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HLA-DR and HLA-DQ polymorphism in human thyroglobulin-induced autoimmune thyroiditis: DR3 and DQ8 transgenic mice are susceptible
HLA-DR and HLA-DQ Polymorphism in Human Thyroglobulin-Induced Autoimmune Thyroiditis: DR3 and DQ8 Transgenic Mice are Susceptible Qiang Wan, Rajal Shah, John C. Panos, Alvaro A. Giraldo, Chella S. David, and Yi-chi M. Kong ABSTRACT: In contrast to H2-based susceptibility to experimental autoimmune thyroiditis (EAT) induced with thyroglobulin (Tg), human leukocyte antigen (HLA) association with Hashimoto’s thyroiditis, the human counterpart, is less clear, and determining association is further complicated by DR/DQ linkage disequilibrium. Previously, we addressed the controversial implication of HLA-DR genes by introducing HLA-DRA/DRB1*0301 (DR3) transgene into endogenous class II negative H2Ab0 mice. EAT induction with either human (h) or mouse (m) Tg demonstrated the permissiveness of DR3 molecules for shared Tg epitopes. Here, we examined the participation of HLA-DQ genes by introducing DQA1*0301/ DQB1*0302 (DQ8) transgene into class II negative Ab0 or class I and II negative 2m(⫺/⫺) Ab0 mice. About 50% and 80% of HLA-DQ8⫹ Ab0 and 2m⫺ Ab0 mice, respectively, developed moderate EAT after hTg immunization, but only minimal response to mTg. The hTg presentation to hTg-primed cells was blocked by anti-DQ
ABBREVIATIONS Ab0 class II negative mice 2m⫺ beta2-microglobulin⫺ (class I negative mice) EAT experimental autoimmune thyroiditis HT Hashimoto’s thyroiditis
INTRODUCTION After induction with thyroglobulin (Tg) in genetically susceptible mice, experimental autoimmune thyroiditis
From the Department of Immunology and Microbiology (Q.W., Y.M.K), Wayne State University School of Medicine, Detroit, MI; the Division of Immunopathology (R.S., J.C.P., A.A.G.), St. John Hospital and Medical Center, Detroit, MI; and the Department of Immunology (C.S.D.), Mayo Clinic, Rochester, MN, USA. Address reprint requests to: Dr. Yi-chi M. Kong, Department of ImmuHuman Immunology 63, 301–310 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.
mAb in vitro. By contrast, HLA-DRB1*1502 (DR2) and *0401 (DR4) transgenes contributed little to hTg induction. Similarly, DQA1*0103/DQB1*0601 or DQA1* 0103/DQB1*0602 (DQ6) transgenic Ab0 mice were unresponsive to hTg induction and carried no detectable influence in DQ8/DQ6 double transgenic mice. Thus, both HLA-DR and -DQ polymorphism exists for hTg in autoimmune thyroiditis. The use of defined single or double transgenic mice obviates the complications seen in polygenic human studies. Human Immunology 63, 301–310 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: experimental autoimmune thyroiditis; EAT; HLA polymorphism in EAT; hTg in HLA transgenic mice; EAT in HLA transgenic mice; HLA polymorphism in EAT
hTg LPS mTg Tg
human thyroglobulin lipopolysaccharide mouse thyroglobulin thyroglobulin
(EAT), an animal model for Hashimoto’s thyroiditis (HT), exhibits mononuclear cells in the thyroid infil-
nology and Microbiology, Wayne State University School, of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201, USA; Tel: (313) 577-1589; Fax: (313) 577-1155; E-mail: [email protected]. Received September 18, 2001; revised December 18, 2001; accepted January 3, 2002.
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trate, Tg autoantibody production, and T-cell proliferative response in vitro [1–3]. In addition to mouse (m) Tg, Tg from heterologous species, such as human (h), can also induce EAT, although thyroiditis is generally less extensive, indicating that mTg contains additional epitopes for the mouse not found on hTg [4, 5]. Susceptibility to both mTg and hTg goes hand-in-hand in conventional strains and has been clearly mapped to H2A class II genes [5, 6]. In contrast, human leukocyte antigen (HLA) association with HT has been controversial, despite improved typing techniques, and delineation in polygenic humans was further complicated by linkage disequilibium [7–12]. For example, the early typing techniques two decades ago were by serology and only polyclonal antibodies to HLA-DR antigens were available for patient studies [8]. Linkage disequilibrium between certain DR and DQ genes were recognized later but its influence was difficult to assess, probably due to additional contributing factors, including ethnic differences, multiple epitopes on Tg [5], and the involvement of other thyroid antigens, such as thyroid peroxidase. Using Tg as the prototype antigen known to induce murine thyroiditis with susceptibility linked to class II genes, we examined single HLA class II transgenic mice in the absence of other class II genes. This protocol would permit the detection of potential susceptible and resistant genes, and the eventual examination of gene complementation when double transgenic mice are evaluated. We first addressed potential HLA-DR association with HT by introducing HLA-DRA/DRB1*0301 (DR3) transgene into resistant B10.M (H2f) or class II negative (Ab0) mice and observed severe thyroid infiltration following immunization with either hTg or mTg [7]. These observations supported some patient studies implicating HLA-DR3 [8 –12]. Moreover, because HLADRB1*1502 (DR2) transgenic mice did not respond to EAT induction by mTg, these studies demonstrated that HLA-DRB1 polymorphism to mTg-induced EAT can be determined by introducing single HLA-DR transgenes [7]. At the time, DRB1 polymorphism in hTg-induced EAT could not be delineated, because the H2Eb molecules present in DR2-transgenic mice, resulting from the introduction of E␣k and DR2 transgenes, responded to hTg, although not to mTg [13]. We report here a different strategy to test DRB1 polymorphism in hTginduced EAT by using a recipient strain that does not express H2E molecules. As to DQ polymorphism, certain HLA-DQ alleles have also been associated with HT in recent years. However, such associations are complicated by linkage disequilibrium with the DRB1 locus, which include DR3, as well as *0401 (DR4) and *0501 (DR5); the latter two have also been implicated in studies of association with
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HT [11,12,14]. On the other hand, in experimental models of other autoimmune diseases, the introduction of single DQA1*0301/DQB1*0302 (DQ8) transgene has led to the demonstration of susceptibility to collagen-induced arthritis [15] and myasthenia gravis [16]. We tested the role of DQ8 transgene in class II-negative Ab0 as well as class I and II negative 2-microglobin(⫺/⫺) Ab0 mice for their response to hTg-induced EAT. HLA-DQ polymorphism was examined by comparing Ab0 mice given the DQ8 transgene or DQA1*0103/DQB1*0601 or *0602 (DQ6) transgene. In addition, we tested the mutual influence of DQ8 and DQ6 expression by examining DQ8/DQ6 double transgenic mice for their susceptibility to hTg-induced EAT. MATERIALS AND METHODS Antigen and Adjuvant The mTg and hTg were prepared from frozen thyroids as described previously by fractionation of thyroid extracts on a Sephadex G-200 column (Pharmacia Inc., Piscataway, NJ, USA) [17]. All preparations were checked for lipopolysaccharide (LPS) contamination with Limulus amebocyte lysate (Associates of Cape Cod, Woods Hole, MA, USA); 20 g antigen contained ⬍ 1-ng LPS [3]. Salmonella enteritidis LPS as adjuvant was prepared by trichloroacetic acid precipitation. Generation of Transgenic Mice All mice were bred and maintained in the Immunogenetics Mouse Core Facility at the Mayo Clinic until shipment. They were housed in specific pathogen-free facility and given acidified, chlorinated water at least 1 week prior to use at 10 –16 weeks of age. The HLA-DQ8 (DQA1*0301/DQB1*0302) transgene in B10.M mice was introduced by mating into two strains of mice deficient in endogenous MHC molecules as described previously [15]. The first was the Ab0 mouse, which expressed neither surface IA (mutant A chain) nor surface IE (nonfunctional Ea gene in this H2b haplotype) molecules [18]. The second strain lacked both surface class I and class II molecules due to the introduction by mating of 2-microglobulin knock-out gene (2m⫺/⫺) [19] into Ab0/DQ8 mice. The breeding scheme to introduce HLADQ6 (DQA1*0103/DQB1*0601 or DQA1*0103/ DQB1*0602) transgene or DQ6/DQ8 double transgene into Ab0 mice has been described [20]. Transgene-positive mice were determined by PCR of PBL with DQ allele-specific oligonucleotide primers and DQ8 or DQ6 expression in PBL by flow cytometry with HLA-DQ␣specific mAb IVD12 [21]. No hybrid MHC molecules formed between H2A␣b with HLA-DQ chain were detected with chain-specific mAbs, as described elsewhere [15]. The HLA-DR3 (DRA/DRB1*0301) trans-
