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Human DQ8 Can Substitute for Murine I-A^g7 in the Selection of Diabetogenic T Cells Restricted to I-A^g71

Li Wen^2,*, F. Susan Wong, Robert Sherwin^* and Conchi Mora

^* Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; Department of Pathology and Microbiology, University of Bristol, Bristol, United Kingdom; and Laboratori Experimental de Diabetis, Hospital Clinic Universitari, Barcelona, Spain

	Abstract

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The strong association of type 1 diabetes with specific MHC class II genes, such as I-A^g7 in nonobese diabetic mice and HLA-DQ8 in humans, suggests that MHC class II molecules play an important role in the development of the disease. To test whether human DQ8 molecules could cross the species barrier and functionally replace their murine homolog I-A^g7, we generated DQ8/BDC2.5 transgenic mice. We have shown that BDC2.5 transgenic T cells are selected on DQ8 in the thymus and cause diabetes in a manner similar to that seen when the T cells are selected on H2^g7. Splenocytes from DQ8/BDC2.5 mice also showed reactivity toward islets in vitro as seen in H-2^g7/BDC2.5 mice. We conclude that DQ8 molecules not only share structural similarity with the murine homolog I-A^g7, but also can cross the species barrier and functionally replace I-A^g7 molecules to stimulate diabetogenic T cells and produce diabetes.

	Introduction

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Type 1 diabetes mellitus (T1DM),³ also known as insulin-dependent diabetes mellitus, is a polygenic disease, but many studies have confirmed that the primary locus defining genetic susceptibility is found encoded within the MHC region on human chromosome 6 (1, 2, 3). Although the extended MHC haplotype appears to be important in disease development, studies have shown that the DQ locus is particularly linked to the pathogenesis of T1DM. The DQ allele that has been most studied in this context is DQA1*0301/DQB1*0302 (DQ8). Much emphasis has been placed on the fact that this allele has an alanine residue at position 57 of the -chain. Other DQ alleles that encode an aspartic acid at this position are neutral or have been linked to protection from diabetes (4, 5).

In the nonobese diabetic (NOD) mouse a widely studied murine model of human T1DM, the MHC is also the most important genetic susceptibility locus. Interestingly, there is considerable sequence homology between DQ8 and the only MHC class II molecule expressed in NOD mice, I-A^g7. Unlike most mouse strains, this I-A molecule is characterized by substitution of a noncharged amino acid for the charged aspartic acid residue at position 57 of the -chain (as in DQ8), in this case, serine (6, 7).

The crystal structure of I-A^g7 has recently been reported (8, 9). Based on these data it has been suggested that I-A^g7 binds peptides promiscuously, and this allows a different repertoire of peptides to be presented compared with the homologous I-A^d (8, 10). It has been inferred that a larger P9 pocket can specifically accommodate both negatively charged residues and residues with bulky side chains. Peptide binding studies have shown a preference for negatively charged residues (11, 12), and this may contribute to the fact that I-A^g7 is associated with diabetes. Similarly, the structure of DQ8 has also been recently crystallized together with an insulin peptide (13), and a striking similarity has been demonstrated between DQ8 and I-A^g7 binding pockets despite some sequence differences. DQ8 has an alanine residue at position 57 and also has a large P9 pocket that seems to have a preference for negatively charged residues at this position (14). Recently, a comparison was made of the peptides that bind to I-A^g7 compared with those bound by DQ8. This study showed that peptides from three putative autoantigens in diabetes, insulin, glutamic acid decarboxylase 65, and heat shock protein 60, can bind to DQ8 and I-A^g7 with similar, although not identical, binding specificity (15).

