A novel pathogenic peptide of thyroglobulin (2208-2227) induces autoreactive T-cell and B-cell responses in both high and low responder mouse strains

Abstract

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208-2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (A(b)), or susceptibility (A(s), E(k)) to EAT. We therefore immunized LR BALB/c (H-2(d)) and C57BL/6 (H-2(b)) strains, as well as HR CBA/J (H-2(k)) and SJL/J (H-2(s)) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1-3), as well as in SJL/J mice (two of six mice, infiltration index 1-2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.

Keywords: T-cell epitopes; experimental autoimmune thyroiditis; high/low responders; pathogenic thyroglobulin peptides; resistant/susceptible strains.

Figure 1

Assessment of p2208 immunogenicity. (a, b) Proliferative responses of peptide-primed lymph node cells (LNCs) from SJL/J (a) and C57BL/6 (b) mice to the antigen shown were evaluated according to their Stimulation indices (SI) and were regarded as positive when ≥ 3. Mice were challenged subcutaneously with 100 nmol of peptide, LNCs were collected 9 days later and were cultured in vitro for 3 days in the presence or absence of the antigens. Background counts per minute (c.p.m.) ranged from 300 to 1000 and the results were statistically significant (P < 0·05). (c) Interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels (pg/ml) in the supernatants of p2208-primed LNCs from SJL/J and C57BL/6 mice, cultured in the presence of the respective peptide. Mice were immunized with 100 nmol of p2208 and 9 days later their LNCs were collected and cultured for 48 hr in the presence of 4·4 μm of p2208. The cytokine concentration was calculated according to the standard curve of the respective cytokine in the manufacturer's protocol. Error bars represent SD of duplicate wells and the results are representative of three independent experiments.

Figure 2

Representative appearance of experimental autoimmune thyroiditis severity after challenge of high responder and low responder mouse strains with p2208. (a) Normal thyroid gland from C57BL/6 or SJL/J mice [infiltration index (I.I.) = 0]; (b) focal infiltration by mononuclear cells in C57BL/6 or SJL/J thyroids (I.I. = 1); (c) SJL/J thyroid (I.I. = 2); (d) C57BL/6 thyroid (I.I. = 3). Magnification: × 40.

Figure 3

IgG responses against p2208 and human thyroglobulin (hTg) in immunized mice. Sera were collected from p2208-immunized SJL/J and C57BL/6 mice and their reactivity was individually assessed (mice 1–6 or 7) against the immunizing peptide (a, c) and hTg (b, d). Samples were incubated at threefold serial dilutions (1/50 to 1/450) and in duplicate wells. The reactivity of all pre-immune sera against hTg or p2208 was undetectable. Data are representative of three independent experiments.

Figure 4

The p2208-specific antibodies reacted with intact human thyroglobulin (hTg). Antibody reactivity against hTg was assessed by ELISA using: (a) pooled antisera from four p2208-immunized mice (•); (b) pooled antisera depleted of anti-p2208 antibodies by passing through an immunoadsorbent column (○); (c) pre-immune pooled sera from the same mice used as controls (×).