Pemphigus vulgaris is a severe autoimmune blistering disease characterized by IgG autoantibodies (auto-abs) against the desmosomal adhesion molecules desmoglein (DSG) 3 and DSG1. Underlying mechanisms leading to blister formation upon binding of DSG-specific IgG auto-abs are not fully understood. Numerous studies showed the pathogenicity of IgG auto-ab binding to the aminoterminal region 1 (EC1) of the DSG3 ectodomain. However, auto-abs in pemphigus vulgaris are polyclonal, including IgG against both aminoterminal- and membrane-proximal epitopes of the DSG3 ectodomain. In this study, the pathogenicity of a previously uncharacterized murine monoclonal IgG antibody, 2G4, directed against the membrane-proximal region (EC5) of the DSG3 ectodomain was characterized and tested in various specificity and functionality assays. The results clearly show that 2G4 is capable of inhibiting intercellular keratinocyte adhesion and of inducing cellular DSG3 redistribution by activation of the p38MAPK signal transduction pathway. In this study, we provide evidence that an IgG auto-abs directed against the membrane-proximal region EC5 of DSG3 induces acantholysis, the hallmark in pemphigus vulgaris. These findings challenge the current concept that IgG auto-abs targeting the NH2-terminal portion of the DSG3 ectodomain are pathogenic only. Our study provides further aspects for a deeper understanding of desmosomal keratinocyte adhesion and improves our insight into the complex auto-ab‒induced blister formation in pemphigus vulgaris.

PV is a severe autoimmune disorder of the mucous membranes and skin, which is caused by IgG auto-antibodies against desmoglein (Dsg)3. Pathogenesis depends on recognition of Dsg3 epitopes by autoreactive T cells, mainly restricted by HLA-DRB1*04:02. We report on the single ascending dose part of TPV11 phase 1 open label study, in which 12 patients (4 cohorts of 3 PV patients each) with anti-Dsg3 IgG, no-to-moderate disease activity, and no immunosuppressives other than ≤10 mg/d of prednisone, received TPM203 at 0.12, 0.36, 1.2 and 3.6μmol of total peptide and were followed-up for 12 weeks. TPM203 is a mixture of 4 different Topas particle conjugates (TPC) each coupled to a distinct Dsg3 T cell epitope, shown to induce tolerance against Dsg3 in an HLA-DRB1*0402-transgenic mouse model of PV. Eleven patients reported at least one adverse event (AE), none of which was severe or serious. Three PV-related AEs were mild. Mild-to-moderate worsening of PV was reported in 2 patients following COVID vaccination. No clinically relevant changes in laboratory parameters were observed. Detectable TPC levels were only found at the two highest doses. A trend towards decreased anti-Dsg3 IgG was found with the first 3 dose levels. There was also a decrease in CD27+ memory B and Th17 cells in contrast to an increase in Th2, Tfh2 and Treg cells (all % change, 95% CI). In 5/12 patients, a decrease of memory T cells and Th17, Th17.1 and Th1 cells was associated with diminished anti-Dsg3 IgG. Thus, escalating single doses of TPM203 were safe and well-tolerated in the studied PV patients. Despite the small patient number and single drug administration, the preliminary data could indicate that this T cell-targeted approach might modulate the immune response in PV patients by increasing Treg and decreasing Th17 and memory B cells.

ZusammenfassungHintergrund und ZielePemphigus gehört zu den seltenen, aber potentiell lebensbedrohlichen Autoimmunerkrankungen. Typisch sind Blasen und Erosionen der Haut und der Schleimhäute. Grundsätzlich unterscheiden wir zwei Subtypen: Pemphigus vulgaris (PV) und Pemphigus foliaceus (PF). In dieser Studie wurden die klinisch‐pathologischen Daten aus einem Register für bullöse Autoimmundermatosen analysiert mit dem Ziel, das Autoantikörperprofil und das Therapieansprechen bei Patienten mit PV und PF genauer zu charakterisieren.Patienten und MethodenIn einer retrospektiven Studie wurden die Daten von 69 Patienten mit PV und PF analysiert. Zur Beurteilung des klinischen Verlaufs, der Remissionen und Rezidive sowie des Schweregrads der Krankheit bei Erstmanifestation und während des gesamten Beobachtungszeitraums diente der ABSIS (Autoimmune Bullous Skin Intensity Score) (ABSIS). Mittels ELISA wurden die Spiegel von Anti‐Desmoglein (Dsg)‐1‐ und Anti‐Dsg‐ IgG‐Autoantikörpern im Serum bestimmt.ErgebnisseDie mittlere Remissionszeit bei Patienten mit PV und PF betrug 63 Wochen. PV‐Patienten mit Schleimhautbeteiligung zeigten eine schnellere Heilung. Bei PV‐Patienten mit moderat oder stark erhöhten Anti‐Dsg1‐IgG‐Autoantikörpern im Serum zu Beginn erfolgte im Lauf des Beobachtungszeitraums ein Absinken der Anti‐Dsg3‐IgG‐Spiegel.SchlussfolgerungenUnsere Studie liefert neue Erkenntnisse zum Krankheitsverlauf bei Patienten mit PV und PF und offenbart, dass ein Phänotyp mit Schleimhautbeteiligung eine stärkere Neigung zur Remission aufweist.

