Heritability of levels of autoantibodies to thyroid antigens using the method of plotting regression of offspring on midparent (ROMP)

Only a few methods can be applied in a simple manner to estimate the genetic control of autoimmunity in humans. Here we examined the heritability of autoantibodies to two thyroid antigens; thyroglobulin (Tg) and thyroperoxidase (TPO, formerly known as thyroid microsomal antigen), using methods of regression of offspring on mid-parental values (ROMP). With the data sets available, affected and unaffected siblings were compared by this rapid screening method using results determined by hemagglutination (HA). The presence of both types of autoantibodies showed positive heritability in patients with Graves' thyrotoxicosis (TT), but it was not observed in chronic lymphocytic or Hashimoto's thyroiditis (CLT) patients. Since these assays have been extensively used over the years by most diagnostic and research laboratories, they should provide some insight as to which quantifiable parameters may be usefully accumulated to help select groups of patients and their families for further genetic study. ROMP may also be useful to determine the sequential appearance of different types of antibody in predicting disease onset in other family members, and in distinguishing maternal and paternal effects on imprinting. The method may be extended to study epitope spreading and other measures of disease progression.     

Thyroglobulin as an autoantigen: what can we learn about immunopathogenicity from the correlation of antigenic properties with protein structure?

Autoantibodies against human thyroglobulin are a hallmark of autoimmune thyroid disease in humans, and are often found in normal subjects. Their pathogenic significance is debated. Several B-cell epitope-bearing peptides have been identified in thyroglobulin. They are generally located away from the cysteine-rich regions of tandem sequence repetition. It is possible that our current epitopic map is incomplete because of the difficulty that proteolytic and recombinant approaches have in restituting conformational epitopes based upon proper pairing between numerous cysteinyl residues. Furthermore, the homology of cysteine-rich repeats with a motif occurring in several proteins, endowed with antiprotease activity, suggests that these regions may normally escape processing and presentation to the immune system, and brings attention to the mechanisms, such as oxidative cleavage, by which such cryptic epitopes may be exposed. A number of T-cell epitope-bearing peptides, endowed with thyroiditogenic power in susceptible mice, were also identified. None of them was dominant, as none was able to prime in vivo lymph node cells that would proliferate or transfer autoimmune thyroiditis to syngeneic hosts, upon stimulation with intact thyroglobulin in vitro. More than half of them are located within the acetylcholinesterase-homologous domain of thyroglobulin, and overlap B-cell epitopes associated with autoimmune thyroid disease, while the others are located within cysteine-rich repeats. The immunopathogenic, non-dominant character of these epitopes also favours the view that the development of autoimmune thyroid disease may involve the unmasking of cryptic epitopes, whose exposure may cause the breaking of peripheral tolerance to thyroglobulin. Further research in this direction seems warranted.     

Autoreactive IgG elicited in mice by the non-dominant but pathogenic thyroglobulin peptide (2495–2511): implications for thyroid autoimmunty

We have previously shown that mice challenged with the rat thyroglobulin (Tg) peptide TgP1 (corresponding to aa 2495–2511 of human Tg) develop experimental autoimmune thyroiditis (EAT) and produce IgG antibodies that cross-react with Tg from various species. It was not clear, however, whether such antibodies were TgP1-specific or were induced secondarily—i.e. by autologous Tg released from the destroyed gland—and therefore directed to determinants other than TgP1. In this study we describe that, 5 weeks after priming with TgP1, the binding of serum IgG on native Tg is completely inhibited by free peptide, suggesting lack of recognition of other determinants on mouse Tg (mTg). In addition, TgP1-induced but not mTg-induced IgG bound better to heat-denatured than intact mTg, a result compatible with the recognition of a linear epitope by the peptide-induced antibodies. Comparison of the IgG subclass distribution among mTg-induced versus TgP1-induced IgG did not reveal qualitative differences, since all subclasses were represented in the order IgG1 > IgG2b > IgG2a > IgG3. Finally, TgP1-specific IgG reacted strongly with the follicular colloid in sections of normal thyroids, indicating the potential to bind to native Tg in vivo. These data: (i) highlight TgP1 as the only, so far, Tg sequence known to generate both EAT and Tg-reactive IgG in mice: and (ii) do not provide evidence for an amplification of the Tg-specific IgG response through the involvement of endogenous autoantigen in EAT.     

