Human anti-thyroid peroxidase (TPO) autoantibodies (aAb) are generated during autoimmune thyroid diseases (AITD). Within recent years, increasing knowledge of the TPO-specific aAb repertoire, gained mainly by the use of combinatorial library methodology, has led to the cloning and sequencing of around 180 human anti-TPO aAb. Analysis of the immunoglobulin (Ig) variable (V) genes encoding the TPO aAb in the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr) reveals major features of the TPO-directed aAb repertoire during AITD. Heavy chain VH domains of TPO-specific aAb from Graves' disease patients preferentially use D proximal IGHV1 genes, whereas those from Hashimoto's thyroiditis are characterized more frequently by IGHV3 genes, mainly located in the middle of the IGH locus. A large proportion of the anti-TPO heavy chain VH domains is obtained following a VDJ recombination process that uses inverted D genes. J distal IGKV1 and IGLV1 genes are predominantly used in TPO aAb. In contrast to the numerous somatic hypermutations in the TPO-specific heavy chains, there is only limited amino acid replacement in most of the TPO-specific light chains, particularly in those encoded by J proximal IGLV or IGKV genes, suggesting that a defect in receptor editing can occur during aAb generation in AITD. Among the predominant IGHV1 or IGKV1 TPO aAb, conserved somatic mutations are the hallmark of the TPO aAb repertoire. The aim of this review is to provide new insights into aAb generation against TPO, a major autoantigen involved in AITD.

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766 -775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.Human thyroid peroxidase (TPO), 1 described previously as the "thyroid microsomal antigen" (1), is a membrane-bound enzyme expressed at the apical pole of thyrocytes (2). TPO generates the functional form of thyroglobulin by iodination and coupling of tyrosine residues (3). During autoimmune thyroid diseases (AITD), TPO represents a major target for the immune system (4, 5), leading to high titer TPO-specific autoantibodies (aAbs) in the sera of patients suffering from Hashimoto's thyroiditis and Graves' disease. Besides their role as efficient and early diagnostic markers of AITD, TPO-specific aAbs also act as effector molecules either through modulating antigen presentation to T cells or by mediating thyroid destruction after complement activation or antibody-dependent cell cytotoxicity (6 -12). Alignment studies and structural homologies have shown that TPO is formed by three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an EGF-like domain, from the N-to the Cterminal extremities (13). Although the structure of each domain has been elucidated in part by three-dimensional modeling (13-16), the full three-dimensional structure of TPO remains unknown, even though low resolution crystals have been obtained (17,18). The flexibility observed for the hinge regions probably make difficult the exact positioning of each domain in relation to the others (15).These observations denote a highly complex structure of TPO, thus explaining the reason why TPO aAbs from patients' sera preferentially recognize discontinuous epitopes on . Different approaches have been used to determine the epitopic regions recognized by anti-TPO aAbs from ...

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The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven ␤-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat ␤-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-κB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3ζ, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Cθ, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cγ1, and p95vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10–30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-κB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects.