Molecular Mechanisms Of T-B Cell Interaction In Pemphigus And The Role Of CD19^hi B Cells In Autoimmune Diseases

Pemphigus is a tissue-specific autoimmune blistering disease that is characterized by suprabasal blisters in skin and mucous membranes.Pemphigus has two major subtypes,pemphigus vulgaris（PV）and pemphigus foliaceus（PF）,which are caused primarily by immunoglobulin G（IgG）autoantibodies against desmoglein 3（Dsg3）and/or desmoglein（Dsg1）,respectively.Anti-Dsg3 and Dsg1 IgG autoantibodies play a pathogenic role in blister formation,and the antibody titers of patients often correlate with the intensity of disease activity.Given the importance of the helper function of CD4⁺T cells in Ab production by B cells,autoreactive CD4⁺T cells are thought to play critical roles on the pathogenesis of pemphigus.In addition,the interaction between antigen-specific T cells and B cells via their co-stimulatory molecules is required for efficient antibody production.However,the underlying molecular mechanisms of how T cell promoting B cell functionality and what kind of B cells is mainly responsible for producing antibodies,the occurrence and development of diseases are not well understood.（1）Molecular mechanisms of T-B cell interaction in pemphigus patientsIn present study,the results showed that the CD4⁺T cells from pemphigus patients were relatively activated with obvious upregulation of ICAM-1,ICOS,CD40L and OX40than those in healthy controls.By using in vitro cell conjugation assay,we observed CD4⁺T cells from pemphigus patients exhibited a higher adhesion capacity to autologous B cells.More interestingly,the increase of adhesion between CD4⁺T-B cells coordinated with the augmented IgG,IgM and specific autoantibody production upon CD4⁺T and B cell co-culture in vitro.In addition,interruption of the interaction between these molecules and their corresponding ligands by different blockade antibodies,disrupted the survival of CD4⁺T and B cells,antibody levels and the generation of CD19^hi B cells which were corelated with antibody production.It was found that the blockade of ICAM-1,CD40L and OX40 displayed much stronger inhibitory effect than blockade of ICOS on the number of both CD4⁺T and B cells,levels of IgG and IgM,as well as the pecentage of CD19^hi B cells.Our results aforementioned provided the direct evidence that T cell activation facilitates B cell functionality both at the early T-B adhesion stage and the induction of B cell differentiation and also uncovered diverse roles of ICAM-1,ICOS,CD40L and OX40 in T cell dependent antibody production.This might lay the molecular foundation to screen optimized therapeutic targets in antibody-driven autoimmune diseases.（2）Properties of CD19^hi B cells and their role in autoimmune disease developmentPemphigus and Systemic Lupus Erythematosus（SLE）are two representatives of pathogenic autoantibody-driven autoimmune diseases systemically and organ-specifically,respectively.Given the involvement of autoantibody in pathogenesis,the mechanisms of B cell differentiation and functionality in autoimmune diseases need to be further investigated.In this study,we identified a unique CD19^hi B cell population in the periphery of pemphigus and SLE patients as well as human tonsils.They could be induced in vitro after co-culturing fully activated CD4⁺T cells with autologous B cells together with higher levels of IgG and IgM production in the culture supernatant.CD19^hi B cells expressed elevated levels of HLA-DR,IgG,IgM and multiple ligands of costimulatory molecules including ICAM-1,ICOSL,CD40,and OX40L when compared with CD19^lo B cells.They were also able to produce extra IgG antibody.Results from gene expression profiling showed that genes involved in multiple signaling pathways for B cells ontogeny,activation,differentiation,survival and antibody production including B cell receptor（BCR）,chemokine,Toll-like receptor（TLR）,MAPK and Jak-STAT signaling pathways were up-regulated in CD19^hi B cells.Enhanced phosphorylation of key molecules involved in key signaling pathways such as Syk,Erk,Btk,NF-κB and Pyk2 were confirmed in CD19^hi B cells from in vitro induction or patients.CD4⁺T cells from SLE and pemphigus patients also reinforced the generation of CD19^hi B after in vitro co-culture.More significantly,the presence of CD19^hi B cells in the periphery was associated with autoantibody levels in SLE and pemphigus patients.Our results aforementioned indicated that CD19^hi B cells existing in the periphery of SLE and pemphigus exhibit activated properties and gene expression profiling and they were generated with the help of activated CD4⁺T cells.With the strong correlation between peripheral CD19^hi B cells and IgG levels in SLE and pemphigus patients,CD19^hi B cells might become a useful indicator for disease development.To summarize our study,the results aforementioned,provided the direct evidence that the activation of CD4⁺T cells enhanced B cells to differentiate into CD19^hi B cells and produce antibodies,further promoted pemphigus development.This process probably initiated through increased adhesion between T and B cells by different upregulated costimulatory molecules in pemphigus patients.At the same time,we also identified a unique CD19^hi B cells,uncovered their generation,phenotype,function,gene expression profiling and potential role in the pathogenesis of the autoimmune diseases.Our results thus dissect the molecular mechanisms of T-B cell interaction during antibody production by B cells and lay the molecular foundation for T-B cell interaction in pathogenesis of pemphigus as well as the screening of potential therapeutic targets in other autoimmune diseases.