Figure 5: Maturation and tolerogenic features of murine and human neonatal skin DC2 are sustained upon commensal uptake.

(A-B) Neonatal mice were colonized with S. epi-ZsGreen and skin was harvested 16h later, with commensal antigen-loaded (ZsGreen⁺) and unloaded (ZsGreen⁻) DCs separately sorted and submitted for scRNAseq. (A) UMAP of all ZsGreen⁺ vs. ZsGreen⁻ DCs. (B) Heatmap of select genes differentially expressed between ZsGreen⁺ vs. ZsGreen⁻ CD301b⁺ DC2 (CD301b status defined based on Mgl2 expression). (C) Spectral flow cytometry of key markers both percent positive and gMFI on positive cells for ZsGreen⁺ vs. ZsGreen⁻ CD301b⁺ DC2 in neonatal skin 16h after S. epi-ZsGreen colonization. Each dot is a biological replicate. 1 of 2 independent experiments. Two-way ANOVA. (D-F) Human neonatal foreskin was incubated 4h with S. epi-ZsGreen then live ZsGreen⁺ and ZsGreen⁻ CD45⁺CD16⁻ HLADR⁺ cells were separately sorted and submitted for scRNAseq. (D) UMAP of all cells combined, (E) UMAP of DC clusters split by ZsGreen status, and (F) heatmaps comparing expression of select genes between ZsGreen⁺ and ZsGreen⁻ DC2. (G) CD301b⁺ or CD301b⁻ DC2 were sorted from D10 SDLN migDC, incubated overnight with S. epi-OVA, then cultured with CTV-labelled OT-II plus IL-2 and TGF-β to measure DC Treg-promoting capacity. (H) Representative plots (pre-gating: Live CD4⁺TCRβ⁺) and (I) and quantification of OT-II Treg cells at 72h. Each dot represents a biological replicate of DCs pooled from independent groups of neonatal mice, data pooled from 2–4 independent experiments. Student’s unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001, ns = not significant.