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TABLE 1 HLA-DR2 (DRB1*1502) transgene does not alter the response of resistant B10.RFB3 mice to hTg induction of thyroiditis Thyroiditisa Transgene expression DR2⫹ DR2⫺ DR3⫹b
Number of mice with % thyroid involvement
Incidence
Anti-Tg (O.D. ⫾ SE)a 1:3200
0
⬎ 0–10
⬎ 10–20
⬎ 20–40
⬎ 40–80
Pos./total
Percent (%)
2.0 ⫾ 0.1 1.9 ⫾ 0.3 1.2 ⫾ 0.2b
13 4 —
2 1 2
— — —
— — 1
— — 1
2/15 1/5 4/4
13 20 100
Mice were immunized intravenously with 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. DR3 transgene was on B10.Ab0 background; anti-Tg titers were presented at 1:100 dilution, and were ⬍ 0.2 at 1:3200. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; mTg ⫽ mouse thyroglobulin; Tg ⫽ thyroglobulin. a
b
gene was introduced into Ab0 mice as described previously and made congenic by backcross to B10 mice [7]. However, because H2Eb molecules are permissive for EAT induction by hTg [13], the HLA-DR2 (DRB1*1502) and HLA-DR4 (DRB1*0401) transgene, which required the conserved H2E␣ chain for expression [7], were introduced into the B10.RFB3 strain by intercross with DR2 or DR4 transgenic Ab0 mice [22], followed by nine-generation backcross to B10.RFB3 mice. The B10.RFB3 strain was chosen because it is of the EAT-resistant f haplotype [7] and lacks a functional Eb (-chain) gene. Thus, functional Ef molecules that might respond to EAT induction by hTg were absent. The progenies were screened by the antiDRB1 mAb L227 [23]. Induction and Assay of EAT All transgenic mice were immunized on days 0 and 7 by tail vein injections of 40 or 100 g mTg or 100 or 200 g hTg, followed each time by 20 g LPS 3-h later. On day 28 sera, spleens, and thyroids were collected. Serum antibody titers to mTg or hTg were assayed by ELISA as described previously [24]. In brief, 96-well flat bottom plates (Immulon II; Dynatech Laboratory, Inc., Chantilly, VA, USA) were coated with Tg (1 g/well). Serum dilutions of 1:100 and 1:3200 were added. After incubation and washing, alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotech Assoc., Inc., Birmingham, AL, USA) was added as second antibody. Standard immune serum and normal mouse serum served as positive and negative controls. The spleens were used for in vitro proliferative assay to Tg (see below). The thyroids were processed, examined histologically and scored as illustrated elsewhere [25]. Percent thyroid infiltration was determined after examining 30 – 60 vertical sections (7–10 step levels) throughout both lobes: 0%, no infiltration; ⬎ 0%–10%, perivascular foci of infiltration; ⬎ 10%–20%, infiltration with follicular destruction; ⬎ 20%– 40%, multiple foci of follicular
destruction; and ⬎ 40%– 80%, extensive thyroid involvement. In Vitro Culture On day 28 postimmunization, spleen cells from mTg or hTg-primed mice were cultured at 37 °C and 5% CO2 air for 5 days in flat bottomed, 96-well tissue culture plates (6⫻105/well) with 40 g/ml Tg, as detailed previously [24, 25]. For blocking of Ag presentation, 20 l of culture supernatants containing anti-DQ␣ (IVD12) or irrelevant mAb was added to 200 l cultures with or without Tg. Culture medium consisted of RPMI 1640 with 25 mM HEPES buffer, supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (all GIBCO BRL, Grand Island, NY, USA), 50 M 2-mercaptoethanol (Sigma, St. Louis, MO, USA), and 1% normal mouse serum. After pulsing with 1.0 Ci of [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA, USA) for the final 18-h culture, the cells were harvested onto glass fiber filters. The [3H]thymidine uptake was presented as cpm (mean ⫾ SE) of triplicate cultures. RESULTS HLA-DRB1 Polymorphism is a Determinant in hTg-Induced EAT Earlier studies have reported that HLA-DRB1 polymorphism can be demonstrated for EAT induced with mTg by comparing DR3- and DR2-transgenic Ab0 mice; only DR3 molecules were found to be permissive [7]. Whereas DR3 molecules also presented hTg to induce EAT, the role of DR2 molecules, which were expressed by the pairing of conserved E␣k chain with the DR2 chain in E␣k- and DR2-transgenic Ab0 mice, could not be determined. The coexpressed H2E (E␣kEb) molecules were found to be permissive for EAT induction by hTg, but not by mTg [13]. We therefore introduced the transgene for DR2 or DR4 with E␣k into B10.RFB3 (H2f) mice, which are EAT-resistant and lack endoge-
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TABLE 2 HLA-DR4 (DRB1*0401) transgene has little effect on the response of resistant B10.RFB3 mice to mTg or hTg induction of thyroiditis Thyroiditisa
Antigen
Transgene expression
Anti-Tg (O.D. ⫾ SE) 1:5000
mTg
DR4⫹ DR4⫺ DR4⫹ DR4⫺
0.4 ⫾ 0.4 0.2 ⫾ 0.2 1.1 ⫾ 0.3 0.7 ⫾ 0.1
a
hTg
Number of mice with % thyroid involvement
Incidence
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent (%)
12 14 12 15
4 4 11 7
1 — — —
5/17 4/18 11/23 7/22
29 22 48 32
Mice were immunized intravenously with 40 g mTg or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera for anti-mTg or anti-hTg and thyroids were assayed on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; mTg ⫽ mouse thyroglobulin.
a
nous E. Table 1 illustrates that the animals remained essentially EAT-resistant whether DR2 molecules were expressed or not, as most mice displayed no thyroid infiltration. Because hTg is a heterologous Tg with foreign epitopes for B10.RFB3 mice, the antibody response remained high and little affected by the presence or absence of DR2 molecules. In contrast, DR3-transgenic B10.Ab0 mice, which had both DR␣ and DR3 without the need for E␣k, developed mild to severe thyroiditis after hTg induction as reported previously [7]. In E␣k- and DR4-transgenic B10.RFB3 mice, both mTg (40 g) and hTg (100 g) were used for immunization at the same dosage previously used for DR2- or DR3-transgenic mice [7]. As listed in Table 2, most animals in both the DR4⫹ and DR4⫺ groups did not respond to mTg immunization; 22%–29% incidence consisted of focal infiltration, except one mouse with mild thyroiditis. A little higher incidence of focal infiltration was observed in the DR4⫹ than the DR4⫺ group
immunized with hTg. Antibody titers to the immunizing antigen were higher in the hTg-immunized group than the mTg-immunized group, again probably due to the foreign epitopes in hTg. Because all the DR3-transgenic mice developed mild to severe EAT in contrast to DR4- and DR2-transgenic mice, DRB1 polymorphism is a determinant for susceptibility to hTg-induced EAT, similar to mTg [7]. HLA-DQ8-Transgenic Class II Negative Ab0 Mice are Moderately Susceptible to hTg-Induced EAT but are Resistant to mTg-Induced EAT A previous article reported that DQ8⫹ Ab0 mice were resistant to EAT induction by mTg [26]. Therefore, DQ8⫹ Ab0, DQ8⫺ Ab0, and control Ab0 mice were immunized with hTg, comparing their responses to mTg. Whereas mTg injection led to minimal thyroid involvement and relatively low antibody production in DQ8⫹ Ab0 mice, hTg immunization generated a higher
TABLE 3 hTg, but not mTg, induces moderate thyroiditis in HLA-DQ8-transgenic class II negative H2Ab0 mice Thyroiditisa
Antigen hTg hTgb mTg (40 g) mTg (100 g)
MHC expression DQ8⫹ Ab0 DQ8⫹ DQ8⫺ DQ8⫹ DQ8⫹
Anti-Tg (O.D. ⫾ SE)a
Number of mice with % thyroid involvement
1:100
1:3200
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent (%)
1.1 ⫾ 0.3 0.3 ⫾ 0.1 1.3 ⫾ 0.3 0.3 ⫾ 0.1 0.3 ⫾ 0.3 0.9 ⫾ 0.6
0.7 ⫾ 0.2 0.2 ⫾ 0.1 0.8 ⫾ 0.2 0.1 ⫾ 0.1 ⬍ 0.2 0.3 ⫾ 0.3
2 4 4 3 7 3
2 1 1 — 1 1
2 — 2 — — —
4/6 1/5 3/7 0/3 1/8 1/4
67 20 43 0 13 25
Incidence
a Mice were immunized intravenously with 40 or 100 g mTg, or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. b Experiment comparing littermates. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
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FIGURE 1 T-cell proliferative response to human thyroglobulin (hTg) was blocked by anti-DQ monoclonal antibody (mAb) in HLA-DQ transgenic Ab0 mice. (A) Spleen cells from DQ8⫹; (B) 2m⫺ DQ8⫹; (C) DQ6(0601)⫹; and (D) DQ8⫹ DQ6(0601)⫹ transgenic Ab0 mice or control Ab0 mice, immunized with 100 g hTg ⫹ 20 g lipopolysaccharide (LPS) on days 0 and 7, were tested on day 28. The [3H]thymidine uptake was measured after 5 days of culture (6⫻105/well). Open bars represent background cpm of cultures in the presence of only anti-DQ␣ (IVD12, supernatant 1:10) or anti-IAb (AF6-120, supernatant 1:10) mAb; black bars represent cpm of cultures in the presence of 40 g/ml hTg plus mAb. Control T-cell proliferation in Ab0 mice was performed with (black bar) or without (open bar) hTg.
