DC enriched CD103. CD11b. CD11c. Spleen. DC enriched. CD11c. DC enriched. CD11c MLN

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1 CD CD11 + CD + CD11 d g CD CD11 + CD + CD11 CD + CD11 + Events (% of max) Events (% of max) CD + CD11 Events (% of max) Spleen CLN MLN Irf fl/fl e Irf fl/fl h Irf fl/fl DC enriched CD CD11 Spleen DC enriched DC enriched CD CLN CD MLN CD11 CD11 int c # DCs (per cells) f # DCs (per cells) i # DCs (per cells) Irf fl/fl NS CD + CD CD11 CD11 + Irf fl/fl NS CD + CD11 Irf fl/fl NS CD + CD11 Spleen CLN MLN CD CD11 + CD + CD11 + Supplementary Figure 1. Selective loss of CD11 + DCs in Irf fl/fl mice (a) Splenic DC populations of wild-type mice were analyzed y flow cytometry for intracellular expression of and protein. Filled grey histograms are isotype controls. and expression was similarly analyzed for DC populations in cutaneous lymph nodes (CLN) (d) and mesenteric lymph nodes (MLN) (g). () Left, DC-enriched cells from spleens of and Irf fl/fl mice were analyzed y flow cytometry for expression of and. Numers indicate percentage of cells within the + int gate. Right, + int DC supopulations were distinguished y expression of CD11 and CD. Numers indicate percentage of cells within each gate. (c) Quantitative analysis of DCs descried in (). Data represent mean ± s.e.m. from - independent experiments, each with pooled splenic tissue from animals. NS = not significant; = P <., two-tailed t-test. (e) Left, DC-enriched cells from CLNs of and Irf fl/fl mice were analyzed y flow cytometry for expression of and. Numers indicate percentage of cells within the + gate. Right, + migratory DC supopulations were distinguished y expression of CD11 and CD. Numers indicate percentage of cells within each gate. (f) Quantitative analysis of DCs descried in (e). Data represent mean ± s.e.m. from at least independent experiments, each with pooled CLN tissue from animals. NS = not significant; = P <., two-tailed t-test. (h, i) Migratory DCs from MLN of and Irf fl/fl mice were analyzed as in panels e and f. Data represent mean ± s.e.m. from independent experiments, each with pooled MLN tissue from animals. NS = not significant; = P <., two-tailed t-test. Nature Immunology: doi:./ni.7

2 MC-OVA cells 1.x.x x Irf fl/fl (OT-II) MHCI (OT-I) (OT-II) MHCI (OT-I) # cells (x ) # cells (x ) 1 CellTrace Violet 1 Supplementary Figure. -dependent CD11 + DCs preferentially prime helper T cell responses to cell-associated antigen CellTrace violet-laeled OT-I and OT-II T cells were co-transferred into and Irf fl/fl mice. Recipient mice were susequently immunized y footpad injection with the indicated numers of necrotic ovalumin-expressing MC (MC-OVA) tumor cells. Histograms show OT-I and OT-II proliferation measured y dye dilution, days after antigen administration. For each dose, paired OT-II and OT-I data from the same mouse is displayed. Data are representative of independent experiments, n = mice per dose in each experiment. Nature Immunology: doi:./ni.7

3 anti-tnp-ova IgG1 (μg/ml) c CD + T cells (per lung) 1 7 Irf fl/fl Time after immunization (d) Irf fl/fl anti-tnp-ova IgE (ng/ml) d Neutrophils (per lung) Time after immunization (d) Naive Challenged Naive Challenged 1 7 Irf fl/fl Irf fl/fl e WT UI LCMV Irf fl/fl LCMV f WT Irf fl/fl IFN-γ CD Viral pfu/liver NS UI LCMV LCVM Supplementary Figure. Selective impairment of T H -dependent immunity in Irf fl/fl mice (a, ) and Irf fl/fl mice were immunized with TNP-OVA and serum antiody titers for IgG1 (a) or IgE () isotypes were determined y ELISA on the indicated days. Data represent antiody titers from individual mice per group with horizontal ars indicating mean values. Data are representative of independent experiments. (c, d) and Irf fl/fl mice were sensitized with TNP-OVA. days after sensitization mice were challenged y administration of neulized TNP-OVA into the airways for 7 consecutive days. h after the last antigen administration lungs were removed. Lungs from naive mice served as controls. Lung infiltrating CD + T cells (c) and neutrophils (d) were enumerated y flow cytometry. Data represent cell counts from individual mice (naive n =, challenged n = ) mean ± s.d.; one experimental repicate. (e, f) and Irf fl/fl mice were infected with LCMV. Uninfected (UI) wild-type (WT) C7BL/ mice served as controls. (e) days after infection splenocytes were stimulated in vitro with LCMV gp peptide. CD + CTLs were analyzed y intracellular flow cytometry for expression of IFN-γ. Representative data from individual mice are shown. Numers indicate percentage of gated CD + IFN-γ + splenocytes. (f) Viral titers in livers of infected mice were determined at day after infection y pfu assay. Data depict titers determined for individual mice, mean indicated y horizontal ars; NS = not significant; one-tailed Mann-Whitney. Data are representative of independent experiments. Nature Immunology: doi:./ni.7

