Peripheral Blood Lymphocyte Transformation to G2s in Patients with Autoimmune Thyroid Disease with and without Ophthalmopathy

Abstract

Thyroid associated ophthalmopathy (TAO) is an autoimmune disease involving the extra ocular muscles and surrounding orbital connective and adipose tissues. The mechanism for the link between ophthalmopathy and thyroid autoimmunity is unknown but current evidence favors an immune reaction against a thyroid and orbital tissue shared antigen such as the novel protein G2s, which is highly expressed in both eye muscles and thyroid, or the TSH receptor (TSHR). Earlier, we showed that serum antibodies against G2s were closely linked to ophthalmopathy. Although lymphocytic infiltration of the eye muscles is a pathologic feature of TAO, it is unclear whether the reaction is in the eye muscle fiber or the surrounding connective tissue. We tested for peripheral blood mononuclear cell sensitization to G2s fusion protein in patients with TAO, Graves' hyperthyroidism or Hashimoto's thyroiditis without evident ophthalmopathy and normal subjects. Results were expressed as counts per min (cpm) and as stimulation indices (SI). Although proliferation tests were positive in 23% of patients with TAO, overall, there were no significant differences between the four groups. Tests were also positive in four out of seven patients with Hashimoto's thyroiditis, suggesting that immune reactivity against G2s could be a marker of this progressive thyroid disorder. There was no significant correlation between T cell reactivity to G2s and serum antibodies against the same protein, measured in enzyme linked immunosorbent assay. Failure to demonstrate significant T lymphocyte sensitization to G2s in the majority of patients with TAO may reflect the small number of sensitized T cells expected to be circulating in the peripheral blood which could be overcome by testing cloned orbital T cells, as available. Another possibility is that the T cell epitope(s) is not present on the 141 amino acid fragment of G2s that we have so far cloned. The finding of positive T cell tests in a small proportion of patients with ophthalmopathy suggests that future studies using cloned orbital T cells and full length G2s, or its dominant epitope, are indicated.

Peripheral blood T lymphocyte sensitisation against calsequestrin and flavoprotein in patients with Graves' ophthalmopathy

“…Hence, the data from the present study provide further evidence that autoimmunity against calsequestrin is closely linked to the eye muscle reaction in patients with GO, and may play a role in its development. The present results confirm that G2s is not a key immunological target, as lymphocyte proliferation to G2s was not significantly different among the patient groups compared to healthy controls, consistent with results from previous T lymphocyte (20) and antibody (5) studies.…”

“…More recently, we showed that a small proportion of patients with TAO had peripheral T cells which were sensitised to recombinant human G2s [20]. In the present study, we have tested for blood T lymphocyte-mediated reactivity against three eye muscle antigens, calsequestrin [21], Fp [22] and a 141 amino acid fragment of the winged-helix transcription factor FOXP1 that we call G2s [23], in patients with Graves' ophthalmopathy (GO), Graves' hyperthyroidism (GH) without ophthalmopathy and age and sex matched normal subjects using a standard lymphocyte proliferation assay.…”

Longitudinal Correlation of Thyroid-Stimulating Immunoglobulin With Clinical Activity of Disease in Thyroid-Associated Orbitopathy

“…34 Several moieties have been suggested as potential antigens in the orbit among them protein G2 and expression of TSHR in orbital tissue. 31,35 The preadipocyte and fibroblast are thought to potentially be the effector cells in TAO and responsible for the connective tissue remodeling that is seen in TAO. 2,31,34,36 Both the preadipocyte and fibroblast are thought to provide a rich source of inflammatory mediators and to contribute to the recruitment and activation of T-cells, all critical elements to initiating and driving the pathogenesis of TAO in which glycosaminoglycan hyaluronan accumulates in orbital cells.…”

Antibodies targeting the calcium binding skeletal muscle protein calsequestrin are specific markers of ophthalmopathy and sensitive indicators of ocular myopathy in patients with Graves’ disease

“…submitted). Fp and G2s antibodies are thus unlikely to be implicated in the pathogenesis of the eye muscle disorder and appear to be secondary to antigen release following eye muscle damage, probably through a T cell‐mediated mechanism [21]. Although not pathogeneic, flavoprotein antibodies are also good markers of eye muscle involvement in patients with non‐specific orbital inflammation [22] and ocular myasthenia gravis [23].…”