The Tim3-galectin 9 pathway induces antibacterial activity in human macrophages infected with Mycobacterium tuberculosis.

T cell Ig and mucin domain 3 (Tim3) is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis in the mouse model of infection. In this study, we sought to determine whether the Tim3-Gal9 pathway plays a similar role in human pulmonary TB. We identified that pulmonary TB patients have reduced expression of Tim3 on CD14(+) monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1ß. Our results establish that Tim3-Gal9 interaction activates human M. tuberculosis -infected macrophages and leads to the control of bacterial growth through the production of the proinflammatory cytokine IL-1ß. Data presented in this study suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1ß, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.

TRP channels and cancer: new targets for diagnosis and chemotherapy.

The Transient Receptor Potential (TRP) channels family consists of seven different subfamilies, namely TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like) that are related to several physiological and pathological processes. Recent years have witnessed an increased interest of research into the connection between TRP channels and cancer, leading to the discovery of tumor-related functions such as regulation of proliferation, differentiation, apoptotis, angiogenesis, migration and invasion during cancer progression. Among the TRP families, TRPCs, TRPMs and TRPVs are mainly related to malignant growth and progression. Depending on the type and stage of the cancer, regulation of TRPs mRNA and protein expression have been reported; these changes may regulate ion-dependent cell proliferation and resistance of cancer cells to apoptotic-induced cell death with consequent cancer promoting effects and resistance to chemotherapic treatments. Considerable efforts have been made to fight cancer cells and targeted therapy seems to be the most promising strategy: in this regard, ion channels belonging to the TRP channel superfamily could play an important role. Aim of this review is to summarize data reported so far on the expression and the functional role of TRP channels during cancer growth and progression, and the relationship with clinico-pathological markers. Moreover, the feasibility of TRP channels as target of chemotherapy and the different approaches by which these channels can be targeted will be analyzed in detail. Deeper investigations are required to understand the role TRP channels in cancer in order to develop further knowledge of TRP proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting.

Macrophage colony-stimulating factor enhances rituximab-dependent cellular cytotoxicity by monocytes.

Recent studies suggest that monocytes are the dominant effectors by which rituximab induces cell death in B-cell lymphoma. Because macrophage colony-stimulating factor (M-CSF) can enhance the cytotoxicity of monocytes, the authors examined whether this growth factor can enhance their ability to kill lymphoma cells in vitro. Monocytes derived from a healthy volunteer were cultured for 48 h in the presence or absence of M-CSF. Monocytes stimul ated with M-CSF were significantly more cytotoxic to Daudi B-cell lymphomas than unstimulated monocytes. Flow cytometry revealed that M-CSF increased monocyte expression of Fcgamma receptors III and I by 1.6- and 1.5-fold, whereas the expression of Fcgamma receptor II remained unchanged. These results suggest that pretreatment with M-CSF can improve the therapeutic efficacy of rituximab against intractable CD20(+) lymphoma.

Recombinant gp120 vaccine-induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptor-bearing effector cells and correlate inversely with HIV infection rate.

Nonneutralizing Abs may play a role in protecting animals and humans from lentiviral infections. We explored the Ab-dependent, cell-mediated virus inhibition (ADCVI) Ab response to recombinant gp120 (rgp120) vaccination in sera from 530 participants in the Vax 004 trial. Serum ADCVI activity was measured against a clinical R5 strain of HIV-1 using peripheral blood mononuclear effector cells from healthy donors. The level of vaccine-induced ADCVI activity correlated inversely with the rate of acquiring HIV infection following vaccination, such that for every 10% increase in ADCVI activity, there was a 6.3% decrease in the hazard rate of infection (p=0.019). Some vaccinated individuals also mounted an ADCVI response against two other clinical R5 strains of HIV-1. However, ADCVI activity correlated poorly with neutralizing or CD4-gp120-blocking Ab activity measured against laboratory strains. Finally, the degree to which the ADCVI Ab response predicted the rate of infection was influenced by polymorphisms at the FcgammaRIIa and FcgammaRIIIa gene loci. These data indicate that rgp120 vaccination can elicit Abs with antiviral activity against clinical strains of HIV-1. However, such activity requires the presence of FcR-bearing effector cells. Our results provide further evidence that ADCVI may play a role in preventing HIV infection.

TNFalpha kinoid vaccination-induced neutralizing antibodies to TNFalpha protect mice from autologous TNFalpha-driven chronic and acute inflammation.

