Autoimmune diseases are the third most common category of diseases in the United States, and their incidence is slowly rising as the population ages. Pemphigus vulgaris (PV) is a debilitating autoimmune disease in which B cells produce antibodies against desmoglein-3 (Dsg3), a transmembrane cell adhesion protein responsible for binding keratinocytes together in the epidermis. The disease can cause widespread blisters and erosions in the skin and mucous membranes, leading to severe pain, super-infection, and possibly death. Like most autoimmune diseases, PV is treated using non-specific immunosuppressants like steroids and B-cell depleting agents (e.g. rituximab). Because these treatments have serious side effects like sepsis and death, there is a dire clinical need for a more tailored approach to treating the disease. To develop effective targeted treatments for pemphigus, it is essential to understand how the pathogenic autoantibodies develop. It has been previously shown that the pathogenic anti-Dsg3 autoantibodies in PV are restricted to the IgG isotype, and specifically to the IgG1 and IgG4 subtypes. IgG4, in particular, is important during active stages of the disease, while IgG1 appears dominant during remission. Our laboratory has previously shown that anti-Dsg3 antibodies in a given patient use a limited number of variable region sequences, with shared variable region gene usage among different patients, implying common mechanisms of disease development.. However, it is unknown how pathogenic antibodies in PV are distributed between the IgG1 and IgG4 fraction, how they are clonally related to each other, and at what point during the development of these antibodies that they become autoreactive. In this proposal, we will use subtype-specific antibody repertoire cloning in order to isolate the anti-Dsg3 IgG1 and IgG4 antibodies from a panel of PV patients with active disease and understand their creation and maturation.
In Aim 1 of this proposal, we will use a subtype-specific phage display procedure to clone IgG1 and IgG4 from patients. We will then use genetic analysis to determine whether the IgG1 and IgG4 clones are clonally related, i.e. whether the IgG4 clones are somatically mutated """"""""children"""""""" of IgG1 clones that share a germ line sequence. This approach will be augmented by using PCR to find IgG1 """"""""parents"""""""" of any IgG4 clones for whom IgG1 relatives are not identified through cloning. In our second aim, we will characterize the fine specificities our anti-Dsg3 IgG1 and IgG4 clones using well-established in vivo blister formation assays, epitope mapping experiments, and surface plasmon resonance to quantitate antigen binding affinity. We will also use a site-directed mutagenesis approach to determine at what stage particular clones became reactive during development by reverting somatic mutations to the germline state. Through these experiments, we can evaluate whether IgG4 is a valid therapeutic target for PV that can effectively capture the disease- relevant B cell populations. Furthermore, we will gain a much better understanding of how autoantibodies arise in PV, and therefore a deeper understanding of the pathogenesis of autoimmune disease.
Autoimmune diseases are caused by immune cells producing antibodies against the body's own components rather than foreign bacteria or viruses, and are currently treated by non-specific destruction of both harmful self-reactive and protective immune cells. The purpose of this proposal is to study pemphigus vulgaris, an autoimmune blistering disease of the skin, to understand what distinguishes the aberrant antibodies from protective, healthy one. This information will help develop a therapy that only targets the self-reactive antibodies while leaving protective ones intact, thus avoiding the potentially fatal side effects (sepsis, death) of current treatments.
|Chen, Jing; Zheng, Qi; Hammers, Christoph M et al. (2017) Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics. Cell Rep 18:237-247|
|Mao, Xuming; Cho, Michael Jeffrey T; Ellebrecht, Christoph T et al. (2017) Stat3 regulates desmoglein 3 transcription in epithelial keratinocytes. JCI Insight 2:|
|Ellebrecht, Christoph T; Bhoj, Vijay G; Nace, Arben et al. (2016) Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science 353:179-84|
|Lo, Agnes S; Mao, Xuming; Mukherjee, Eric M et al. (2016) Pathogenicity and Epitope Characteristics Do Not Differ in IgG Subclass-Switched Anti-Desmoglein 3 IgG1 and IgG4 Autoantibodies in Pemphigus Vulgaris. PLoS One 11:e0156800|
|Cho, Michael Jeffrey; Ellebrecht, Christoph T; Hammers, Christoph M et al. (2016) Determinants of VH1-46 Cross-Reactivity to Pemphigus Vulgaris Autoantigen Desmoglein 3 and Rotavirus Antigen VP6. J Immunol 197:1065-73|