incidence with some mice developing mild infiltration and higher antibody levels (Table 3). However, only 43%– 67% of the animals from both experiments had thyroid involvement. T-cell proliferative response to mTg or crossreaction to hTg was not observed in either mTg-immunized group (data not shown). In contrast, there is a strong proliferative response to hTg. Class II presentation of hTg was blocked by mAb to DQ␣, but not by the control mAb to IAb (Figure 1A). It appears that DQ8 molecules can present hTg epitopes not shared by mTg, unlike DR3 molecules which can present epitopes shared by both Tgs [7]. Absence of Class I Genes in HLA-DQ8-Transgenic Ab0 Mice Results in Higher Incidence of EAT Earlier studies in CBA/J mice with mTg-induced EAT have reported that both CD4⫹ and CD8⫹ T cells are involved in thyroiditis development [27–29]. On the other hand, little difference in the incidence or severity of thyroiditis was discerned in 2m⫺ CBA mice deficient in CD8⫹ cells, indicating a compensatory role for CD4⫹ cells and macrophages [30]. To determine the requirement for CD8⫹ cells in EAT development in DQ8transgenic Ab0 mice, we immunized 2m⫺ DQ8⫹ Ab0 mice with hTg, using mTg as low responder control. In Table 4 (experiment 1), all four mice developed focal to mild thyroiditis; as expected, 2m⫺ DQ8⫺ Ab0 mice did not respond. In experiment 2, a higher incidence (73%) of 2m⫺ DQ8⫹ mice manifested thyroid infiltra-
tion, compared with DQ8⫹ mice. However, a minority revealed no thyroid infiltration, as in DQ8⫹ mice here and in Table 3. Antibody titers were observed in all the mice, with the exception of those in experiment 1, which were unexpectedly low for mice with higher than normal CD4⫹ T cell repertoire. Similar to DQ8⫹ mice, the T-cell proliferative response was blocked by mAb to DQ␣ (Figure 1B). To determine whether the incidence and severity of thyroiditis could be increased by doubling the hTg dose to 200 g and changing the adjuvant from LPS to CFA, we immunized both DQ8⫹ and 2m⫺ DQ8⫹ mice. Table 5 illustrates that, whereas the incidence of EAT remained higher in 2m⫺ DQ8⫹ mice than in DQ8⫹ mice, the overall response in each strain was little affected by the higher dose or the adjuvant. Many mice still did not display thyroid infiltration, despite high antibody levels. The data in DQ8-transgenic mice confirmed the findings in conventional, EATresistant strains that antibody levels do not correlate with thyroiditis development [1, 7]. HLA-DQ6-Transgenic Class II Negative Ab0 Mice are Resistant to Both mTg and hTg Immunization We next tested DQ6-transgenic mice to determine if DQ polymorphism was a determining factor in EAT-susceptibility. Two DQB1 genes were paired with the same DQA1 gene in two groups of Ab0 mice and immunized with hTg, using mTg as low responder control [26]. Table 6 reveals that only 0601⫹ (2/20) mice developed
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Q. Wan et al.
TABLE 4 hTg induction in HLA-DQ8-transgenic class I negative (2m⫺/⫺) H2Ab0 mice increases the incidence of thyroiditis Thyroiditisa
Experiment number
Antigen
1
mTg hTg
2
hTg
MHC expression 2m⫺ DQ8⫹ 2m⫺ DQ8⫹ 2m⫺ DQ8⫺ DQ8⫹ 2m⫺ DQ8⫹ Ab0
Number of mice with % thyroid involvement
Anti-Tg (O.D. ⫾ SE)a 1:100
1:3200
0
0.8 ⫾ 0.5 0.2 ⫾ 0.2 4 0.1 ⫾ 0.0 ⬍ 0.2 — ⬍ 0.2 ⬍ 0.2 4 ⬎ 1.8 ⫾ 0.2 1.7 ⫾ 0.1 4 1.7 ⫾ 0.1 0.3 ⫾ 0.2 3 0.3 ⫾ 0.1 ⬍ 0.2 8
Incidence
⬎ 0–10
⬎ 10–20
⬎ 20–40
Pos./total
Percent (%)
— — — 5 — —
1 3 — 1 7 —
— 1 — — 1 —
1/5 4/4 0/4 6/10 8/11 0/8
20 100 0 60 73 0
a Mice were immunized intravenously with 40 g mTg, or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
focal infiltration, whereas 0602⫹ mice (0/5) were negative. Moreover, similar to mTg-immunized mice, hTg was not immunogenic in 0602⫹ mice; little antibody production was detected and no proliferative response to hTg was detected (data not shown). By contrast, T cells from 0601⫹ mice responded strongly to hTg in vitro and its presentation was blocked by mAb to DQ␣ (Figure 1C). The differential response to hTg with thyroiditis in DQ8⫹ versus DQ6⫹ mice suggests that HLA-DQ polymorphism is a determinant factor in the extent of EAT susceptibility. To determine if the presence of a resistant DQ6 transgene would influence thyroiditis development in DQ8transgenic Ab0 mice, DQ8/DQ6 (0601) double transgenic mice were immunized with 100 or 200 g hTg. Table 7 illustrates that the DQ8-mediated response was
little affected by the presence of DQ6 molecules. Antibody production (Table 7) and T-cell proliferative response (Figure 1D) remained in the range observed for single DQ8-transgenic groups. DISCUSSION The complications of DR/DQ linkage disequilibrium, varied typing techniques over the years, and ethnic differences have contributed to the controversy in HLA association with HT [31]. To address the question of HLA association, we have used HLA class II-transgenic mice, without endogenous murine class II molecules, and hTg as the putative autoantigen. Although hTg possesses shared as well as foreign epitopes for the mouse as seen from studies of conventional mouse strains, it is primar-
TABLE 5 Neither higher immunizing dose of hTg nor change of adjuvant increases the severity of thyroiditis in HLA-DQ8-transgenic Ab0 mice Thyroiditisa
Adjuvant
hTg dose
MHC expression
LPS
100 g
DQ8⫹ 2m⫺DQ8⫹ DQ8⫹ 2m⫺DQ8⫹ DQ8⫹ 2m⫺DQ8⫹ DQ8⫹ 2m⫺DQ8⫹
200 g CFA
100 g 200 g
Number of mice with % thyroid involvement
Anti-Tg (O.D. ⫾ SE)a
Incidence
1:100
1:3200
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent (%)
1.9 ⫾ 0.1 1.6 ⫾ 0.1 1.8 ⫾ 0.2 1.7 ⫾ 0.1 ⬎ 1.3 ⫾ 0.1 ⬎ 1.3 ⫾ 0.1 ⬎ 1.2 ⫾ 0.1 ⬎ 1.3 ⫾ 0.1
1.6 ⫾ 0.1 0.2 ⫾ 0.2 1.4 ⫾ 0.2 0.3 ⫾ 0.2 1.3 ⫾ 0.1 1.2 ⫾ 0.1 1.2 ⫾ 0.1 1.3 ⫾ 0.1
2 3 4 1 5 4 9 2
— 3 — 4 6 7 2 1
— — 3 2 1 3 — 3
0/2 3/6 3/7 6/7 7/12 10/14 2/11 4/6
0 50 43 86 58 71 18 67
Mice were immunized either with 100 or 200 g hTg, and 20 g LPS intravenously or subcutaneously with hTg in CFA into alternate thigh and hind footpad on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: CFA ⫽ complete Freund’s adjuvant; HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
a
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TABLE 6 HLA-DQ6 (0601 or 0602) transgenic H2Ab0 mice are resistant to both mTg and hTg induction of thyroiditis Thyroitidisa
Antigen mTg hTg
Number of mice with % thyroid involvement
Anti-Tg (O.D. ⫾ SE)a
Incidence
MHC expression
1:100
1:3200
0
⬎ 0–10
Pos./total
Percent
DQ6⫹(0601) DQ6⫹(0602) DQ6⫹(0601) DQ6⫹(0602)
0.3 ⫾ 0.3 0.2 ⫾ 0.0 1.7 ⫾ 0.2 0.1 ⫾ 0.1
⬍0.2 ⬍0.2 1.0 ⫾ 0.1 ⬍0.2
6 2 20 5
— — 2 —
0/6 0/2 2/22 0/5
0 0 9 0
a Mice were immunized intravenously with 40 g mTg, or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
ily the shared, thyroiditogenic epitopes, some of which are known, that are responsible for thyroid infiltration [4, 5, 24]. Thus, based on thyroid infiltration, a hallmark for susceptibility, rather than antibody production and T-cell proliferative response to hTg, we have determined the relative capacity of several DR and DQ alleles to support EAT induction. The data demonstrate that DR and DQ polymorphism is a determinant for hTg-induced EAT. In the case of DRB1 alleles, whereas the DR3⫹ line was established with a DRA transgene [32], the expression of DR2 and DR4 chains required the aid of an Ea transgene to provide a DR␣ chain homologue, which also resulted in the co-expression of H2E molecules [7]. Because H2E molecules are permissive for hTg-induced EAT [13], we used an EAT-resistant strain, B10.RFB3, which lacks a functional Eb gene as transgene recipient. Even with high antibody titers in this resistant strain due to the many foreign epitopes on hTg, DR2 and DR4 molecules did not contribute significantly to thyroiditis development (Tables 1 and 2); only focal infiltration was observed in some mice and none in the majority, compared with DR3-transgenic mice (Table 1) [7]. More-
over, this DRB1*0401 gene carried a mutation at two residues in the 2 domain to provide better interaction with mCD4 [33], which could have led to the very high anti-hTg titers detectable at 1:5000, but the low thyroid pathology was consistent with a low responder strain of the f haplotype [7]. HLA-DQ genes are highly polymorphic at both the DQA1 and DQB1 loci. We concentrated on DQ8 and DQ6 genes which have been studied in several other autoimmune diseases. Unlike DR3 molecules, which are highly permissive for both hTg- and mTg-induced EAT [7], DQ8⫹ Ab0 mice responded poorly to EAT induction after mTg immunization. After hTg induction, the incidence increased to ⬃50%, averaging several groups (Tables 3–5), and a few animals showed moderate mononuclear cell infiltration involving ⬎ 10%–20% of the thyroid (Table 3). However, there were always a sizable numbers of animals in each experiment with no thyroid infiltration, despite the doubling of hTg dosage and the use of a strong adjuvant such as CFA (Table 5). These differences in thyroiditogenic response were apparently unrelated to the expression of DQ8 molecules in these mice, since each animal was monitored for DQ8 expres-
