Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Chazenbalk, Gregorio D.; Jaume, Juan Carlos; McLachlan, Sandra M.; Rapoport, Basil

doi:10.1074/jbc.272.30.18959

Cited by 110 publications

(87 citation statements)

References 64 publications

(67 reference statements)

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“…(It should be emphasized that because of the low serum concentration of TSHR autoantibodies [21,22], this approach cannot be used for clinical assays in GD.) As a first step therefore, we assessed serum antibody binding to ELISA wells coated with a secreted form of the TSHR, TSHR-289 [15]. Surprisingly, wells coated with either unpurified or partially purified TSHR-289 (but not with BSA) gave high OD values for sera from mice injected with RT4.15HP fibroblasts, regardless of Significantly greater than for TSHR-RT-injected, but not for RTinjected, mice (P , 0´05; analysis of variance).…”

Section: Resultsmentioning

confidence: 99%

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Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

Yan

Guo

Pichurin

et al. 2000

Clinical and Experimental Immunology

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SUMMARYAKR/N mice injected with fibroblasts expressing MHC class II (RT4.15HP cells) and the TSH receptor (TSHR) develop antibodies similar to those in Graves' disease. We were unable to analyse the subclass of these antibodies because of unexpectedly high non-specific binding by ELISA or flow cytometry. The non-specific binding reflected generalized immune activation which occurred even when the fibroblasts did not express the TSHR. However, the IgG subclasses were determined for thyroid peroxidase (TPO) antibodies induced using TPO-expressing RT4.14HP cells and found to be IgG2a . IgG1. This Th1 pattern is consistent with spontaneous secretion of interferon-gamma (but not IL-4 or IL-10) by splenocytes from injected mice. The Th1 bias was related to fibroblast injection because conventional immunization of the same mouse strain with purified TPO and adjuvant induced a Th2 response (IgG1 .. IgG2a). Further, untransfected fibroblasts themselves induced powerful, non-specific proliferative responses when used as antigen-presenting cells (APC) in vitro. Flow cytometry revealed that the RT4.15HP fibroblasts (and TSHR-and TPO-transfected derivatives) expressed B7-1. Unexpected constitutive expression of this key molecule may bypass the requirement for up-regulation of other costimulatory molecules involved in T cell stimulation. Our data support the concept that RT4.15HP fibroblasts present the TSHR (or TPO), at least for initiating the immune response. However, the accompanying generalized immune stimulation creates difficulties for analysis of TSHR-specific T and B lymphocytes. On the other hand, extension of the model to TPO, an easier antigen to study, will facilitate analysis of murine T cell responses likely to resemble those in human thyroid autoimmunity.

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Section: Resultsmentioning

confidence: 99%

“…ELISA for mouse antibodies to TSHR or TPO ELISA wells were coated with the following antigens: a secreted form of the human TSHR ectodomain (TSHR-289) [15,16], human TPO [13], and, as a control, bovine serum albumin (BSA; Sigma). TPO and BSA were used at approx.…”

mentioning

confidence: 99%

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

Yan

Guo

Pichurin

et al. 2000

Clinical and Experimental Immunology

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“…TSHR A-subunit cDNA (amino acid residues 1-289) (13) with a 6-histidine tag at the C terminus was excised from pSV 2 -ECE-TSHR-289-6H-dhfr (14) with EcoRI and XbaI (Invitrogen Life Technologies) and subcloned into the same sites in pcDNA3 (Invitrogen Life Technologies). This intermediate plasmid (pcDNA3-289), containing a KpnI site 5Ј to the insert, was mutagenized (QuikChange; Stratagene) to introduce a KpnI site 3Ј to the bovine growth hormone poly(A) tail.…”

Section: Transgenic Mice With the Human Tshr A-subunit Targeted To Thmentioning

confidence: 99%

Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization

Pichurin

Chen

Chazenbalk

et al. 2006

The Journal of Immunology

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The thyrotropin receptor (TSHR), the major autoantigen in Graves’ disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves’ hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, “non-self” regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.

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“…TSHR-289, a variant of the receptor expressed in eukaryotic cells that corresponds approximately to the extracellular A subunit (19), was affinity purified from culture medium (20). ELISA wells were coated overnight with 100 l of TSHR-289 protein (1 g/ml) and incubated with mouse sera (1/300 dilution).…”

Section: Elisa For Tshr Absmentioning

confidence: 99%

Prevention of Autoantibody-Mediated Graves’-Like Hyperthyroidism in Mice with IL-4, a Th2 Cytokine

Nagayama

Mizuguchi

Hayakawa

et al. 2003

The Journal of Immunology

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Graves’ hyperthyroidism has long been considered to be a Th2-type autoimmune disease because it is directly mediated by autoantibodies against the thyrotropin receptor (TSHR). However, several lines of evidence have recently challenged this concept. The present study evaluated the Th1/Th2 paradigm in Graves’ disease using a recently established murine model involving injection of adenovirus expressing the TSHR (AdCMVTSHR). Coinjection with adenovirus expressing IL-4 (AdRGDCMVIL-4) decreased the ratio of Th1/Th2-type anti-TSHR Ab subclasses (IgG2a/IgG1) and suppressed the production of IFN-γ by splenocytes in response to TSHR Ag. Importantly, immune deviation toward Th2 was accompanied by significant inhibition of thyroid-stimulating Ab production and reduction in hyperthyroidism. However, in a therapeutic setting, injection of AdRGDCMVIL-4 alone or in combination with AdCMVTSHR into hyperthyroid mice had no beneficial effect. In contrast, coinjection of adenoviruses expressing IL-12 and the TSHR promoted the differentiation of Th1-type anti-TSHR immune responses as demonstrated by augmented Ag-specific IFN-γ secretion from splenocytes without changing disease incidence. Coinjection of adenoviral vectors expressing IL-4 or IL-12 had no effect on the titers of anti-TSHR Abs determined by ELISA or thyroid-stimulating hormone-binding inhibiting Ig assays, suggesting that Ab quality, not quantity, is responsible for disease induction. Our observations demonstrate the critical role of Th1 immune responses in a murine model of Graves’ hyperthyroidism. These data may raise a cautionary note for therapeutic strategies aimed at reversing Th2-mediated autoimmune responses in Graves’ disease in humans.

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Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Cited by 110 publications

References 64 publications

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization

Prevention of Autoantibody-Mediated Graves’-Like Hyperthyroidism in Mice with IL-4, a Th2 Cytokine

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