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

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

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

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

Characterization of MGL2⁺ dDCs in skin-draining LNs.

<p>Flow cytometry analysis of cells from skin-draining LNs for DC markers. (A) LN cells were analyzed for the expression of MGL2 or CD103 and MHCII. (B) MHCII^high cells from naïve mice are shown as “Naive” and FITC⁺ cells from sensitized mice (1 day after FITC painting) are shown as “Sensitized.” MHCII^high cells or FITC⁺ cells were analyzed for the expression of MGL2, EpCAM, CD103, or Langerin. (C) CD11c⁺ cells from naïve mice, shown as “Naïve,” and FITC⁺ cells from sensitized mice, shown as “Sensitized,” were analyzed for the expression of MGL2, B220, mPDCA-1, CD4, or CD8á. (A–C) The experiments were independently performed three times.</p

Immune response in mice after transfers of MGL2⁺ dDCs <i>in vivo</i>.

<p>In the following panels, data obtained from untreated mice, mice that received FITC⁺MGL2⁺ cells, and mice that received FITC⁺CD103⁺ cells are indicated as “Ctrl,” “MGL2,” and “CD103,” respectively. (A) FITC-specific ear swelling 1 day after elicitation is shown in µm. (B) Relative quantity of mRNA for <i>Ifng, Il4, Il17a, Il5, Il13</i> and <i>Foxp3</i> was measured by the quantitative real-time PCR in LN cells collected from recipient mice 1 day after elicitation. The values are shown as the mean ± SD from three independent measurements, and the levels of significance for the differences are: *** for p<0.005 and n.s. for not significant. (C) Results of flow cytometry analysis of intracellular cytokines in CD4⁺ T cells from LNs collected from recipient mice 1 day after elicitation are shown. The numbers in the panels indicate the percentages of IFN-γ⁺IL-4^–CD4⁺ T cells, IFN-γ^–IL-4⁺CD4⁺ T cells, IFN-γ⁺IL-17A^–CD4⁺ T cells, and IFN-γ^–IL-17A⁺CD4⁺ T cells in the total CD4⁺ T cells. (D) The percentages of IFN-γ⁺IL-4^–CD4⁺ T cells, IFN-γ^–IL4⁺CD4⁺ cells, and IFN-γ^–IL-17A⁺CD4⁺ T cells in total CD4⁺ T cells are illustrated in bar graphs. Values in the Y-axis indicate the mean ± SD from three separate experiments, * indicates that the difference is statistically significant (p<0.05) and n.s. indicates that the difference is not statistically significant. (E) FITC-specific IgG1 and IgG2a in the sera of mice that received a transfer of FITC⁺MGL2⁺ cells or FITC⁺CD103⁺ cells were determined. The y-axis indicates the relative antibody binding to FITC. Mean ± SD from 9 separate experiments are shown, and *, ***, and n.s. indicate p<0.05, p<0.005, and difference not statistically significant, respectively. (A–E) The experiments were independently performed more than three times.</p

The numbers of Treg cells increased in the tumor tissues in MUC1.Tg mice.

<p>SL4-MUC1 cells (1×10⁶ cells/50 µL) were injected into the space under the cecal serosa of B6 mice or MUC1.Tg mice. Five or ten days after injection, mice were sacrificed, the cecal tumors, spleens, and MLNs were resected and single cell suspensions were prepared. (A–G) Cells were stained with CD4, CD25, and Foxp3 and analyzed by flow cytometry. CD4⁺CD25⁺Foxp3⁺ cells were considered Treg cells and CD4⁺CD25⁺Foxp3⁻ cells were considered Teff cells. (A, B) Percentages of Treg cells (A) and Teff cells (B) in CD4⁺ cells in the tumor tissues are shown. (C) Absolute numbers of Treg cells in the tumor tissues are shown. (D–F) Treg/Teff ratios in tumor tissues (D), spleens (E) and MLNs (F) are calculated and shown. Data from B6 mice are indicated as open circles, and data from MUC1.Tg mice are indicated as filled circles. Lines indicate the median values. (G) A representative profile of CD4 and Foxp3 of cells from cecal tumor in MUC1.Tg mice is shown. (H) Cells from tumor tissues in B6 or MUC1.Tg mice were stimulated by PMA and ionomycin for 4 hours and stained with CD4, Foxp3, and IL-10. The cells in CD4⁺ gate were shown. Statistical analysis was performed using the Mann-Whitney U-test (n.s.: not significant). A representative result from three independent experiments (A–G) or two independent experiments (H) is shown.</p

MUC1.Tg mice appeared to elicit MUC1-specific peripheral tolerance.

<p>T cells from mice vaccinated with MUC1 DNA or T cells from mice immunized with control vectors (3×10⁶ cells) were mixed with B16-F10-MUC1 cells (5×10⁴ cells) and subcutaneously injected into naïve B6 or MUC1.Tg mice. Survival rates of the mice are shown. Statistical analysis was performed using the log rank test (n.s.: not significant). A representative result from three independent experiments is shown.</p

Treg cells in MUC1.Tg mice contained MUC1-specific populations.

<p>Antigen specificity of Treg cell suppression was investigated <i>in vitro</i>. (A) MUC1-specific T cell hybridoma VF5 cells (1×10⁵ cells) were co-cultured with BM-DCs (2×10⁴ cells) pulsed with MUC1 peptide and CD4⁺ T cells as indicated. (B) VF5 cells (1×10⁵ cells) were cultured alone or co-cultured with BM-DCs (2×10⁴ cells) pulsed with or without MUC1 peptide (+/−MUC1p). (C) OVA-primed CD4⁺ T cells (1×10⁵ cells) were co-cultured with BM-DCs (2×10⁴ cells) pulsed with OVA protein and CD4⁺ T cells as indicated. (D) OVA-primed CD4⁺ T cells (1×10⁵ cells) were cultured alone or co-cultured with BM-DCs (2×10⁴ cells) pulsed with or without OVA (+/−OVA). (E) OVA-specific OT-II T cells (4.5×10⁴ cells) were co-cultured with BM-DCs (4.5×10³ cells) pulsed with OVA peptide and/or MUC1 peptide and CD4⁺ T cells obtained from MUC1.Tg mice as indicated. (F) OT-II T cells (4.5×10⁴ cells) were cultured alone or co-cultured with BM-DCs (4.5×10³ cells) pulsed with OVA peptide and/or MUC1 peptide. Twenty-four hours (A–D) or 48 hours (E, F) after the initiation of co-cultures, supernatants were collected, and the concentration of IL-2 was measured by ELISA. Data are shown as the mean±SD (n = 3 or 4). Statistical analysis was performed using the Student’s t-test (n.s.: not significant). A representative result from three independent experiments is shown.</p