Fig 2: Characterization analysis of ADSCs by flow cytometry. ADSCs were positive for mesenchymal stem cell surface antigen CD29, CD44 and CD90, but negative for hematopoietic surface antigen CD34 and CD45, and also negative for vascular endothelial cell antigen CD31

Fig 3: Single-Cell Survey Reveals Heterogeneity of cDC2s, Related to Figure 1A. Representative histogram showing expression of T-bet (RFP) in splenic cells from Tbx21RFP-cre mice.(B). Expression of T-bet in CD11b+XCR1+ DCs from the intestinal lamina propria. Data representative of > 5 independent experiments, with at least 3 mice per experiment.(C). Expression of T-bet in splenic myeloid cells. Cells were defined as: (i) Ly-6Chi monocytes (Lin –Ly6C+Ly6G–CD11b+CX3CR1+); neutrophils (Lin–Ly6C+Ly6G+); macrophages (Lin–CD64+Ly6C–). Lineages (Lin) were defined as: CD3e, CD90.2, CD19, CD49b and Siglec F. Each circle represents an individual mouse, error bars represent mean ± SEM.(D). Left: Gating strategy for single-cell sorting. DCs were defined as Lin(CD3, CD19, CD90)–Ly6C–CD64–CD11c+MHCII+. Two populations were sampled: RFP+ DCs and RFP– DCs (encompassing XCR1+ cDC1s, CD11b+RFP– and CD11b–XCR1– DCs). Right: Post-sort purity of RFP+ and RFP– cells. Contaminating population of Ly6C+ cells identifiable on post-sort purity (lower panel).(E). Similarity of splenic CD11c+MHCII+ cells to reference myeloid cells (ImmGen Consortium) Colors represent the Pearson correlation between the mean gene expression from the dendritic cell cluster in the rows and the bulk reference transcriptome in the columns.(F). Top 20 positive and negative gene loadings of PC1 for T-bet+ cDC2 clusters after cell-cycle correction (left panel). Scatterplot of PC1 and PC2 for T-bet+ cDC2 clusters after cell-cycle correction (right panel).

Fig 4: Restoration of Foxp3 expression in peripheral Treg “wannabes” rescues immune activation in male Foxp3LSL mice.a, Experimental Design. 2-week-old mice were treated with 4-hydroxytamoxifen (4-OHT) while being continuously treated with FTY720 to block thymic output. A Rosa26loxP-STOP-loxP-tdTomato (R26LSL-tdTomato) recombination reporter allele was introduced to the mice to enable labeling of all CD4 T cells undergoing Cre-mediated recombination induced by 4-OHT. b, Number of Treg cells in the thymus (left), and percentage of tdTomato+ cells among Foxp3-Thy1.1- CD4SP thymocytes (right), from FTY720-treated mice and untreated controls on day 14. Two-tailed unpaired t-tests with multiple hypothesis testing correction using the Holm-Sidak method. c, d, Numbers of activated, proliferating, and cytokine-producing conventional CD4 and CD8 T cells in the spleen (c) and lymph nodes (d). One-way ANOVA with Tukey’s multiple comparison test. All error bars denote mean ± s.e.m. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Fig 5: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen(A) Derivation of splenic CD11b+ DC subsets from bone marrow (BM) progenitors. Shown are splenic cDC2s 7 days post transfer. Data representative of MDP and CDP recipients (n = 3) or pre-DC recipients (n = 6) from 3 independent experiments.(B) Analysis of YFP in BM progenitors from RorcCreRosa26lsl-YFP mice (top) or RFP expression in cells from Tbx21RFP-cre mice (bottom).(C) t-SNE embedding of diffusion map of T-bet+, Tbet– cDC2, and Siglec-H+ pre-DC clusters identified in Figure 1E.(D) Palantir branch probabilities from Siglec-H+ pre-DC to T-bet+ cDC2 (top) or Tbet– cDC2 (bottom) terminal states. Gene expression trends in pseudo-time along corresponding trajectories.(E) Expression of genes identified as varying significantly along the trajectory from the pre-DC cluster to T-bet+ or Tbet– cDC2.(F) Expression of Siglec-H in Lin–CD90–Ly6C–CD64–CD11c+MHCII+ cells. Far right: overlay of Siglec-H+ DCs against all MHCII+CD11c+ DCs.(G) Representative plot showing percentage of YFP+ cells in splenic cDC1 and cDC2 populations in RorcCreRosa26lsl-YFP mice.(H) Heatmap showing expression of genes differentially expressed between bulk T-bet+ and Tbet– cDC2s across T-bet+, Tbet–, and Ror?t fate-mapped (fm) cDC2s.(I) Frequency of T-bet+ cDC2s within the spleen, MLN, large intestine lamina propria (LI), and small intestine lamina propria (SI) at indicated time point post birth. n = 3 per time point, error bars represent mean ± SEM. For the day 7 analysis SI and LI tissues were pooled from 2 mice per sample.(J) Generation of T-bet+ cDC2s in mice treated with a broad-spectrum antibiotic cocktail (AVKM) or H2O (control). Each symbol represents one mouse. Error bars represent mean ± SEM.See also Figure S4 and Table S5.