026 Detection of rare autoreactive T cell subsets in patients with pemphigus vulgaris by the CD154 activation assay

Analysis of T cell proliferative responses to antigenic or mitogenic stimuli are based on e.g. the incorporation tritiated thymidine BrdU, CFSE labeling and detection secreted cytokines by ELISA ELISPOT assay. There inherent drawbacks these methods rather long ex vivo expansion, stimulation protocols inability distinguish specific populations assumption importance certain cytokines. Rapid identification quantification low- frequent autoreactive cells, however, presents a main goal in autoimmune diseases such as pemphigus vulgaris (PV). In this study, we characterized expression intracellular CD154 marker for activated CD4+ subsets PV patients healthy controls (HC) after both polyclonal antigen-specific stimulation. Polyclonal using PHA resulted significantly higher HC compared pointing at general exhaustion caused treatment clinical stage disease. contrast, upon with human desmoglein (Dsg) 3, major autoantigen PV, increased cells from HC. Furthermore, active disease showed subset-specific differences Th well CXCR5+ follicular helper (Tfh) numbers Dsg3-specific Tfh17 Th17 comparison remittent suggesting predominant involvement IL-17-secreting acute stages PV. addition, cytokine staining revealed significant increase IL-21, known inducer immunoglobulin production inhibitor regulatory CD154+ cells. summary, here show that analyzing allows rare discrimination different Tfh according their stage.