Modulation of diabetes in NOD mice by GAD65-specific monoclonal antibodies is epitope specific and accompanied by anti-idiotypic antibodies

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96.11) specific to the 65-kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non-obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope-specific, as only b96.11 showed this therapeutic property, while a GAD65-specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96.11 or b78 to NOD mice was accompanied by the generation of anti-idiotypic antibodies. Importantly, the induced anti-idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96.11 in the anti-idiotypic antibody, supporting an immunomodulatory role for GAD65-specific autoantibodies, as originally postulated by Jerne.     

Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice.

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid-specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio-iodinated TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody titre. Furthermore, high TGPO autoantibody titres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n = 48) nor with complement-dependent cytotoxicity (n = 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n = 6) or human TPO (n = 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG- and TPO-immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross-reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG-immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.     

Bacteria and Primary Biliary Cirrhosis

Infectious agents have been postulated to play a pathogenic role in the loss of immunological tolerance and the induction of primary biliary cirrhosis, an immune-mediated cholestatic liver disease characterized by progressive destruction of the small intrahepatic bile ducts and subsequent cirrhosis and liver failure. This review discusses emerging issues implicating infectious agents such as Escherichia coli, mycobacteria, chlamydia, helicobacter species, lactobacilli, Novosphingobium aromaticivorans, and betaretroviruses in the pathogenesis of primary biliary cirrhosis. We also review the immunopathological mechanisms responsible for the induction of the disease with special emphasis on the role of molecular mimicry and microbial/self immunological cross-reactivity.     

Thyroid peroxidase and the induction of autoimmune thyroid disease

Animal models of autoimmune thyroid disease are associated with thyroglobulin (Tg) as autoantigen whereas in man the autoimmune response to microsomal antigen/thyroid peroxidase (TPO) appears to play a major role in thyroiditis. Consequently, we have compared the ability of TPO and Tg to induce thyroid autoantibodies and thyroid damage in mice known to be susceptible (CBA/J) or resistant (BALB/c) to thyroiditis induced using murine Tg. Groups of three to five mice were immunized twice using Freund's complete adjuvant with 80-100 micrograms highly purified porcine (p) TPO, pTg, rat (r) Tg, human Tg, bovine serum albumin (BSA) or BSA + 0.2 micrograms pTg (the level of Tg contamination of TPO). Four weeks after immunization with TPO, plasma from CBA/J (but not BALB/c) mice contained IgG class antibodies which bound to TPO-coated tubes in the presence or absence of excess Tg (and could therefore be clearly distinguished from Tg antibodies) but there was no evidence of thyroiditis in either strain of mice. In contrast, in CBA/J mice immunized with rTg and, to a lesser extent in mice that had received pTg, thyroid tissue was infiltrated with lymphoid cells and/or neutrophils and antibodies to pTg (but not pTPO) were present. Our observations demonstrate that induction of TPO antibody alone is insufficient to lead to thyroiditis in CBA/J mice. Further, these studies emphasize the complex interactions between MHC and different thyroid antigens in the processes leading to thyroid destruction.     

Molecular heterogeneity of antigen- or idiotype-induced anti-thyroglobulin monoclonal autoantibodies.