In view of the considerable similarities seen between DQ8 and I-A^g7, we examined whether the human and mouse MHC molecules most closely associated with diabetes could select for diabetogenic mouse CD4 T cells. We took advantage of the fact that the BDC2.5 T cell transgenic mouse (16) requires I-A^g7 for selection, but diabetes occurs at a relatively higher frequency and earlier when this MHC molecule is expressed on a C57BL/6 genetic background (17). To test our hypothesis that DQ8 might be able to substitute for I-A^g7 and induce positive selection of the BDC2.5 T cells, we crossed BDC2.5 TCR transgenic mice to our HLA-DQ8 transgenic mice deficient in mouse MHC class II so that the only MHC class II molecules present were the transgenic DQ8. We used these mice to study selection of T cells and development of diabetes. Our studies have shown that DQ8 not only shares structural similarity with I-A^g7 but also is able to functionally substitute for I-A^g7, thereby providing in vivo biological evidence supporting the crystal structure of DQ8 molecules recently reported by Lee et al. (13). We believe that this animal model could be a very useful tool for studying MHC-TCR interactions in diabetes and, most importantly, for the development of effective immunotherapy.

	Materials and Methods

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Generation of DQ8⁺/BDC2.5 transgenic mice

HLA-DQA1*0301/DQB1*0302 (DQ8) transgenic, murine MHC class II molecule-deficient (mII^-) C57BL/6 mice were generated as described previously (18). The BDC2.5 TCR transgenic mouse on the B6.H-2^g7 (H-2^g7/BDC2.5) genetic background was provided by Drs. C. Benoist and D. Mathis (Harvard School of Medicine, Boston, MA) (17). DQ8 mice were then bred with H-2^g7/BDC2.5, and their offspring that were positive for DQ8 and/or BDC2.5 transgene(s) were intercrossed. The following eight types of mice were used for this study that were derived from the intercross: 1) DQ8⁺BDC2.5⁺ (mII^-H-2^g7-), 2) H-2^g7+BDC2.5⁺ (mII^-), 3) DQ8⁺H-2^g7+BDC2.5⁺ (mII^-), 4) DQ8^-BDC2.5⁺ (mII^-H-2^g7-), 5) DQ8^-H-2^g7-BDC2.5⁺, 6) DQ8⁺BDC2.5^- (the same as DQ8 mice), 7) H-2^g7+BDC2.5^- (the same as H-2^g7 congenic mice), and 8) DQ8⁺H2^g7+BDC2.5^-. The expressions of the DQ8, murine class II I-A^b molecules, and H-2^g7 were screened for by flow cytometric analysis of PBL using mAbs to DQ (IVD12; American Type Culture Collection, Manassas, VA), I-A^b (212.A1; BD PharMingen, San Diego, CA), K^d (SF1-1.1; BD PharMingen), and I-A^g7 (10.2.16; C. Janeway, Jr., Yale University, New Haven, CT), respectively. The BDC2.5 transgene was screened for by PCR (16) and was confirmed by flow cytometry using mAb to V4 (BD PharMingen).

The mice were housed in specific pathogen-free facilities, and all experiments were undertaken in accordance with approved Yale animal care and use committee protocols.

RT-PCR for detection of TCRV mRNA

Total RNA was prepared by TRIzol (Life Technologies, Grand Island, NY) from purified CD4⁺ T cells using magnetic beads. Approximately 1 µg RNA was reverse transcribed into single-strand cDNA using an oligo(dT) primer (Amersham Pharmacia Biotech, Piscataway, NJ) and Moloney murine leukemia virus reverse transcriptase (Life Technologies). One microliter of the cDNA (from 50 µl) was amplified with primers specific for V 1–10 and C. The PCR were performed with hot start, followed by 35 cycles of denaturing at 94°C, annealing at 55°C, extension at 72°C, and a final extension for 7 min. PCR products were analyzed on 1.5% agarose gels. Primers were synthesized in the Keck Facility of Yale University, and the primer sequences are (5'3'): V1, -AGCAGAGCCCAGAATCCCTC-; V2, -GACAGCAATTCTGAACTGCA-; V3, -CGACACCTTATCTGTTCTGA-; V4, -CCCGGAGAAGGTCCACAGCT-; V5, -GGACAAGGATTCACTGTCCT-; V6, -CACAACCAGATTCAATGGAA-; V7, -AGAACTCACCCTGGACTGTT-; V8, -CTCAACGAAGCCCCTCAGGT-; V9, -GCTGCGACGTTCCTTAGTGA-; V10, -CGCAGCTCTTTGCACATTTC-; and C, -ATATCTTGGCAGGTGAAGCTTGT.

