Pathogenesis Of Pemphigus Vulgaris Research Articles

Single-Cell Transcriptomes and Immune Repertoires Reveal the Cell State and Molecular Changes in Pemphigus Vulgaris.

The etiology and pathogenesis of pemphigus vulgaris (PV) entail intricate interactions between immune cells and epithelial cells. However, the specific subtypes of immune cells involved in PV, along with their respective roles, remain elusive. Likewise, the precise functions and mechanisms by which glucocorticoids affect cell types within the disease context require further elucidation. To address these knowledge gaps, we performed 5' single-cell RNA sequencing, combined with V(D)J enrichment on buccal mucosal lesions and peripheral blood samples from treatment-naive patients with PV, in conjunction with post-treatment peripheral blood samples obtained after oral prednisone treatment. Our findings suggest that the IL-1α signaling pathway, myeloid APCs, inflammatory CD8+ resident memory T cells, and dysfunctional CD4+ regulatory T cells are involved in the pathogenesis of PV. Part of these findings were validated by immunohistochemical assays and multiplex immunofluorescence assays. Furthermore, our results highlight the significant impact of prednisone treatment on monocytes and mucosal-associated invariant T cells while revealing a limited effect on CD4+ regulatory T cells. Additionally, we present the CDR3 amino acid sequence of BCR related to PV disease and investigate the characteristics of TCR/BCR clonotypes. In conclusion, our study provides a comprehensive understanding of PV, particularly focusing on the mucosal-dominant type, and sheds light on the effects of glucocorticoids within the PV context. These insights hold promise for the development of new therapeutic strategies in this autoimmune disorder.

Evaluation of E-Cadherin Expression in Patients with Pemphigus Vulgaris via Immunohistochemistry

Pemphigus is a group of autoimmune bullous diseases that can affect the skin and mucous membranes, and it is vital to recognize the exact pathogenesis of this disease. This study aimed to investigate the role of E-cadherin in the pathogenesis of pemphigus vulgaris (PV) and compare the expression of E-cadherin in the lesions of PV patients with that in healthy individuals’ skin. Thirty tissue samples from histopathologically confirmed PV patients as the case group and 30 skin samples from healthy individuals as the control group were evaluated for E-cadherin expression via the immunohistochemical method. Data analysis was performed using SPSS software version 25; chi-squared and Fisher’s exact tests were used to examine the relationship between qualitative variables. Immunohistochemical staining revealed decreased E-cadherin expression in the basal and suprabasal layers of the epidermis of PV patients compared with healthy individuals ( P < 0.001 ). E-cadherin expression was 1+ in 53.3% of patients, 2+ in 40% of patients, and 3+ in only one (3.3%) patient. On the other hand, the expression of E-cadherin in other layers of the epidermis was 1+ in one patient, 2+ in five patients (25%), and 3+ in 14 patients (70%). Also, the expression of E-cadherin in all layers of the epidermis was 3+ in all controls. E-cadherin expression in the basal and suprabasal layers of the epidermis appears to be lower in patients with PV compared with controls. Therefore, E-cadherin immunohistochemical staining helps diagnose PV along with other diagnostic methods. Moreover, these findings may shed light on the role of E-cadherin as a potential target for disease treatment aiming at disease stabilization. However, more studies are needed to clarify this issue.