TABLE 7 HLA-DQ6 has little effect on hTg-induced thyroiditis in HLA-DQ8/DQ6(0601)-double transgenic class II negative H2Ab0 mice Thyroiditisa
hTg dose
MHC expression
100 g 200 g
DQ8⫹ DQ6⫹ DQ8⫹ DQ6⫹
Anti-Tg (O.D. ⫾ SE)a
Number of mice with % thyroid involvement
1:100
1:3200
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent
1.6 ⫾ 0.2 1.3 ⫾ 0.3
0.7 ⫾ 0.1 0.6 ⫾ 0.3
23 1
2 3
1 1
3/26 4/5
11 80
Incidence
Mice were immunized intravenously with 100 or 200 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide. a
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sion by FACS analysis prior to use and expression in ⬃20% of the PBL was quite uniform (data not shown). With CIA, the incidence in DQ8⫹ Ab0 mice was ⬃70% [15]. Interestingly, in CD8-deficient, 2m⫺ DQ8⫹ Ab0 mice, there was a notable increase in the incidence of thyroiditis. Moreover, in first examination, it would appear that thyroid infiltration was more pronounced in 2m⫺ DQ8⫹ Ab0 mice than in DQ8⫹ Ab0 mice (Table 4, experiment 2). However, even with the use of higher doses of hTg and CFA as adjuvant, the extent of thyroid infiltration did not rise above 20% and the two strains showed quite similar responses (Table 5). Because of the sizable number of mice in the different groups not displaying any thyroid infiltration, the differences between groups in thyroid involvement are not statistically significant by a nonparametric test (Mann-Whitney). Similar to 2m⫺ CBA mice in which EAT pathology was not diminished [30] despite the known involvement of both CD4⫹ and CD8⫹ cells in wild type mice [27–29], CD4⫹ cells apparently can compensate for the lack of cytotoxic CD8⫹ cells in mediating destruction in conjunction with macrophages [27]. That DQ8-transgenic mice developed only moderate thyroiditis and in only 50%–70% of animals with or without the CD8 subset are in distinct contrast to DR3transgenic mice with 83%–100% incidence and many up to 80% thyroid involvement [7]. At least three reasons might help explain the low and nonuniform response of DQ8-transgenic mice. First, the T-cell receptor (TCR) repertoire selection and deletion by DR3 and DQ8 molecules are different. For example, similar to H2E molecules, albeit with different efficiency, DR3 molecules mediate the deletion of V5⫹ and 11⫹ T cells, but with lower selection of V6⫹ cells; in addition, DR3⫹ mice also have low V13⫹ cells [32]. In contrast, a sampling of the V repertoire in DQ8⫹ mice did not reveal any notable differences from conventional H2A⫹E⫺ mice [15]. Second, DQ8 molecules likely bind and present different Tg epitopes from DR3 as well as a more limited number. hTg and mTg have ⬃73% homology [34] and both induce EAT in DR3⫹ mice, indicating the recognition of shared thyroiditogenic epitopes [7]. In DQ8⫹ mice, mTg is hardly thyroiditogenic, suggesting a more limited presentation of epitopes primarily on hTg. From studies with mTg in CBA mice, we have described that mTg contains both shared as well as unique thyroiditogenic epitopes [5, 24]. It is possible that hTg also contains hTg-specific, thyroiditogenic epitopes processed by DQ8⫹ antigen-presenting cells. This presentation is DQ-restricted; the response to hTg was blocked in vitro by anti-DQ mAb (Figure 1). Because the only humanized component in these mice is the DQ8 transgene,
Q. Wan et al.
antigen presentation may be less efficient with mCD4 cells, if DQ8 molecules have a higher affinity for hCD4⫹ than mCD4⫹ T cells. We therefore tested DQ8⫹ hCD4⫹ mCD4-knockout mice, but the incidence and severity of EAT after hTg immunization were unchanged (data not shown). Third, from adoptive transfer studies, we know that a certain number of donor Tg-reactive T cells are required to achieve and maintain thyroid inflammation [2, 24]. However, in DQ8⫹ mice, even with twice the number of hTg-activated cells commonly used for thyroiditis transfer, only 4/10 recipient mice exhibited thyroid involvement similar to actively immunized mice (data not shown). Since there were more than adequate activated cells transferred without increasing the incidence of thyroiditis, DQ8/peptide complexes do not seem to engage sufficient thyroiditogenic T cells to reach above the threshold required for infiltration. This observation favors the idea of a limited number of cross-reactive epitopes presentable by DQ8 molecules. The need for additional costimulatory molecules may also play a role in enhancing T cell activation by DQ8/peptide complexes. For DQ8-transgenic mice to develop spontaneous diabetes in the NOD mouse, B7-1 expression on pancreatic  cells appeared necessary, but still only 81% of the mice had diabetes [35]. HLA-DQ polymorphism may be a determining factor for EAT, similar to DRB1. DQ6-transgenic mice were resistant to both hTg and mTg induction, as reported for myasthenia gravis [16] and collagen-induced arthritis [36]. With hTg, the DQA1*0103/DQB1*0601 allele supported antibody production and T cell response in vitro, but *0602 did not, whereas mTg was not immunogenic in either strain (Table 6). Thus, one advantage of a single transgenic model may be the detection of minor allelic differences in Tg reactivity. In human autoimmune disease, DQA1*0102/DQB1*0602 has been reported to play either a protective (Type I diabetes) or a predisposing (multiple sclerosis) role (see [37]). A protective role of murine class II genes can be shown in EAT by transgenic technology. The introduction of an Ea transgene into susceptible As mice reduced thyroiditis severity following mTg immunization [26]. Conversely, the Ab class II gene also reduced thyroid infiltration in hTg-induced EAT in Ea-transgenic mice [13]. To determine if the EAT-resistant DQ6 (0601) allele would influence the response to hTg in DQ8⫹ mice, we immunized DQ8/DQ6 double transgenic mice with hTg. The extent of thyroid infiltration was not different from DQ8⫹ mice given the low and high hTg dose, although the incidence was 11% (3/26) and 80% (4/5), respectively. However, because of the lack of effect in the high-dose, albeit small, group, we tentatively concluded that DQ6 had little protective role in hTg-
HLA-DR and -DQ Polymorphism in EAT
induced EAT in DQ8⫹ mice. DQ8/DQ6 double transgenic mice may show gene complementation in other autoimmune diseases. Whereas DQ8⫹ mice are susceptible to collagen-induced arthritis and DQ6⫹ mice are resistant, auricular chondritis after Type II collagen immunization has been described in the double transgenic mice [20]. In summary, by using single HLA class II transgenic mice, DRB1 and DQ polymorphism has been shown for EAT induced with hTg. Whereas DR3 and DQ8 alleles were susceptible, DR2, DR4 and DQ6 alleles were resistant. Although the transgenic mouse model harbors only one human component, the HLA class II molecules, and hTg is not a self autoantigen, hTg and mTg share many thyroiditogenic epitopes as shown in conventional strains. Yet, in contrast to conventional strains, where susceptibility to self Tg and heterologous Tgs goes handin-hand, DQ8⫹ mice develop moderate thyroiditis only after hTg induction, whereas DR3⫹ mice were susceptible to both hTg and mTg [7]. The discernible differences in DQ8 and DR3 permissiveness for hTg and mTg likely reflect differences in epitope presentation by HLA molecules and their influence on epitope recognition by the resultant TCR repertoire. Thyroid infiltration due to effector T-cell recognition could facilitate the search for shared, thyroiditogenic epitopes on hTg. Moreover, because DR3 and DQ8 are not known to be in linkage disequilibrium, unlike DR3 with DQ2 and DR4 with DQ8 [37], it would be interesting to test them as double transgenes in mice to determine if their coexpression and interaction would affect EAT development. ACKNOWLEDGMENTS
Sincerest thanks are extended to Ms. J. Hanson and her staff for the breeding and care of transgenic mice, Ms. N. MaplesVolhardt and A.M. Mazurco for excellent histology sections, Dr. J. Flynn for reviewing the manuscript, and Dr. C. Jeffries for S. enteritidis LPS. This work was supported in part by the National Institutes of Health Grants DK45960 (Y. M. Kong) and AI14764 for HLA transgenic mice (C. S. David), and a grant from the St. John Hospital and Medical Center (Y. M. Kong). Presented in part at the Experimental Biology 1999 meeting in Washington, D.C., April 17–21, 1999 (FASEB J 13:A1000, 1999).