Isolation of pancreatic islets

The pancreas was placed in a sterile vial containing full-strength collagenase (0.15%; Roche, Indianapolis, IN) and placed in a shaking water bath at 37°C for 20 min. The reaction was then quenched with HBSS (Life Technologies) containing 0.0375% BSA (Life Technologies), 8.75 µmol/liter HEPES, 0.25% DNase (Sigma-Aldrich, St. Louis, MO), 12.5 U/ml penicillin, and 12.5 µg/ml streptomycin, after which the digestion was repeated using a half-strength collagenase (0.075%) solution. Thereafter, islets were hand-picked using a dissecting microscope; this procedure was repeated two or three times until the islets were free of acinar tissue.

Purification of T cells

Total splenocytes, after removing erythrocytes, were incubated with magnetic beads conjugated with goat anti-mouse IgG/IgM (PerSeptive Diagnostics, Cambridge, MA) on ice with gentle agitation for 45 min. B cells were removed by using a magnetic plate (PerSeptive Diagnostics). The purity of the CD3⁺ population using this method is routinely >90%.

Purification of CD4⁺ T cells

Total splenocytes after removing erythrocytes were incubated with anti-CD8 mAb (53-6.72, rat IgG2b) on ice for 30 min and washed once with cold Click’s medium. The cells were then incubated with magnetic beads conjugated with goat anti-mouse IgG/IgM and goat anti-rat IgG (PerSeptive Diagnostics) on ice with gentle agitation for 45 min. B cells and CD8⁺ cells were removed using a magnetic plate (PerSeptive Diagnostics). The purity of the CD4⁺ population using this method is routinely >90%. Erythrocytes were routinely removed by briefly exposing the cells to hypotonic solution, followed whenever necessary by further Ficoll (ICN Biomedicals, Costa Mesa, CA) separation.

Proliferation and cytokine assays

Splenic CD4 T cells (10⁵/well) were assayed for antigenic response against islets. Secreted cytokine proteins (IL-4, IL-6, IL-10, and IFN-) from those responses were measured by ELISA using mAbs and recommended protocols (BD PharMingen).

Immunohistology

Pancreas, kidney, liver, and salivary gland from all mice used in this study were examined by immunohistochemistry as described previously (19).

Microsatellite analysis of mice used in this study

Genetic background can influence immune responses. Therefore, to examine whether the mice used in the study were on the same genetic background, we performed microsatellite analysis using 10 genetic markers that were described for the original H-2^g7/BDC2.5 colony (17). These markers span mouse chromosomes 1–4, 6, 7, and 15 (17). The mapping results are identical at the loci for the C57BL/6 strain (Massachusetts Institute of Technology database).

	Results

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Selection of BDC2.5 T cells by HLA-DQ8 molecule: the human homolog of I-A^g7

