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

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

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

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

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