Fig 2: Two gating strategies for DC subsets generated from murine BM with Flt3L. Total BM cells were cultured for 7 (A) or 10 (B) days in a complete medium supplemented with Flt3L. (A) Before harvesting of the cells, calibration beads were added. Cells were then collected and processed for flow cytometry. Cells are first identified based on size and granularity by plotting the forward (FSC-A) versus side scatter (SSC-A). Doublets are then exclude based on the FSC-W and FSC-H, and live cells are identified as PI-negative. On single living cells, populations are determined as follows: CDP are CD11c- Siglec-H-; pre-DC are CD11c+ MHC-II- Siglec-H-; pDC are CD11cint Siglec-H+ CCR9+; cDC are CD11chi MHC-II+ Siglec-Hlow/- and can be further divided into cDC1 (XCR1+ Sirp-a-) and cDC2 (XCR1- Sirp-a+). (B) Cells were collected and processed for flow cytometry. Living, single cells are selected (not shown) and analyzed for CD11c expression. CD11c+ cells are next split into pDC (B220hi CD11b-) and cDC (B220-). cDC are further subdivided into cDC1 (Sirp-a- CD11blo CD24hi) and cDC2 (Sirp-a+ CD11b+ CD24lo). Numbers near the gates represent percentage of parent gate.

Fig 3: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen, Related to Figure 5(A). Gating strategy for the identification of DC progenitors in the bone marrow (BM)(B). Palantir pseudo-time analysis of differentiation potential and branch probabilities from the Siglec-H+ pre-DC state to T-bet+ cDC2 and T-bet– cDC2 terminal states.(C). Plots showing Palantir differentiation potential (y axis) along Palantir pseudo-time (x axis) for Siglec-H+ DC and T-bet+ cDC2s (top) or Siglec-H+ DC and T-bet– cDC2 clusters (bottom)(D). Plots showing the top two diffusion component embeddings for Siglec-H+ DC and T-bet+ cDC2 clusters (top) or Siglec-H+ DC and Tbet– cDC2 clusters (bottom). Black arrow indicates Siglec-H+ DC cluster cells adjacent to cells from the proliferative T-bet+ cDC2 clusters 6 and 8.(E). Top panel: plots showing probability of each cell being within 20 nearest neighbors of randomly sampled shortest paths from the Siglec-H+ DC to the indicated end points. Middle panel: plots showing the proportion of cells belonging to Siglec-H+ DC, T-bet+ cDC2, or T-bet– cDC2 from 20 nearest neighbors of randomly sampled shortest paths. Bottom: plots showing diffusion distance step sizes for each step along the indicated shortest paths (bottom panel). Colors illustrate cluster membership.(F). Graph showing AUC (x axis) for genes differentially expressed between Siglec-H+ DC cluster (cluster 11) and all other cDC2 clusters. EMD on the y axis. Dashed lines represents µEMD ± 3sEMD.(G). Gating strategy for FACS-isolation of MHCII+ ILC3s: Lin = CD3, CD19, CD49b, Siglec-F.(H). Heatmap reports scaled expression of 3550 differentially expressed genes (log2FC > 1, FDR < 0.01) between ILC3s and Ror?t fm cDC2s. Selected genes listed to the right.(I). Representative flow cytometric analysis of phenotypes of splenic progeny from Tbx21RFP-cre CD45.2+Ly6C-CD64–MHCII+CD11c+Siglec-H+ pre-DCs adoptively transferred into sub-lethally irradiated CD45.1 recipient mice 7 days earlier (data from one experiment with n = 3).J. Sort purified T-bet+ or T-bet– cDC2 were cultured for 24hrs in the presence of LPS, CpG, TNF-a or IFN-?. Representative overlay histogram showing the expression of RFP(T-bet) at 24hrs. Data representative of 2 (TNF-a) or 4 (all other cytokines/TLR agonists) independent experiments, n = 2-3.

Fig 4: 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.