Serum Prolactin Concentration in Patients with Pemphigus Vulgaris

Background: Pemphigus vulgaris (PV) is an autoimmune bullous disorder characterized clinically by blisters and erosions of the skin and/or mucous membranes. Serum prolactin plays a role in the pathogenesis of pemphigus vulgaris. Objective: The study aims to observe the serum prolactin concentration in patients with pemphigus vulgaris. Methods: This cross-sectional study was conducted at the Department of Dermatology & Venereology of Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh, between May 2017 and April 2018. A total of 35 patients were included in the study. A consecutive type of sampling technique was used. The dermatological examination was done to ascertain the extent of involvement of the disease. Results: Among 35 patients, 31.4% belonged to 30-50 years age group, while 62.9% belonged to >50 years and 5.7% patients belonged to <30 years ae group. The mean age was found 42.7±11.8 years with ranged from 19-65 years. The majority of the patients (57.1%) were male, and 42.9% patients were female. It was observed that 30(85.7%) had vesicles, 25(71.4%) had bulla, 35(100%) had erosions and 6(17.1%) had other types of lesions. 28 patients (80.0%) had scalp, 32(91.4%) had extremity, 35(100.0%) had trunk and 31(88.6%) had mucous membrane as the sites of involvement. The mean duration of disease was 9.0±12.7 months with a range from 0.5 to 36 months. It was observed that 9 patients (25.7%) had hyperprolactinemia, while 26 patients (74.3%) had normal levels of prolactin. The mean serum prolactin level was found 28.7±16.7 ng/ml with a range from 1.64 to 51.04 ng/ml. Conclusion: Since serum prolactin plays a role in the pathogenesis of pemphigus vulgaris, it may offer a novel therapeutic target for treatment of PV. Thus, we may reduce morbidity and mortality rate in PV patients by modifying their serum prolactin levels. Mugda Med Coll J. 2023; 6(1): 20-24

Type 2 T-Cell Responses against Distinct Epitopes of the Desmoglein 3 Ectodomain in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucous membranes caused by IgG autoantibodies that predominantly target two transmembrane desmosomal cadherins: desmoglein (DSG)1 and DSG3. DSG-specific T cells play a central role in PV pathogenesis because they provide help to autoreactive B cells for autoantibody production. In this study, we characterized DSG3-specific peripheral T cells in a cohort of 52 patients with PV and 41 healthy controls with regard to cytokine profile and epitope specificity. By ELISpot analysis, type 2 T cells reactive with the DSG3 ectodomain were significantly increased in patients with PV compared with those in healthy controls. By dextramer analysis, CD4+ T cells specific for an epitope within the extracellular domain of DSG3, DSG3(206-220), were found at significantly higher frequencies in patients with PV than in HLA-matched healthy controls. T-cell recognition of two distinct DSG3 epitopes, that is, DSG3(206-220) and DSG3(378-392), correlated significantly, suggesting a synergistic effect in B-cell help. Immunization of HLA-DRB1∗04:02-transgenic mice with PV with the same set of DSG3 peptides induced pathogenic DSG3-specific IgG antibodies, which induced loss of keratinocyte adhesion invitro. Thus, DSG3 peptide-specific T cells are of particular interest as surrogate markers of disease activity and potential therapeutic targets in PV.

Dominance and improved survivability of human γδT17 cell subset aggravates the immunopathogenesis of pemphigus vulgaris.

Human γδ T cells are highly enriched in epithelial cell-dominated compartments like skin. Nonetheless, their function in the pathogenesis of pemphigus vulgaris (PV), an autoimmune skin disorder, is lacking. Therefore, we investigated the functional expression of human γδT cell subsets along with their homing chemokine receptor-ligand and inflammatory cytokines in the immunopathogenesis of PV. Estimation of the frequency of γδT cell subsets by flow cytometry revealed four major subsets of γδ T cells (γδT1, γδT2, γδT17, γδTreg) in both control and PV circulation. The elevated frequency of γδT17 cells producing IL17 and expressing CCR6 receptor suggests their inflammatory and migratory potential in PV. In vitro culture of γδ T cells from patients showed increased mRNA expression of inflammatory cytokines IL17, RORγt, IL23, IL1, and co-stimulatory markers, CD27 and CD70. These findings correlated the role of IL1 and IL23 cytokines that alleviate the Th17 population in PV. Cytotoxic activities of γδ T cells were higher and inflammatory γδT17 cells were localized in the skin of PV whereas γδTreg cells associated TGFβ and FOXP3 were lowered. Hyperinflammatory phenotype of the γδT17 cell subset and its migratory potential might be aiding in the pathogenesis of PV, whereas γδTreg cells fail to suppress these inflammatory responses. Hence, γδT17 cell-associated markers can be targeted for identifying novel therapeutics in PV.

