Immunocompetent Full-Skin Test System with Primary Immune Cells for Functional and Predictive Characterization of Contact Allergens
by Marvin Nüsken
Date of Examination:2025-07-02
Date of issue:2025-08-18
Advisor:Prof. Dr. Michael P. Schön
Referee:Prof. Dr. Michael P. Schön
Referee:Prof. Dr. Holger Reichardt
Persistent Address:
http://dx.doi.org/10.53846/goediss-11451
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Description:Dissertation Marvin Nüsken
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Abstract
English
The skin is a complex organ composed of many different cell types that interact with each other, including various types of immune cells. Contact dermatitis is the most common skin disease and can be divided into irritative contact dermatitis (ICD) and allergic contact dermatitis (ACD). In Germany, 12.7 % of the population exhibits clinically relevant contact allergy, while sensitization rates reach 20 %. ACD is a T-cell-mediated, delayed-type hypersensitivity reaction of the skin to an external substance that usually requires repeated exposure to the allergen. This exposure activates epidermal antigen-presenting cells upon barrier penetration. In light of the EU’s 2013 ban on animal testing for cosmetics and consumer products, full-thickness (FT) skin models that incorporate components of the immune system are a promising alternative. The first part of this thesis focuses on establishing the integration of primary immune cells into an FT skin model, consisting of primary fibroblasts. It describes the development and immunological characterization of such models, utilizing autologous primary keratinocytes and fibroblasts from the same donor. Barrier integrity was confirmed via filaggrin, involucrin, hornerin, and loricrin expression analysis, supported by histological evaluation. This expression was comparable to that of healthy human skin. Elevated expression of cytokeratin-6 and increased secretion of IL-6, CXCL-8, and CCL2 indicate a phenotype resembling early wound healing or inflammation. A wide range of approaches for incorporating immune cells were assessed, including the addition of predifferentiated Langerhans cells (LCs) and naïve peripheral blood mononuclear cells (PBMCs), which together form a composite immune microenvironment. Flow cytometry and immunohistochemical staining (CD3, CD14, CD20, CD45, CD68) were used to confirm the quantification and localization of immune cells. LC epidermotropism was notably inhibited by the regularly added hydrocortisone in most culture media, a phenomenon that was reversible upon its withdrawal. In the second phase, immunocompetent skin models were exposed to two important contact allergens, Lyral (a fragrance) and nickel sulfate (a metal), in two different vehicles (PBS, petrolatum). As skin-resident immune cells from allergic patients were not used, the focus was on activating healthy donor cells and evaluating the inflammatory potential of the test substances. Exposure to allergens resulted in the upregulation of T cell-activating cytokines (such as IL-1β, IL-12p70, IL-6), which was modulated by the application vehicle. Nickel exhibited stronger activation of immune cells when applied in PBS than in petrolatum. Significant variations were observed within the treatment groups, suggesting that the activation outcomes may vary due to different immune cells and keratinocyte donors. Future work will evaluate both sensitization and elicitation phases, including pre-exposure and restimulation protocols. Incorporating patient-derived immune cells will enhance the model’s translational relevance. The established model could serve as a diagnostic platform and a non-animal testing system to evaluate allergenic and immunomodulatory properties of novel compounds in pharmaceutical and cosmetic development. Importantly, personalized medicine applications with patient-derived cells can be used to test for individually tailored therapeutic approaches.
Keywords: Skin; Allergy; Langerhans cells; Contact allergy; Skin model; Organoid; Nickel
