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.

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis.

In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed "Inverted Repeat Binding Protein (IRBP) 18" and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.

Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

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.

The dual nature of interleukin-10 in pemphigus vulgaris.

The immunomodulatory cytokine interleukin-10 (IL-10) plays beneficial but also potentially detrimental roles in inflammation, infection, and autoimmunity. Recent studies suggest a regulatory role for IL-10-expressing B cells in the autoimmune blistering disease pemphigus vulgaris. Here we review the studies on IL-10 in pemphigus vulgaris and discuss the potential pathophysiological significance of these findings in comparison to prior studies of IL-10 in other human conditions. A better understanding of the complex roles of IL-10 in immune regulation may improve our understanding of pemphigus pathogenesis and treatment.

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.

CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.

Chromosomal rearrangements often occur at genomic loci with DNA secondary structures, such as common fragile sites (CFSs) and palindromic repeats. We developed assays in mammalian cells that revealed CFS-derived AT-rich sequences and inverted Alu repeats (Alu-IRs) are mitotic recombination hotspots, requiring the repair functions of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) and the Mre11/Rad50/Nbs1 complex (MRN). We also identified an endonuclease activity of CtIP that is dispensable for end resection and homologous recombination (HR) at I-SceI-generated "clean" double-strand breaks (DSBs) but is required for repair of DSBs occurring at CFS-derived AT-rich sequences. In addition, CtIP nuclease-defective mutants are impaired in Alu-IRs-induced mitotic recombination. These studies suggest that an end resection-independent CtIP function is important for processing DSB ends with secondary structures to promote HR. Furthermore, our studies uncover an important role of MRN, CtIP, and their associated nuclease activities in protecting CFSs in mammalian cells.

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint.

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.