Detection of herpes simplex viruses in the oral lesions of patients with pemphigus vulgaris: Is it diagnostic or predictive of disease severity?

Some studies emphasise the relationship between the herpes simplex virus (HSV) and pemphigus. Although the possible role of HSV in the pathogenesis of pemphigus and the severity of the disease is obscure, we aimed to evaluate the presence of herpes simplex viruses (HSV 1/2) in the oral lesions of patients with pemphigus vulgaris and also assess its association with disease severity and types of lesions. A retrospective study was conducted on collected data in the form of collecting paraffin blocks, slides, and relevant pathology reports and referring to patients' medical records. A questionnaire containing details on the degree of skin, scalp, and mucosal involvement (Pemphigus Disease Area Index (PDAI)) was fulfiled. The immunoassay result was also collected to check the anti-desmoglein 3 and 1 antibodies (using ELISA technique). In this study, 52 patients of pemphigus vulgaris with oral lesions (case) and 52 patients with oral lesions not related to the disease (control) were evaluated. HSV1 was detected in 13.5% of oral pemphigus lesions and 1.9% of the control group (p=0.0598). There were no positive cases of HSV2 in either group. There was no significant association between the positivity of HSV1 and the site of lesions (p=1.00) or disease severity (p=0.28). However, we found a strong correlation between the PDAI disease severity score with the titre of the AntiDsg3 antibody (r=0.487, p=0.001) and AntiDsg1 antibody (r=0.309, p=0.026). This study demonstrated a significant prevalence of HSV1 in oral pemphigus lesions, and acyclovir therapy may play a significant role in managing these patients. However, HSV's role in the pathogenesis of pemphigus vulgaris cannot be clearly determined.

The dysregulation of circulating innate lymphoid cells is related to autoantibodies in pemphigus vulgaris.

Pemphigus vulgaris (PV) is a typical autoimmune disease caused by autoantibodies. Innate lymphoid cells (ILCs) are non-antigen-dependent populations composed of different subsets, also known as "mirror cells" of T cells, which play a crucial role in immune inflammatory diseases. However, the characteristics of ILCs in PV are unclear. Patients diagnosed with PV along with healthy controls in Second Xiangya Hospital of Central South University were studied. Flow cytometry was used to detect the proportion of ILC subsets in the peripheral blood. Anti-desmoglein (DSG) 1 and anti-DSG3 antibody levels of PV patients were detected. Thirty-eight PV patients and 37 healthy controls were enrolled. There were no differences in sex or age between the two groups. Compared with controls, ILCs/CD45+ lymphocytes were significantly decreased in patients with PV. The frequency of ILC1s increased in patients with PV and was positively correlated with anti-DSG3 antibodies. However, the frequency of ILC3s decreased in patients with PV and was negatively correlated with anti-DSG1 antibodies. After methylprednisolone treatment, ILC1/ILC levels significantly decreased. Circulating ILC subsets are associated with PV pathogenesis. Upregulated ILC1s seem to correlate positively with PV severity and can be restored after treatment.

The genetic basis of pemphigus vulgaris

AbstractThe severe autoimmune blistering disease pemphigus vulgaris (PV) is most probably mainly due to autoantibodies directed against desmosomal components of the epithelium, disrupting cell‐cell adhesion. The precise mechanisms underlying the development of the disease are still unknown, and immunosuppressive medications such as corticosteroids, which are linked to potentially serious adverse effects, are still the mainstay of treatment. Ethnic susceptibility and familial incidence hint to a genetic component to the pathophysiology of PV, which, if identified, could further improve our understanding of PV pathogenesis and lead to the discovery of new therapeutic targets for this potentially fatal condition. In this article, we review the evidence supporting a genetic component in PV and discuss the clinical and therapeutic implications of these discoveries.

ERK/MEK Pathway Regulates Th17 Cell Differentiation in Patients with Pemphigus Vulgaris.

