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. 2009 Feb 3;106(5):1524-9.
doi: 10.1073/pnas.0812247106. Epub 2009 Jan 23.

Antibody to Langerin/CD207 localizes large numbers of CD8alpha+ dendritic cells to the marginal zone of mouse spleen

Juliana Idoyaga  1 , Nao Suda, Koji Suda, Chae Gyu Park, Ralph M Steinman
Affiliations

Antibody to Langerin/CD207 localizes large numbers of CD8alpha+ dendritic cells to the marginal zone of mouse spleen

Juliana Idoyaga et al. Proc Natl Acad Sci U S A. .

Abstract

Dendritic cells (DCs) are strategically positioned to take up antigens and initiate adaptive immunity. One DC subset expresses CD8alphaalpha in mice and is specialized to capture dying cells and process antigens for MHC class I "cross-presentation." Because CD8(+) DCs also express DEC205/CD205, which is localized to splenic T cell regions, it is thought that CD8(+) DCs also are restricted to T zones. Here, we used a new antibody to Langerin/CD207, which colabels isolated CD8(+) CD205(+) DCs, to immunolabel spleen sections. The mAb labeled discrete cells with high levels of CD11c and CD8. Surprisingly most CD207(+) profiles were in marginal zones surrounding splenic white pulp nodules, and only smaller numbers were in T cell areas, where CD205 colabeling was noted. Despite a marginal zone location, CD207(+) DCs lacked identifying molecules for 3 different types of macrophages, localized in proximity and, in contrast to macrophages, marginal zone DCs were poor scavengers of soluble and particulate substrates. After stimulation with microbial agonists, Langerin expression disappeared from the marginal zone at 6-12 h, but was greatly expanded in the T cell areas, and by 24-48 h, Langerin expression disappeared. Therefore, anti-Langerin antibodies localize a majority of CD8(+) DCs to non-T cell regions of mouse spleen, where they are distinct from adjacent macrophages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
L31 mAb specifically recognizes CD8+ DCs. Fixed and permeabilized B cell depleted BALB/c splenocytes were analyzed by multicolor flow cytometry (Fig. S1). (A) Expression of Langerin, DEC205, and CD8 in CD11c high gated cells (Fig. S1, gate g). (B) RPMs (Fig. S1, gate j) were evaluated for Langerin, DEC205, MMR, and CD68. (C–E) Langerin and DEC205 expression was analyzed in (C) plasmacytoid DCs (Fig. S1, gate k), (D) monocytes (Fig S1, gate n), and (E) granulocytes (Fig. S1, gate l). One experiment representative of 3 is shown.
Fig. 2.
Fig. 2.
Langerin+ cells predominate in the MZ and are distinct from macrophages. (A and B) Splenic sections of BALB/c mice were stained in green for F4/80 (A) or CD169 (B), red for Langerin, and blue for B220. (Scale bar: 200 μm.) (C–G) Higher magnification of splenic RP and MZ with Langerin+ cells in red. RPMs were stained with F4/80 (C–E, green), MMMs with CD169 (F and G, green), and MZMs with SIGNR1 (E and G, blue). B220 (blue) delineates WP in C, D, and F. (Scale bar: 100 μm.)
Fig. 3.
Fig. 3.
L31 anti-Langerin mAb localizes most CD8α+ DCs to the MZ. (A) BALB/c spleen sections were stained for CD11c (green), Langerin (red), and B220 (blue). Langerin+ CD11c+ cells in the MZ (Upper) and T cell area (Lower). (B) CD8+ DCs (green) costained for Langerin (red) in the MZ (Upper) and T cell area (Lower). (C) Cryosections were stained for DEC205 (red), Langerin (green), and B220 (blue). (Bottom) Magnified region of Middle (white square). (Scale bar: 100 μm.)
Fig. 4.
Fig. 4.
Weak phagocytosis of bloodborne substrates by Langerin+ CD8+ marginal zone DCs. Pseudodot plots of CD19-depleted splenocytes 30 min or 3 h after injecting BALB/c mice with PBS or different substrates, indicated in the center as follows: YG-PS, BSA, E. coli, or S. aureus. Dendritic cell (CD11chi) phagocytosis was assessed in CD8+ and CD8− subsets (Fig. S1, gate g); also, the uptake of bloodborne particles by red pulp macrophages (Fig. S1, gate j) was evaluated.
Fig. 5.
Fig. 5.
Polystyrene, E. coli, and S. aureus accumulate in RP and MZ macrophages but not Langerin+ cells. (A) Cryosections of spleen were examined 30 min after the injection of YG-PS. In addition to the green YG-PS, primarily in the MZ, sections were stained with L31 mAb, followed by Alexa 555 anti-rat Ig (red) and Alexa 647 SIGNR1 (blue). (Lower) Magnified region of Upper (*). MZ indicates marginal zone; T, T cell area. (Scale bar: Upper, 150 μm; Lower, 100 μm.) (B) As in A, but sections were stained with α-CD11c (Upper, red) or α-CD8 (Lower, red) and Alexa 647 B220 (blue). (Scale bar: 100 μm.) (C) As in A, but sections were stained with α-F4/80 (Upper, red) or α-CD169 (Lower, red) and Alexa 647 α-SIGNR1 (blue). (Scale bar: 100 μm.) (D and E) Mice were inoculated with Alexa 488-labeled E. coli (D) or S. aureus (E) i.v. for 30 min. Sections were stained with α-Langerin (Left, red), α-F4/80 (Center, red), or α-CD169 (Right, red), and Alexa 647-B220 (Left, blue) or SIGNR1 (Center and Right, blue). (Scale bar: 100 μm.)
Fig. 6.
Fig. 6.
Poly(IC) induces marked changes in Langerin expression. BALB/c mice were injected i.v. with YG-PS 30 min before inoculation of PBS (Left) or 50 μg of poly(IC) i.p (Center and Right). At 12 h (Left and Center) or 48 h (Right) later, spleen sections were stained with L31 mAb, followed by Alexa 555 anti-rat Ig (red) and Alexa 647 anti-B220 (blue). MZ indicates marginal zone; T, T cell area. (Scale bar: 200 μm.)
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