Juan Carlos Jaume

Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves' patients' sera.

Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained ∼20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves’ patients’ sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70–100% of TSHR autoantibody activity in all 18 Graves’ sera studied. In summary, carboxyl-terminal truncation of the human TSHR ectodomain generates a secreted protein with complex carbohydrate that neutralizes autoantibodies in Graves’ patients’ sera. Antigenically active TSHR will be valuable for future studies on the diagnosis, pathogenesis, and immunotherapy of Graves’ disease.

Endogenous expression levels of autoantigens influence success or failure of DNA immunizations to prevent type 1 diabetes: addition of IL-4 increases safety

Administration of autoantigens through DNA immunizations or via the oral route can prevent progression of islet destruction and lower the incidence of type 1 diabetes in animal models. This beneficial effect is mediated by autoreactive regulatory CD4 lymphocytes, and it is known that their induction depends on the precise dose and route of antigen administration. However, it is not clearwhich endogenous factors determine when such immunizations lead to activation of regulatory versus aggressive autoreactive lymphocytes and how a deleterious outcome can be avoided. Here we describenovel observations made in an animal model for virally induced type 1 diabetes, showing that the endogenous expression levels of the islet antigens and glutamic acid decarboxylase determine whetherimmunization with these antigens is beneficial or detrimental. Lower expression levels in β‐cells support immune regulation resulting in induction of autoreactive, regulatory cells characterized by increased IL‐4 production (Th2‐like), whereas higher levels favor Th1‐like autoaggressive responses characterized by augmented IFN‐γ generation. Co‐immunization with an IL‐4‐expessing plasmid reduces the risk of augmenting autoaggression and in this way increases the safety margin of this immune‐based therapy. Our findings will be of importance for designing safe antigen‐specific interventions for human type 1 diabetes.

Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fcε receptor by a recombinant human autoantibody Fab converted to IgE

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.

Thyroid peroxidase autoantibody epitopic ‘fingerprints’ in juvenile Hashimoto's thyroiditis: evidence for conservation over time and in families

In Hashimotos thyroiditis, the humoral component is manifest by autoantibodies to thyroid peroxidase (TPO). Epitopic ‘fingerprinting’ of polyclonal serum TPO autoantibodies has been facilitated by the molecular cloning and expression as Fab of a repertoire of human TPO autoantibody genes. To investigate whether TPO autoantibody fingerprints are (i) stable over long periods of time (∼15 years), and (ii) inherited, we studied a cohort of nine patients with juvenile Hashimoto’s thyroiditis and 21 first degree relatives of four of these patients. Fingerprints were determined by competition between four selected Fab and serum autoantibodies for binding to 125I‐TPO. Regardless of titre, the TPO epitopic profile was stable in 10/12 individuals whose TPO autoantibody levels were sufficient for analysis on two or three occasions over 12–15 years. Although the TPO epitopic fingerprint profiles in two families raised the possibility of inheritance, overall the data from all four families did not reveal an obvious pattern of genetic control. In no family was the TPO epitopic fingerprint associated with HLA A, B or DR. In conclusion, TPO autoantibody epitopic fingerprints are frequently conserved over many years. Studies on additional families are necessary to establish whether or not the epitopic profiles of TPO autoantibodies are inherited.

Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominant region

The nature of the autoantibody repertoire to the dominant autoantigen in human autoimmune thyroid disease is controversial. There is evidence that autoantibodies to thyroid peroxidase (TPO) interact with overlapping conformational epitopes in an immunodominant region and binding to denatured (DN) protein is decreased. Contrary data demonstrate TPO autoantibody reactivity with DN‐TPO or polypeptide fragments. However, none of the TPO‐specific, human monoclonal autoantibodies isolated to date preferentially recognize denatured autoantigen. We therefore searched an immunoglobulin gene phage display library for human autoantibodies that bind TPO denatured by reduction and alkylation (DN‐TPO). Thyroid‐infiltrating B cells from a typical TPO autoantibody‐positive patient were the source of mRNA for library construction. Surprisingly, the library enriched after panning on DN‐TPO, as well as a panel of individual clones, preferentially bound native (N)‐TPO. Of 13 clones selected using DN‐TPO or N‐TPO, 12 clones recognized the TPO immunodominant region. Moreover, regardless of selection with N‐TPO or DN‐TPO, their heavy and light chains were encoded by similar VDJ and Vκ combinations. One clone (DN4), isolated using DN‐TPO, did not interact with the TPO immunodominant region and its H chain derives from a different VH gene. Although DN4 binds specifically to TPO, its affinity is low, unlike the high affinities of other human TPO autoantibodies. In conclusion, human monoclonal autoantibodies that preferentially recognize denatured TPO could not be isolated from an immunoglobulin gene library despite selection with denatured protein. Our findings demonstrate the bias of the human B cell repertoire towards recognition of an immunodominant region on the conformationally intact form of a major thyroid autoantigen.

Influence of the Light Chain Repertoire on Immunoglobulin Genes Encoding Thyroid Autoantibody Fab from Combinatorial Libraries

The diversity of the immunoglobulin heavy (H) and light (L) gene libraries used to construct a combinatorial library is an important parameter in determining the characteristics of antigen-specific Fab obtained from the library. To investigate the role of library diversity, we compared Fab specific for the autoantigen thyroid peroxidase (TPO) isolated from two different combinatorial libraries. Both libraries contained the same H chain genes. The original combinatorial library (H/R) utilized kappa chains generated using a single kappa variable region oligonucleotide primer. We constructed a second combinatorial library (H/D) containing kappa chains amplified with a diverse panel of variable region primers. From the the original H/R library, only two groups of TPO-specific Fab had been obtained, involving two H chain types (V1-3B and hv1L1) but only one kappa chain type (012). In contrast, among the seven TPO Fab characterized from the second library (H/D) we observed five different VH/VL combinations, comprising three types of H chains (V1-3B, VH26 and DP7) and four types of kappa chains (O12, L12, L2/hv328H5 and B3). Besides differences in VH and VL genes, as well as VH/VL combinations, the new TPO Fab used different D regions and JH and JK elements. Nevertheless, the new kappa Fab resembled previously isolated TPO Fab in terms of their affinity for TPO (Kd approximately 10(-9)M) and preferential recognition of conformationally intact autoantigen. In summary, our studies demonstrate that the diversity of the L chain library repertoire, while having little effect on immunological properties, has a major influence on the genes encoding antigen-specific Fab selected from a combinatorial library. For the successful isolation of rare but clinically important autoantibodies (such as to the TSH receptor) by the combinatorial library approach, library diversity is likely to be a major factor.