Thyroxine-binding antibodies inhibit T cell recognition of a pathogenic thyroglobulin epitope

Abstract

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5' iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549-2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the A(k)-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.

FIGURE 1

The 3.47 T cell hybridoma clone specifically recognizes the Tg peptide T4(2553) and is A^k-restricted. TA3 cells were used as APC. Points indicate mean of triplicate wells and denote radioactive thymidine uptake (cpm) by the IL-2-dependent CTLL cells. A, IL-2 secretion following activation of the 3.47 clone by the Tg peptides is shown. B, 3.47 T cell activation is shown in the presence of increasing concentrations of mAbs specific for A^k or E^k Ags and 150 nM of T4(2553) peptide. Control cultures, without mAbs, yielded a mean of 13,120 cpm. C, Activation of 3.47 T cells by T4(2553) but not other T4-containing peptides of Tg. D, Competitive inhibition of 3.47 T cell activation in the presence of 37.5 nM of T4(2553) and increasing amounts of inhibitor peptides. Control culture cpm mean without inhibitory peptide is 14,830; without Ag is 530.

FIGURE 2

The 3.47 clone recognizes a Tg determinant modified by iodine atoms. TA3 cells were used as APC. A, Lack of 3.47 T cell activation by the thyronine-containing analog peptide T0(2553). B, Competitive inhibition of 3.47 activation in the presence of 25 nM of T4(2553) and increasing amounts of the competitor analog peptide T0(2553). Data denote cpm mean of triplicate wells and are representative of three separate experiments. Control culture cpm mean values are the same as in Fig. 1D.

FIGURE 3

A and B, Reactivity of 55H8 and 91A1 mAbs to the indicated Tg peptides encoding hormonogenic regions. ELISA plates were coated at 1 µg of peptide per well. C and D, Competitive inhibition of mAb binding to mouse Tg using 1.0 µg/ml 55H8 (C) or 5.0 µg/ml 91A1 (D) in the presence of increasing amounts of free T4 or Tg peptide competitors. Points indicate mean of triplicate wells; SD values were <10% of the mean. OD values in the absence of inhibitory peptides were 1.48 (C) and 0.947 (D).

FIGURE 4

The mAbs 55H8 (A) and 91A1 (B) differ in their fine specificity. ELISA reactivities were assessed using Tg, or conjugates of T3 or T4 with RSA (1 µg/well) in the presence of increasing concentrations of mAbs shown.

FIGURE 5

The T4-specific mAb 55H8 blocks recognition of the T4(2553) peptide by the 3.47 clone. The APC line TA3 (A) or thioglycollate-stimulated peritoneal macrophage from CBA mouse (B) were pulsed with T4(2553) (2 µg/ml) for 6 h, washed and cultured with 3.47 cells in the presence of increasing concentration of the mAbs as shown. C, TA3 cells were pulsed with T4(2553) as in A, and were then fixed with 0.05% glutaraldehyde (F) for 30 s, or left untreated (N). Inhibition of the 3.47 cell activation was monitored in the presence of increasing concentrations of the mAbs shown. Points indicate the cpm mean of triplicate wells. Mean values of control wells in the absence of mAbs were (cpm): 14,097 (A); 31,555 (B), and 23,450 (C). Background values in the absence of T4(2553) peptide ranged from 80 to 400 cpm. Similar results were obtained in three separate experiments.

FIGURE 6

Activation of T4(2553)-specific pathogenic T cells is blocked by the 55H8 mAb. A, T4(2553)-primed LNC were cultured with mitomycin C-treated splenocytes that were pulsed with 10 µg/ml T4(2553) peptide for 6 h, washed and subsequently blocked with 60 µg/ml mAb 55H8, 91A1, or 3B3 (▨) or no mAb (■) for 1 h. LNC proliferation was measured by adding [³H]thymidine during the last 18 h of a 4-day culture. Splenocytes not pulsed with peptide were used as controls (□). B, Adoptive transfer of EAT by 10⁷ T4(2553)-primed LNC following their in vitro restimulation with mitomycin C-treated, T4(2553)-pulsed syngeneic (CBA/J) splenocytes in the presence of 100 µg/ml 55H8 and 91A1 mAbs. EAT severity was scored on day 13 after transfer and the data were analyzed by the nonparametric Wilcoxon rank-sum test.