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease characterized by autoantibodies (auto-ab) against the desmosomal adhesion molecules desmoglein3 (Dsg3) and Dsg1. Binding of Dsg specific auto-ab to target structures induces an interruption of the desmosomal integrity which ultimately results in the clinical manifestation of flaccid blisters and erosions in PV patients. Underlying mechanisms inducing blister formation upon binding of Dsg-specific auto-ab are largely unknown. Numerous studies demonstrated the pathogenicity of auto-ab specific for the amino-terminal region (extracellular domain 1, EC1) of Dsg3. However, the Dsg3 specific auto-ab response in PV patients is polyclonal, including auto-ab directed against both amino- and membrane proximal epitopes. In this study, the pathogenicity of a murine monoclonal antibody directed against the membrane-proximal region (EC5) of the Dsg3 ectodomain was analysed. This Dsg3-specific antibody was isolated from the supernatant of a Dsg3-specific B-cell hybridoma and tested in various specificity and functional assays as well as in-vivo. Results clearly demonstrate that this murine auto-ab specifically binds human Dsg3 and is capable of inhibiting intercellular keratinocyte adhesion accompanied by the activation of the p38 MAPK signal transduction pathway. Here, for the first time, we can demonstrate that a specific auto-ab directed against the membrane-proximal region EC5 of human Dsg3 exhibit pathogenic activity similar to the well characterized EC1-specific antibody AK23 without the Ca2+ dependency. Our results deliver new aspects of a more defined understanding of auto-ab-induced blister formation in PV.

BACKGROUND AND OBJECTIVES:Pemphigus diseases are potentially life-threatening and rare autoimmune bullous disorders characterized by blisters and erosions of the skin and mucous membranes. These disorders can be largely divided into two major subtypes: pemphigus vulgaris (PV) and pemphigus foliaceus (PF). The objective of this study was to evaluate the autoantibody profile and response to therapy of PV and PF patients by analyzing the clinicopathological data from a registry for bullous autoimmune dermatoses.PATIENTS AND METHODS:In a retrospective study, data from 69 patients with PV and PF were included in the analysis. The Autoimmune Bullous Skin Intensity Score (ABSIS) was used to assess the clinical course, remissions, relapses and severity of the disease at first manifestation and throughout the observation period. ELISA was performed to assess levels of anti-desmoglein (Dsg)-1 and anti-Dsg3 IgG serum autoantibodies.RESULTS:The mean remission time in PV and PF patients was 63 weeks. PV patients with mucosal involvement showed a more favorable healing process. In PV patients with a moderate/high anti-Dsg1 IgG serum level at baseline, anti-Dsg3 IgG levels decreased during the observation period.CONCLUSIONS:Our study provides additional insights into the clinical course of patients with PV and PF, revealing that a mucosal phenotype is associated with a higher tendency towards remission.

