Fig 2: Gating strategy for skin ILCs and myeloid cells(A) The gating strategy for ILCs. Lineage markers include CD3e, CD5, CD19, CD11b, Cd11c, FceRIa, NK1.1 and CD2. Lin+ Thy1– cells are myeloid cells. The majority of Lin+ Thy1+ cells are T cells. Lineage– Thy1+ cells are identified as a mixture of ILC2s and ILC3s. From this gate, GATA3high cells are ILC2 and GATA3low ROR?thigh cells are ILC3s.(B) The gating strategy of skin myeloid cells. Lineage markers include CD3e, CD19, NK1.1 and Siglec-F. Gate for CD45+ lineage– (Siglec-F–, CD3e–, CD19– and NK1.1–) to exclude eosinophils, T cells, B cells and NK cells. Lineage– CD11b+ Ly6c+ cells are identified as monocytes. From the CD11b+ Ly6c– gate, macrophages (EpCAM– CD64+), Langerhans cells (CD64– EpCAM+ MHC II+) and dermal dendritic cells (CD64– EpCAM– MHC II+ CD11c+) are identified.

Fig 3: Cold exposure modulates monocytes in the circulation(A) Volcano plot showing the up- and downregulated transcripts by RNA sequencing of monocytes that were MACS (anti-PE) and FACS (CD115-PE+) sorted from blood of 2-week cold-exposed (10°C) mice compared to room-temperature mice.(B) 15 most enriched Gene Ontology biological processes of monocytes as in (A). Genes were considered differentially regulated with p < 0.05. Shown are –log(p value).(C) Blood immune cells of mice as in (A) visualized using UMAP and clustered using FlowSOM algorithm in R. Ly6Chi monocytes (green), Ly6Clo monocytes (yellow), neutrophils (brown), eosinophils (orange), and others. Heatmap shows median relative expression of all panel markers.(D) Flow cytometry analysis of blood cells from mice as in (A). Percentage of Ly6C high (hi), intermediate (int), and low (lo) monocytes of total, single CD45+ cells.(E) Percentage of blood MHCII+ cells of Ly6Chi monocytes (left), MHCII+ Ly6Chi monocytes of total MHCII+ cells (middle), and total MHCII+ cells of total, single CD45+ cells (right) of mice as in (A).(F) Blood cell analysis after 1 week of daily i.p. injected beta 3 adrenoreceptor agonist CL316,243 or vector (PBS). Percentage of MHCII+ cells of Ly6Chi monocytes (left), MHCII+ Ly6Chi monocytes of total MHCII+ cells (middle), and total MHCII+ cells of CD45+ cells (right).(D–F) Each dot represents one animal. Shown is mean ± SD. Significance was calculated using Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Pool of three experiments (D and E).

Fig 4: T-bet– cDC2s Are Distinct from Monocytes(A) Expression of Zbtb46 in bulk sorted DC subsets.(B) Representative plot showing expression of CSF1R in cDC2s and summary graph (right). Each symbol represents one mouse.(C) Representative plot showing expression of CX3CR1 in cDC2s and summary graph (right). Each symbol represents one mouse.(D) Expression of CCR2 (GFP) in splenic DCs. T-bet– cDC2s identified as CLEC12A+Esam– and T-bet+ cDC2s as CLEC12A+Esam– cDC2s. Monocytes gated as Lin–Ly6C+CD11b+CX3CR1+. Each symbol represents one mouse.(E) Frequency of monocytes and indicated cDC2 subset in CCR2–/– mice and CCR2+/– littermates.Error bars represent mean ± SEM; p values calculated using Student’s t test.

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.