N = 5) and ß1 integrin (ITGB1, B, N = 3)) on the surface of B16F10-derived EVs detected by small particle flow cytometry. * p < 0.05, ** p < 0.01, Student's t-test. (C) Representative immunofluorescence images of subcapsular (left) and medullary (right) regions of naïve and B16F10 tumour-draining inguinal LNs stained for Lyve-1 (red) and VCAM-1 (green). (D) Example histograms and quantification of VCAM-1 expression in LN LEC subsets by flow cytometry (N = 3). *** p < 0.001, **** p < 0.0001, one-way ANOVA with Sidak's post-test. (E) Re-analysed single-cell RNA sequencing data of human LN LECs (Takeda et al., 2019) demonstrating enrichment of VCAM-1 transcripts in fLECs (LEC II) and mLECs (LEC IV). (F, G) B16F10-derived EVs labelled with DiD were injected into tamoxifen-treated Prox1-CreERT2 x Vcam1fl/fl mice. Example histograms (left panels) and quantification (right panels) of DiD uptake in popliteal LN LECs (F) and MSMs (G) (Control = wildtype mice injected with control liposomes; N = 3–4 pools of 2–3 mice each / condition). * p < 0.05, unpaired Student's t-test

Fig 3: The effect of altered gp130 signalling on MDSCs during intestinal inflammation.Wild type (WT), gp130757F/F, LysMcre/STAT3flox and gp130757F/F LysMcre/STAT3flox mice were treated with the acute (A–E) or chronic (F) DSS protocol to induce colitis and compared with untreated mice (H2O). (A) Cell populations from spleens (SPL), mesenteric lymph nodes (MLN), intraepithelial lymphocytes (IEL) and lamina propria mononuclear cells (LPMC) of wild type mice were characterized by flow cytometry for subpopulations of CD11b+Ly6ClowLy6G+CD49d- granulocytic MDSCs (G-MDSC) and CD11b+Ly6ChighLy6G-CD49d+ monocytic MDSCs (M-MDSC). (B) Colonic cell population from IELs and LPMCs of untreated WT mice (H2O) and DSS-treated WT and gp130757F/F mice were characterized by flow cytometry for subpopulations of CD11c+F4/80-Ly6G-Ly6C- dendritic cells (DC), CD11c-CD11b+Ly6G-F4/80+ monocytes (Mo), CD11b-F4/80+Ly6C-Ly6G- macrophages (MF) and CD11b+Ly6G+Ly6ClowF4/80-CD11c– granulocytes (Gr). (C) Colonic cell population from IELs and LPMCs of DSS-treated wild type (WT) and gp130757F/F mice were characterized by flow cytometry for subpopulations of CD11b+Ly6ClowLy6G+CD49d- granulocytic MDSCs (G-MDSC) and CD11b+Ly6ChighLy6G-CD49d+monocytic MDSCs (M-MDSC). (D) Arginase activity was determined in homogenized colon tissue of DSS-treated or untreated (H2O) wild type (WT) and gp130757F/F mice (Di) and also in polymorphonuclear Gr1highLy6G+ MDSCs isolated from spleens of wild type (WT) and gp130757F/F mice (Dii). (E) Granulocytic Gr1highLy6G+ MDSCs (G-MDSCs) were isolated from spleens of wild type (WT), gp130757F/F, LysMcre/STAT3flox and gp130757F/F LysMcre/STAT3flox mice and 1 × 106 cells were cultured for 24 hours. Cell culture supernatants were analysed by luminex array. (F) Cell populations from lamina propria mononuclear cells (LPMCs) were isolated from the colon of mice with chronic DSS-induced colitis and characterized by flow cytometry for MDSC subpopulations (CD11b+CD45+CD11c-/int, Gr1high/low). *indicates P < 0.05 and **indicates P < 0.01 (compared to untreated control or wild type); #indicates P < 0.05 (compared to LysMcre/STAT3flox or gp130757F/F mice). Bars represent the mean±SEM of at least three independent experiments (n = 5–12 per group).