Background: T(H)2 cells were thought to be a pivotal factor for initiation of the autoimmune blistering disease pemphigus. However, the role of other T-cell subsets in pemphigus pathogenesis remained unclear. Objective: We aimed to characterize the exact phenotype of T cells responsible for the development of pemphigus. Methods: Whole transcriptome shotgun sequencing was performed to determine differential gene expression in pemphigus lesions and skin of healthy individuals. The cutaneous cytokine signature was further evaluated by real-time quantitative PCR. In peripheral blood, the distribution of T H cell and folliclular helper (T-FH) cell subsets was analyzed by flow cytometry. Finally, the capacity of T-H and T-FH cell subsets to induce desmoglein (Dsg)-specific autoantibodies by memory B cells was evaluated in coculture experiments. Results: Transcriptome analysis of skin samples identified an IL-17A-dominated immune signature in patients with pemphigus, and Kyoto Encyclopedia of Genes and Genomes pathway analysis confirmed the dominance of the IL-17A signaling pathway. Increased expression of IL17A and associated cytokines was also detected by real-time quantitative PCR comparing lesional with perilesional or healthy skin. Interestingly, utilization of flow cytometry showed that patients with active pemphigus had elevated levels of circulating IL-17(+), T(H)17, T(FH)17, and T(FH)17.1 cells. Notably, levels of T(H)17 and T(FH)17 cells correlated with levels of Dsg-specific CD19(+) CD27(+) memory B cells, and patients with acute pemphigus showed higher levels of Dsg3-autoreactive T(FH)17 cells. Coculture experiments revealed T(FH)17 cells as primarily responsible for inducing Dsg-specific autoantibody production by B cells. Conclusion: Our findings show that T(FH)17 cells are critically involved in the pathogenesis of pemphigus and offer novel targets for therapeutic intervention.

Pemphigus vulgaris (PV) is an autoimmune blistering disease, in which autoantibodies against epidermal cadherins, such as desmoglein (Dsg)1 and Dsg3, lead to the development of blisters and erosions on the skin and mucous membranes. Autoreactive CD4+ T cells are essential for the induction and perpetuation of the disease by interaction with B cells producing autoantibodies. PV has a strong genetic association with certain human leucocyte antigen (HLA) alleles with HLA-DRB1*04:02 and LA-DQB1*05:03 being the most prevalent in patients. Recently, genome-wide association studies have provided a new approach to identify single nucleotide polymorphisms, alongside the known association with HLA alleles. Loss of tolerance against Dsgs and other autoantigens is a critical event in the pathogenesis of PV. Epitope spreading contributes to the progression of PV, leading to an extension of the Dsg-specific autoimmune response to other molecular epitopes of autoantigens, such as desmocollins or muscarinic receptors. Alterations in CD4+CD25+ FoxP3+ regulatory T cells are thought to contribute to the development of PV representing a suitable target for therapeutic interventions. Several CD4+ T-cell subsets and cytokines are involved in the pathogenesis of PV, while Th2 cells are the extensively studied population. Recently, other T cell subsets like T follicular helper cells and Th17 have gained attention as new potential players in PV pathogenesis. The involvement of local autoantibody production in the lesional skin of PV patients in tertiary lymphoid organs is currently discussed but not yet clarified. In this study, we reviewed the current knowledge about the development, characteristics and function of autoreactive T cells in pemphigus and present current new T cell-targeted therapeutic approaches.

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.

Bullous pemphigoid (BP) is a prototypic autoimmune disorder of the elderly, characterized by serum IgG autoantibodies, namely anti-BP180 and anti-BP230, directed against components of the basal membrane zone that lead to sub-epidermal loss of adhesion. Pruritus may be indicative of a pre-clinical stage of BP, since a subset of these patients shows serum IgG autoantibodies against BP230 and/or BP180 while chronic pruritus is increasingly common in the elderly population and is associated with a variety of dermatoses. Clinical and experimental evidence further suggests that pruritus of the elderly may be linked to autoimmunity with loss of self-tolerance against cutaneous autoantigens. Thus, the objective of this study was to determine autoreactive T cell responses against BP180 in elderly patients in comparison to patients with BP. A total of 22 elderly patients with pruritic disorders, 34 patients with bullous or non-bullous BP and 34 age-matched healthy controls were included in this study. The level of anti-BP180 and anti-BP230 IgG serum autoantibodies, Bullous Pemphigoid Disease Area Index (BPDAI), and pruritus severity were assessed for all patients and controls. For characterization of the autoreactive T cell response, peripheral blood mononuclear cells were stimulated ex vivo with recombinant BP180 proteins (NH 2 - and COOH-terminal domains) and the frequencies of BP180-specific T cells producing interferon-γ, interleukin (IL)-5 or IL-17 were subsequently determined by ELISpot assay. Patients with BP showed a mixed Th1/Th2 response against BP180 while autoreactive Th1 cells were identified in a minor subset of elderly patients with pruritic disorders. Furthermore, our T cell characterization revealed that therapeutic application of topical clobetasol propionate ointment in BP patients significantly reduced peripheral blood BP180-specific T cells, along with clinically improved symptoms, strongly suggesting a systemic immunosuppressive effect of this treatment.

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.