Detection of rare autoreactive T cell subsets in patients with pemphigus vulgaris

Abstract

Analysis of T lymphocyte proliferation and activation after antigenic or mitogenic stimulation is a vital parameter used in the diagnosis of various immuno-deficiencies and during the monitoring of treatment responses. Most applied techniques are based on the incorporation of tritiated thymidine (3H-TdR) or ELISPOT analysis, both rely on rather time-consuming/-intensive ex vivo protocols or encompass inherent drawbacks such as the inability to distinguish specific cell populations (3H-TdR, ELISPOT) or focus on a single cytokine (ELISPOT). Here we aimed at characterizing the rapid expression of intracellular CD154 (CD40L) as a marker for rare antigen-specific CD4+ T cells in pemphigus vulgaris (PV). Upon stimulation with human desmoglein (Dsg) 3, the major autoantigen in PV, the expression of CD154 was significantly increased in PV patients compared to healthy controls (HC) and correlated with anti-Dsg3 IgG titers. Patients with active disease showed higher numbers of Dsg3-reactive CD4+ T cells in CXCR5+ T follicular helper cells. In remittent PV and HC, CXCR5+CD4+ T cells remained largely unaffected by Dsg3. IL-17 and IL-21 expression were significantly induced only in CD154+CD4+ T cells from PV patients, lending themselves as potential novel treatment targets. Additionally, stimulation with immunodominant Dsg3-derived epitopes strongly induced a CD4+ T cell response via CD40-CD154 interaction similar to the human Dsg3 protein. We here established a rapid ex vivo assay allowing the detection of Dsg3-reactive CD4+ T cells from activated systemically available PBMCs, which further supports the crucial concept of antigen-specific T cells in the pathogenesis of PV.

Targeting therapy in pemphigus: Where are we now and where are we going?

“…In PV, pathogenic memory B cells express anti -Dsg3 B cell receptors (BCRs) [ 39 ]. The researchers reasoned that by expressing Dsg3 as the extracellular domain of a chimeric immunoreceptor, cytotoxicity would become specific for only those B cells bearing anti -Dsg3 BCRs, providing targeted therapy for PV without general immunosuppression [ 40 ]. Such a strategy would directly eliminate surface immunoglobulin (sIg)+ anti -Dsg3 memory B cells and indirectly eliminate sIg−Dsg3-specific short-lived plasma cells that produce the disease-causing antibodies [ 41 ].…”

“…In this study, serum levels of IL-17 in PV patients showed significant differences compared to healthy controls. Polakova et al found a significant induction of IL-17 and IL-21 in CD154 + CD4 + T cells from PV patients compared to HC, meanwhile, CD154- CD4 + T cells remained mainly unaffected regarding their cytokine expression after antigenic stimulation [ 40 ]. These findings strongly suggest that CD154 as a specific activation marker in PV, expressed by antigen-specific CD4 + T cells, is critical during the pathogenesis of PV [ 40 ].…”

“…Polakova et al found a significant induction of IL-17 and IL-21 in CD154 + CD4 + T cells from PV patients compared to HC, meanwhile, CD154- CD4 + T cells remained mainly unaffected regarding their cytokine expression after antigenic stimulation [ 40 ]. These findings strongly suggest that CD154 as a specific activation marker in PV, expressed by antigen-specific CD4 + T cells, is critical during the pathogenesis of PV [ 40 ]. These results showed that peripheral blood T cell subsets of patients with active pemphigus are dominated by IL-17-producing Th and Tfh cell subsets.…”

“…These results showed that peripheral blood T cell subsets of patients with active pemphigus are dominated by IL-17-producing Th and Tfh cell subsets. Moreover, upregulation of IL-17 was seen to associate with antigen-specific activation, therefore lending themselves as potential therapeutic targets in pemphigus [ 40 ]. Zou et al found an over representation of CD4 + tissue-resident memory T (T RM ) cells which accumulated significantly in pemphigus skin lesions.…”

Mouse models of pemphigus: valuable tools to investigate pathomechanisms and novel therapeutic interventions

“…While several T-cell subsets with a strong Th2 and Th17 polarization are undoubtedly involved in the pathogenesis of pemphigus ( 15 , 16 ), identifying immunodominant self-peptides and characterization of autoreactive T cells is still challenging. Indirect characterization via activation markers such as CD154 presents as a reliable method to detect antigen-reactive T cells, while other methods such as ELISPOT or incorporation of 3 H-thymidine are unable to distinguish specific cell populations ( 3 H-T, ELISPOT) or focus on a single cytokine secreted by activated T cells (ELISPOT) ( 17 ). With the advent of MHC class II–peptide technology a more comprehensive understanding of antigen-specific T cells in the immune pathogenesis of several HLA class II–linked autoimmune disorders is now achievable ( 18 ).…”