Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells.

Abstract

SummaryAlthough thyroglobulin (Tg), the thyroid prohormone, is well known as a T cell dependent autoantigen in human and experimental autoimmune thyroid disease, very little is known about the molecular basis of Tg recognition by T cells. In this paper, we have characterized the epitopes recognized by .two clonotypically distinct, murine Tg autoreactive T cell hybridomas, CH9 and ADA2 . In vitro iodination of a Tg preparation which was deficient in in vivo organified iodine was first used to confirm our previous observation that these T cells recognize iodination-related epitopes in the Tg molecule . Affinity chromatography of tryptic peptides derived from normally iodinated human Tg revealed that these epitopes were exclusively located in thyroxine (T4) containing peptides. Through the use of synthetic T4-containing peptides, representing the four major hormonogenic sites in Tg, we demonstrated that both CH9 and ADA2 recognize an epitope containing the T4 at position 2553 in human Tg. Sets of overlapping 5mer to 12mer peptides around this T4 showed that the most potent peptide was a 9mer beginning at Asp 2551. The T4 was shown to be a critical residue, since its replacement with any of the 20 naturally occurring amino acids produced only nonstimulatory peptides. Since the T cell hybridomas could also be stimulated by major histocompatibility complex class II positive (interferon-, y-treated) thyroid epithelial cells in vitro, and their parent T cell lines can induce thyroiditis on adoptive transfer, the T4-containing Tg sequence described here is implicated as a pathogenic epitope in murine thyroid autoimmunity. T he production of the thyroid hormones thyroxine (T4),t tri-iodothyronine (T3) and reverse T3 (rT3) is dependent on the organification of iodine into thyroglobulin (Tg), the major protein product of the thyroid (1, 2) . This involves thyroid peroxidase catalyzed iodination of tyrosine residues in Tg to form mono-and di-iodotyrosines and their subsequent crosslinking to form the iodothyronines T3 and T4. These mature Tg molecules are stored in a colloidal form in the lumen of thyroid follicles. Secretion of T4 and T3 involves the endocytosis and subsequent proteolysis of colloidal Tg, which releases the hormone residues for diffusion into the circulation . The recent cloning of the genes coding for Tg from several species has allowed the precise localization 1 Abbreviations used in this paper.

Delineation of Five Thyroglobulin T Cell Epitopes with Pathogenic Potential in Experimental Autoimmune Thyroiditis

“…Interestingly, p306 is localized within the I.4 domain with type 1 homology, whereas p1579 as well as p2102 are encompassed, respectively, in the IIIa.1 and IIIa.3 domains that express type 3 homology (Table III). The fifth pathogenic peptide, p2596, maps in the vicinity of the (2549 -2560) epitope, which encompasses a thyroxine molecule at position 2553 (26).…”

“…First, one could maintain that immunodominant T cell epitopes in Tg exist but remain unidentified because 1) the sheer size of Tg prevents their easy detection, 2) such epitopes do not contain hormonogenic sites but are normally iodinated, and 3) detection is precluded by limitations of algorithm-based approaches in epitope mapping (here and in Refs. 21 and 32) or inherent restrictions in using cloned Tg-reactive T cell hybridomas for screening the antigenicity of overlapping Tg peptides, a method followed by Champion et al (26) for the discovery of the pathogenic (2549 -2560) sequence. Second, the theoretical possibility exists that all A k -restricted Tg peptides interact with MHC with similar affinity and activate T cell precursors of low frequency, thus not allowing a clear-cut hierarchy of immunodominance to emerge.…”

“…The peptides (824 -838) with three proline residues flanking the motifs, and (837-851) with a proline residue inside a motif, were not considered for further study because of concerns that Pro may drastically affect the secondary structure. Peptides (2490 -2504) and (2543-2557) were shown previously to contain EAT-causing T cell epitopes (21,26,33). With these considerations in mind, we proceeded to synthesize the eight motif A and B-containing peptides (p110, p226, p306, p1579, p1826, p2026, p2102, and p2596; Table I) and examine their immunopathogenic properties.…”

Iodide induces thyroid autoimmunity in patients with endemic goitre: a randomised, double-blind, placebo-controlled trial

“…Uptake and metabolism of iodine is crucial to the development of thyroiditis, as administration of perchlorate and mononitrotyrosine (to inhibit iodine transport and promote thyroidal iodine loss respectively) in ovo virtually prevents thyroiditis in these birds, thus implying a role for physiological concentrations of iodine (33). Response of murine T cell hybridomas to thyroglobulin is also directly related to its iodine content (35,36).…”

T cell responses to natural human proteins in primary biliary cirrhosis

“…In vitro study of the immunological basis of many human autoimmune diseases has been hampered by failure to identify, and to isolate and purify, the relevant autoantigens in their natural form. Several different approaches have been used to compensate for the non-availability of natural human autoantigen in the study of autoreactive B and T cell responses, including the use of sequence-specific peptides [1][2][3], of recombinant proteins over-expressed in bacteria [4][5][6], and of equivalent proteins purified from tissue obtained from other species [7,8]. None of these approaches is wholly satisfactory, each having theoretical and practical disadvantages.…”