title: Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fc epsilon receptor by a recombinant human autoantibody Fab converted to IgE.
abstract: Fc epsilon receptor (CD23)-mediated capture of IgE-antigen complexes by B cells provides a powerful antigen presenting system. Our goal was to develop a system using high affinity, human, organ-specific monoclonal autoantibodies for antigen capture by B cells. For this purpose, we converted a recombinant human autoantibody to TPO from a Fab (SP1.4) to an IgE molecule. Sera from all patients with autoimmune thyroid disease contain autoantibodies with the same epitope as SP1.4. The SP1.4 H and L chain V region genes were spliced by overlap PCR to a mammalian, non-immunoglobulin signal peptide and transferred to expression vectors for human IgG1 and kappa, respectively. After inserting the IgE constant region genes into the H chain vector, the kappa and IgE H chain vectors were expressed in SP2/0 cells. SP1.4-IgE retains its high affinity (Kd) for TPO (approximately 2 x 10(-10) M), recognizes the same epitope as Fab SP1.4 and, importantly binds to a different epitope than does Fab TR1.9. Binding of preformed complexes of SP1.4-IgE and biotinylated TPO to EB virus transformed B cells (EBVL) was weakly detectable by flow cytometry and was displaced by unlabeled TPO. SP1.4-IgE/125I-TPO complex binding to EBVL was much more clearly evident, was also inhibited by the addition of unlabeled TPO, and was greatly reduced by preincubation of the EBVL with anti-CD23. Further, autologous EBVL preincubated with SP1.4-IgE/TPO complexes stimulated proliferation of TPO-specific T cells. IgE autoantibody-mediated antigen focusing to B cells is unlikely to operate in vivo but is, instead, a powerful investigative tool. In conclusion, SP1.4-IgE is the first monoclonal human autoantibody to be developed for IgE-mediated antigen presentation to T cells by EBVL. Recombinant human autoantibodies converted to IgE, possibly in combinations if their epitopes permit simultaneous binding to the same molecule, provide a unique system to generate human T cell lines and clones specific for peptides naturally processed from internalized high affinity autoantibody/autoantigen complexes.
authors.affiliation: Thyroid Molecular Biology Unit (111T), Veterans' Administration Medical Center, San Francisco, CA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Guo
authors.forename: S
authors.initials: S
authors.lastname: Quaratino
authors.forename: J C
authors.initials: JC
authors.lastname: Jaume
authors.forename: G
authors.initials: G
authors.lastname: Costante
authors.forename: M
authors.initials: M
authors.lastname: Londei
authors.forename: S M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: B
authors.initials: B
authors.lastname: Rapoport
mesh_terms: D017951:Antigen Presentation; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D011961:Receptors, Fc / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D007140:Immunoglobulin Fab Fragments; D011961:Receptors, Fc; D011994:Recombinant Proteins; D007073:Immunoglobulin E