The proinflammatory cytokine TNFalpha is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis and Crohn's disease. As an alternative to anti-human TNFalpha (hTNFalpha) mAbs and other hTNFalpha blocker approved drugs, we developed an active anti-hTNFalpha immunotherapy, based on a vaccine comprised of a keyhole limpet hemocyanin-hTNFalpha heterocomplex immunogen (hTNFalpha kinoid) adjuvanted in incomplete Freund's adjuvant. In mice transgenic for hTNFalpha (TTg mice), hTNFalpha kinoid vaccination elicited high titers of Abs that neutralized hTNFalpha bioactivities but did not result in a cellular response to hTNFalpha. The vaccine was safe and effective in two experimental models. Kinoid-immunized but not control TTg mice resisted hTNFalpha-driven shock in one model and were prevented from spontaneous arthritis, inflammatory synovitis, and articular destruction in a second model. These data demonstrate an anti-cytokine induction of autoimmune protection against both acute and chronic hTNFalpha exposure. They show that active vaccination against a human cytokine can be achieved, and that the immune response can be effective and safe.

Targeting fibroblast growth factor-inducible-14 signaling protects from chronic relapsing experimental autoimmune encephalomyelitis.

The TNF-related weak inducer of apoptosis (TWEAK) is a TNF family member mediating proinflammatory effects by its receptor fibroblast growth factor-inducible-14 (Fn14). We studied the role of TWEAK/Fn14 in experimental autoimmune encephalomyelitis (EAE) by protein vaccination with TWEAK and Fn14 and recombinant TWEAK-DNA, respectively. TWEAK-DNA vaccination worsened the clinical course of EAE and increased central nervous system (CNS) inflammation. TWEAK increased the secretion of CCL2 [monocyte chemotactic protein-1 (MCP-1)] by CNS endothelial cells and astrocytes in vitro, suggesting CCL2 as a critical mediator of TWEAKs proinflammatory effects. Vaccination with the extracellular domain of TWEAK or with Fn14 resulted in the induction of specific inhibitory antibodies and an amelioration of EAE signs in two different models in rats and mice. Spinal cord inflammatory infiltrates were significantly diminished. Purified IgG from TWEAK- or Fn14-vaccinated rats prevented TWEAK-induced production of CCL2 by endothelial cells. Blocking Fn14 signaling represents a novel approach with potential for the treatment of CNS autoimmunity.

Induction of neutralising antibodies restricts the use of human granulocyte/macrophage colony stimulating factor for vaccine studies in rhesus macaques.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a valuable adjuvant to enhance induction of cellular immune responses in rodents. Less information is available regarding its use as an adjuvant in primates or humans. We explored recombinant human GM-CSF for potential vaccine studies in rhesus macaques and focused on its effect on peripheral monocytes as progenitors of dendritic cells and its potential immunogenicity. Application of human GM-CSF to nine animals led to an average 32-fold increase in monocyte numbers. This was not observed upon re-treatment, which coincided with GM-CSF-specific neutralising antibodies. These also neutralised the activity of rhesus macaque GM-CSF. The data underscore the need to use species-specific GM-CSF for immunomodulation in primates.

Active immunization against murine TNFalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection.

New lines of treatment targeting cytokines have been successfully developed recently and are now widely used in therapy. They are based on passive administration of cytokine inhibitors either soluble receptors or mAbs and the major example is TNFalpha in rheumatoid arthritis (RA). Since a few years, our group has developed a novel alternative approach targeting cytokines by using active immunization against biologically inactive but immunogenic cytokine derivatives. In the present work, we present a new aspect of this research, based on immunization against specific cytokine peptides chosen by molecular modelling. We could elicit a significant humoral response against four TNFalpha peptides by active immunization, and show that the Abs generated cross-reacted with the native cytokine with good titers as determined by ELISA. Interestingly, during coimmunization experiments with couples of peptides, one showed a clear immunodominant effect over the other. Overall, we could not show the neutralization of TNFalpha biological activity in vitro by the immunized sera, but it seems that it is not a prerequisite to observe clinical efficacy. Indeed, using the LPS/galactosamine-induced shock, we could demonstrate that one of the four peptides tested conferred a clinical protection. These results validate the feasibility and efficacy of active immunization against cytokine peptides, and confirm that active immunization against cytokines could represent in the future an alternative to passive immunization in many diseases.

Blocking effects of serum reactive antibodies induced by glatiramer acetate treatment in multiple sclerosis.

