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The skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here, we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it.

Desmoglein 3 chimeric autoantibody receptor T cells (DSG3-CAART) expressing the pemphigus vulgaris (PV) autoantigen DSG3 fused to CD137-CD3ζ signaling domains, represent a precision cellular immunotherapy approach for antigen-specific B cell depletion. Here, we present definitive preclinical studies enabling a first-in-human trial of DSG3-CAART for mucosal PV. DSG3-CAART specifically lysed human anti-DSG3 B cells from PV patients and demonstrated activity consistent with a threshold dose in vivo, resulting in decreased target cell burden, decreased serum and tissue-bound autoantibodies, and increased DSG3-CAART engraftment. In a PV active immune model with physiologic anti-DSG3 IgG levels, DSG3-CAART inhibited antibody responses against pathogenic DSG3 epitopes and autoantibody binding to epithelial tissues, leading to clinical and histologic resolution of blisters. DSG3 autoantibodies stimulated DSG3-CAART IFN-γ secretion and homotypic clustering, consistent with an activated phenotype. Toxicology screens using primary human cells and high-throughput membrane proteome arrays did not identify off-target cytotoxic interactions. These preclinical data guided the trial design for DSG3-CAART and may help inform CAART preclinical development for other antibody-mediated diseases.

Pemphigus is a chronic and severe autoimmune bullous disease caused by autoantibodies targeting adhesion molecules between keratinocytes. It requires 2–3 years on average to manage the disease. To date, although Rituximab combined with short-term systemic glucocorticoids was accepted as first-line therapy, systemic glucocorticoids remain the primary therapeutic option for pemphigus patients, successfully decreasing morbidity and mortality from pemphigus. However, novel therapeutic strategies are desirable due to the low efficacy in some subset of patients and the long-term severe adverse effects of traditional therapies. Recently, immunotherapy has proved to be encouraging for disease control or cure. Based on the current understanding of the immune mechanisms of pemphigus, we review the immune targets and corresponding agents applied in practice or under clinical trials. The goals of the novel treatments are to improve the quality of life of pemphigus patients by improving efficacy and safety, minimizing side effects, achieving fast disease control, or curing the disease.

Background: Pemphigus vulgaris (PV) is a potentially life-threatening mucocutaneous autoimmune blistering disease characterized by suprabasal acantholysis, causing painful mucocutaneous blisters and erosions. Current mainstay therapy for pemphigus is systemic corticosteroids in combination with or without immunosuppressive adjuvants, which may cause severe adverse effects and seriously impact on the quality of life in pemphigus patients. The objective of this study was to evaluate the efficacy and safety of thalidomide therapy in patients with PV. Methods: This study examined six PV patients from June 5, 2017, to November 11, 2018, in the dermatology department of Peking Union Medical College Hospital. Treatment with thalidomide was applied at a dose of 50–100 mg/day for disease control. Results: The mean age of the six patients (two male and four female patients) at the time of thalidomide therapy initiation was 50.2 years (range: 38–67 years), and the total duration of follow-up after thalidomide therapy was 13.2 months (range: 5–25 months). All patients responded favorably to thalidomide treatment, and three patients showed a dramatic reduction in anti-Dsg3 autoantibodies in the serologic examinations within 1 year. Five patients were found to have mucosal involvement. Mild adverse effects were noted in three patients, which could be managed after the application of symptomatic treatment and did not interfere with the pemphigus therapy. Conclusion: These results demonstrate that thalidomide could be an effective and safe option for PV patients, especially those who are concerned about steroid-induced severe complications, and have mucosal diseases.

By the cellular and molecular approaches, such as microarray analysis, flow cytometry, immunofluorescence or immunohistochemistry, proteomics, metabolomics, and single-cell RNA sequencing, combined with state-of-art data analysis (bioinformatic analysis), characteristic cellular and molecular features or signatures could be identified in specific skin conditions. Particularly, rituximab combined with short-term systemic glucocorticoids has been adapted as first-line therapy for treating pemphigus patients with excellent efficacies (4). [...]a cell-based chimeric antigen receptor T-cell (CAR-T) treatment has demonstrated excellent efficacy in treating pemphigus patients in pre-clinical studies (5). [...]the roles of immunoglobulin E (IgE) in comorbid psoriasis and atopic dermatitis AD are overviewed.

