Unraveling Mechanisms of Autoimmune Skin Blistering: Applying Single-Cell Transcriptomics to Pemphigus B Cells

Clinical Implications•Single-cell RNA sequencing of antigen-specific B cells results in novel, potentially druggable targets for therapy.•CD137L/CD137 signaling and BATF are critically involved in patients with active pemphigus.•Integration of published omics datasets on patients with pemphigus may hold great promise. •Single-cell RNA sequencing of antigen-specific B cells results in novel, potentially druggable targets for therapy.•CD137L/CD137 signaling and BATF are critically involved in patients with active pemphigus.•Integration of published omics datasets on patients with pemphigus may hold great promise. Autoimmunity against the skin is typically attributed to autoantibodies (with their corresponding B cells producing them) or T cells that react against autoantigens, resulting in disease that can be clinically and immunologically differentiated into distinct patterns in most patients (Gudjonsson et al., 2020Gudjonsson J.E. Kabashima K. Eyerich K. Mechanisms of skin autoimmunity: cellular and soluble immune components of the skin.J Allergy Clin Immunol. 2020; 146: 8-16Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Among those, autoimmune skin conditions with potentially life-threatening blistering encompass entities from the pemphigus and pemphigoid groups, with the respective autoantigens being well-defined at the molecular level, facilitating research and diagnosis (Hammers and Stanley, 2016Hammers C.M. Stanley J.R. Mechanisms of disease: pemphigus and bullous pemphigoid.Annu Rev Pathol. 2016; 11: 175-197Crossref PubMed Scopus (198) Google Scholar). In patients with pemphigus, autoantibodies against desmosomal transmembrane adhesion proteins desmoglein (DSG) 3 and DSG1 are formed and result in pathologies on the skin and/or mucous membranes that are explained by the presence or absence of those autoantibody reactivities: patients with mucosal-dominant pemphigus vulgaris (mPV) feature anti-DSG3 antibodies, whereas patients with mucocutaneous pemphigus vulgaris (mcPV) display anti-DSG3 and anti-DSG1 antibodies. In pemphigus foliaceus (PF), only the skin is affected, and patients show only anti-DSG1 antibodies in circulation. Intracellular signaling processes are involved in targeted keratinocytes, as evidenced by various cell biologic studies, complementing the direct steric effects mediated by these pathogenic autoantibodies (Hammers and Stanley, 2016Hammers C.M. Stanley J.R. Mechanisms of disease: pemphigus and bullous pemphigoid.Annu Rev Pathol. 2016; 11: 175-197Crossref PubMed Scopus (198) Google Scholar). Although the role and pathogenicity of autoantibodies in these patients are increasingly understood, the characteristics of the B cells that produce them are not fully understood or elucidated. In their article published in the Journal of Investigative Dermatology, Egami et al., 2023Egami S. Watanabe T. Fukushima-Nomura A. Nomura H. Takahashi H. Yamagami J. et al.Desmoglein-specific B-cell–targeted single-cell analysis revealing unique gene regulation in patients with pemphigus.J Invest Dermatol. 2023; 143: 1919-1928.e16Abstract Full Text Full Text PDF Google Scholar performed state-of-the-art single-cell transcriptomic analysis on autoimmune antigen-specific memory B cells from patients with pemphigus to unravel new pathways of pathophysiologic relevance. Over the past decade, much progress has been made in decoding autoimmune mechanisms at all stages of this paradigmatic model disease, with important input stemming from omics-based high-throughput technologies: antibody and/or B-cell receptor repertoires of patients with pemphigus were studied in great detail and partly over time by antibody phage display (Payne et al., 2005Payne A.S. Ishii K. Kacir S. Lin C. Li H. Hanakawa Y. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Google Scholar), Epstein−Barr virus−transformed hybridoma cells (Di Zenzo et al., 2012Di Zenzo G. Di Lullo G. Corti D. Calabresi V. Sinistro A. Vanzetta F. et al.Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface.J Clin Invest. 