Glatiramer acetate (GA) is a treatment option for multiple sclerosis. Although its mechanism of action remains unclear, evidence has emerged supporting the role of GA as an immunomodulatory drug that regulates T-cell function. It has been demonstrated that long-term GA treatment induces a serum antibody response; however, the functional properties of these 'reactive antibodies' are unknown. It has been speculated that GA-induced antibodies may have a blocking effect that can inhibit the immunologic activity of GA. This study was conducted to determine whether serum antibodies induced by GA treatment can block the in vitro immunoregulatory effects of GA on T-cell proliferation and cytokine production. Forty-two patients with relapsing-remitting multiple sclerosis who were treated with GA for 1-5 years were examined for GA antibody titres using enzyme-linked immunoabsorbent assay (ELISA). Thirty-three percent of patients developed high antibody titres [antibody binding index (ABI) = 16-64] and 14% had low antibody titres (ABI = 4) after 1 year on treatment. Results showed that purified GA antibodies blocked the stimulatory effects of GA on GA-specific T-cell lines of Th0 cytokine profile. The increase in interleukin-10 (IL-10) and IL-4 levels and the decrease in IL-12 and tumour necrosis factor-alpha levels, normally seen with GA stimulation, were reversed in the presence of GA antibodies. The study has important implications in our understanding of the potential role of high-titre GA antibodies in the treatment of multiple sclerosis.

Anti-interleukin-18 therapy in murine models of inflammatory bowel disease.

Interleukin (IL)-18 is a cytokine with a broad array of effector functions, the most prominent of which is to act synergistically with IL-12 in interferon-gamma production and the induction of a strong T-helper-1-mediated immune response. In addition, IL-18 also upregulates the production of proinflammatory cytokines such as IL-1 and tumor necrosis factor-alpha. Analysis of IL-18-deficient mice revealed an important role of IL-18 in the activation of macrophages and natural killer cells in the context of infection with intracellular bacteria or parasites. In humans, it was reported that IL-18 is elevated at sites of inflammation in inflammatory bowel disease (IBD), particularly in Crohn's disease, suggesting a possible role for IL-18 in the development and persistence of IBD. In this review we summarize recent findings on the functional role of IL-18 in the pathogenesis of colitis with a special focus on murine models of IBD. The neutralizing mouse anti-mouse IL-18 antibodies generated in our group should facilitate the evaluation of the efficiency of therapeutic blockade of endogenous IL-18 in chronic mouse models of colitis besides the use of recombinant forms of the inhibitory IL-18-binding protein.

CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice.

Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49.

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.

Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum.

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.

T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies.

Allergen-specific immunotherapy represents one of the few curative approaches toward type I allergy. Up to 25% of allergic patients are sensitized against the major birch pollen allergen, Bet v 1. By genetic engineering we produced two recombinant (r) Bet v 1 fragments comprising aa 1-74 and aa 75-160 of Bet v 1, which, due to a loss of their native-like fold, failed to bind IgE Abs and had reduced allergenic activity. Here we show that both fragments covering the full Bet v 1 sequence induced human lymphoproliferative responses similar to rBet v 1 wild type. The C-terminal rBet v 1 fragment induced higher lymphoproliferative responses than the N-terminal fragment and represented a Th1-stimulating segment with high IFN-gamma production, whereas the N-terminal fragment induced higher IL-4, IL-5, and IL-13 secretion. Immunization of mice and rabbits with rBet v 1 fragments induced IgG Abs, which cross-reacted with complete Bet v 1 and Bet v 1-related plant allergens and strongly inhibited the IgE binding of allergic patients to these allergens. Thus, our results demonstrate that hypoallergenic T cell epitope-containing rBet v 1 fragments, despite lacking IgE epitopes, can induce Abs in vivo that prevent the IgE binding of allergic patients to the wild-type allergen. The overall demonstration of the immunogenic features of the hypoallergenic rBet v 1 fragments will now enable clinical studies for safer and more efficient specific immunotherapy.

A monoclonal antibody against the 66-kDa protein expressed in mouse spleen and thymus inhibits Ly-6A.2-dependent cell-cell adhesion.