To test whether human DQ8 molecules could cross the species barrier and functionally replace their murine homolog I-A^g7, DQ8 mice were bred with BDC2.5 TCR transgenic mice on the H-2^g7 congenic (I-A^g7, K^dD^b) C57BL/6 background (B6.H-2^g7/BDC2.5), and their progeny were intercrossed. Eight types of mice were used for the study that were derived from a cohort of breeding, and the genotypes of these mice are summarized in Table I. It is interesting that DQ8 molecules are able to positively select BDC2.5 T cells in the absence of I-A^g7 (Fig. 1). The selection appears to be less complete compared with I-A^g7 in both the thymus and periphery, as more CD4⁺ T cells were seen in H-2^g7 congenic mice than in DQ8⁺ mice (Table II). CD4⁺ T cells are present in reduced numbers, and this appears to be a general phenotype of human HLA transgenic lines that are deficient for murine endogenous MHC regardless of the presence or the absence of a TCR transgene. The weaker positive selection seen in DQ8/BDC2.5 mice is possibly due to the murine CD4 coreceptor not interacting as strongly with human MHC class II as with mouse MHC class II molecules (20). However, to examine whether CD4⁺ T cells in DQ8/BDC2.5 mice express BDC2.5 TCR (V4V1), we stained the cells with anti-V4 mAb and analyzed them by flow cytometry. The cells were also stained with a mixture of anti-V2, -V3.2, and -V8.3 to indirectly identify the -chain of BDC2.5 TCR because there is no clonotypic Ab available to directly examine either the BDC2.5 TCR or V1. Similar to their counterpart H2^g7/BDC2.5 mice, most CD4 T cells in DQ8/BDC2.5 mice express the BDC2.5 TCR, as shown in Table II. There is no evidence that the expression of V or V in the selected T cell population differs in DQ8/BDC2.5 mice compared with H2^g7/BDC2.5 mice (data not shown). However, it appears that there is a greater variation in the number of BDC2.5 T cells found in DQ8/BDC2.5 mice than was seen in H2^g7/BDC2.5 mice (Table II). The selection of BDC2.5 TCR (identified as positive for V4) in DQ8/BDC2.5 mice could be grouped into complete, relatively complete, and incomplete, as shown in Fig. 2, whereas the frequency of incomplete BDC2.5 selection was less in H2^g7/BDC2.5 mice (data not shown). It is interesting that there were very few T cells that stained positively for non-V1 (estimated by staining with the mixture of available V Abs) even in the mice with incomplete selection of BDC2.5 T cells (data not shown). To circumvent this limitation, we performed RT-PCR from both thymus and spleen of DQ8/BDC2.5 mice using a panel of 10 V-specific primers for the detection of those V expression at mRNA level. Apart from the strong amplification of V1 in the RT-PCR, we did not obtain obvious amplification of the other Vs tested (data not shown). This result, however, still cannot rule out the possibility that other Vs were used, because there are >100 functional V gene segments in the mouse, and we have no way of detecting all of these. We also analyzed the usage of other Vs in the V4-negative population of the mice expressing incomplete BDC2.5 TCR using a panel of mAbs specific for V2, -3, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, and -17. It appears that non-BDC2.5 T cells use a relatively diverse V repertoire with dominant V5 usage (20% of non-BDC2.5 T cells; data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Selection of CD4+ T cells in DQ8/BDC2.5 and H-2g7/BDC2.5 mice. Lymphocytes isolated from spleen (six left panels) and thymus (two right panels) in DQ8/BDC2.5 (upper panels) and H-2g7/BDC2.5 (lower panels) mice (6–8 wk of age). Cells were stained with 1) the B cell marker B220 (PE) in combination with anti-HLA-DQ (FITC; first panel from left), 2) mouse MHC class I molecules Kd (PE) and Kb (FITC; second panel from left), and 3) CD4 (PE) and CD8 (FITC; middle and right panels).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Expression pattern of BDC2.5 T cells in DQ8/BDC2.5 mice. Lymphocytes isolated from thymus (upper panels) and spleen (lower panels) in DQ8/BDC2.5 mice (6–8 wk of age) were stained for CD4 and V4. Cells were gated on positive staining for CD4, and the expression of BDC2.5 TCR was identified using anti-V4 mAb. Three individual mice are shown. The expression of V4+ cells was analyzed in the cells gated on CD4+. The left panels illustrate that 100% of CD4 single-positive thymocytes expressed transgene V4, and 95% of CD4+ splenocytes were positive for transgene V4 from the same mouse. The middle panels demonstrate that 100% of CD4 single-positive cells expressed transgene V4 in the thymus, whereas 83% of CD4+ cells expressed transgene V4 in the spleen of the same mouse. The right panels show that 46 and 55% of CD4+ T cells expressed the BDC2.5 V4 receptor in the thymus and spleen, respectively, of the same mouse.