T helper (Th) cells are involved in the pathogenesis of pemphigus vulgaris (PV). However, the mechanism still needs more exploration. This study aimed to evaluate the molecular mechanism of the dysregulation of Th17 cells in the peripheral blood of patients with PV. Serum levels of IL-17 and anti-Dsg3 titres in patients with PV were analysed using ELISA. The mRNA expression of retinoic acid orphan receptor γt (RORγt) in CD4+ T cells was detected using reverse transcription-quantitative PCR (qPCR). The number of Th17 cells was examined using flow cytometry. Western blot analysis and immunofluorescent staining were also performed to investigate the expression levels of ERK/MAPK signalling proteins and Th17 lineage-associated proteins. The proportion of Th17 cells and Th17 spectrum-associated proteins (p-STAT3, RORγt and IL-17) were upregulated in CD4+ cells in PV patients. The increased transcriptional levels of RORγt and IL-17 correlated positively with the severity of PV. Elevated phosphorylation of the ERK signalling factors was found in the collected CD4+ T cells in PV patients. The inhibition of the ERK signalling pathway significantly reduced the differentiation of Th17 cells in PV patients in vitro. Th17 cells are essential in the dysregulation of PV, and ERK signalling is involved in Th17-type immunity and promotes the development of PV. The study here provides us with a potential therapeutic target for PV.

Identification of Six microRNAs as Potential Biomarkers for Pemphigus Vulgaris: From Diagnosis to Pathogenesis.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune bullous disease. The role of microRNA (miRNA, miR) in the diagnosis and pathogenesis of PV remains unknown. This study aims to provide potential miRNA biomarkers for PV diagnosis and therapy options. Serum samples were obtained from 22 PV patients, 15 mucous membrane pemphigoid (MMP) patients, and 10 normal controls (NC). Total RNA was extracted from the serum samples, and 12 selected miRNAs were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic analyses including target gene prediction and enrichment analysis were performed. Twelve miRNAs were increased in the serum of the PV group compared with the NC group, in which six miRNAs had good efficacy to diagnose PV from MMP with the area under the receiver operator characteristic curves of 0.970 to 0.988. A series test for the combination of miR-584-5p and miR-155-5p reached the sensitivity and specificity of 95.5% and 100%. Bioinformatic analysis revealed target gene enrichment in the cell adhesion pathways, immune-relating pathways, and P38 mitogen-activated protein kinases signaling pathway. The study provides new insights and targets of miRNAs for the precise diagnosis and the exploration of pathogenesis for PV, which may serve as a reference for further research into autoimmune bullous diseases.

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

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.

Assessment of tissue E-cadherin and its proteolytic serum fragment in pemphigus vulgaris before and after remission: Acase-control study.

E-cadherin is a classic cadherin that mediates keratinocyte adhesion. To assess the tissue expression of E-cadherin and its proteolytic serum fragment (soluble E-cadherin) in pemphigus vulgaris (PV) before and after clinical remission compared with controls. Thirty-seven PV patients and thirty controls were enrolled. Pemphigus disease area index (PDAI) was calculated for patients at baseline and after remission. Punch biopsy specimens were taken from patients before, and after remission, and from controls for assessment of tissue E-cadherin by immunofluorescence. Similarly, serum samples were collected for assessment of serum soluble E-cadherin by ELISA. Presence, intensity, and mean intensity of tissue E-cadherin were significantly reduced in PV patients before treatment compared with controls (p< 0.001). Detected E-cadherin showed mainly a basal and suprabasal distribution with cell surface and a cytoplasmic expression. Serum E-cadherin was significantly higher in patients before treatment compared with controls (p= 0.006). With remission, tissue E-cadherin presence, intensity, mean intensity, and serum E-cadherin showed statistically significant improvement (p= 0.003, <0.001, <0.001, and 0.003 respectively). Tissue E-cadherin presence and serum E-cadherin level reached values equivalent to the controls (p= 0.49 and 0.44, respectively). Disruption of tissue E-cadherin and upregulation of serum soluble E-cadherin can contribute to the pathogenesis of PV. Clinical remission of PV is associated with normalization of tissue and serum E-cadherin.

