Preliminary Exploration. Fc¦Ár Expression Analysis Of The Subcellular Localization Of Neutrophils And Fc¦Ár Spliceosome & Depends On Fc¦Ár Bispecific Molecules In The Treatment Of Autoimmune Diseases

These work cotain three parts.In the first part,we investigated the locoliazation of FcαR in neutrophil and the isoforms of FcαR.The human FcαRI(CD89) is expressed on cells of myeloid lineage and plays an important role in host defense.Neutrophils make up the majority of FcαRI-positive cells.Previous reports suggested that FcαR was stored in neutrophil intracellular pools and it could be quickly mobilized once neutrophils were activated.However,the subcellular localization of FcαR in neutrophils has not yet been defined.In this study,we identified that FcαR was stored in secretory vesicles and tertiary granules of neutrophils by flow cytometry analysis,ELISA,confocal microscopy and Western blotting.The molecular mass of FcαR in secretory vesicles was different from that in tertiary granules.FcαR stored in tertiary granules had a molecular mass of 50-70 kDa,whereas FcαR in secretory vesicles and membranes had a molecular mass of 55-75 kDa.After treatment by PNGase F,an enzyme that removes N-glycosylation,FcαR from both secretory vesicles and tertiary granules revealed a core protein of 32 kDa,which was the same as the backbone of full length of FcαR.A smaller FcαR variant with a core protein of 29-30 kDa was found in tertiary granules but not in secretory vesicles.The nature of the small variant is not clear at present and remains to be further investigated. Human promyelocytic leukemic cell line,HL-60 cells,can be induced to differentiate into a mature,neutrophilic-like pbenotype by dimethyl sulfoxide(DMSO).Previous study suggested that FcαR was expressed on DMSO-differentiated HL-60 cells.However,at least 10 isoforms of FcαR have been identified so far.In present study,we investigated whether different isoforms of FcαR are expressed at different differentiation stages of HL60.We demonstrated that a.3 isoform of FcαR was expressed on HL-60 cells at early differentiation stage,and two isoforms were expressed at later differentiation stage. In the second and third parts,we investigated the role of bespesific moleucule in autoimmune disease therapy.In recent years,Fc receptor-based bispecific molecules have become more and more important in therapy of disease.Here,we try to develop a new treatment for myasthenia gravis by depletion of autoreactive B cells via bispecific molecule.We designed a bispecific molecule composed of self antigen and mAb specific to FcαR,which could bind autoreactive B cell expressing mIg against self antigen(target cells) and FcαR bearing effector cells simultaneously.This bispecific molecule can take target cells and effector cells together,so help effecter cells(e.g.neutrophils,U937 cells, MDM) mediated depletion of autoreactive B cells selectively through phagocytosis, degranulation,et al.Autoreactive B cell produced autoantibodies recognizing AChR is the major pathogenic factor of myasthenia gravis.So,we first established autoreactive B cells expressing mIg recognizing AChR.Functional extracellular domain of AchRα1 subunit was expressed, renatured,and then used to immunize Lewis rats.One of the three immunized rats developed clinical symptoms of EAMG.Then anti-AchR monoclonal antibodies were produced.Hybridomas expressing surface immunoglobulin(mIg) could be used as target cells in the assessment of FcαR mediated target cell killing by effector cells.Then,we constructed bispecific molecule MIP8aF(ab')₂-19p by chemically linking F(ab') fragments of MIP8a recognizing FcαR and a 19 amino acid peptide recognized by mIg expressed on hybidoma 11H12.FACS analysis showed that this bispecific molecule could bind FcαR-positive U937 cells and 11H12 cells simultaneously.Using a flow cytometry-based assay and confocal imaging,we show that BiM could mediate phagocytosis of hybidoma 11H12 by macrophages.Based on these studies,we propose that FcαR-based BiM may be a new promising immunotherapy for patients with MG.