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5.
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7.
8. 9.
10.
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13.
14.
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ABBREVIATIONS Ab0 class II negative mice 2m⫺ beta2-microglobulin⫺ (class I negative mice) EAT experimental autoimmune thyroiditis HT Hashimoto’s thyroiditis
INTRODUCTION After induction with thyroglobulin (Tg) in genetically susceptible mice, experimental autoimmune thyroiditis
From the Department of Immunology and Microbiology (Q.W., Y.M.K), Wayne State University School of Medicine, Detroit, MI; the Division of Immunopathology (R.S., J.C.P., A.A.G.), St. John Hospital and Medical Center, Detroit, MI; and the Department of Immunology (C.S.D.), Mayo Clinic, Rochester, MN, USA. Address reprint requests to: Dr. Yi-chi M. Kong, Department of ImmuHuman Immunology 63, 301–310 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.
mAb in vitro. By contrast, HLA-DRB1*1502 (DR2) and *0401 (DR4) transgenes contributed little to hTg induction. Similarly, DQA1*0103/DQB1*0601 or DQA1* 0103/DQB1*0602 (DQ6) transgenic Ab0 mice were unresponsive to hTg induction and carried no detectable influence in DQ8/DQ6 double transgenic mice. Thus, both HLA-DR and -DQ polymorphism exists for hTg in autoimmune thyroiditis. The use of defined single or double transgenic mice obviates the complications seen in polygenic human studies. Human Immunology 63, 301–310 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: experimental autoimmune thyroiditis; EAT; HLA polymorphism in EAT; hTg in HLA transgenic mice; EAT in HLA transgenic mice; HLA polymorphism in EAT
hTg LPS mTg Tg
human thyroglobulin lipopolysaccharide mouse thyroglobulin thyroglobulin
(EAT), an animal model for Hashimoto’s thyroiditis (HT), exhibits mononuclear cells in the thyroid infil-
nology and Microbiology, Wayne State University School, of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201, USA; Tel: (313) 577-1589; Fax: (313) 577-1155; E-mail: [email protected]. Received September 18, 2001; revised December 18, 2001; accepted January 3, 2002.
0198-8859/02/$–see front matter PII S0198-8859(02)00360-9
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trate, Tg autoantibody production, and T-cell proliferative response in vitro [1–3]. In addition to mouse (m) Tg, Tg from heterologous species, such as human (h), can also induce EAT, although thyroiditis is generally less extensive, indicating that mTg contains additional epitopes for the mouse not found on hTg [4, 5]. Susceptibility to both mTg and hTg goes hand-in-hand in conventional strains and has been clearly mapped to H2A class II genes [5, 6]. In contrast, human leukocyte antigen (HLA) association with HT has been controversial, despite improved typing techniques, and delineation in polygenic humans was further complicated by linkage disequilibium [7–12]. For example, the early typing techniques two decades ago were by serology and only polyclonal antibodies to HLA-DR antigens were available for patient studies [8]. Linkage disequilibrium between certain DR and DQ genes were recognized later but its influence was difficult to assess, probably due to additional contributing factors, including ethnic differences, multiple epitopes on Tg [5], and the involvement of other thyroid antigens, such as thyroid peroxidase. Using Tg as the prototype antigen known to induce murine thyroiditis with susceptibility linked to class II genes, we examined single HLA class II transgenic mice in the absence of other class II genes. This protocol would permit the detection of potential susceptible and resistant genes, and the eventual examination of gene complementation when double transgenic mice are evaluated. We first addressed potential HLA-DR association with HT by introducing HLA-DRA/DRB1*0301 (DR3) transgene into resistant B10.M (H2f) or class II negative (Ab0) mice and observed severe thyroid infiltration following immunization with either hTg or mTg [7]. These observations supported some patient studies implicating HLA-DR3 [8 –12]. Moreover, because HLADRB1*1502 (DR2) transgenic mice did not respond to EAT induction by mTg, these studies demonstrated that HLA-DRB1 polymorphism to mTg-induced EAT can be determined by introducing single HLA-DR transgenes [7]. At the time, DRB1 polymorphism in hTg-induced EAT could not be delineated, because the H2Eb molecules present in DR2-transgenic mice, resulting from the introduction of E␣k and DR2 transgenes, responded to hTg, although not to mTg [13]. We report here a different strategy to test DRB1 polymorphism in hTginduced EAT by using a recipient strain that does not express H2E molecules. As to DQ polymorphism, certain HLA-DQ alleles have also been associated with HT in recent years. However, such associations are complicated by linkage disequilibrium with the DRB1 locus, which include DR3, as well as *0401 (DR4) and *0501 (DR5); the latter two have also been implicated in studies of association with
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HT [11,12,14]. On the other hand, in experimental models of other autoimmune diseases, the introduction of single DQA1*0301/DQB1*0302 (DQ8) transgene has led to the demonstration of susceptibility to collagen-induced arthritis [15] and myasthenia gravis [16]. We tested the role of DQ8 transgene in class II-negative Ab0 as well as class I and II negative 2-microglobin(⫺/⫺) Ab0 mice for their response to hTg-induced EAT. HLA-DQ polymorphism was examined by comparing Ab0 mice given the DQ8 transgene or DQA1*0103/DQB1*0601 or *0602 (DQ6) transgene. In addition, we tested the mutual influence of DQ8 and DQ6 expression by examining DQ8/DQ6 double transgenic mice for their susceptibility to hTg-induced EAT. MATERIALS AND METHODS Antigen and Adjuvant The mTg and hTg were prepared from frozen thyroids as described previously by fractionation of thyroid extracts on a Sephadex G-200 column (Pharmacia Inc., Piscataway, NJ, USA) [17]. All preparations were checked for lipopolysaccharide (LPS) contamination with Limulus amebocyte lysate (Associates of Cape Cod, Woods Hole, MA, USA); 20 g antigen contained ⬍ 1-ng LPS [3]. Salmonella enteritidis LPS as adjuvant was prepared by trichloroacetic acid precipitation. Generation of Transgenic Mice All mice were bred and maintained in the Immunogenetics Mouse Core Facility at the Mayo Clinic until shipment. They were housed in specific pathogen-free facility and given acidified, chlorinated water at least 1 week prior to use at 10 –16 weeks of age. The HLA-DQ8 (DQA1*0301/DQB1*0302) transgene in B10.M mice was introduced by mating into two strains of mice deficient in endogenous MHC molecules as described previously [15]. The first was the Ab0 mouse, which expressed neither surface IA (mutant A chain) nor surface IE (nonfunctional Ea gene in this H2b haplotype) molecules [18]. The second strain lacked both surface class I and class II molecules due to the introduction by mating of 2-microglobulin knock-out gene (2m⫺/⫺) [19] into Ab0/DQ8 mice. The breeding scheme to introduce HLADQ6 (DQA1*0103/DQB1*0601 or DQA1*0103/ DQB1*0602) transgene or DQ6/DQ8 double transgene into Ab0 mice has been described [20]. Transgene-positive mice were determined by PCR of PBL with DQ allele-specific oligonucleotide primers and DQ8 or DQ6 expression in PBL by flow cytometry with HLA-DQ␣specific mAb IVD12 [21]. No hybrid MHC molecules formed between H2A␣b with HLA-DQ chain were detected with chain-specific mAbs, as described elsewhere [15]. The HLA-DR3 (DRA/DRB1*0301) trans-
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TABLE 1 HLA-DR2 (DRB1*1502) transgene does not alter the response of resistant B10.RFB3 mice to hTg induction of thyroiditis Thyroiditisa Transgene expression DR2⫹ DR2⫺ DR3⫹b
Number of mice with % thyroid involvement
Incidence
Anti-Tg (O.D. ⫾ SE)a 1:3200
0
⬎ 0–10
⬎ 10–20
⬎ 20–40
⬎ 40–80
Pos./total
Percent (%)
2.0 ⫾ 0.1 1.9 ⫾ 0.3 1.2 ⫾ 0.2b
13 4 —
2 1 2
— — —
— — 1
— — 1
2/15 1/5 4/4
13 20 100