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.

Pemphigus vulgaris (PV) is characterized by IgG1 and IgG4 autoantibodies to desmoglein (Dsg) 3, causing suprabasal blistering of skin and mucous membranes. IgG4 is the dominant autoantibody subclass in PV and correlates with disease activity, whereas IgG1 can be associated with remittent disease. It is unknown if switching the same variable region between IgG4 and IgG1 directly impacts pathogenicity. Here, we tested whether three pathogenic PV monoclonal antibodies (mAbs) from three different patients demonstrate differences in antigen affinity, epitope specificity, or pathogenicity when expressed as IgG1 or IgG4. F706 anti-Dsg3 IgG4 and F779 anti-Dsg3 IgG1, previously isolated as heterohybridomas, and Px43, a monovalent anti-Dsg3/Dsg1 IgG antibody isolated by phage display, were subcloned to obtain paired sets of IgG1 and IgG4 mAbs. Using ELISA and cell surface staining assays, F706 and F779 demonstrated similar antigen binding affinities of IgG1 and IgG4, whereas Px43 showed 3- to 8-fold higher affinity of IgG4 versus IgG1 by ELISA, but identical binding affinities to human skin, perhaps due to targeting of a quaternary epitope best displayed in tissues. All 3 mAb pairs targeted the same extracellular cadherin (EC) domain on Dsg3, caused Dsg3 internalization in primary human keratinocytes, and caused suprabasal blisters in human skin at comparable doses. We conclude that switching IgG1 and IgG4 subclasses of pathogenic PV mAbs does not directly affect their antigen binding or pathogenic properties.

The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Triton-insoluble fraction of cell lysates within 2hours. Immunofluorescence and antibody ELISA studies suggest that pathogenic mAbs cause internalization of cell-surface Dsg3 but not Dsc3 through early endosomes. Electron microscopy demonstrated a lack of well-formed desmosomes in keratinocytes treated with pathogenic compared to nonpathogenic mAbs. In contrast, pathogenic mAbs caused late depletion of Dsg3 from preformed desmosomes at 24hours, with effects on multiple desmosomal proteins including Dsc3 and plakoglobin. Together, these studies indicate that pathogenic PV mAbs specifically cause internalization of newly synthesized Dsg3 during desmosome assembly, correlating with their pathogenic activity. Monovalent human PV anti-Dsg mAbs reproduce the effects of polyclonal PV IgG on Dsg3 and will facilitate future studies to further dissect the cellular mechanisms for the loss of cell adhesion in pemphigus.

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.

The aim of this study was to confirm the association of RHOB and FAM167A-BLK gene polymorphisms with susceptibility to systemic sclerosis (SSc) in a Chinese Han population. A total of 248 SSc patients and 251 healthy controls of Chinese Han ethnicity, which visited the department of dermatology of Peking Union Medical College Hospital, were included in the study. Six selected single nucleotide polymorphisms (SNPs) in the RHOB and FAM167A-BLK regions were selected as markers and were genotyped using a MassARRAY system, which is based on the matrix-assisted laser desorption/ionization time of flight mass spectrometry technique. Three SNPs in the coding regions of the RHOB and FAM167A-BLK genes displayed an association with SSc: (1) rs1062292T, which is a newly discovered SNP in the RHOB gene (P = 0.03, odds ratio [OR] = 1.62, 95% confidence interval (CI) = 1.05-2.50), (2) rs2736340T (P = 0.03, OR = 1.39, 95%CI = 1.03-1.85), and (3) rs13277113A (P = 0.04, OR = 1.34, 95%CI = 1.01-1.76), both in the FAM167A-BLK gene. Our results support previous findings that vaiants in the RHOB and FAM167A-BLK genes may be associated with susceptibility to SSc. However, the loci of the SNPs in RHOB region that displayed an association with SSc are quite different from the loci which were identified in studies of Caucasian populations. Our results confirm that RHOB and FAM167A-BLK polymorphisms exist in Chinese Han SSc patients. Therefore, variants of the RHOB and FAM167A-BLK genes are promising genetic markers for SSc.