Induction of FITC-specific CHS in mice adoptively transferred with FITC⁺ MGL2⁺ DDC.

<p>(A) The left panels show the gating to sort FITC⁺ MGL2⁺ cells from the MACS-purified CD11c⁺ cells. The purity of FITC⁺ MGL2⁺ DDCs was approximately 98%. Ear swellings in mice transferred with FITC⁺ MGL2⁺ DDCs (“MGL2⁺”, <i>p</i><0.005), transferred with glutaraldehyde-fixed FITC⁺MGL2⁺ DDC (“fixed MGL2⁺”, difference not statistically significant), sensitized directly with FITC painting (“FITC painted”, <i>p</i><0.005), or untreated (“naive”) are shown in the graph. Each bar indicates mean±SD obtained from three independent recipients respectively. (B) The left panels show the gating strategy to sort FITC⁺MGL2⁺DDCs from the day 1 LNs or FITC⁺MGL2⁻DCs from the day 4 LNs. The purity of sorted cells was >90% when re-analyzed by FACS. Ear swelling in mice transferred with 5×10⁴ cells of FITC⁺MGL2⁺DDCs from the day 1 LNs (d1 DDC), glutaraldehyde-fixed FITC⁺MGL2⁺DDCs from the day 1 LNs (fixed d1 DDC) or FITC⁺MGL2⁻DCs from the day 4 LNs (d4 non-DDC) is shown in the right panel. The mice sensitized by FITC painting (FITC painted) and left untreated (naive) were used as positive and negative controls respectively. Each bar indicates the mean value from two recipients and the data are representative of two independent experiments.</p

Hapten incorporation and migration kinetics of MGL2⁺ DDC after FITC sensitization revealed by the flow cytometric analysis.

<p>(A) FITC fluorescence associated with MGL2⁺ DDCs in LNs. MGL1⁺ cDC (blue) or MGL2⁺ DDCs (red), but not MGL1⁺ pDCs (purple) or cells nonspecifically bound by mAb URA-1 (orange) incorporate FITC. (B) Kinetics of MGL2⁺ DDCs in LNs after FITC sensitization. Top panels indicate the FITC fluorescence and staining with anti-MGL2 mAb on DCs purified from naive LNs (left) or LNs 24 h (middle) or 96 h (right) after sensitization. The bottom panel shows the ratio of total FITC⁺ DCs (closed diamond), FITC⁺ MGL2⁺ DDCs (open square) and FITC⁺ MGL2⁻ DCs (closed triangle) calculated by each quadrant shown in top panels. The data are shown as Mean±SD.</p

Immunohistochemical localization of MGL2⁺ DDCs, LCs and LN conduits.

<p>(A) Distribution of MGL2 (red) and Langerin (blue) in a draining (top) or a non-draining (bottom) LNs 24 h after FITC (green) sensitization. Arrows indicate venule-like structures in the CR. Arrowheads indicate the Langerin⁺ cells loaded with FITC. Almost all the MGL2⁺ DDCs were FITC⁺ and shown in yellow when merged. B: B cell follicles. Scale bar, 100 µm. (B) Distribution of MGL2 (red), PNAd (HEV), Langerin (LC) or ER-TR7 antigen (FRN) (blue in each panel) in the sensitization process. Areas indicated by the squares are magnified in the lower panels. FITC is shown in green. CR associated with HEV in a naive LN is adjacent to MGL2⁺ DDC only at its follicular side but not at the cortical side. Arrowheads indicate association of MGL2⁺ DDCs with ER-TR7⁺ FRN around HEV-associated CR. Scale bar, 100 µm.</p

Distribution of MGL2⁺ cells in draining LNs during the sensitization with FITC revealed by immunohistochemical analysis.