BDC2.5 T cells selected by DQ8 molecules retain Ag specificity

Both the parental BDC2.5 T cell clone and BDC2.5 T cells from the BDC2.5 TCR transgenic mouse respond to pancreatic islets (21, 22, 23, 24). The response is restricted to NOD MHC (I-A^g7), whereas the Ag (islet) is not unique to the NOD mouse strain, as BDC2.5 cells are able to respond to islets from many mouse strains (24). To test whether BDC2.5 CD4 T cells selected by DQ8 molecules retain ligand specificity, we performed proliferation assays using islets isolated from DQ8/B6 and NOD mice. B6.H-2^g7/BDC2.5 mice were also examined as positive controls. As shown in Fig. 3, splenic CD4 T cells from DQ8/BDC2.5 mice exhibited proliferative responses to islets from both DQ8/B6 and NOD mice; the response was slightly greater when the CD4 T cells were stimulated with DQ8/B6 islets (Fig. 3A). As expected, CD4 T cells from H-2^g7/BDC2.5 mice also responded to islets of both NOD and DQ8/B6 strains (Fig. 3B). It is noteworthy that a better response was observed in B6.H-2^g7/BDC2.5 T cells toward NOD islets, whereas the reverse pattern was seen in DQ8/BDC2.5 mice, which respond more strongly to DQ8/B6 islets. The response was accompanied by IFN- production (Fig. 3C). The islet reactivity of DQ8/BDC2.5 and H-2^g7/BDC2.5 mice was restricted to DQ8 and I-A^g7, respectively, as demonstrated by Ab inhibition assays (82 and 87% inhibition, respectively). There was no cross-inhibition observed; namely anti-I-A^g7 mAb 10.2.16 did not block islet reactivity of DQ8/BDC2.5 T cells, nor did the anti-DQ mAb IVD12 block the islet response of H-2^g7/BDC2.5 T cells. This is not surprising given the fact that the two mAbs recognize very different epitopes (25, 26, 27). In keeping with this, anti-I-A^g7 did not stain B220⁺ cells from DQ8 transgenic mice, nor did anti-DQ mAb IVD12 stain B220⁺ cells from NOD mice or B6.H-2^g7 congenic mice (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Islet reactivity and IFN- production of CD4+ BDC2.5 T cells from DQ8/BDC2.5 and H-2g7/BDC2.5 mice. CD4+ T cells (105/well) from either DQ8/BDC2.5 (A) or H-2g7/BDC2.5 (B) mice were cultured in medium (Click’s medium containing 5% heat-inactivated FCS) alone or with irradiated islets isolated from either DQ8/B6 or young male NOD mice (as indicated) in the presence of irradiated syngeneic splenocytes (5 x 104/well) as APCs. The cultures were pulsed with 0.5 µCi [3H]thymidine after 72 h. C, IFN- production of CD4+ BDC2.5 T cells from DQ8/BDC2.5 and H-2g7/BDC2.5 mice in response to DQ8/B6 and young male NOD islets, respectively, was measured by ELISA. IL-4, IL-6, and IL-10 were undetectable.

The studies shown above demonstrated that the human homolog of NOD I-A^g7, the DQ8 molecule, could positively select BDC2.5 T cells, and this selection retains the BDC2.5 TCR Ag specificity toward islets. To investigate the diabetogenicity of BDC2.5 T cells selected by DQ8 molecules, we performed a natural history study. Eight groups of mice were used in this study, and the observation period was 7 mo (Table III). Like H-2^g7/BDC2.5 mice, DQ8/BDC2.5 mice also developed diabetes with similar kinetics, although the overall incidence of diabetes in DQ8/BDC2.5 mice was slightly, but not significantly, lower (20%) than that seen in H-2^g7/BDC2.5 mice (28%). BDC2.5 mice expressing both MHC molecules, DQ8 and H-2^g7, had a similar incidence of diabetes (18%) as DQ8/BDC2.5 (20%; Fig. 4). The incidence of diabetes was not statistically significant among the three groups (by Student’s t test, p = 0.48 for DQ8/BDC2.5 vs H-2^g7/BDC2.5, p = 0.08 for DQ8/BDC2.5 vs DQ8/H-2^g7/BDC2.5, and p = 0.1 for H-2^g7/BDC2.5 vs DQ8/H-2^g7/BDC2.5), and there was no obvious sex preference in diabetes development (Table III). It is interesting that two DQ8/BDC2.5 mice with incomplete selection of BDC2.5 T cells also developed diabetes (data not shown). Neither the BDC2.5 transgene-negative mice expressing DQ8 or H-2^g7 nor the BDC2.5 transgene-positive mice expressing neither DQ8 nor H-2^g7 developed diabetes (Table III). The mice with the latter genotype (DQ8^-H-2^g7-BDC2.5⁺) were generally smaller than other types of mice in this study, and their life span was shorter (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Diabetes development in DQ8/BDC2.5 and H-2g7/BDC2.5 mice. Three groups of mice were used in the study as indicated. Diabetes was determined by monitoring of urinary glucose and was confirmed by blood glucose (>250 mg/dl). The observation was terminated when the mice were 7 mo of age.

Pancreata from mice that developed diabetes were analyzed for lymphocytic infiltration by immunohistochemistry staining. Unlike normal NOD mice, in which CD4, CD8 and B cells are the three main types of infiltrating lymphocytes, examination of diabetic H-2^g7/BDC2.5 pancreatic islets revealed heavy CD4 T cell infiltrates and very few CD8 T cells (Fig. 5). Like NOD mice, many B220⁺ B cells are also found in the islet infiltrates where CD4 T cells are located (Fig. 5). Similar to H-2^g7/BDC2.5 mice, diabetic DQ8/BDC2.5 islets also showed very few CD8 T cells in the infiltrates (Fig. 5). However, there were also fewer CD4 T cells in the infiltrates despite the fact that the mice were diabetic (Fig. 5).

View larger version (76K): [in this window] [in a new window]   FIGURE 5. Immunohistochemistry staining of pancreatic sections of diabetic mice. Upper panels, Diabetic H-2g7/BDC2.5 mouse; lower panels, diabetic DQ8/BDC2.5 mouse. Islet infiltrates were stained for B220+ B cells (left panels), CD4+ T cells (middle panels), and CD8+ T cells (right panels).

	Discussion

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Intrathymic positive selection of BDC2.5 T cells by HLA-DQ8 was not as efficient as that on I-A^g7 molecules as a relative higher fraction of mice (two of seven examined) was found expressing <70% V4 (Fig. 2 and Table II), whereas fewer H2^g7/BDC2.5 mice (one of six analyzed) showed a similar expression pattern (data not shown). Because a reduced number of CD4 (V4⁺) single-positive cells was seen in DQ8/BDC2.5 thymocytes, this may result in a reduced number of mature CD4 BDC2.5 T cells in the periphery, as well as in the islets. It is possible that the murine CD4 coreceptor recognizes the DQ8 molecule with low affinity, in other words, the murine CD4 coreceptor does not interact as strongly with human MHC class II as with mouse MHC class II molecules (20). It is also plausible that the relatively weak positive selection and antigenic response in DQ8/BDC2.5 mice are due to the differences between I-A^g7 and DQ8 molecules. Although there are striking similarities between I-A^g7 and DQ8 molecules, there are several differences in sequence between the two molecules. The obvious sequence differences close to position 57 are at residues 55–57 and 61 of the -chain (proline 55-proline 56-alanine 57 and tyrosine 61 for DQ8 and arginine 55-histidine 56-serine 57 and tryptophan 61 for I-A^g7, respectively). Proline and tryptophan at positions 56 and 61 of the -chain are conserved in most alleles of both species (8, 13, 31). It is possible that all three residues, histidine, serine, and tyrosine at positions 56, 57, and 61, are required for the best fit of the ligand of BDC2.5 TCR and lead to optimal positive selection. The crystal structure of I-A^g7 indicates that the substitution of histidine for proline at position 56, which immediately precedes position 57, opens the side of the P9 pocket that contains the -helix of the -chain (8). This suggests that the neighboring residues are very important for antigenic peptide binding by shaping and accommodating the unique character of the I-A^g7 P9 pocket. As the DQ8 -chain lacks histidine and tyrosine at positions 56 and 61, respectively, and the noncharged residue at position 57 is alanine rather than serine, these differences may account for less efficient positive selection in the DQ8/BDC2.5 mice. Interestingly, NOD mice carrying a mutated I-A^g7 transgene that encodes proline and aspartic acid at positions 56 and 57, respectively, were protected from developing diabetes (32, 33, 34). Although transgenic NOD mice with single positional (56 or 57) mutations were not generated in one of those studies (33), the fact that the double mutation of I-A^g7 inhibits diabetes suggests that both residues 56 and 57 are important. The difference in -chains at position 56 between DQ8 and I-A^g7 may also explain the less potent proliferative response of peripheral mature CD4⁺ T cells to islet autoantigen in DQ8/BDC2.5 mice. However, it is interesting that the selection difference does not seem to contribute to disease development, as DQ8/BDC2.5 mice developed spontaneous diabetes with similar kinetics to B6.H-2^g7/BDC2.5 mice even with fewer positively selected cells in the thymus and a less potent antigenic response in the periphery. Due to the lack of reagents to identify the BDC2.5 -chain (V1), we used a mixture of anti-V mAbs (V2, V3.2, and V8.2) to analyze the BDC2.5 -chain indirectly. Surprisingly, very few non-V4⁺ T cells expressed the Vs for which staining was performed. This suggests that non-V4⁺ T cells either still use the BDC2.5 -chain (V1) or express V chains other than V2, V3.2, and V8.2. As more reagents are available for detection of Vs, we found that a diverse V repertoire was expressed in the non-V4⁺ T cells. It is not clear whether these non-BDC2.5 T cells play a down-regulatory role in diabetes development in our DQ8/BDC2.5 model. This question could be answered by the generation of DQ8/BDC2.5 mice with either RAG deficiency or TCR C or C deficiency, as shown in the parental H2^g7/BDC2.5 model. However, this would be beyond the scope of the current study.

