Volume 21, Issue 5, Pages 1203-1214 (October 2017) A Discrete Subset of Monocyte-Derived Cells among Typical Conventional Type 2 Dendritic Cells Can Efficiently Cross-Present  Jianpeng Sheng, Qi Chen, Irene Soncin, See Liang Ng, Klaus Karjalainen, Christiane Ruedl  Cell Reports  Volume 21, Issue 5, Pages 1203-1214 (October 2017) DOI: 10.1016/j.celrep.2017.10.024 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Identification of a CD24+CD11chiMHCIIhiF4/80hiCD169+ APC Subset (A) Gating and staining strategy used to delineate distinct myeloid cell subpopulations (cDC1, cDC2, F4/80hi Mϕ, and F4/80hi APC) in the small intestine, lungs, spleen, and mesenteric and mediastinal lymph nodes. (B) t-SNE analysis of cDC1, cDC2, F4/80hi Mϕ, and F4/80hi APC in the small intestine, lungs, spleen, and lymph nodes based on multi-color flow cytometry. Data are representative of nine mice from three independent experiments. Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Phenotypic Comparison between cDC2s and F4/80hi APCs (A) cDC2 and F4/80hi APCs isolated from the small intestine, lungs, spleen, and mesenteric and mediastinal lymph nodes were gated according to the strategy shown in Figure 1A. Both subsets were tested for expression of core Mϕ markers, including CD64, CD169, MERTK, and CX3CR1; core DC markers, including CLEC4A4/DCIR2 and PLET1; and the monocyte markers LY6C and CCR2. Red histograms show surface marker expression on cDC2 and F4/80hi APCs, and negative controls are indicated in gray. Data are representative of eight independent mice. (B) Intranasal CFSE instillation schedule is shown in the top left panel. Mediastinal lymph node subsets were gated as migratory CD103+CD11b− cDC1, CD103−CD11b+ cDC2, and CD169+ F4/80hi subsets, as well as resident CD8+CD11b− cDC1 and CD8−CD11b+ cDC2 populations (top right panel). CCR7 expression and CFSE labeling are shown in the corresponding histograms (bottom panels). Data are representative of six mice from two independent experiments. Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Fate-Mapping Analysis Delineates Distinct Origins for cDC2s and F4/80hi APCs (A and B) YFP labeling index of four distinct myeloid cell subpopulations determined in two different fate-mapping mouse models: LysMCre/R26 (A) and inducible KitMerCreMer/R26 mice (B). cDC1s, cDC2s, F4/80hi Mϕs, and F4/80hi APCs were gated according to the strategy shown in Figure 1A. Representative flow cytometry analysis illustrates labeling efficiency in the small intestine, lungs, spleen, and mesenteric and mediastinal lymph nodes of LysMCre/R26 mice (A). YFP expression (x axis) is plotted against CD11c or F4/80 (y axis). KitMerCreMer/R26 inducible fate-mapping mice were treated with tamoxifen, and cells obtained from lung were analyzed after 3 months of chase (B). Representative dot plots and histograms show the YFP labeling efficiency of cDC1s, cDC2s, F4/80hi Mϕs, and F4/80hi APCs. Data are representative of five mice from two independent experiments. Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 F4/80hi APCs Are Derived from Ccr2-Dependent Bone Marrow Progenitors For competitive bone marrow chimera experiments, CD45.1+ WT (1 × 106) and CD45.2+ Ccr2−/− (3 × 106) mouse bone marrow leukocytes were mixed and transferred into lethally irradiated CD45.1+/CD45.2+ recipient mice. Reconstituted chimeric mice were analyzed after 4 months by flow cytometry (top left). Representative proportions of different donor cells in bone marrow stem cell (LSK) and progenitor (MDP and CDP) compartments are shown (top right panel). Bar charts (bottom) represent the CD45.1+/CD45.2+ ratio of LSK, MDP, and CDP progenitors in the bone marrow (BM) and of cDC2s and F4/80hi APCs in the small intestine (SI), lungs, spleen (SP), and mesenteric (mLN) and mediastinal (MLN) lymph nodes. Data represent mean ± SEM (n = 14 mice from three independent experiments). Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 F4/80hi APCs and cDC2s Display Distinct Cytokine Requirements CSF1 signaling was neutralized by repetitive injections of anti-CSF1R blocking antibody for two consecutive weeks. Csf2rb−/− mice were used to assess the requirement of CSF2 signaling. The levels of F4/80hi Mϕs and F4/80hi APCs were determined according to the gating strategy shown in Figure 1A. Bar charts indicate cell number (×103) of F4/80+ cells in the small intestine, lungs, and spleen and of CD169+ cells in mediastinal and mesenteric lymph nodes (LN). Data represent mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Bonferroni test. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001. WT mice, n = 7; Csf2rb−/− mice, n = 3–5; CSF-1R-antibody-treated mice, n = 3–6; mice from two independent experiments. Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 F4/80hi APCs Are Enriched in the Tumor Microenvironment Leukocytes obtained from lung metastases of B16 melanoma or from intestinal adenomas and surrounding tissues were analyzed according to the gating strategy in Figure 1A. CD24 gating was included in the analysis but omitted from the plot for clarity. (A) Schematic representation of the B16 melanoma lung metastasis model (top). Flow cytometry representation of myeloid cells in B16 tumor and tumor-free lung tissue (bottom left) and bar chart analysis of F4/80hi APCs in B16 tumors and the surrounding lung tissue. Data are shown as mean ± SEM. Statistical significance was determined using an unpaired Student’s t test. ∗∗∗∗p < 0.0001; n = 9 mice from two independent experiments. (B) Schematic representation of the intestinal adenoma model of APCMin/+ mouse. Adult APCMin/+ mice were treated with dextran sodium sulfate (DSS) in the drinking water for 1 week. Cells were isolated and analyzed 3–4 weeks post-treatment (top). Intestinal adenomas and surrounding lamina propria were collected and analyzed independently. Flow cytometry representation of myeloid cells in adenomas and intestinal lamina propria (bottom panel, left), and bar chart analysis of F4/80hi APCs detected in adenomas and intestinal lamina propria, respectively. Data represent mean ± SEM. Statistical significance was determined using an unpaired Student’s t test. ∗∗∗∗p < 0.0001; n = 5 mice from two independent experiments. Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 F4/80hi APCs Are Phagocytic and Able to Cross-Present Antigen (A) Phagocytic capacity of different Mϕ and DC subpopulations. 18–21 days after injection of control B16 or B16-GFP melanoma cells into WT mice, melanoma lung foci were analyzed (top). cDC1s, cDC2s, F4/80hi Mϕs, and F4/80hi APCs were gated according to the strategy shown in Figure 6. B16-GFP melanoma cell uptake is depicted by red histograms. Gray full histograms represent background levels obtained from mice inoculated with control B16 cells. Data are representative of 12 independent mice. (B) F4/80hi APCs can cross-present in vivo. DT-injected Clec9A-DTR mice and anti-CSF1R-treated WT mice were injected with 4 × 106 irradiated OVA-expressing B16 cells followed 2–3 hr later by injection with 1 × 106 CFSE-labeled OVA-specific CD8+ T cells. Two days later, the percentage of dividing CD8+ T cells was determined by measuring CFSE staggering. On the left, representative histograms illustrating CFSE labeling of isolated CD8+ T cells from WT, Clec9A-DTR, and anti-CSF1R-treated WT mice. Bar chart on the right indicates mean + SEM of the percentage of undivided cells. Statistical significance was determined using one-way ANOVA followed by Bonferroni test. ∗∗∗p < 0.005 and ∗∗∗∗p < 0.0001; n = 3 mice for each experimental group. (C) Ex vivo cross-priming assay. B16-OVA tumor inoculation and co-culture schedule is shown in the top panel. Dividing CD8+ T cells were determined by measuring CFSE staggering. On the left, representative histograms illustrating the CFSE-labeling of co-culture with no DCs, cDC1s, and F4/80hi APCs. Bar chart on the right shows mean + SEM of the percentage of undivided cells. Statistical significance was determined using one-way ANOVA followed by Bonferroni test. ∗∗∗∗p < 0.0001; n = 4–5 mice for each experimental group. Cell Reports 2017 21, 1203-1214DOI: (10.1016/j.celrep.2017.10.024) Copyright © 2017 The Author(s) Terms and Conditions