<p>(A) Serial sections of LNs stained with anti-MGL1, anti-MGL2 or anti-MGL1/2 cross-reactiv mAbs. MGL1 was strongly stained in the sinuses in both naive and FITC sensitized (24 h after sensitization) LNs. Arrows indicate the increased signals in the cortex after the sensitization, though the anti-MGL1 staining in the cortex was weaker than in the sinuses. Scale bar, 250 µm. (B) Serial LN sections 4 h after sensitization stained with mAbs to MØ/DC markers. Restricted localization of MGL2⁺ cells to the outer T cell cortex was unique compared to other APCs (MHCII) including B cells (B220), MØs (CD11b and F4/80), DCs (CD11c) or MGL1⁺ cells. S: subcapsular sinus, M: medulla, T: T cell cortex, B: B cell follicle. Scale bar, 200 µm. (C) Confocal microscopic observations of the distributions of MGL1 (mAb LOM-8,7) or MGL2 (mAb URA-1) (blue in each panel) with MØ markers (red) in the LN 24 h after FITC sensitization. FITC used is shown in green. Scale bar, 25 µm (×400).</p

Expression of MGL1 and MGL2 in a DDC subset in skins of naive mice.

<p>(A) Binding of anti-MGL1 and anti-MGL2 mAbs to cryosections of skin (blue in each panel). The dashed line in the control panel (Ctrl. IgG) indicates the dermal-epidermal junction. Nuclei are shown in red. Scale bar, 100 µm. E: epidermis, D: dermis, F: hair follicle. (B) Surface staining by anti-MGL1 and anti-MGL2 mAbs of epidermal and dermal cell suspensions. (C) Intracellular staining of CD11c and surface staining of MHCII and MGL2 in the dermal cell suspension. Virtually all MGL2⁺ cells expressed CD11c. The proportion of MGL2⁺ cells in MHCII^hi CD11c⁺ DDC (cells in the rectangular gate) was 88.5±6.8%.</p

Characterization of MGL2⁺ dDCs in skin.

<p>Flow cytometry analysis of skin cell suspensions for DC markers. MHCII⁺ cells were analyzed for the expression of MGL2, EpCAM, CD103, and Langerin. The experiments were independently performed three times.</p

A Th2-type humoral response is induced by targeting MGL2⁺ dDCs <i>in vivo</i>.

<p>(A) One day after the injection of biotinylated anti-MGL2 mAbs or rat IgG2a (isotype control) into <i>Mgl2</i>^+/+ or <i>Mgl2</i>^–/– mice, cells from draining LNs were subjected to flow cytometry analysis for biotin residues internalized into CD11c⁺ DCs using PE-SAv. (B) Flow cytometry analysis of surface MGL2 on the cells, gated for the levels of PE-SAv binding and CD11c circled in the panel A. (C) Antibodies specific for rat IgG2a in sera were detected 1 week after the injection of rat anti-MGL2 mAbs or rat IgG2a (isotype control) into <i>Mgl2</i>^+/+ mice or <i>Mgl2</i>^–/– mice. (D) Sera obtained 1 week after the injection of anti-MGL2 mAbs into <i>Mgl2</i>^+/+ mice were assessed for antibody isotypes that were specific for rat IgG2a. (A–D) The experiments were independently performed three times.</p

Paucity in IL-12 production by MGL2⁺ dDCs.

<p>(A) Flow cytometry analysis of intracellular IL-12_p40 in MGL2⁺ dDCs, MGL2^–CD103^– skin-derived DCs, and CD103⁺ dDCs in skin-draining LNs under naïve or sensitized conditions. MGL2⁺ dDCs are shown as “MGL2,” MGL2^–CD103^– DCs are shown as “DN” and CD103⁺ dDCs are shown as “CD103.” Cells from naïve mice are shown as “Naïve,” and cells from sensitized mice are shown as “Sensitized.” Areas shaded in gray indicate the staining pattern with an isotype control antibody. The numbers represent the mean fluorescence intensity (MFI) of each skin-derived DC subset. (B) The MFI of IL-12_p40 for each skin-derived DC subset is shown in panel A in bar graphs. Data are shown as the mean ± SD of three biological replicates (n = 3); n.s. indicates that the difference is not statistically significant. (A–B) The experiments were independently performed three times. (C) Concentrations of IL-12_p40 and IL-12_p70 in culture supernatant of MHCII^highMGL2⁺ dDCs, MHCII^highCD103⁺ dDCs, FITC⁺MGL2⁺ dDCs, and FITC⁺CD103⁺ dDCs were measured by the Bio-Plex Suspension Array System and are shown in the panels as “Naive MGL2,” “Naïve CD103,” “Sensitized MGL2,” and “Sensitized CD103,” respectively. The experiments were independently performed two times.</p