Mice were immunized intravenously with 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. DR3 transgene was on B10.Ab0 background; anti-Tg titers were presented at 1:100 dilution, and were ⬍ 0.2 at 1:3200. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; mTg ⫽ mouse thyroglobulin; Tg ⫽ thyroglobulin. a
b
gene was introduced into Ab0 mice as described previously and made congenic by backcross to B10 mice [7]. However, because H2Eb molecules are permissive for EAT induction by hTg [13], the HLA-DR2 (DRB1*1502) and HLA-DR4 (DRB1*0401) transgene, which required the conserved H2E␣ chain for expression [7], were introduced into the B10.RFB3 strain by intercross with DR2 or DR4 transgenic Ab0 mice [22], followed by nine-generation backcross to B10.RFB3 mice. The B10.RFB3 strain was chosen because it is of the EAT-resistant f haplotype [7] and lacks a functional Eb (-chain) gene. Thus, functional Ef molecules that might respond to EAT induction by hTg were absent. The progenies were screened by the antiDRB1 mAb L227 [23]. Induction and Assay of EAT All transgenic mice were immunized on days 0 and 7 by tail vein injections of 40 or 100 g mTg or 100 or 200 g hTg, followed each time by 20 g LPS 3-h later. On day 28 sera, spleens, and thyroids were collected. Serum antibody titers to mTg or hTg were assayed by ELISA as described previously [24]. In brief, 96-well flat bottom plates (Immulon II; Dynatech Laboratory, Inc., Chantilly, VA, USA) were coated with Tg (1 g/well). Serum dilutions of 1:100 and 1:3200 were added. After incubation and washing, alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotech Assoc., Inc., Birmingham, AL, USA) was added as second antibody. Standard immune serum and normal mouse serum served as positive and negative controls. The spleens were used for in vitro proliferative assay to Tg (see below). The thyroids were processed, examined histologically and scored as illustrated elsewhere [25]. Percent thyroid infiltration was determined after examining 30 – 60 vertical sections (7–10 step levels) throughout both lobes: 0%, no infiltration; ⬎ 0%–10%, perivascular foci of infiltration; ⬎ 10%–20%, infiltration with follicular destruction; ⬎ 20%– 40%, multiple foci of follicular
destruction; and ⬎ 40%– 80%, extensive thyroid involvement. In Vitro Culture On day 28 postimmunization, spleen cells from mTg or hTg-primed mice were cultured at 37 °C and 5% CO2 air for 5 days in flat bottomed, 96-well tissue culture plates (6⫻105/well) with 40 g/ml Tg, as detailed previously [24, 25]. For blocking of Ag presentation, 20 l of culture supernatants containing anti-DQ␣ (IVD12) or irrelevant mAb was added to 200 l cultures with or without Tg. Culture medium consisted of RPMI 1640 with 25 mM HEPES buffer, supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (all GIBCO BRL, Grand Island, NY, USA), 50 M 2-mercaptoethanol (Sigma, St. Louis, MO, USA), and 1% normal mouse serum. After pulsing with 1.0 Ci of [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA, USA) for the final 18-h culture, the cells were harvested onto glass fiber filters. The [3H]thymidine uptake was presented as cpm (mean ⫾ SE) of triplicate cultures. RESULTS HLA-DRB1 Polymorphism is a Determinant in hTg-Induced EAT Earlier studies have reported that HLA-DRB1 polymorphism can be demonstrated for EAT induced with mTg by comparing DR3- and DR2-transgenic Ab0 mice; only DR3 molecules were found to be permissive [7]. Whereas DR3 molecules also presented hTg to induce EAT, the role of DR2 molecules, which were expressed by the pairing of conserved E␣k chain with the DR2 chain in E␣k- and DR2-transgenic Ab0 mice, could not be determined. The coexpressed H2E (E␣kEb) molecules were found to be permissive for EAT induction by hTg, but not by mTg [13]. We therefore introduced the transgene for DR2 or DR4 with E␣k into B10.RFB3 (H2f) mice, which are EAT-resistant and lack endoge-
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TABLE 2 HLA-DR4 (DRB1*0401) transgene has little effect on the response of resistant B10.RFB3 mice to mTg or hTg induction of thyroiditis Thyroiditisa
Antigen
Transgene expression
Anti-Tg (O.D. ⫾ SE) 1:5000
mTg
DR4⫹ DR4⫺ DR4⫹ DR4⫺
0.4 ⫾ 0.4 0.2 ⫾ 0.2 1.1 ⫾ 0.3 0.7 ⫾ 0.1
a
hTg
Number of mice with % thyroid involvement
Incidence
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent (%)
12 14 12 15
4 4 11 7
1 — — —
5/17 4/18 11/23 7/22
29 22 48 32
Mice were immunized intravenously with 40 g mTg or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera for anti-mTg or anti-hTg and thyroids were assayed on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; mTg ⫽ mouse thyroglobulin.
a
nous E. Table 1 illustrates that the animals remained essentially EAT-resistant whether DR2 molecules were expressed or not, as most mice displayed no thyroid infiltration. Because hTg is a heterologous Tg with foreign epitopes for B10.RFB3 mice, the antibody response remained high and little affected by the presence or absence of DR2 molecules. In contrast, DR3-transgenic B10.Ab0 mice, which had both DR␣ and DR3 without the need for E␣k, developed mild to severe thyroiditis after hTg induction as reported previously [7]. In E␣k- and DR4-transgenic B10.RFB3 mice, both mTg (40 g) and hTg (100 g) were used for immunization at the same dosage previously used for DR2- or DR3-transgenic mice [7]. As listed in Table 2, most animals in both the DR4⫹ and DR4⫺ groups did not respond to mTg immunization; 22%–29% incidence consisted of focal infiltration, except one mouse with mild thyroiditis. A little higher incidence of focal infiltration was observed in the DR4⫹ than the DR4⫺ group
immunized with hTg. Antibody titers to the immunizing antigen were higher in the hTg-immunized group than the mTg-immunized group, again probably due to the foreign epitopes in hTg. Because all the DR3-transgenic mice developed mild to severe EAT in contrast to DR4- and DR2-transgenic mice, DRB1 polymorphism is a determinant for susceptibility to hTg-induced EAT, similar to mTg [7]. HLA-DQ8-Transgenic Class II Negative Ab0 Mice are Moderately Susceptible to hTg-Induced EAT but are Resistant to mTg-Induced EAT A previous article reported that DQ8⫹ Ab0 mice were resistant to EAT induction by mTg [26]. Therefore, DQ8⫹ Ab0, DQ8⫺ Ab0, and control Ab0 mice were immunized with hTg, comparing their responses to mTg. Whereas mTg injection led to minimal thyroid involvement and relatively low antibody production in DQ8⫹ Ab0 mice, hTg immunization generated a higher
TABLE 3 hTg, but not mTg, induces moderate thyroiditis in HLA-DQ8-transgenic class II negative H2Ab0 mice Thyroiditisa
Antigen hTg hTgb mTg (40 g) mTg (100 g)
MHC expression DQ8⫹ Ab0 DQ8⫹ DQ8⫺ DQ8⫹ DQ8⫹
Anti-Tg (O.D. ⫾ SE)a
Number of mice with % thyroid involvement
1:100
1:3200
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent (%)
1.1 ⫾ 0.3 0.3 ⫾ 0.1 1.3 ⫾ 0.3 0.3 ⫾ 0.1 0.3 ⫾ 0.3 0.9 ⫾ 0.6
0.7 ⫾ 0.2 0.2 ⫾ 0.1 0.8 ⫾ 0.2 0.1 ⫾ 0.1 ⬍ 0.2 0.3 ⫾ 0.3
2 4 4 3 7 3
2 1 1 — 1 1
2 — 2 — — —
4/6 1/5 3/7 0/3 1/8 1/4
67 20 43 0 13 25
Incidence
a Mice were immunized intravenously with 40 or 100 g mTg, or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. b Experiment comparing littermates. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
HLA-DR and -DQ Polymorphism in EAT
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FIGURE 1 T-cell proliferative response to human thyroglobulin (hTg) was blocked by anti-DQ monoclonal antibody (mAb) in HLA-DQ transgenic Ab0 mice. (A) Spleen cells from DQ8⫹; (B) 2m⫺ DQ8⫹; (C) DQ6(0601)⫹; and (D) DQ8⫹ DQ6(0601)⫹ transgenic Ab0 mice or control Ab0 mice, immunized with 100 g hTg ⫹ 20 g lipopolysaccharide (LPS) on days 0 and 7, were tested on day 28. The [3H]thymidine uptake was measured after 5 days of culture (6⫻105/well). Open bars represent background cpm of cultures in the presence of only anti-DQ␣ (IVD12, supernatant 1:10) or anti-IAb (AF6-120, supernatant 1:10) mAb; black bars represent cpm of cultures in the presence of 40 g/ml hTg plus mAb. Control T-cell proliferation in Ab0 mice was performed with (black bar) or without (open bar) hTg.
