Absence of anti-pendrin auto-antibodies in the sera of Tunisian patients with autoimmune thyroid diseases.

BACKGROUND: We previously demonstrated that the PDS gene is involved in the genetic susceptibility to autoimmune thyroid diseases (AITD) in Tunisia. In the same population, we now investigated the presence of anti-pendrin auto-antibodies (aAbs) in AITD patients' sera. METHODS: Thirty seven Tunisian AITD patients and 19 healthy subjects from families previously linked to the PDS gene, 75 unrelated patients and 20 healthy unrelated subjects were included in our study. The detection of anti-pendrin aAbs in patients' sera was performed by ELISA using membrane protein extracts of CHO cells expressing pendrin (CHO-hPDS) and by immunofluorescence using transient COS-7 cells expressing a GFP tagged pendrin. CHO cells transfected with human TPO in the same ELISA conditions were used as positive control. RESULTS: The majority of AITD patients' sera were positive for the presence of anti-TPO aAbs. In contrast, no reactivity was detected with CHO-hPDS membrane protein extracts. Likewise, no significant immunostaining was found on transfected COS-7cells upon exposure to patients' and controls' sera. CONCLUSIONS: Our data point to the absence of anti-pendrin aAbs in Tunisian AITD patients' sera.

Antithyroperoxidase antibody-dependent cytotoxicity in autoimmune thyroid disease.

CONTEXT: Thyroid antibody-dependent cytotoxicity has been reported in autoimmune thyroid disease (AITD). Indeed, the role of thyroperoxidase (TPO) autoantibodies (aAbs) in complement-mediated damage by binding to TPO expressed on the surface of human thyroid cells was demonstrated, whereas their activity in antibody-dependent cell cytotoxicity (ADCC) is not well established. OBJECTIVE: The aim of this study was to define the partners involved in antibody and complement-dependent cytotoxicity (CDC) in AITD and characterize which effector cells are involved in cytotoxicity mediated by anti-TPO aAbs using a chromium release assay. RESULTS: The relative capability of anti-TPO aAbs to mediate ADCC using human thyroid cells in culture varies from 11 to 74.5%, depending on the effectors cells used. The human monocyte cell line HL60 gives a better lysis than the THP-1 cell line as effector cells. It seems obvious that the mechanism of ADCC is mediated quite exclusively by FcgammaRI. Indeed, the two effector cell lines differ by the level of the FcgammaRI expression (91.83% for HL-60 cells and 22.55%t for the THP-1). In addition to ADCC, the anti-TPO aAbs mediate the destruction of thyrocytes by CDC (56%). CONCLUSIONS: These results demonstrate that anti-TPO aAbs can damage cultured thyroid cells by ADCC and CDC mechanisms. The monocytes, via their FcgammaRI, are important effector cells in ADCC mediated by anti-TPO aAbs and may contribute with T cells to the destruction of thyroid gland in AITD.

In vitro antitumoral activity of baculovirus-expressed chimeric recombinant anti-CD4 antibody 13B8.2 on T-cell lymphomas.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule. The recombinant IgG1 antibody 13B8.2 was previously shown to inhibit HIV-1 replication and to abrogate the one-way mixed-lymphocyte reaction and block proliferation of CD3-stimulated peripheral blood CD4 lymphocytes from healthy donors. Before testing this recombinant anti-CD4 antibody in in vivo preclinical trials, in vitro mechanisms of action of rIgG1 13B8.2 were assessed using various CD4 T-cell lymphomas. The baculovirus-expressed rIgG1 13B8.2 antibody led to 14% to 40% proliferation inhibition of the lymphoblastic leukaemia-derived SUP-T1, the acute T lymphoma-derived CCRF-CEM and Jurkat, and the cutaneous T-Cell lymphoma-derived HUT-78 cell lines, but it did not affect the cell cycle nor induce cell apoptosis. rIgG1 antibody 13B8.2 bound the C1q fraction, leading to 9% to 17% complement-mediated lysis of the HUT-78, H9, Sup-T1, and the CCRF-CEM cell lines. No correlation was observed between cell sensitivity to rIgG1 13B8.2-triggered complement-dependent lysis and CD35-, CD46-, CD55-, and CD59-surface expression on T lymphoma cells. Using fluorescence-activated cell sorter analysis, the antibody was shown to bind to FcgammaRI/CD64-transfected IIA1.6, FcgammaRII/CD32-transfected CDw32L, and FcgammaRIII/CD16-transfected Jurkat CD16 cell lines. In correlation with these findings, rIgG1 13B8.2 induced 11% to 31% antibody-dependent cell-mediated cytotoxicity of the CCRF-CEM, SUP-T1, A2.01 CD4, and Jurkat cell lines. These convincing results on the activity of the recombinant chimeric anti-CD4 antibody 13B8.2 have led us to perform in vivo preclinical study in a murine xenograft model of CD4 lymphomas.