The incidence of spontaneous diabetes in H-2^g7/BDC2.5 mice in our study is lower (28%) than that found in the original colony (58%) (17). Microsatellite analysis suggests that mice used in this study were not different from those in the original colony. However, it is possible that some unidentified genetic loci may be different between the two colonies. In contrast, biological heterogeneity has been well recognized in both pure in-bred animals as well as animals that express TCR transgenes. For example, NOD mice neither develop diabetes at the same time point, nor develop 100% diabetes. The heterogeneity is also expressed in several TCR transgenic systems (16, 17, 35, 36, 37), including our current study. We have noticed that individual differences may be present in both intrathymic-positive and -negative selection and peripheral maturation/regulation levels (unpublished observations). However, the discrepancy seen in diabetes incidence between the original H-2^g7/BDC2.5 colony and our colony may also be due to environmental factors, as the original H-2^g7/BDC2.5 colony was housed in a different animal facility.

Our study using an in vivo model demonstrates that human HLA-DQ8 molecules can cross a species barrier and functionally replace I-A^g7 molecules to produce spontaneous diabetes. Our study also provides in vivo biological evidence supporting the recent reports of crystal structures of I-A^g7 and DQ8 molecules. This suggests that diabetes susceptibility in human and mouse might result from similar Ag processing and presentation. We believe that our model system will provide us with a useful tool for testing specific therapeutic interventions for human type 1 diabetes.

	Acknowledgments

 
We thank Drs. J. Katz and D. Mathis for the original B6.H2g7/BDC2.5 mice, Dr. C. A. Janeway, Jr., for his generous support (reagents and equipment) and critical review of the manuscript, Ning-yuan Chen and Jie Tang for excellent technical assistance, and Tamara Dlugos for isolation of islets.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants R01AI44427 and P01DK53015 core B and Diabetes Endocrinology Research Center Grant DK97004 for islet isolation. L.W. is the recipient of a Career Development Award from the Juvenile Diabetes Research Foundation. C.M. is the recipient of an Advanced Postdoctoral Fellowship from the Juvenile Diabetes Research Foundation. F.S.W. previously held a Career Development Award from the Juvenile Diabetes Research Foundation and is currently supported by a Wellcome Trust Senior Fellowship in Clinical Science.

² Address correspondence and reprint requests to Dr. Li Wen, Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, Box 208020, New Haven, CT 06520-8020. E-mail address: li.wen{at}yale.edu

³ Abbreviations used in this paper: T1DM, type 1 diabetes mellitus; NOD, nonobese diabetic.

Received for publication September 10, 2001. Accepted for publication January 30, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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