Mitochondrial Autoantibodies and the Role of Apoptosis in Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocutaneous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of epidermal keratinocytes termed apoptolysis. Mitochondrial damage by antimitochondrial antibodies (AMA) has proven to be a critical link in this process. AMA act synergistically with other autoantibodies in the pathogenesis of PV. Importantly, absorption of AMA inhibits the ability of PVIgGs to induce blisters. Pharmacologic agents that protect mitochondrial function offer a new targeted approach to treating this severe immunoblistering disease.

A potential reciprocal emergence of regulatory T cells in oral lichen planus and pemphigus vulgaris: A meta-analysis

Oral lichen planus (OLP) and pemphigus vulgaris (PV) are 2 common inflammatory mucocutaneous diseases of immune-based etiology. Evidence indicates regulatory T (Treg) cells maybe play a role in the pathogenesis of OLP and PV, which are caused by aberrant immune responses. In this report, 7 eligible case–control studies containing 517 OLP patients and 261 healthy controls (HC) were identified. The level of Tregs was significantly higher in OLP patients than HC (mean difference: 1.79; 95%CI: 0.99, 2.60). On the other hand, 7 eligible case–control studies containing 169 PV patients and 121 HC were identified. Conversely, the level of Tregs was significantly lower in PV patients than HC (mean difference: −2.49; 95%CI: −3.90, −1.08). Collectively, this analysis for the first time reported reciprocal emergence of Tregs in OLP and PV using meta-analysis, and provided an interesting insight into a previously undescribed the linkage of the 2 mucocutaneous diseases.

Role of ADAM10 and ADAM17 in the Regulation of Keratinocyte Adhesion in Pemphigus Vulgaris.

The severe autoimmune blistering disease Pemphigus vulgaris (PV) is mainly caused by autoantibodies (IgG) against desmoglein (Dsg) 3 and Dsg1. The mechanisms leading to the development of blisters are not fully understood, but intracellular signaling seems to play an important role. Sheddases ADAM10 and ADAM17 are involved in the turnover of the desmosomal cadherin Dsg2 and ADAM10 has been shown to contribute to acantholysis in a murine pemphigus model. In the present study, we further examined the role of ADAM10 and ADAM17 both in keratinocyte adhesion and in the pathogenesis of PV. First, we found that inhibition of ADAM10 enhanced adhesion of primary human keratinocytes but not of immortalized keratinocytes. In dissociation assays, inhibition of ADAM10 shifted keratinocyte adhesion towards a hyperadhesive state. However, ADAM inhibition did neither modulate protein levels of Dsg1 and Dsg3 nor activation of EGFR at Y1068 and Y845. In primary human keratinocytes, inhibition of ADAM10, but not ADAM17, reduced loss of cell adhesion and fragmentation of Dsg1 and Dsg3 immunostaining in response to a PV1-IgG from a mucocutaneous PV patient. Similarly, inhibition of ADAM10 in dissociation assay decreased fragmentation of primary keratinocytes induced by a monoclonal antibody against Dsg3 and by PV-IgG from two other patients both suffering from mucosal PV. However, such protective effect was not observed in both cultured cells and ex vivo disease models, when another mucocutaneous PV4-IgG containing more Dsg1 autoantibodies was used. Taken together, ADAM10 modulates both hyperadhesion and PV-IgG-induced loss of cell adhesion dependent on the autoantibody profile.

Downregulation of aquaporin 3 in patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease. Aquaporin 3 (AQP3) is a glycerol/water channel involved in several physiological functions. Evaluation of the tissue expression and localization of AQP3 in the skin of PV patients. Twenty-seven PV patients and 30 controls were included. The patients were subjected to history taking, clinical evaluation, Autoimmune Bullous Skin Disorder Intensity Score and 4-mm punch biopsy. The biopsies were stained using anti-human AQP3 antibody and the immunofluorescence pattern and intensity were evaluated using a scoring system and ImageJ software analysis. AQP3 was expressed in the basal epidermis in 27 (100%) and in the suprabasal epidermis in 19 PV patients (70.4%). It was expressed in all controls in basal and suprabasal layers. Intensity of AQP3 immunofluorescence was strong in 2 (7.4%), moderate in 19 (70.4%) and weak in 6 patients (22.2%) while it was strong in 18 (60%) and moderate in 12 controls (40%). AQP3 expression was significantly lower in patients than controls in the suprabasal epidermis (p= 0.001). Patients with extensive disease had significantly weaker AQP3 intensity than those with marked disease (p= 0.005) Downregulation of AQP3 in patients with PV, especially in the suprabasal layers and in extensive clinical disease, suggests a potential role of AQP3 in the pathogenesis of PV.

