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

ESDR026 - Detection of rare autoreactive T cell subsets in patients with pemphigus vulgaris by the CD154 activation assay

50th Annual Meeting of the European Society for Dermatological Research. VIRTUAL MEETING