NativeSkin®, an immunocompetent human skin model to study antigen uptake and processing by Langerhans cells

USING NATIVESKIN IMMUNOCOMPETENT EX VIVO SKIN MODEL

Allergen applied to the skin during epicutaneous immunotherapy (EPIT) with Viaskin® patches seemed to induce tolerance in sensitized mice (Dioszeghy, Clin Exp Allergy 2014). Safety and adequate dose for treatment with Viaskin® Peanut in peanut allergic patients was studied in earlier Phase 1 and Phase 2 clinical trials and completed Phase 3 safety and efficacy trial. In preliminary studies in sensitized mice, DBV Technologies showed that allergen delivered by Viaskin® is mainly takenup by Langerhans cells (LCs) and transported to regional lymph nodes. The aim of this study was to determine whether similar allergen uptake occurred in an ex vivo human skin model.

Graphic showing the experimental workflow for the treatment of skin biopsies with Viaskin® placebo and Viaskin® peanut during 24 hours
Treatment with Viaskin® placebo Viaskin® peanut (A-647) during 24H

Experimental Workflow

The experiment was performed on 2 donors and in triplicate by condition. Viaskin® patches were applied on the skin after the production of NativeSkin® models.

No major alteration of the skin after Viaskin® patches applications during 24h

For each condition, representative pictures of models and the epidermis/dermis sections were imaged. Results for donor 2 are shown and are similar to those obtained for donor 1. On macroscopical pictures, it is possible to observe allergen solubilization due to perspiration.

Skin biopsies photos and histology. Macroscopical and histological evaluation of tissue integrity during treatment
Macroscopical and histological evaluation of tissue integrity during treatment
Graphics showing allergen penetration and colocalization with Langerhans cells on tissue sections
Study of allergen penetration and colocalization with Langerhans cells on tissue sections

Allergen penetration in the epidermis & colocalization with Langerhans cells

Representative pictures for donor 2, on merged pictures, cell nuclei stained with DAPI (Grey), LC with anti-CD1a (Blue) and anti-CD207 (Green) and peanut (Red). On Peanut pictures, allergen particles appear in black, blue arrows point Peanut in the epidermis, and orange arrows target allergen colocalizing with CD1a positive cells (visible in black on the last line).

Graphics showing quantification of allergen penetration and colocalization with Langerhans cells
Quantification of allergen penetration and colocalization with Langerhans cells

Variable kinetics of penetration between donors

Peanut in the epidermis (Blue) and colocalization with LC (orange) were detected using Fiji for donors 1 and 2 on skin cross-sections.
Results are presented for Viaskin® Peanut. The analysis was performed for Viaskin® Placebo, no Peanut signal was detected.

Colocalization of allergen with Langerhans cells after Viaskin® treatment

Representative pictures for donor 1 (A) and donor 2 (B) of penetration of peanut in Langerhans cells positive for in situ CD207 immunostaining of the epidermis of NativeSkin® models treated with Viaskin® Peanut during 24 hours. Global view of 3D reconstruction. White circles allow to localize peanut and CD207 positive cells. In the center, detailed view of positive Langerhans cells after 24 hours of treatment. CD207 signal was removed to visualize internalization of antigen.

Observation of in situ immunostaining on detached epidermal sheet after 24H of treatment with Viaskin® peanut using confocal microscopy
Observation of in situ immunostaining on detached epidermal sheet after 24H of treatment with Viaskin® peanut using confocal microscopy
After Viaskin application, similar detection of cytokines between peanut and placebo could explain the absence of inflammation occuring during treatment
After Viaskin® application, similar detection of cytokines between peanut and placebo could explain the absence of inflammation occuring during treatment

Measurements of cytokines released in the culture medium during 24h of treatment with Viaskin®

Cytokines detected in culture medium (after 24 hours of treatment with Viaskin®). A human 27-Plex® kit (Bioplex®, Biorad) was used for the quantitative detection of cytokine in culture medium. Only 3 cytokines were detected above the limit of quantification: IL-6, IL-8, MCP1. Results are presented for donor 1 and are similar for donor 2. The bars represent the mean ± SD of triplicates.

Conclusion

NativeSkin® model is a relevant model to investigate the local immune response related to skin treatment. It allows confirming the role of LCs in the antigen uptake and processing after Viaskin® application on human skin. Particularly, we showed that Viaskin® Peanut applied on ex vivo human skin leads to the antigen penetration in the epidermis and its uptake by Langerhans cells within 24 hours (with variable kinetics depending on the donor).

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