incidence with some mice developing mild infiltration and higher antibody levels (Table 3). However, only 43%– 67% of the animals from both experiments had thyroid involvement. T-cell proliferative response to mTg or crossreaction to hTg was not observed in either mTg-immunized group (data not shown). In contrast, there is a strong proliferative response to hTg. Class II presentation of hTg was blocked by mAb to DQ␣, but not by the control mAb to IAb (Figure 1A). It appears that DQ8 molecules can present hTg epitopes not shared by mTg, unlike DR3 molecules which can present epitopes shared by both Tgs [7]. Absence of Class I Genes in HLA-DQ8-Transgenic Ab0 Mice Results in Higher Incidence of EAT Earlier studies in CBA/J mice with mTg-induced EAT have reported that both CD4⫹ and CD8⫹ T cells are involved in thyroiditis development [27–29]. On the other hand, little difference in the incidence or severity of thyroiditis was discerned in 2m⫺ CBA mice deficient in CD8⫹ cells, indicating a compensatory role for CD4⫹ cells and macrophages [30]. To determine the requirement for CD8⫹ cells in EAT development in DQ8transgenic Ab0 mice, we immunized 2m⫺ DQ8⫹ Ab0 mice with hTg, using mTg as low responder control. In Table 4 (experiment 1), all four mice developed focal to mild thyroiditis; as expected, 2m⫺ DQ8⫺ Ab0 mice did not respond. In experiment 2, a higher incidence (73%) of 2m⫺ DQ8⫹ mice manifested thyroid infiltra-
tion, compared with DQ8⫹ mice. However, a minority revealed no thyroid infiltration, as in DQ8⫹ mice here and in Table 3. Antibody titers were observed in all the mice, with the exception of those in experiment 1, which were unexpectedly low for mice with higher than normal CD4⫹ T cell repertoire. Similar to DQ8⫹ mice, the T-cell proliferative response was blocked by mAb to DQ␣ (Figure 1B). To determine whether the incidence and severity of thyroiditis could be increased by doubling the hTg dose to 200 g and changing the adjuvant from LPS to CFA, we immunized both DQ8⫹ and 2m⫺ DQ8⫹ mice. Table 5 illustrates that, whereas the incidence of EAT remained higher in 2m⫺ DQ8⫹ mice than in DQ8⫹ mice, the overall response in each strain was little affected by the higher dose or the adjuvant. Many mice still did not display thyroid infiltration, despite high antibody levels. The data in DQ8-transgenic mice confirmed the findings in conventional, EATresistant strains that antibody levels do not correlate with thyroiditis development [1, 7]. HLA-DQ6-Transgenic Class II Negative Ab0 Mice are Resistant to Both mTg and hTg Immunization We next tested DQ6-transgenic mice to determine if DQ polymorphism was a determining factor in EAT-susceptibility. Two DQB1 genes were paired with the same DQA1 gene in two groups of Ab0 mice and immunized with hTg, using mTg as low responder control [26]. Table 6 reveals that only 0601⫹ (2/20) mice developed
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TABLE 4 hTg induction in HLA-DQ8-transgenic class I negative (2m⫺/⫺) H2Ab0 mice increases the incidence of thyroiditis Thyroiditisa
Experiment number
Antigen
1
mTg hTg
2
hTg
MHC expression 2m⫺ DQ8⫹ 2m⫺ DQ8⫹ 2m⫺ DQ8⫺ DQ8⫹ 2m⫺ DQ8⫹ Ab0
Number of mice with % thyroid involvement
Anti-Tg (O.D. ⫾ SE)a 1:100
1:3200
0
0.8 ⫾ 0.5 0.2 ⫾ 0.2 4 0.1 ⫾ 0.0 ⬍ 0.2 — ⬍ 0.2 ⬍ 0.2 4 ⬎ 1.8 ⫾ 0.2 1.7 ⫾ 0.1 4 1.7 ⫾ 0.1 0.3 ⫾ 0.2 3 0.3 ⫾ 0.1 ⬍ 0.2 8
Incidence
⬎ 0–10
⬎ 10–20
⬎ 20–40
Pos./total
Percent (%)
— — — 5 — —
1 3 — 1 7 —
— 1 — — 1 —
1/5 4/4 0/4 6/10 8/11 0/8
20 100 0 60 73 0
a Mice were immunized intravenously with 40 g mTg, or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
focal infiltration, whereas 0602⫹ mice (0/5) were negative. Moreover, similar to mTg-immunized mice, hTg was not immunogenic in 0602⫹ mice; little antibody production was detected and no proliferative response to hTg was detected (data not shown). By contrast, T cells from 0601⫹ mice responded strongly to hTg in vitro and its presentation was blocked by mAb to DQ␣ (Figure 1C). The differential response to hTg with thyroiditis in DQ8⫹ versus DQ6⫹ mice suggests that HLA-DQ polymorphism is a determinant factor in the extent of EAT susceptibility. To determine if the presence of a resistant DQ6 transgene would influence thyroiditis development in DQ8transgenic Ab0 mice, DQ8/DQ6 (0601) double transgenic mice were immunized with 100 or 200 g hTg. Table 7 illustrates that the DQ8-mediated response was
little affected by the presence of DQ6 molecules. Antibody production (Table 7) and T-cell proliferative response (Figure 1D) remained in the range observed for single DQ8-transgenic groups. DISCUSSION The complications of DR/DQ linkage disequilibrium, varied typing techniques over the years, and ethnic differences have contributed to the controversy in HLA association with HT [31]. To address the question of HLA association, we have used HLA class II-transgenic mice, without endogenous murine class II molecules, and hTg as the putative autoantigen. Although hTg possesses shared as well as foreign epitopes for the mouse as seen from studies of conventional mouse strains, it is primar-
TABLE 5 Neither higher immunizing dose of hTg nor change of adjuvant increases the severity of thyroiditis in HLA-DQ8-transgenic Ab0 mice Thyroiditisa
Adjuvant
hTg dose
MHC expression
LPS
100 g
DQ8⫹ 2m⫺DQ8⫹ DQ8⫹ 2m⫺DQ8⫹ DQ8⫹ 2m⫺DQ8⫹ DQ8⫹ 2m⫺DQ8⫹
200 g CFA
100 g 200 g
Number of mice with % thyroid involvement
Anti-Tg (O.D. ⫾ SE)a
Incidence
1:100
1:3200
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent (%)
1.9 ⫾ 0.1 1.6 ⫾ 0.1 1.8 ⫾ 0.2 1.7 ⫾ 0.1 ⬎ 1.3 ⫾ 0.1 ⬎ 1.3 ⫾ 0.1 ⬎ 1.2 ⫾ 0.1 ⬎ 1.3 ⫾ 0.1
1.6 ⫾ 0.1 0.2 ⫾ 0.2 1.4 ⫾ 0.2 0.3 ⫾ 0.2 1.3 ⫾ 0.1 1.2 ⫾ 0.1 1.2 ⫾ 0.1 1.3 ⫾ 0.1
2 3 4 1 5 4 9 2
— 3 — 4 6 7 2 1
— — 3 2 1 3 — 3
0/2 3/6 3/7 6/7 7/12 10/14 2/11 4/6
0 50 43 86 58 71 18 67
Mice were immunized either with 100 or 200 g hTg, and 20 g LPS intravenously or subcutaneously with hTg in CFA into alternate thigh and hind footpad on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: CFA ⫽ complete Freund’s adjuvant; HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
a
HLA-DR and -DQ Polymorphism in EAT
307
TABLE 6 HLA-DQ6 (0601 or 0602) transgenic H2Ab0 mice are resistant to both mTg and hTg induction of thyroiditis Thyroitidisa
Antigen mTg hTg
Number of mice with % thyroid involvement
Anti-Tg (O.D. ⫾ SE)a
Incidence
MHC expression
1:100
1:3200
0
⬎ 0–10
Pos./total
Percent
DQ6⫹(0601) DQ6⫹(0602) DQ6⫹(0601) DQ6⫹(0602)
0.3 ⫾ 0.3 0.2 ⫾ 0.0 1.7 ⫾ 0.2 0.1 ⫾ 0.1
⬍0.2 ⬍0.2 1.0 ⫾ 0.1 ⬍0.2
6 2 20 5
— — 2 —
0/6 0/2 2/22 0/5
0 0 9 0
a Mice were immunized intravenously with 40 g mTg, or 100 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide; MHC ⫽ major histocompatibility complex; mTg ⫽ mouse thyroglobulin.