To define the molecular basis of the cognitive interaction in experimental autoimmune thyroiditis (EAT), we sequenced the variable regions of monoclonal autoantibodies to thyroglobulin (Tg), specific or not for the F40D peptide, a Tg peptide capable of inducing EAT in CBA/J mice. Three MoAbs were obtained by immunization with syngeneic Tg of CBA/J (3B8G9, 2F6F2) or C57Bl/6 (4D11F4) mice. 3B8G9 was specific for F40D peptide, whereas 2F6F2 and 4D11F4 were not. Two others were raised in CBA/J mice by manipulation of idiotypic pathways: B12 resulted from the immunization with one Ab2 beta, bearing the internal image of one F40D epitope, and TA2 from the immunization with F40D-specific cytotoxic HTC2 T cells. B12 and TA2 were both specific for F40D. All hybridomas expressed different members of the J558 VH family, except 3B8G9 which expressed a Q52 VH gene segment. These data led us to hypothesize that regulatory anti-id autoantibodies used members of one VH family located in the 5'-end of the VH locus, whereas EAT-associated autoantibodies used a member of one of the most D-proximal VH family. As expected, no homologies were found when anti-F40D monoclonal autoantibodies were compared with two other monoclonal autoantibodies displaying a different epitopic specificity. Among the anti-F40D monoclonal autoantibodies, one histidine residue located in position 35 of the CDR1 region was constantly found. Moreover, TA2 and B12 exhibited two common amino acids in their CDR3 regions, one glycine and one tyrosine, in positions 98 and 99, respectively. Striking homologies were found between TA2 and one anti-polyGAT MoAb, and between 3B8G9 and some anti-phenyloxazolone (phOx) monoclonal autoantibodies. Lastly, the VK sequence from 4D11F4 was identical at the amino acid level to the VK sequence from another monoclonal autoantibody, 81B1, which was previously raised towards syngeneic Tg in CBA/J mice. Our data imply that anti-idiotypic regulatory circuits in EAT might be generated by a heterogeneous population of B cells rather than obtained by a single dominant B cell population.     

Meta-Analysis: The Relationship Between CTLA-4 +49 A/G Polymorphism and Primary Biliary Cirrhosis and Type I Autoimmune Hepatitis

Objectives: CTLA-4 exon-1 +49A?>?G (rs231775) polymorphism has been reported to influence the risk for primary biliary cirrhosis (PBC) as well as type I autoimmune hepatitis (AIH-1) in many studies; however, the results still remain controversial and ambiguous. This study aimed to determine more precise estimations for the relationship between CTLA-4 +49 A?>?G polymorphism and the risk for PBC and AIH-1 by using a meta-analysis.

Design and Methods: PubMed, EMBASE and MEDLINE were searched. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the strength of the association.

Results: Fifteen studies including 3661 patients with PBC and 4427 controls as well as seven studies including 1270 patients with AIH-1 and 1614 controls were identified. Our pooled analysis revealed that G allele of CTLA-4 gene +49A/G polymorphism may confer an increased risk of PBC in overall (p?=?0.001, OR?=?1.29; 95% CI?=?1.13–1.47) and Caucasians (p?=?0.001, OR?=?1.32; 95% CI?=?1.21–1.44). At genotypic level, the codominant, dominant and recessive models showed no significant association with PBC. With respect to AIH-1, the AG genotype demonstrated a trend for association with increased risk of AIH-1 (p?=?0.04, AG vs. AA, OR?=?1.20; 95% CI?=?1.01–1.43). However, the CTLA-4 alleles as well as genotypes in dominant and recessive models were not associated with a risk for AIH-1 in both Caucasians and Asians.

Conclusions: This meta-analysis concluded that the CTLA-4 G allele and the AG genotype were associated with an increased risk for PBC and AIH-1, respectively, suggesting the CTLA-4 +49 A/G polymorphism as a candidate of susceptibility locus to PBC and AIH-1.     

Antibodies to acetylcholinesterase cross-reacting with thyroglobulin in myasthenia gravis and Graves's disease.

In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimoto's thyroiditis (HT), 53 with Graves' disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti-AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti-Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti-AChE and anti-Tg activities (MG, 18%; P < 0.001; and GD, 15%; P < 0.001, versus CR, 0%). This bi-reactivity was exhibited by anti-AChE antibodies cross-reacting with Tg (anti-AChE/Tg activity); (i) serum anti-AChE activity was effectively inhibited by soluble Tg, and (ii) affinity-purified anti-Tg antibodies cross-reacted with AChE. Cross-reactivity seems to be a property of pathological (auto)antibodies; induced (rabbit) antibodies to AChE or Tg were highly monospecific. Analysis of clinical data showed that increased IgG anti-AChE/Tg activity was well associated with: (i) overlapping GD in MG (P < 0.02), and (ii) ophthalmopathy in GD (P < 0.01). In contrast, no correlation was noted in MG between anti-AChE activity units and anti-Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti-AChE/Tg antibodies remains to be elucidated.     