The Ly-6 locus encodes several cell surface proteins of 10-12 kDa. Some members of this multigene family may function in cell signaling and/or cell adhesion processes. T lymphocytes overexpressing Ly-6A.2 (one member of the Ly-6 gene family) protein homotypically aggregate when cultured in vitro. Further analysis of this homotypic aggregation suggests that Ly-6A.2 participates in cell-cell adhesion. These observations indicated the presence of a Ly-6 ligand(s) on the surface of lymphoid cells. In this study we report generation of a hamster mAb, 9AB2, that blocks Ly-6A.2-dependent cell-cell adhesion. The 9AB2 Ab recognizes a 66-kDa glycoprotein with unique tissue expression. The 9AB2 mAb does not bind Ly-6A.2, but coimmunoprecipitates Ly-6A.2 molecule. Moreover, 9AB2 Ag-expressing thymocytes specifically bind to Chinese hamster ovary cells overexpressing Ly-6A.2 protein, and this binding is specifically blocked by 9AB2 and anti-Ly-6A.2 Abs. These results suggest that the 66-kDa protein recognized by 9AB2 mAb is the putative ligand for Ly-6A.2.

Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152).

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.

Development of a monoclonal antibody blocking-ELISA for detection of antibodies against Maedi-Visna virus.

A monoclonal antibody (MAb) blocking ELISA (Blck-ELISA) was developed to detect antibodies against Maedi-Visna virus (MVV) in sheep sera. The assay employs a MAb directed against the envelope protein p90 of the virus in a sandwich blocking procedure. To determine whether the MAb was a potential antibody for developing a Blck-ELISA, a collection of three hundred sera obtained from several sheep flocks known to be infected with MVV were used to examine the sensitivity of the Blck-ELISA. A total of 50 serum samples originating from a flock free of MVV were tested to assess the specificity of the assay. The results were compared with a commercial indirect ELISA (I-ELISA) and samples giving a conflicting or doubtful result were tested by immunoblot. The Blck-ELISA proved to be specific, sensitive and it showed high reproducibility and low variability.

The production of MLR-blocking factors after lymphocyte immunotherapy for RSA does not predict the outcome of pregnancy.

PROBLEM: The questions of whether production of mixed lymphocyte reaction-blocking factors (MLR-BFs) after immunotherapy with lymphocytes for recurrent spontaneous abortion (RSA) has prognostic value and whether cytotoxic antibodies are also involved were tested. METHOD OF STUDY: A prospective study with 33 patients who had a history of two or more abortions, lacking MLR-BFs, was carried out. The patients received immunizations with lymphocytes and 6 weeks or later were tested for seroconversion of MLR-BFs. Seventeen of these thirty-three patients were evaluated for antipaternal cytotoxic antibodies. The results were correlated with the outcome of the next pregnancy after treatment. RESULTS: Eighty percent of the 33 patients had a live child. Of those patients having success, only 50% produced MLR-BFs. Of those patients having a new loss, five did and two did not produce MLR-BF (P > 0.05). Regarding the 17 patients tested for cytotoxic antibodies, 4 of the 5 patients who tested positive had a new abortion, whereas only 1 of 12 whose tests remained negative did not have gestational success (P < 0.01). CONCLUSION: The presence of MLR-BFs is not a prognostic criterium for the outcome of pregnancy after alloimmunotherapy, and, consequently, it is not a good diagnostic tool for RSA of alloimmune cause.

Production and characterization of monoclonal antibodies against the leukemia inhibitory factor low affinity receptor, gp190.

Leukemia inhibitory factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT1) act through transmembrane receptors which share the gp190 glycoprotein chain. The understanding of its involvement in the biology of these cytokines is of importance since these systems have recently been shown to participate in major inflammatory and neoplastic processes such as myelomatosis (Rose-John, S., Heinrich, P.C., 1994. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300, 281). In addition, this family of receptors also shares the gp130 transducing chain, with the IL6 and IL11 receptors. Because IL6 and gp130 were the first members to be discovered, most of the available reagents are directed at them. In this respect, monoclonal antibodies have played a major role in elucidating these receptor/ligand interactions and exploring the pathophysiological aspects of their biology. So far, no such reagents have been described for the gp190. We now report the production and characterization of 16 monoclonal antibodies directed against human gp190. They were obtained using recombinant chimeric or truncated proteins produced in a eukaryotic CHO cell line. One was able to block the biological activity of LIF. Because gp190 comprises two hematopoietin binding domains, crude epitope mapping was possible using the same reagents. While more of these antibodies are required, the present set validate the technological approach used for their preparation and should improve our understanding of this class of cytokines.

Characterisation of the antibody response to the extracellular region of recombinant thyrotropin receptor.

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.