2012; 122: 3781-3790Crossref PubMed Scopus (131) Google Scholar), and single-cell cloning after sorting (Cho et al., 2019Cho A. Caldara A.L. Ran N.A. Menne Z. Kauffman R.C. Affer M. et al.Single-cell analysis suggests that ongoing affinity maturation drives the emergence of pemphigus vulgaris autoimmune disease.Cell Rep. 2019; 28: 909-922.e6Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), yielding information on clonal diversity, processes of affinity maturation, antigen-binding properties, and synergistic effects of mAbs in mediating tissue pathology. By including next-generation sequencing approaches into these repertoire analyses, subclass-specific autoantibody repertoires (i.e., IgG4/IgG1, IgA1/IgA2) could be studied, providing new insights into their origins and formation in human disease (Ellebrecht et al., 2018Ellebrecht C.T. Mukherjee E.M. Zheng Q. Choi E.J. Reddy S.G. Mao X. et al.Autoreactive IgG and IgA B cells evolve through distinct subclass switch pathways in the autoimmune disease pemphigus vulgaris.Cell Rep. 2018; 24: 2370-2380Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Proteomics-based deconvolution of pemphigus autoantibody repertoires finally allowed for unprecedented tracking of single-serum autoantibodies over time in patients, suggesting a much more clonally diverse and diverse landscape of autoantibodies in pemphigus (Chen et al., 2017Chen J. Zheng Q. Hammers C.M. Ellebrecht C.T. Mukherjee E.M. Tang H.Y. et al.Proteomic analysis of pemphigus autoantibodies indicates a larger, more diverse, and more dynamic repertoire than determined by B cell genetics.Cell Rep. 2017; 18: 237-247Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Early genomic works applied microarrays to identify signatures of pemphigus vulgaris (PV)−associated differentially expressed genes (DEGs), comparing PBMCs from patients of differing clinical activity with those from controls (Dey-Rao et al., 2013Dey-Rao R. Seiffert-Sinha K. Sinha A.A. Genome-wide expression analysis suggests unique disease-promoting and disease-preventing signatures in pemphigus vulgaris.Genes Immun. 2013; 14: 487-499Crossref PubMed Scopus (27) Google Scholar). Similar techniques were then used on single antigen-specific B cells to identify molecular effects of treatment, revealing pattern differences in IL1β and CD27 gene expression in the corticosteroid and rituximab treatment groups (Hébert et al., 2019Hébert V. Petit M. Maho-Vaillant M. Golinski M.L. Riou G. Derambure C. et al.Modifications of the transcriptomic profile of autoreactive B cells from pemphigus patients after treatment with rituximab or a standard corticosteroid regimen.Front Immunol. 2019; 10: 1794Crossref PubMed Scopus (16) Google Scholar) or increases in the surface expression of the KCNN4 potassium channel on B cells after rituximab administration (Caillot et al., 2018Caillot F. Derambure C. Berkani N. Riou G. Maho-Vaillant M. Calbo S. et al.Long-term increase of Kcnn4 potassium channel surface expression on B cells in pemphigus patients after rituximab treatment.J Invest Dermatol. 2018; 138: 2666-2668Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Total RNA sequencing of peripheral blood CD3−CD19+ lymphocytes from patients with pemphigus beautifully confirmed murine experimental data on reduced FcγRIIB signaling as an important driver for pathogenic class switching of autoantibodies also in human disease development (Nomura et al., 2022Nomura H. Wada N. Takahashi H. Kase Y. Yamagami J. Egami S. et al.IgM to IgG class switching is a necessary step for pemphigus phenotype induction in desmoglein 3-specific B cell receptor knock-in mouse.J Immunol. 2022; 208: 582-593Crossref PubMed Scopus (3) Google Scholar). A trove of new transcriptomics data has been generated recently by studying lesional pemphigus skin samples that, in ectopic lymphoid-like structures, contain autoreactive B and T cells. Total RNA sequencing−based and microarray-based analyses not only led to the identification of an IL-17+ T-cell−governed autoimmune response (extending the concept of pemphigus just being a classical T helper 2−driven disease [Holstein et al., 2021Holstein J. Solimani F. Baum C. Meier K. Pollmann R. Didona D. et al.Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.J Allergy Clin Immunol. 2021; 147: 2358-2369Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar]) but also to new potential epigenetic regulators (Huang et al., 2022Huang Z.X. Qu P. Wang K.K. Zheng J. Pan M. Zhu H.Q. Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators.J Transl Med. 2022; 20: 182Crossref PubMed Scopus (6) Google Scholar) and new insights into the in vivo biology of autoimmune B cells (Zhou et al., 2020Zhou S. Liu Z. Yuan H. Zhao X. Zou Y. Zheng J. et al.Autoreactive B cell differentiation in diffuse ectopic lymphoid-like structures of inflamed pemphigus lesions.J Invest Dermatol. 2020; 140: 309-318.e8Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Single-cell−sequencing technologies allow for the detection of pathologic dysregulations even in small subpopulations of cells, with regard to changes on the genetic, transcriptional, epigenetic, or proteomic levels, depending on the methodology applied. This technique can either be applied to complex samples (with preserved cellular heterogeneity) or to samples enriched for specific cell types, such as B cells. This latter approach was applied by Egami et al., 2023Egami S. Watanabe T. Fukushima-Nomura A. Nomura H. Takahashi H. Yamagami J. et al.Desmoglein-specific B-cell–targeted single-cell analysis revealing unique gene regulation in patients with pemphigus.J Invest Dermatol. 