Caspase Inhibition as a Possible Therapeutic Strategy for Pemphigus Vulgaris: A Systematic Review of Current Evidence.

Simple SummaryPemphigus vulgaris is a potentially fatal disease characterised by blister formation affecting the skin and mouth. The mechanisms of blister formation may involve a biological process called apoptosis—a type of cell death—and some death-associated molecules known as caspases. Our review of the existing literature shows that caspase inhibitors exhibit an inhibitory effect on PV-induced apoptosis formation in vitro. In particular, activity of caspase 1 and caspase 3 is essential for the development of PV in vitro and in vivo. However, a majority of in vivo studies assessing caspase inhibition in PV models have a high risk of bias.Background: Pemphigus vulgaris (PV) is an IgG-mediated autoimmune disease characterised by epithelial cell–cell detachment (acantholysis) resulting in mucocutaneous blistering. The exact pathogenesis of blister formation is unknown and this has hampered the development of non-steroidal, mechanism-based treatments for this autoimmune disease. This systematic review aims to investigate the role of caspases in the pathogenesis of PV to inform the choice of more targeted therapeutic agents. Methods: A systematic search of MEDLINE/PubMed and Scopus databases was conducted to identify eligible studies. Multiple phases of inclusion and exclusion of the primary articles were conducted in pairs, and studies were recorded and analysed according to the latest version of the preferred reporting items for systematic reviews and meta-analyses (PRISMA). Risk of bias assessment was conducted for extracted in vivo animal intervention studies using SYRCLE’s risk of bias tool. Results: Eight articles from a total of 2338 in vitro, in vivo, and human studies met the inclusion criteria, with a high degree of inter-rater reliability. By and large, the results show that caspase activation was pathogenic in experimental PV because pan-caspase inhibitors could block or reduce PV acantholysis and blistering in vitro and in vivo, respectively. The pathogenic pathways identified involved caspase-1 and caspase-3. One study failed to show any improvement in the PV model with a caspase inhibitor. The majority of animal studies had high or unclear risk of bias. Conclusion: There are consistent data pointing towards a pathogenic role of caspase activation in PV acantholysis. However, high-quality evidence to confirm that caspase inhibition can prevent PV-induced blistering in vivo is limited. Therefore, further research is required to test the preclinical efficacy of caspase inhibitors in PV.

004 Up-regulation of ST18 drives pemphigus vulgaris pathogenesis: a perpetuum mobile model

Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease. We previously showed that a genetic variant within the ST18 promoter promotes ST18 up-regulation in a p53/p63-dependent manner and is associated with a 6-fold increased risk to develop PV. ST18 was also found to be overexpressed in the skin of PV patients. In addition, it has been shown that desmoglein 3 (DSG3) down-regulation is associated with increased p53 expression and activity. Based on these data, using a combination of reporter assays, Western blotting, confocal immunofluorescence microscopy, we investigated the possibility that ST18, DSG3 and p53 may be jointly involved on the pathogenesis of PV.

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

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.

011 Investigating the role of skin-resident T cells in pemphigus vulgaris in humans

Pemphigus vulgaris (PV) is an autoimmune blistering disease, in which autoantibodies against desmogleins (Dsg) 1 and Dsg3 interfere with epidermal cell-cell adhesion, thereby causing blister formation and erosions of the skin and/or mucous membranes. Autoreactive Dsg-specific T cells play a central role in the PV pathogenesis. However, little is known about T cells in PV skin lesions and a detailed phenotypical and functional analysis is currently missing. Therefore, we aimed to comprehensively characterize T cells residing in lesional and perilesional skin of PV patients as well as matched peripheral blood compared to healthy controls.