Fig 2: Antigen-based classification of lymphatic endothelial cells (LECs) and identification of marker genes.(a) Day 14 LECs were classified into antigen-high and antigen-low using a two-component Gaussian mixture model. A uniform manifold approximation and projection (UMAP) is shown for antigen-low and antigen-high cells. T cells, B cells, and epithelial cells are shown in white (Other). (b) Distribution of antigen counts for antigen-low (light blue) and antigen-high (dark blue) cells. Dotted lines indicate mean counts for each population. Identification of genes associated with ovalbumin (ova)-low and ova-high for each cell type is available in Figure 5—source data 1. (c) The fraction of cells belonging to each LEC type for antigen-low and antigen-high populations. (d) UMAPs show expression of genes significantly enriched in the antigen-high population (scale is log-normalized counts). (e) Expression (log-normalized counts) of antigen-high markers in each cell type. (f) Mice were injected in the footpad with nystatin (dose) and 1 hr later ova488/polyI:C/aCD40. After 24 hr, mice were euthanized and draining popliteal lymph node (LN) removed, stained for LEC markers (CD45, PDPN, CD31, PDL1), and gated as in Figure 3—figure supplement 4. Shown are representative flow plots and quantification of geometric mean fluorescence intensity (gMFI) from naive (black bar), vehicle control (red bar), and nystatin treated (blue bar). Three mice per group were evaluated, and experiment was performed three independent times with similar results. Nystatin treatment reduces ova488 signal in LECs relative to vehicle (p=0.02; Wilcoxon rank-sum test). Error bars indicate standard error of the mean (SEM). Figure 5—source data 1.Genes associated with ovalbumin (ova)-low and ova-high cells for dendritic cells (DCs), FRCs, and lymphatic endothelial cells (LECs).Ova-low and ova-high cells were identified independently for DCs, FRCs, and LECs using a Gaussian mixture model implemented with the R package mixtools. Differentially expressed genes were identified as described for Figure 4—source data 1.

Fig 3: Phenotypic characterization of human umbilical cord mesenchymal stem cells. (A) The phenotype of mesenchymal stem cells (MSCs) was identified, including positive markers CD90, CD105, CD73, CD44, and CD29 and negative markers CD45 and HLA-DR. (B) Isotype antibodies were used as controls.

Fig 4: Flow cytometry analysis of corneal dendritic cell (DC) phenotype. Using a gating strategy (A), the CD86+/CD68+ CD45+CD11c+ DCs were isolated and examined for each saline treatment. The modal histograms (B) show the CD86 and CD68 staining intensity of the concatenated DC populations in each treatment group, with the bold line delineating the CD86 and CD68 gating threshold. The percentage of CD86+ DCs (C) increased in corneas after topical application of 450 mOsm/L and 600 mOsm/L saline compared to 350 mOsm/L saline, whereas CD68+ DCs (D) did not differ between groups. Data are presented as mean ± SEM. Asterisks denote statistical significance between groups (**P < 0.01, ***P < 0.001). ns, not statistically significant.

Fig 5: MSC characterization and in vitro differentiation capacity.a Flow cytometry results show that MSCs from both young and aged donor mice were uniformly positive for MSCs markers CD44 and Sca-1 and negative for hematopoietic stem cells markers CD34 and CD45 and endothelial marker CD31 (n = 6). b MSCs from young and aged donor mice display fibroblast-like morphology on bright field microscopy (left panels; scale bar = 20 µm). Following exposure to differentiation media MSCs from both young and aged mice showed osteogenic (middle panels) and adipogenic (right panels) differentiation as detected by Von Kossa and oil red O staining, respectively. c Quantitative assessment of calcium deposition showed significantly less calcium deposition in aged MSCs compared to young following exposure to differentiation media (p = 0.05; t test; n = 3 each group). d There was no significant difference in lipid content between groups (t test; n = 3 each). MSCs cultured in control medium served as negative control for both conditions. Error bars show s.d.