The Chemical Pathway to Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a female predominant chronic disease of autoimmune pathogenesis and unknown etiology, although data suggest that genetic predisposition and environmental factors concur to its onset. Among nongenetic factors, several lines of evidence spanning from geoepidemiology to experimental findings support the role of xenobiotics, i.e., chemicals that are capable to induce molecular mimicry through cross reactivity. Indeed, specific xenobiotics are hypothesized to substitute lipoic acid residues on PBC-specific autoepitopes thus triggering autoimmunity. This is supported by data obtained with patient sera reactivities as well as animal models. The scenario is further complicated by the possibility that xenobiotic-metabolizing bacteria might also play a role. We will review the available evidence in this intriguing and rapidly growing field of research and critically discuss its potential implications.     

Detection of four diabetes specific autoantibodies in a single radioimmunoassay: an innovative high-throughput approach for autoimmune diabetes screening

Highly sensitive and specific radioimmunoassays have been validated for autoantibodies reacting with the four major autoantigens identified so far in autoimmune diabetes. However, the analysis of this large number of autoantigens has increased the costs and time necessary for complete autoantibody screenings. Our aim was to demonstrate that it is possible to detect the immunoreactivity against a combination of four different autoantigens by a single assay, this representing a rapid, low-cost first approach to evaluate humoral autoimmunity in diabetes. By using this novel multi-autoantigen radioimmunoassay (MAA), in subsequent steps we analysed 830 sera, 476 of known and 354 of unknown diabetes-specific immunoreactivity, collected from various groups of individuals including type 1 and type 2 diabetes patients, autoantibody-positive patients with a clinical diagnosis of type 2 diabetes (LADA), prediabetic subjects, individuals at risk to develop autoimmune diabetes, siblings of type 1 diabetic patients, coeliac patients and healthy control subjects. All sera reacting with one or more of the four autoantigens by single assays also resulted positive with MAA, as well as eight of 24 type 1 diabetic patients classified initially as autoantibody-negative at disease onset based on single autoantibody assays. In addition, MAA showed 92% sensitivity and 99% specificity by analysing 140 blinded sera from type 1 diabetic patients and control subjects provided in the 2010 Diabetes Autoantibody Standardization Program. MAA is the first combined method also able to evaluate, in addition to glutamic acid decarboxylase (GAD) and tyrosine phosphatase (IA)-2, insulin and islet beta-cell zinc cation efflux transporter (ZnT8) autoantibodies. It appears to be particularly appropriate as a first-line approach for large-scale population-based screenings of anti-islet autoimmunity.     

Hashimoto’s thyroiditis: TGAb,TPOAb, TRAb and recovery from hypothyroidism

Hashimoto described four patients with goiter. The histology of the goiter was characterized by diffuse lymphocytic infiltration, fibrosis and epithelial cell destruction. Thyroglobulin antibody (TGAb) and thyroid peroxidase antibody (TPOAb) have been used to diagnose Hashimoto’s thyroiditis. Patients with positive TGAb and/or TPOAb have been assumed to have Hashimoto’s thyroiditis. Approximately 10% of those with positive TGAb and/or TPOAb have hypothyroidism. There are two types of autoimmune thyroiditis: goitrous Hashimoto’s thyroiditis and atrophic thyroiditis. The latter patients have blocking antibody (thyroid-stimulating hormone [TSH]-stimulation blocking antibody [TSBAb]). TSBAb is a TSH-receptor antibody (TRAb). TSBAb causes thyroid atrophy and hypothyroidism. TGAb and/or TPOAb do not necessarily cause hypothyroidism. Hypothyroid patients with Hashimoto’s thyroiditis usually receive life-long l-thyroxine therapy. However, spontaneous recovery from hypothyroidism has been reported. Patients who had Hashimoto’s hypothyroidism and then Graves’ hyperthyroidism (and vice versa), have also been reported. Hashimoto’s hypothyroidism and Graves’ hyperthyroidism could be the opposite spectrums of one disease.     

