Induction of murine thyroiditis by a non dominant E(k)-restricted peptide of human thyroglobulin

Abstract

We have previously shown that the human thyroglobulin (hTg) 20-mer peptide p2340 (aa 2340-2359) contains an epitope recognized by Tg-reactive B cells in patients with Graves' disease. The presence of several Ek-binding motifs within p2340 prompted us to examine whether this peptide can stimulate a T-cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2k) mice. The peptide was found to be immunogenic at the T-cell level since it induced specific proliferative responses as well as interleukin-2 and interferon-gamma secretion in secondary cultures of peptide-primed lymph node cells (LNC). The p2340-specific proliferation was blocked almost completely by an Ek-specific monoclonal antibody (mAb) but was unaffected by a control Ak-specific mAb. Peptide-primed LNC did not respond to intact hTg and conversely, LNC primed in vivo with hTg did not respond to p2340 in culture, suggesting that p2340 contains non-dominant T-cell epitope(s). Direct subcutanaeous challenge of AKR/J mice (n = 9) with p2340 in adjuvant, elicited mild to moderate EAT (infiltration index of 1-2) and strong p2340-specific immunoglobulin G responses in all mice tested. These data delineate a new thyroiditogenic sequence within the carboxyl terminal region of hTg.

Figure 1

Alignment of the 20-mer human Tg peptide p2340 with its mouse homologue (mTg) indicating the relative positions of E^k-binding motifs. (a) Boxed sequences delineate motifs identified by pattern searches according to Altuvia et al. Motif B (aa 2344–2352): [AFILPVW]-X-[ACFILMPTVWY]-{DEAGS}-[KRHQY]-X-{NQ}-{NQFHWY}-[DEKNQR]; motif C (aa 2342–2348): [AILSTV]-{NQ}-[ACFILMPTVWY]-{DE}-[ACFILMPTVWY]-{NQ}-[DEKNQR]; motif D (aa 2345–2350) and (aa 2347–2352): [ACFILMPTVWY]-X-[ACFILMPTVWY]-{DE}-{DE}-{HKR}-{NQFHWY}-{DENQFHWY}-[KR]; motif E (aa.2342–2346): A-{HKR}-[ACFILMPTVWY]-{FHWY}-[KRWCHNQY]. [ ] denotes inclusion and { } denotes exclusion of the indicated aa at this position, X indicates any aa; (b) Overlapping E^k-binding motifs delineated by arrows according to the algorithm of Leighton et al. in which two hydrophobic (A,V,I,L), short chain (S,T), or aromatic (Y,W,F) residues are localized six to eight positions before a basic residue (K,R,H). The aa substitutions between hTg and mTg sequences are underlined.

Figure 2

(a and b) Proliferative in vitro response of p2340-primed (a) or hTg-primed (b) LNC against the antigens shown. Inguinal LNC were pooled from two AKR/J mice s.c. primed 10 days earlier with 100 nmol of peptide (a), or 100 µg of hTg (b) in CFA. Background c.p.m. varied from 2400 to 2900 c.p.m. The results are representative of three independent experiments. (c) Inhibition of p2340-specific LNC proliferation in vitro in the presence of 2·5 µm p2340 and 40 µg/ml of I-A^k-specific or I-E^k-specific mAbs; the standard errors of the mean percentage inhibition values from triplicate wells were 4·2 and 5·7, respectively. Inguinal LNC were obtained from AKR/J mice primed as in (a). (d) Cytokine release in culture supernatants of p2340-primed LNC obtained as in (a) in the presence of 5 µm of the antigens shown. Cytokine concentrations were extrapolated from standard curves of sandwich ELISA as described in Materials and Methods. The data are representative of three independent experiments.

Figure 3

Histological appearance of thyroids of AKR/J mice: (a) normal gland, 100×; (b) and (c) interstitial or focal accumulation of mononuclear cell infiltrates after mouse challenge with p2340 in CFA. (b) II = 1, 200×; (c) II = 2, 200×.

Figure 4

Representative IgG responses against the antigens shown in 5 week immune sera of AKR/J mice (n = 4) immunized with 100 nmol of p2340, as in Table 1. Reactivity was determined by an alkaline phosphatase-based ELISA as described in Materials and Methods.