024 Comparative analysis of ex vivo assays aimed at identifying Desmoglein 3 reactive CD4+ T cells in pemphigus vulgaris

Abstract

Autoreactive CD4+ T cells against the desmosomal protein, Desmoglein 3 (Dsg3) play a central role in the pathogenesis of pemphigus vulgaris (PV). Accordingly, descriptive and functional analyses of these cells ex vivo are of great interest. Here, we investigated the phenotype and pathogenicity of CD4+ T cells within peripheral blood mononuclear cells of PV patients. Flow cytometric analysis enabled phenotyping of CD4+ T cells and even identification of Dsg3-reactive CD4+ T cells by using fluorochrome-labeled HLA-DRB1*04:02 multimers loaded with immunogenic Dsg3-peptides. Proliferation of CD3+CD4+ T cells was studied by CFSE-dye dilution in response to in vitro stimulation with Dsg3 protein or peptides. ELISpot analysis characterized cytokine- secretion profiles of CD4+ T cells in response to Dsg3-stimulation. In contrast to healthy controls, PV patients showed an increased number of activated CD4+ T cells with a stimulatory capacity. Dsg3-reactive CD4+ T cells were detected at low frequencies within PBMCs of PV patients. CD4+ T cells proliferated in response to specific Dsg3-peptides and especially IL-5 secretion was enhanced in PV even correlating with the clinical course. Our findings characterize Dsg3 reactive CD4+ T cells contributing to PV pathogenesis and thus serve as basis for the development of targeted therapies in the future.