4 Y-Ae ( gmfi) 1 WT CD CD11 + WT CD + CD11 Mouse Human Resident Migratory Blood CD CD11 BDCA1 CD CD BDCA Eα (mg).. 1. Supplementary Figure. as a regulatory determinant of enhanced p formation in murine and human DCs (a) Efficiency of p formation y CD11 + CD (red) or CD11 CD + (lue) CLN migratory DC susets was assessed after sucutaneous administration of the indicated doses of Eα antigen in wild-type (WT) C7BL/ mice. DCs were enriched and then stained, 1 h after injection of Eα, with Y-Ae antiody and analyzed y flow cytometry. Data are from two independent experiments, n = mice per dose, and represent geometric mean fluorescence intensity (gmfi) ± s.e.m. = P <., one-tailed t-test. () Differential gene expression analysis in paired mouse and human DC susets. Mouse resident DCs are represented y splenic CD + CD (CD) and CD CD + (CD) susets. Mouse migratory DCs are represented y cutaneous lymph node CD CD11 + (CD11) and CD + CD11 (CD) susets. Human DCs isolated from lood are represented y BDCA1 + and BDCA + susets. The murine data is from the ImmGen dataase 17 whereas the human data is from E-TABM- (ref. ). Expression of indicated genes is displayed as fold-increase (red) or decrease (lue) relative to its average expression in paired DC susets. Nature Immunology: doi:./ni.7

5 Total DCs CD11 Events (% max) Total DCs Irf fl/fl CD11 Events (% max) Supplementary Figure. FltL-generated BMDCs express aundant ut not and are not a suitale model for -dependent CD11 + DCs BMDCs were differentiated from (a) and Irf fl/fl () hematopoietic progenitors y culture in the presence of FltL. BMDCs were analyzed y flow cytometry for their expression of and. Numers indicate percentages of cells within the + + DC gate. + + DC supopulations were distinguised y their expression of CD11. Numers indicate percentage of cells within each gate. CD11 + (red) and CD11 (lue) DC populations were analyzed y flow cytometry for intracellular expression of or protein. Filled grey histograms are isotype controls. Data are representative of independent experiments. Nature Immunology: doi:./ni.7

6 events (% max) Irf fl/fl events (% max) Iso US LPS CD11 MHCI CD CD c OVA (μg/ml) 1 Irf fl/fl # cells (x ) 1 CellTrace Violet 1 1 (OT-II) 1 Supplementary Figure. regulates the DC maturation program and antigen presentation (a-c) BMDCs were differentiated from hematopoietic progenitors y culture in the presence of GM-CSF and IL-. (a) + DC populations were analyzed y flow cytometry for intracellular expression of or protein. Filled grey histograms are isotype controls. Data are representative of independent experiments. () BMDCs derived from or Irf fl/fl mice were left unstimulated (US, dashed histograms) or stimulated overnight with LPS (solid histograms), then analyzed y flow cytometry for expression of the indicated markers (see also Fig. a). Isotype controls are shown in filled grey histograms. Data are representative of at least independent experiments. (c) and Irf fl/fl BMDCs were loaded with the indicated concentrations of ovalmin (OVA) then stimulated with LPS. Antigen-loaded + DCs were isolated y magnetic separation and used to stimulate OT-II T cells for days. Histograms show OT-II proliferation as measured y dilution of CellTrace violet dye. Data are representative of at least independent experiments. Nature Immunology: doi:./ni.7

7 PU.1 BATF JunB PU.1 BATF JunB c AICE EICE PU.1 BATF JunB Supplementary Figure 7. directly targets key genes for antigen presentation (a-c) Wild-type C7BL/ BMDCs differentiated with GM-CSF and IL- were stimulated for h with LPS., PU.1, BATF, or JunB-ound chromatin fragments were immunoprecipitated and sujected to high-throughput sequencing (ChIPseq). Sequence tracks displaying peaks for each of the aove transcription factors are shown for Ctss (a), H-Dm (), and Ciita (c) genes. Boxed peaks containing Ets-IRF (EICE) or AP-1-IRF (AICE) composite motifs are highlighted. Two independent ChIPseq replicates showed greater than % overlap in called inding sites. Nature Immunology: doi:./ni.7

8 MSCV- MSCV MSCV- MSCV- MSCV- Events (% max) CD CD MSCV MHCI (OT-I) OVA (μg/ml) MSCV- # cells (x ) MSCV CellTrace Violet Supplementary Figure. and comparaly induce aspects of DC maturation (a) Irf fl/fl hematopoietic progenitors were transduced with MSCV-IRES-huCD (MSCV) or its - or -expressing derivatives and then differentiated into DCs with GM-CSF and IL- (see Fig. a). Complemented BMDCs were analyzed y flow cytometry for expression of,, CD, and CD. Histograms represent staining for MSCV control (lack), MSCV- (red), and MSCV- (lue) complemented BMDCs. Data are representative of at least independent experiments. () Complemented BMDCs were loaded with the indicated concentrations of ovalumin (OVA) protein and then stimulated overnight with LPS. Transduced + cells were purifed y flow cytometry and used to stimulate laeled OT-I T cells for days. T cell proliferation was analysed y dye dilution. Data are representative of independent experiments. Nature Immunology: doi:./ni.7