ily the shared, thyroiditogenic epitopes, some of which are known, that are responsible for thyroid infiltration [4, 5, 24]. Thus, based on thyroid infiltration, a hallmark for susceptibility, rather than antibody production and T-cell proliferative response to hTg, we have determined the relative capacity of several DR and DQ alleles to support EAT induction. The data demonstrate that DR and DQ polymorphism is a determinant for hTg-induced EAT. In the case of DRB1 alleles, whereas the DR3⫹ line was established with a DRA transgene [32], the expression of DR2 and DR4 chains required the aid of an Ea transgene to provide a DR␣ chain homologue, which also resulted in the co-expression of H2E molecules [7]. Because H2E molecules are permissive for hTg-induced EAT [13], we used an EAT-resistant strain, B10.RFB3, which lacks a functional Eb gene as transgene recipient. Even with high antibody titers in this resistant strain due to the many foreign epitopes on hTg, DR2 and DR4 molecules did not contribute significantly to thyroiditis development (Tables 1 and 2); only focal infiltration was observed in some mice and none in the majority, compared with DR3-transgenic mice (Table 1) [7]. More-
over, this DRB1*0401 gene carried a mutation at two residues in the 2 domain to provide better interaction with mCD4 [33], which could have led to the very high anti-hTg titers detectable at 1:5000, but the low thyroid pathology was consistent with a low responder strain of the f haplotype [7]. HLA-DQ genes are highly polymorphic at both the DQA1 and DQB1 loci. We concentrated on DQ8 and DQ6 genes which have been studied in several other autoimmune diseases. Unlike DR3 molecules, which are highly permissive for both hTg- and mTg-induced EAT [7], DQ8⫹ Ab0 mice responded poorly to EAT induction after mTg immunization. After hTg induction, the incidence increased to ⬃50%, averaging several groups (Tables 3–5), and a few animals showed moderate mononuclear cell infiltration involving ⬎ 10%–20% of the thyroid (Table 3). However, there were always a sizable numbers of animals in each experiment with no thyroid infiltration, despite the doubling of hTg dosage and the use of a strong adjuvant such as CFA (Table 5). These differences in thyroiditogenic response were apparently unrelated to the expression of DQ8 molecules in these mice, since each animal was monitored for DQ8 expres-
TABLE 7 HLA-DQ6 has little effect on hTg-induced thyroiditis in HLA-DQ8/DQ6(0601)-double transgenic class II negative H2Ab0 mice Thyroiditisa
hTg dose
MHC expression
100 g 200 g
DQ8⫹ DQ6⫹ DQ8⫹ DQ6⫹
Anti-Tg (O.D. ⫾ SE)a
Number of mice with % thyroid involvement
1:100
1:3200
0
⬎ 0–10
⬎ 10–20
Pos./total
Percent
1.6 ⫾ 0.2 1.3 ⫾ 0.3
0.7 ⫾ 0.1 0.6 ⫾ 0.3
23 1
2 3
1 1
3/26 4/5
11 80
Incidence
Mice were immunized intravenously with 100 or 200 g hTg and 20 g LPS 3-h later on days 0 and 7. The sera and thyroids were obtained on day 28. Abbreviations: HLA ⫽ human leukocyte antigen; hTg ⫽ human thyroglobulin; LPS ⫽ lipopolysaccharide. a
308
sion by FACS analysis prior to use and expression in ⬃20% of the PBL was quite uniform (data not shown). With CIA, the incidence in DQ8⫹ Ab0 mice was ⬃70% [15]. Interestingly, in CD8-deficient, 2m⫺ DQ8⫹ Ab0 mice, there was a notable increase in the incidence of thyroiditis. Moreover, in first examination, it would appear that thyroid infiltration was more pronounced in 2m⫺ DQ8⫹ Ab0 mice than in DQ8⫹ Ab0 mice (Table 4, experiment 2). However, even with the use of higher doses of hTg and CFA as adjuvant, the extent of thyroid infiltration did not rise above 20% and the two strains showed quite similar responses (Table 5). Because of the sizable number of mice in the different groups not displaying any thyroid infiltration, the differences between groups in thyroid involvement are not statistically significant by a nonparametric test (Mann-Whitney). Similar to 2m⫺ CBA mice in which EAT pathology was not diminished [30] despite the known involvement of both CD4⫹ and CD8⫹ cells in wild type mice [27–29], CD4⫹ cells apparently can compensate for the lack of cytotoxic CD8⫹ cells in mediating destruction in conjunction with macrophages [27]. That DQ8-transgenic mice developed only moderate thyroiditis and in only 50%–70% of animals with or without the CD8 subset are in distinct contrast to DR3transgenic mice with 83%–100% incidence and many up to 80% thyroid involvement [7]. At least three reasons might help explain the low and nonuniform response of DQ8-transgenic mice. First, the T-cell receptor (TCR) repertoire selection and deletion by DR3 and DQ8 molecules are different. For example, similar to H2E molecules, albeit with different efficiency, DR3 molecules mediate the deletion of V5⫹ and 11⫹ T cells, but with lower selection of V6⫹ cells; in addition, DR3⫹ mice also have low V13⫹ cells [32]. In contrast, a sampling of the V repertoire in DQ8⫹ mice did not reveal any notable differences from conventional H2A⫹E⫺ mice [15]. Second, DQ8 molecules likely bind and present different Tg epitopes from DR3 as well as a more limited number. hTg and mTg have ⬃73% homology [34] and both induce EAT in DR3⫹ mice, indicating the recognition of shared thyroiditogenic epitopes [7]. In DQ8⫹ mice, mTg is hardly thyroiditogenic, suggesting a more limited presentation of epitopes primarily on hTg. From studies with mTg in CBA mice, we have described that mTg contains both shared as well as unique thyroiditogenic epitopes [5, 24]. It is possible that hTg also contains hTg-specific, thyroiditogenic epitopes processed by DQ8⫹ antigen-presenting cells. This presentation is DQ-restricted; the response to hTg was blocked in vitro by anti-DQ mAb (Figure 1). Because the only humanized component in these mice is the DQ8 transgene,
Q. Wan et al.
antigen presentation may be less efficient with mCD4 cells, if DQ8 molecules have a higher affinity for hCD4⫹ than mCD4⫹ T cells. We therefore tested DQ8⫹ hCD4⫹ mCD4-knockout mice, but the incidence and severity of EAT after hTg immunization were unchanged (data not shown). Third, from adoptive transfer studies, we know that a certain number of donor Tg-reactive T cells are required to achieve and maintain thyroid inflammation [2, 24]. However, in DQ8⫹ mice, even with twice the number of hTg-activated cells commonly used for thyroiditis transfer, only 4/10 recipient mice exhibited thyroid involvement similar to actively immunized mice (data not shown). Since there were more than adequate activated cells transferred without increasing the incidence of thyroiditis, DQ8/peptide complexes do not seem to engage sufficient thyroiditogenic T cells to reach above the threshold required for infiltration. This observation favors the idea of a limited number of cross-reactive epitopes presentable by DQ8 molecules. The need for additional costimulatory molecules may also play a role in enhancing T cell activation by DQ8/peptide complexes. For DQ8-transgenic mice to develop spontaneous diabetes in the NOD mouse, B7-1 expression on pancreatic  cells appeared necessary, but still only 81% of the mice had diabetes [35]. HLA-DQ polymorphism may be a determining factor for EAT, similar to DRB1. DQ6-transgenic mice were resistant to both hTg and mTg induction, as reported for myasthenia gravis [16] and collagen-induced arthritis [36]. With hTg, the DQA1*0103/DQB1*0601 allele supported antibody production and T cell response in vitro, but *0602 did not, whereas mTg was not immunogenic in either strain (Table 6). Thus, one advantage of a single transgenic model may be the detection of minor allelic differences in Tg reactivity. In human autoimmune disease, DQA1*0102/DQB1*0602 has been reported to play either a protective (Type I diabetes) or a predisposing (multiple sclerosis) role (see [37]). A protective role of murine class II genes can be shown in EAT by transgenic technology. The introduction of an Ea transgene into susceptible As mice reduced thyroiditis severity following mTg immunization [26]. Conversely, the Ab class II gene also reduced thyroid infiltration in hTg-induced EAT in Ea-transgenic mice [13]. To determine if the EAT-resistant DQ6 (0601) allele would influence the response to hTg in DQ8⫹ mice, we immunized DQ8/DQ6 double transgenic mice with hTg. The extent of thyroid infiltration was not different from DQ8⫹ mice given the low and high hTg dose, although the incidence was 11% (3/26) and 80% (4/5), respectively. However, because of the lack of effect in the high-dose, albeit small, group, we tentatively concluded that DQ6 had little protective role in hTg-
HLA-DR and -DQ Polymorphism in EAT
induced EAT in DQ8⫹ mice. DQ8/DQ6 double transgenic mice may show gene complementation in other autoimmune diseases. Whereas DQ8⫹ mice are susceptible to collagen-induced arthritis and DQ6⫹ mice are resistant, auricular chondritis after Type II collagen immunization has been described in the double transgenic mice [20]. In summary, by using single HLA class II transgenic mice, DRB1 and DQ polymorphism has been shown for EAT induced with hTg. Whereas DR3 and DQ8 alleles were susceptible, DR2, DR4 and DQ6 alleles were resistant. Although the transgenic mouse model harbors only one human component, the HLA class II molecules, and hTg is not a self autoantigen, hTg and mTg share many thyroiditogenic epitopes as shown in conventional strains. Yet, in contrast to conventional strains, where susceptibility to self Tg and heterologous Tgs goes handin-hand, DQ8⫹ mice develop moderate thyroiditis only after hTg induction, whereas DR3⫹ mice were susceptible to both hTg and mTg [7]. The discernible differences in DQ8 and DR3 permissiveness for hTg and mTg likely reflect differences in epitope presentation by HLA molecules and their influence on epitope recognition by the resultant TCR repertoire. Thyroid infiltration due to effector T-cell recognition could facilitate the search for shared, thyroiditogenic epitopes on hTg. Moreover, because DR3 and DQ8 are not known to be in linkage disequilibrium, unlike DR3 with DQ2 and DR4 with DQ8 [37], it would be interesting to test them as double transgenes in mice to determine if their coexpression and interaction would affect EAT development. ACKNOWLEDGMENTS
Sincerest thanks are extended to Ms. J. Hanson and her staff for the breeding and care of transgenic mice, Ms. N. MaplesVolhardt and A.M. Mazurco for excellent histology sections, Dr. J. Flynn for reviewing the manuscript, and Dr. C. Jeffries for S. enteritidis LPS. This work was supported in part by the National Institutes of Health Grants DK45960 (Y. M. Kong) and AI14764 for HLA transgenic mice (C. S. David), and a grant from the St. John Hospital and Medical Center (Y. M. Kong). Presented in part at the Experimental Biology 1999 meeting in Washington, D.C., April 17–21, 1999 (FASEB J 13:A1000, 1999).
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