2023; 143: 1919-1928.e16Abstract Full Text Full Text PDF Google Scholar on peripheral blood samples from three patients with pemphigus (one with PF, one with mcPV, and one with mPV), resulting in 103 DSG-specific sorted B cells and 128 non−DSG-specific sorted B cells as input material for single-cell RNA sequencing. For the one patient with PF, additional DSG1-specific B cells (31) were single-cell sorted after treatment, for comparison with 37 cells before treatment from that patient. The authors show that with their sorting and sequencing approach, B-cell receptor repertoire data can be obtained, also serving as an internal technical control for their experimentation. The authors then go on to identify, among upregulated DEGs, signatures of CD137L-mediated T-cell costimulation (with CD137L expression on antigen-specific B cells confirmed by flow cytometry), B-/T-cell differentiation (e.g., BATF), and inflammation (e.g., S100A8/A9, CCL3). In light of earlier published works and their translational potential, these findings are particularly intriguing. CD137L/CD137 signaling is involved in follicular T helper cell activation, a cell type recently implicated in pemphigus pathophysiology (Holstein et al., 2021Holstein J. Solimani F. Baum C. Meier K. Pollmann R. Didona D. et al.Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.J Allergy Clin Immunol. 2021; 147: 2358-2369Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Moreover, the transcription factor BATF is known to be expressed in B cells, directly controlling activation-induced cytidine deaminase and thus acting as a regulator of class-switch recombination and somatic hypermutation (Ise et al., 2011Ise W. Kohyama M. Schraml B.U. Zhang T. Schwer B. Basu U. et al.The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells.Nat Immunol. 2011; 12: 536-543Crossref PubMed Scopus (265) Google Scholar). Comparing DEGs before and after immunosuppressive treatment in a patient with PF, the authors identified, as expected, downregulation of genes and signaling implied in B-cell−mediated antibody production (i.e., CD27, peroxisome proliferator–activated receptor, APRIL), consistent with improvements in clinical activity and decreased serum antibody titers from treatment with corticosteroids and azathioprine. Studying cellular mechanisms of autoimmunity is challenging, especially so when rare diseases such as PV or PF are concerned; yet, at the same time, these conditions are very well-characterized with regard to their autoantigens, positioning them as model diseases for (theoretically) unbiased identification of new pathways and, potentially, treatment targets. As shown, Egami et al., 2023Egami S. Watanabe T. Fukushima-Nomura A. Nomura H. Takahashi H. Yamagami J. et al.Desmoglein-specific B-cell–targeted single-cell analysis revealing unique gene regulation in patients with pemphigus.J Invest Dermatol. 2023; 143: 1919-1928.e16Abstract Full Text Full Text PDF Google Scholar succeeded in establishing an appropriate methodology for the isolation of antigen-specific B cells and subsequent single-cell RNA sequencing, holding great potential for translation into personalized medicine and studying more patients with PV, PF, and other dermatologic autoimmune diseases as well. From this and other works, it also becomes clear that the resulting datasets are becoming increasingly complex (and less intuitively accessible), requiring sophisticated bioinformatic analyses and the close collaboration of clinician scientists, basic researchers, informaticians, and statisticians. Finally, another future challenge (and chance) lies in the integration of various single-cell and non-single-cell omic datasets concerning one particular disease or group of diseases from laboratories worldwide, and to conclude and appropriately test promising treatment targets resulting from these strides. Christoph M. Hammers: http://orcid.org/0000-0001-5631-2415 The author states no conflict of interest. Desmoglein-Specific B-Cell−Targeted Single-Cell Analysis Revealing Unique Gene Regulation in Patients with PemphigusJournal of Investigative DermatologyVol. 143Issue 10PreviewAutoreactive B cells are assumed to play a critical role in pemphigus; however, the characteristics of these cells are not yet fully understood. In this study, 23 pemphigus vulgaris or pemphigus foliaceus samples were used to isolate circulating desmoglein (DSG)-specific B cells. Transcriptome analysis of the samples was performed at the single-cell level to detect genes involved in disease activity. DSG1- or DSG3-specific B cells from three patients’ differentially expressed genes related to T cell costimulation (CD137L) as well as B-cell differentiation (CD9, BATF, TIMP1) and inflammation (S100A8, S100A9, CCR3), compared with nonspecific B cells from the same patients. Full-Text PDF Open Access