In vitro differentiation and phenotypic profiling of Langerhans cell subsets derived from human peripheral blood...

Langerhans cells (LCs) are primary immune sentinels at barrier surfaces, acting as the first line of defence against foreign pathogens. However, their low abundance within the human epidermis severely limits their availability for functional and mechanistic studies. To address this challenge, we sought to provide a versatile experimental framework for generating, identifying, and characterizing distinct subsets of LCs derived from in vitro differentiation of human peripheral monocytes. LCs' heterogeneity has long been considered, with recent studies classifying them into four subsets: effector LCs (LC1) and regulatory LCs (LC2) present at steady state, along with activated LCs (aLC) and migratory LCs (migLC) found during skin inflammation. In this study, LC1, LC2, and aLC/migLC subsets were identified by flow cytometric profiling of CD207, CD1a, CD83, and CD197. LC phenotype was further confirmed by examining HLA-DR, CD14, CD209, CD1b, and CD1c expression. Additionally, the phagocytic capacity of LCs relative to distinct macrophage polarizations was evaluated. From the in vitro-generated LCs, the subsets LC1, LC2, and a joint aLC/migLC cluster were identified. LC1 constituted the predominant population, appearing at higher proportions than both LC2 and the aLC/migLC cluster. The proportions of LC2 and aLC/migLC remained relatively consistent across donors, whereas LC1 abundance showed greater variation, suggesting a higher donor-dependent heterogeneity. Overall, LCs exhibited the lowest phagocytic capacity compared with the different macrophage polarizations. Our study defines LC subsets that can be generated from peripheral monocytes in vitro and provides a framework for deeper exploration of human LC biology, subset heterogeneity, and function in both normal and pathological skin.