Cytokine Profile in the Liver of Primary Biliary Cirrhosis

We characterized the cytokine profile in the liver of patients with primary biliary cirrhosis (PBC). Total RNA was extracted from the biopsy specimens of 9 patients with early-stage PBC, 10 with chronic hepatitis C (CHC), and 4 normal controls. cDNA was prepared and amplified with a polymerase chain reaction using primers for interferon (IFN)- and interleukin (IL)-2, -4, -5, -6, -10, -12 (p40), and -15. Cytokines such as IFN- and IL-5, -6, -10, -12, and -15 were expressed in most cases of PBC. Expression rates of IL-5 and IL-6 were higher than in CHC and controls. The higher expression rate of IL-5 in PBC was associated with eosinophil infiltration. IL-2 and IL-4 were rarely detected. Semiquantitative analysis revealed that the expression of IFN- and IL-10 was reversed in PBC and CHC: high IFN- and low IL-10 in PBC and high IL-10 and low IFN- in CHC. These results suggest that cytokine expression is skewed in PBC and both Th1 and Th2 cytokines may play a role in the pathogenesis.     

Epithelioid Hemangioendothelioma of the Liver in Primary Biliary Cirrhosis

We report a case of primary epithelioid hemangioendothelioma of the liver occurring in a patient with primary biliary cirrhosis, stage III. The hepatic tumor was found incidentally by imaging techniques and was surgically resected under a tentative diagnosis of metastatic carcinoma. The resected tumor (1.8±1.6 cm) showed Wtcal histologic features of epithelioid hemangioendd jelioma. The tumor cells were positive for factor VIII related antigen and were stained with Ulex europaeus lectin I. Ultrastruc-turally, the tumor cells showed cytoplasmic vacuoles, tight junctions, basal lamina, pinocytotic vesicles, bundles of thin filaments (approximately 10 nm in diameter) and Weibel Palade bodies. The non tumorous part of the liver showed features of primary biliary cirrhosis, stage III. This is the first reported case of epithelioid hemangioendothelioma occurring in a liver with primary biliary cirrhosis. Acta Pathol Jpn 39: 607 611, 1989.     

Pathogenicity of a human thyroglobulin peptide (2340-2359) in mice with high or low genetic susceptibility to thyroiditis

We have previously identified a 20-mer peptide of human thyroglobulin (hTg), p2340 (aa2340-2359), which induced experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) and HLA-DR3 transgenic mice. In this study, we investigated the thyroiditogenic potential of p2340 in 'high responder' CBA/J (H-2(k)) and SJL/J (H-2(s)) or 'low responder' C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Mice were immunized subcutaneously with 100 nmol of p2340 in complete Freund's adjuvant (CFA) and both the proliferative capacity of their lymph node cells in the presence of p2340 or intact Tg and the production of peptide-specific antibodies were investigated. The p2340 peptide was found to contain B-cell and non-dominant T-cell epitope(s) in all strains tested. Moreover, it elicited EAT in CBA/J (2/6, infiltration index (I.I.) 1) and SJL/J (5/5, I.I. 1-3) mice after direct challenge and in BALB/c (4/7, I.I. 1) and C57BL/6 (1/5, I.I. 1) after adoptive transfer of p2340-primed lymph node cells. P2340 is the first Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction in a genetically resistant host.     

Multiplicity of the antibody response to GAD65 in Type I diabetes

Type I diabetes (TID) is an autoimmune disease characterized in part by the presence of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65), among other pancreatic islet antigens. We investigated the independent epitope specificities of these GAD65 antibodies (GAD65Ab) and their combinations in the sera of new onset TID patients and first-degree relatives positive for GAD65Ab. For our analysis, we used four GAD65-specific recombinant Fabs (rFabs) that recognize different conformational determinants of GAD65 located throughout the molecule, including the N-terminal, the middle and the C-terminal regions. We used these epitope-specific rFabs in competition assays to determine the binding specificity of the autoantibodies found in patient sera. Among the 61 sera from newly diagnosed GAD65Ab-positive TID patients GAD65 binding was competed for 23 sera by all four rFabs, 29 by at least two rFabs, and in nine sera were displaced by one or no rFab. In contrast, none of the 24 sera from GAD65Ab-positive first-degree relatives of TID patients were displaced by all four rFabs. When using all four rFabs simultaneously to compete with GAD65Ab binding, binding of sera from TID patients was reduced by an average of 70%. A significantly weaker competition was observed when evaluating sera of GAD65Ab-positive first-degree relatives (P < 0.0001).     

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

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.