Epidermal Langerhans cells rapidly capture and present antigens from C-type lectin-targeting antibodies deposited in the dermis - PubMed (original) (raw)

Epidermal Langerhans cells rapidly capture and present antigens from C-type lectin-targeting antibodies deposited in the dermis

Vincent Flacher et al. J Invest Dermatol. 2010 Mar.

Abstract

Antigen-presenting cells can capture antigens that are deposited in the skin, including vaccines given subcutaneously. These include different dendritic cells (DCs) such as epidermal Langerhans cells (LCs), dermal DCs, and dermal langerin+ DCs. To evaluate access of dermal antigens to skin DCs, we used mAb to two C-type lectin endocytic receptors, DEC-205/CD205 and langerin/CD207. When applied to murine and human skin explant cultures, these mAbs were efficiently taken up by epidermal LCs. In addition, anti-DEC-205 targeted langerin+ CD103+ and langerin- CD103- mouse dermal DCs. Unexpectedly, intradermal injection of either mAb, but not isotype control, resulted in strong and rapid labeling of LCs in situ, implying that large molecules can diffuse through the basement membrane into the epidermis. Epidermal LCs targeted in vivo by ovalbumin-coupled anti-DEC-205 potently presented antigen to CD4+ and CD8+ T cells in vitro. However, to our surprise, LCs targeted through langerin were unable to trigger T-cell proliferation. Thus, epidermal LCs have a major function in uptake of lectin-binding antibodies under standard vaccination conditions.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1

Figure 1. Langerhans cells in situ are specifically targeted by mAb in murine skin explant cultures

(a): Whole skin explants from BALB/c mice were incubated for 30′ to 48h in culture medium containing NLDC145 (anti-mouse DEC-205) hybridoma supernatant. Epidermis was then separated from dermis, fixed, and targeting mAb was revealed with anti-rat IgG secondary Ab. Arrowheads indicate dermal cells targeted in situ. Arrows point at DC-filled dermal lymphatic vessels. (b): Whole skin explants from BALB/c mice were incubated for 18 h in culture medium containing NLDC145 or LODNP16 (isotype control) hybridoma supernatants, then epidermis was separated, fixed, and stained with anti-rat IgG Ab. (c): Whole skin explants from BALB/c or C57BL/6 mice were incubated for 4h with 5μg/mL of the indicated purified mAb, then epidermis was separated, fixed, and stained with anti-rat IgG Ab. Scale bars = 100μm. Results are representative of three independent experiments.

Figure 2

Figure 2. Langerhans cells and dermal DC migrating out of murine skin explants transport the targeting mAb

Whole skin explants were incubated for 3h in medium containing L31 (anti-langerin), NLDC145 (anti-DEC-205), or IgG2a isotype control, washed in PBS and cultured for 3 days. Targeting mAb was detected in permeabilised migratory cells by anti-rat IgG Ab. Cells were counterstained with 929F3 mAb to an epitope on the cytosolic domain of langerin in (a) or 929F3 anti-langerin plus anti-CD103 in (b). Histograms in (b) show expression of the targeting mAb on subsets gated as depicted in the density plot on the left. Filled histograms: IgG2a; empty histograms: anti-DEC-205. Results are representative of three independent experiments.

Figure 3

Figure 3. Langerhans cells are targeted within the epidermis following intradermal injection of anti-lectin antibodies in vivo

Different targeting mAb were injected intradermally into the ear skin of C57BL/6 mice. Epidermal sheets were prepared 18 h after injection of 0.2, 1, or 5 μg of NLDC145 (a) and 24h or 5 days after injection of 1 μg IgG2a, L31, or NLDC145 (b). The capture of the injected mAb was visualized with anti-rat IgG Ab. (c): 18h after injection of 1μg targeting mAb, MHC class II (upper panel) and targeting mAb (lower panel) were revealed on epidermal sheets from langerin−/− mice. (d): 4 h after injection of 1μg targeting mAb into ear skin of BALB/c mice, whole skin explants were cultured for 3 days. The targeting mAb was detected in permeabilised migratory cells (gated on langerin+ cells; mAb 929F3). Filled histograms: IgG2a; empty histograms: mAb as indicated. Scale bars = 100μm. Results are representative of three independent experiments.

Figure 4

Figure 4. Langerhans cells present DEC-205–targeted antigen following in vivo uptake

4h after i.d. injection of 0.5μg of OVA-coupled anti-DEC-205, or indicated control reagents, epidermal sheets were prepared and cultured for 3 days. Migratory epidermal LC were then cultured with CFSE-labelled CD8+ (a) or CD4+ (b) T cells purified from OT-II or OT-I mice, respectively. After 4 days, T cell proliferation was evaluated by dilution of CFSE. Upper panels show T cell proliferation observed at different T/LC ratios, and lower panels display typical FACS stainings. Results are representative of two independent experiments.

Figure 5

Figure 5. Human skin DC bind and transport targeting mAb

Human whole skin explants were incubated for 4h in medium containing DCGM4 (anti-langerin), MG38 (anti-DEC-205), or IgG1 isotype control, then cultured for 4 days. Targeting mAb were detected in permeabilised migratory cells by means of anti-mouse IgG secondary Ab. Cells were counterstained with anti-CD1a or anti-langerin. n.d., not determined. Results are representative of two independent experiments.

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