The key residues in the immunodominant region 353-363 of human thyroid peroxidase were identified.

Auto-antibodies (aAbs) to thyroid peroxidase (TPO) interact with a restricted immunodominant region (IDR) divided into two overlapping regions A and B. Among the five major regions structuring the IDR/B, regions 210-225, 353-363, 549-563, 713-720 and 766-775, region 353-363 constitutes an important anchor point for the binding of TPO-specific aAbs in sera from Hashimoto's and Graves' patients. We combined site-directed mutagenesis and expression of TPO mutants in stably transfected CHO cells to precisely define the critical residues in that region. By using flow cytometry and ELISA, we identified four amino acid residues, H353, D358, S359 and R361, that contribute to the interaction between human TPO and anti-TPO aAbs. This identification of these contributing amino acid residues in the IDR allowed us to more precisely depict contours of the IDR.

Biological activities on T lymphocytes of a baculovirus-expressed chimeric recombinant IgG1 antibody with specificity for the CDR3-like loop on the D1 domain of the CD4 molecule.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody (mAb) 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule and which inhibits HIV-1 replication. Chimeric rIgG1 antibody 13B8.2 blocked, in a dose-dependent manner, antigen presentation through inhibition of subsequent IL-2 secretion by stimulated T cells. The one-way mixed lymphocyte reaction was abrogated by previous addition of baculovirus-produced rIgG1 13B8.2 in the T-cell culture. Anti-proliferative activity of rIgG1 was demonstrated on CD3-activated CD4+ T lymphocytes from healthy donors, such effect being associated with reduced IL-2 secretion of activated T cells. On the other hand, no proliferation inhibition was observed on CD4+ T lymphocytes activated with phorbol ester plus ionomycin, suggesting that rIgG1 13B8.2 preferentially acts on a proximal TCR-induced signaling pathway. Treatment of DBA1/J human CD4-transgenic mice with 100 microg of recombinant antibody for three consecutive days led to in vivo recovery of rIgG1 antibody 13B8.2 both coated on murine T lymphocytes and free in mouse serum, without CD4 depletion or down-modulation. These findings predict that the baculovirus-expressed chimeric rIgG1 anti-CD4 antibody 13B8.2 is a promising candidate for immunotherapy.

New insights into the conformational dominant epitopes on thyroid peroxidase recognized by human autoantibodies.

Human anti-thyroperoxidase (TPO) autoantibodies (aAbs) are a major hallmark of autoimmune thyroid diseases. Their epitopes are discontinuous and mainly restricted to an immunodominant region (IDR) consisting of two overlapping regions (IDR/A and B). To shed light on the relationship between these regions, we first performed competitive studies using all available reference anti-TPO antibodies. Interestingly, we showed that human IDR/A- and B-specific anti-TPO aAbs recognized essentially the same regions on the TPO molecule. However, our data also indicated that IDR/A-specific human aAbs strongly recognized the region containing residues 599-617, whereas the IDR/B-specific aAbs bind to several regions as well as region 599-617. Next, we scanned this key region to identify the residues involved in the immunodominant autoepitope. Using peptide spot technology together with competitive ELISA experiments, we demonstrated that residues (604)ETP-DL(609) play a major role in the anti-peptide P14 epitope and that IDR/A-specific human anti-TPO aAbs, either expressed as recombinant Fab or obtained from Graves' disease patients, specifically recognize the sequences (597)FCGLPRLE(604) and (611)TAIASRSV(618). All together our data emphasize that both the IDRs involve the same surface area on human TPO, but the differential usage of one or the other regions leads to different inhibition patterns in competitive experiments. In conclusion, our data help to resolve the long-sought issue on the molecular immunology of the two IDRs on TPO and provide new clues to design efficient peptides that may be part of a combinatorial treatment aiming at delaying development of autoimmune thyroiditis when used prophylactically.