The Correlation Of Regulatory B Cells And Antoimmune Bullous Diseases

Autoimmune blistering diseases （AIBDs） is a heterogeneous group of disorders withblisters and erosions characterized by the presence of autoantibodies that target distinctadhesion molecules of the epidermis or dermoepidermal basement membrane zone. Theyare stubborn and tend to recurrent attacks. Bullous pemphigoid （BP） and pemphigusvulgaris （PV） are the most frequent AIBDs associated with definite autoantibodies. Inthese two conditions, autoreactive B cells and CD4⁺T lymphocytes mainly recognizedistinct epitopes of Dsg1and Dsg3in PV or BP180and BP230in BP, respectively. The180-kDa protein is considered to be the main pathogenic antigen in BP. Thenoncollagenous16A （NC16A） domain in the juxtamembranous extracellular part isconsidered to have the major pathogenic epitope for BP.Recent studies have demonstrated that the pathogenic autoantibody was a keypathogenic factor in autoimmune blistering disease. But it is not clear that how togenerate antibodies and what the role is that Breg, Treg and Tfh play in AIBD.It has been generally concerned whether B cells contained multiple regulatorysubsets like T cells, and made a lot of research progress. A subset of IL-10-competentregulatory B cells have been labeled as the phenotypically unique CD1dhiCD5⁺CD19hi.The immunosuppressive function of regulatory B cells has been found in several murinemodels, such as T1D, CHS and CIA. In2011, a research team from Duke Universityconfirmed that regulatory B cells （CD19⁺IL10⁺） are predominantly found within theCD24^hiCD27⁺B cell subpopulation through IL-10-dependent pathways in human. What isthe relationship of Breg cells and autoimmune blistering diseases? Whether Breg cellsaffect the level of antibody production? Through detecting the levels of Breg cells in theperipheral blood of patients, we observe whether Breg cells are involved in the pathogenesis of AIBDs. And to further explore the mechanism of Breg cells in thepathogenesis of antibody production of AIBDs, so as to provide a new strategy to thetreatment of AIBDs.Objective: To explore the relationship between Breg cells and AIBDs anddetermine the negative immunoregulatory function of Breg in BP patients. And to furtherexplore the mechanism of Breg cells in the pathogenesis of antibody production ofAIBDs.Methods:1) To collect the peripheral blood of pemphigus vulgaris and bullouspemphigoid patients and healthy controls. One part of the blood sample wasextracted PBMC, one part of the whole blood was extracted RNA and theother serum was saved;2) To abserve the frequency of Breg cells in AIBDs patients by flowcytometry after fluorescent antibody staining;3) To detect the level of IL-10in the serum of patients and healthy controls byElisa method and the level of IL-10mRNA in the peripheral blood byrealtime-PCR;4) To co-incubate Breg (CD19⁺CD24^hiCD27⁺) cells and CD4⁺CD25-cells bybead sorting and flow cytometry sorting from health control and BPpatients, then detect the negative immunoregulatory function of Breg cellsfor effector T cells;5) Prokaryotic expression of pathogenic antigen BP180-NC16A， thenBP180-NC16A stimulus PBMC of the bullous pemphigoid patients for72hwhen the presence or absence of Breg cells, then detect the levels ofantibodies in the culture supernatant.Results: We found that the the frequency of Breg cells (CD19⁺CD24^hiCD27⁺) inblisters patients was significantly higher than that in healthy control. The IL-10levels ofprotein and RNA in peripheral blood of autoimmune blistering patients compared withhealthy control group have no statistically significant difference. Breg cells from healthy people inhibited IFN-γ，TNF-α and IL-4produced by CD4⁺T cells and the proliferationof CD4⁺T cells. Breg cells from BP patients lost this suppressing function. B cells fromBP patients have an antigen-specific reaction for BP180-NC16A antigen. The pathogenicantibody levels were significantly elevate secreted by B cells. And the level of antibodyproduction had no significant change in the culture supernatant of PBMC deleted Bregcells compared with not deleted Breg cells after BP180-NC16A stimulation.Conclusion: The frequence of Breg cells increased significantly in the peripheralblood of PV and BP patients, but the negative immunoregulatory function of Bregs wasdefective. Breg cells are unable to effectively inhibit the activity of helper T cells and Bcells to produce antibodies. That may be an important mechanism of pathogenicautoantibodies production in AIBDs.