Autoreactive IgG and IgA B Cells Evolve through Distinct Subclass Switch Pathways in the Autoimmune Disease Pemphigus Vulgaris.

Lineage analysis of autoreactive B cells can reveal the origins of autoimmunity. In the autoimmune disease pemphigus vulgaris (PV), desmoglein 3 (DSG3) and DSG1 autoantibodies are predominantly of the IgG4 subclass and less frequently of IgG1 and IgA subclasses, prompting us to investigate whether anti-DSG IgG4 B cells share lineages with IgG1, IgA1, and IgA2. Combining subclass-specific B cell deep sequencing with high-throughput antibody screening, we identified 80 DSG-reactive lineages from 4 PV patients. Most anti-DSG IgG4 B cells lacked clonal relationships to other subclasses and preferentially targeted DSG adhesion domains, whereas anti-DSG IgA frequently evolved from or to other subclasses and recognized a broader range of epitopes. Our findings suggest that anti-DSG IgG4 B cells predominantly evolve independently or diverge early from other subclasses and that IgA is most often not the origin of IgG autoreactivity in PV. These data provide insight into how autoreactivity diversifies across B cell subclasses.

Stat3 regulates desmoglein 3 transcription in epithelial keratinocytes.

Pemphigus vulgaris (PV) is an epithelial blistering disease caused by autoantibodies to the desmosomal cadherin desmoglein 3 (DSG3). Glucocorticoids improve disease within days by increasing DSG3 gene transcription, although the mechanism for this observation remains unknown. Here, we show that DSG3 transcription in keratinocytes is regulated by Stat3. Treatment of primary human keratinocytes (PHKs) with hydrocortisone or rapamycin, but not the p38 MAPK inhibitor SB202190, significantly increases DSG3 mRNA and protein expression and correspondingly reduces phospho-S727 Stat3. Stat3 inhibition or shRNA-knockdown also significantly increases DSG3 mRNA and protein levels. Hydrocortisone- or rapamycin-treated PHKs demonstrate increased number and length of desmosomes by electron microscopy and are resistant to PV IgG-induced loss of cell adhesion, whereas constitutive activation of Stat3 in PHKs abrogates DSG3 upregulation and inhibits hydrocortisone and rapamycin's therapeutic effects. Topical hydrocortisone, rapamycin, or Stat3 inhibitor XVIII prevents autoantibody-induced blistering in the PV passive transfer mouse model, correlating with increased epidermal DSG3 expression and decreased phospho-S727 Stat3. Our data indicate that glucocorticoids and rapamycin upregulate DSG3 transcription through inhibition of Stat3. These studies explain how glucocorticoids rapidly improve pemphigus and may also offer novel insights into the physiologic and pathophysiologic regulation of desmosomal cadherin expression in normal epidermis and epithelial carcinomas.

Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics.

In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity.

Determinants of VH1-46 Cross-Reactivity to Pemphigus Vulgaris Autoantigen Desmoglein 3 and Rotavirus Antigen VP6.

Shared VH1-46 gene usage has been described in B cells reacting to desmoglein 3 (Dsg3) in the autoimmune disease pemphigus vulgaris (PV), as well as B cells responding to rotavirus capsid protein VP6. In both diseases, VH1-46 B cells bearing few to no somatic mutations can recognize the disease Ag. This intriguing connection between an autoimmune response to self-antigen and an immune response to foreign Ag prompted us to investigate whether VH1-46 B cells may be predisposed to Dsg3-VP6 cross-reactivity. Focused testing of VH1-46 mAbs previously isolated from PV and rotavirus-exposed individuals indicates that cross-reactivity is rare, found in only one of seven VH1-46 IgG clonotypes. High-throughput screening of IgG B cell repertoires from two PV patients identified no additional cross-reactive clonotypes. Screening of IgM B cell repertoires from one non-PV and three PV patients identified specific cross-reactive Abs in one PV patient, but notably all six cross-reactive clonotypes used VH1-46. Site-directed mutagenesis studies indicate that amino acid residues predisposing VH1-46 Abs to Dsg3 reactivity reside in CDR2. However, somatic mutations only rarely promote Dsg3-VP6 cross-reactivity; most mutations abolish VP6 and/or Dsg3 reactivity. Nevertheless, functional testing identified two cross-reactive VH1-46 Abs that both disrupt keratinocyte adhesion and inhibit rotavirus replication, indicating the potential for VH1-46 Abs to have both pathologic autoimmune and protective immune functions. Taken together, these studies suggest that certain VH1-46 B cell populations may be predisposed to Dsg3-VP6 cross-reactivity, but multiple mechanisms prevent the onset of autoimmunity after rotavirus exposure.

Pathogenicity and Epitope Characteristics Do Not Differ in IgG Subclass-Switched Anti-Desmoglein 3 IgG1 and IgG4 Autoantibodies in Pemphigus Vulgaris.

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.

Tailoring the Treatment of Melanoma: Implications for Personalized Medicine.

Oncology has been revolutionized by the ability to selectively inhibit the growth of cancerous cells while ostensibly avoiding the disruption of proteins and pathways necessary for normal cellular function. This paradigm has triggered an explosion of targeted therapies for cancer, creating a burgeoning billion-dollar industry of small molecules and monoclonal antibodies [1]. Largely due to these new treatments, spending on cancer pharmaceuticals has surpassed $100 billion worldwide [2]. In particular, the treatment of melanoma, a deadly and fast-spreading form of skin cancer, has been transformed by these new targeted therapies. In this mini-review, we summarize the progress made in the field of personalized treatment of melanoma, with an emphasis on targeted therapies. We then outline future directions for treatment, including novel cell-mediated therapies and new potential targets.

Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients.

Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies (Abs) from four PV patients and identify pathogenic VH1-46 autoAbs from all four patients. Unexpectedly, VH1-46 autoAbs had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs. Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 Abs require few to no mutations to acquire Dsg3 autoreactivity, which may favour their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.