Variable influences of iodine on the T-cell recognition of a single thyroglobulin epitope

Abstract

We have previously shown that iodotyrosyl formation within certain innocuous thyroglobulin (Tg) peptides confers on them immunopathogenic properties. In this report, we generated a panel of T-cell hybridoma clones specific for the immunogenic 16 mer Tg peptide p179 (amino acids 179-94) or its iodinated analogue (I-p179), with a view to examining the effects of a single iodine atom at the Y192 amino acid residue on T-cell recognition. We found that the peptide p179 was subdominant, and its binding to both A(k) and E(k) molecules was not significantly influenced by iodine. T-cell receptor (TCR) engagement was unaffected by the bulky iodine atom in two clones that responded to both analogues but it was sterically hindered in two other clones that recognized only p179. One clone was reactive only to I-p179, suggesting that the iodine atom is an integral part of its TCR ligand. Truncation analysis localized the determinant seen by all clones within the 11 mer peptide p184 (amino acids 184-194), suggesting that the cross-reactive clones were not activated by a minimal epitope lacking Y192 and that the negative influence of iodine was not the result of a flanking residue effect. These results demonstrate, at the clonal level, variable influences of a single iodine atom on the recognition of a single Tg peptide. Iodination of tyrosyl-containing, immunopathogenic Tg peptides may have unpredictable effects at the polyclonal level, depending on the extent of iodination at the particular site, and the relative number or effector function of autoreactive T-cell clones that are switched on or off by the neoantigenic determinant.

Figure 1

p179 contains a subdominant epitope. CBA/J mice (three mice per group) were challenged with either Tg (a) or p179 (b). Antigen-specific proliferation of LNCs, in the presence of Tg (□), p179 (○) or I-p179 (•) was examined 9 days later. Data show the mean ± SD of S.I. values of triplicate wells and are representative of two or three experiments. Background c.p.m. varied from 3499 to 5653.

Figure 2

Iodine modification of p179 does not significantly alter its MHC-binding properties. (a) Amino acid sequence of p179 indicating the A^k-binding (underlined) and E^k-binding (bold) motifs. (b) Activation of the E^k-restricted clone 8F9 by its p2496 ligand (25 nm), in the presence of the inhibitor peptides p179 and I-p179. The A^k-restricted Tg peptide p1826 was used as a negative control. (c) Activation of the A^k-restricted clone 10C1 by its I-p304 ligand (0·15 µm), in the presence of increasing concentrations of the inhibitor peptides p179 and I-p179. The E^k-restricted Tg peptide p2496 was used as a negative control. IL-2 secretion by the activated T-cell hybridomas was assessed by CTLL proliferation.

Figure 3

Variable influences of a single iodine atom on TCR recognition. Hybridoma clones 1E7 (a), 2D5 (b) and 2H2 (c) were generated from p179-primed LNCs and clones 4A11 (d) and 1B3 (e) were generated from I-p179-primed LNCs. The LK35.2 cell line was used as APC. Data represent mean ± SD of c.p.m. for triplicate wells.

Figure 4

TCR Vβ family utilization examined by RT-PCR, using Vβ-specific primers. M, markers; B, blank control; G, GAPDH.

Figure 5

(a) Peptide panel used for truncation analysis. Peptides 7 and 8 represent the full amino acid sequence of p179 and I-p179, respectively. Boxes denote iodotyrosyls. (b) to (f) Activation of 1E7, 2D5, 2H2, 4A11 and 1B3 clones in the presence of 10 µm of indicated peptides. The LK35.2 cell line was used as APC. Data depict mean ± SD c.p.m. of triplicate wells.