A Unique Dermal Dendritic Cell Subset That Skews the Immune Response toward Th2

<div><p>Dendritic cell (DC) subsets in the skin and draining lymph nodes (LNs) are likely to elicit distinct immune response types. In skin and skin-draining LNs, a dermal DC subset expressing macrophage galactose-type C-type lectin 2 (MGL2/CD301b) was found distinct from migratory Langerhans cells (LCs) or CD103⁺ dermal DCs (dDCs). Lower expression levels of Th1-promoting and/or cross-presentation-related molecules were suggested by the transcriptome analysis and verified by the quantitative real-time PCR analysis in MGL2⁺ dDCs than in CD103⁺ dDCs. Transfer of MGL2⁺ dDCs but not CD103⁺ dDCs from FITC-sensitized mice induced a Th2-type immune response <i>in vivo</i> in a model of contact hypersensitivity. Targeting MGL2⁺ dDCs with a rat monoclonal antibody against MGL2 efficiently induced a humoral immune response with Th2-type properties, as determined by the antibody subclass. We propose that the properties of MGL2⁺ dDCs, are complementary to those of CD103⁺ dDCs and skew the immune response toward a Th2-type response.</p></div

Encyclopedic transcriptome analysis of MGL2⁺ dDCs and CD103⁺ dDCs and results of quantitative real-time PCR for the transcripts suggested by the transcriptome analysis to have differential levels.

<p>(A–D) Reciprocal two-dimensional plots of the results of transcriptome analysis among MHCII^highMGL2⁺ cells, MHCII^highCD103⁺ cells, FITC⁺MGL2⁺ cells and FITC⁺CD103⁺ cells. In these panels, MHCII^highMGL2⁺ dDCs are indicated as “Naïve MGL2⁺ dDC,” MHCII^highCD103⁺ dDCs are indicated as “Naïve CD103⁺ dDC,” FITC⁺MGL2⁺ dDCs are indicated as “Sensitized MGL2⁺ dDC,” and FITC⁺CD103⁺ dDCs are indicated as “Sensitized CD103⁺ dDC.” (A) All transcripts of cells from naïve mice and cells from sensitized mice are shown. The numbers in the parentheses indicate the number of transcripts expressed 3 times greater than the other dDC subset. (B–D) The numbers of transcripts in selected categories are plotted. In the comparisons, the names of the transcripts are indicated when they fit the following criteria: (1) the number is greater than 15 in MGL2⁺ dDCs or in CD103⁺ dDCs, and (2) the difference in the number is 5-fold or greater both before and after sensitization. The diagonal lines represent the border for 3-fold differences. (B) Transcripts of cytokines, chemokines and TNF ligand superfamily members are shown. (C) Transcripts of cytokine receptors, chemokine receptors, and TNF receptor superfamily members are shown. (D) Transcripts of C-type lectins, TLRs and NLRs are shown. (E) The quantitative real-time PCR analysis of the expression of indicated genes (<i>Cxcl2, Cxcl3, Ccl1, Il12b, Xcr1, Naip2, Clec4n, Clec9a</i> and <i>Tlr3</i>). They were chosen from the categories indicated above (B), (C), and (D) and the differences between MGL2⁺ dDCs and CD103⁺ dDCs based on the transcriptome analysis, appeared to be significant under both untreated and sensitized conditions (Figs. 3B–D). (A–E) Transcriptome analysis was performed once. The quantitative real-time PCR analysis was independently performed more than two times.</p