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The Journal of Immunology, 2002, 168: 3050-3056.
Copyright © 2002 by The American Association of Immunologists

A 320-Kilobase Artificial Chromosome Encoding the Human HLA DR3-DQ2 MHC Haplotype Confers HLA Restriction in Transgenic Mice1

Zhenjun Chen*, Nadine Dudek*, Odilia Wijburg*, Richard Strugnell*, Lorena Brown*, Georgia Deliyannis*, David Jackson*, Frank Koentgen{dagger}, Tom Gordon{ddagger} and James McCluskey2,*

* Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; {dagger} Ozgene Pty, Nedlands, Western Australia, Australia; and {ddagger} Department of Immunology, Allergy, and Arthritis, Flinders Medical Center, Bedford Park, South Australia, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ~320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (A{beta}null/null) and expressing only murine CD4. A broad range of TCR V{beta} families was used in the peripheral T cell repertoire, which was also purged of V{beta}5-, V{beta}11-, and V{beta}12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the A{beta}null/null background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human histocompatibility leukocyte Ag molecules present antigenic peptides to T lymphocytes resulting in specific immune responses (1). Autoimmune diseases such as type 1 insulin-dependent diabetes mellitus (IDDM)3 and systemic lupus erythematosus are strongly associated with particular HLA class II alleles (2, 3) or with combinations of alleles either in cis (haplotypes) (4) or in trans (4, 5, 6). Although population data (7) and studies of transgenic mice (8, 9) have identified some of the individual genes that are responsible for disease susceptibility in MHC haplotypes, some studies conclude that disease risk is conferred by combinations of HLA DR and DQ genes (6, 10, 11, 12, 13) and may involve genes within the class III region of the MHC (12, 14). This has led to suggestions that disease susceptibility may depend upon the MHC haplotype context rather than any particular gene within the haplotype (4, 6). Of the HLA haplotypes associated with autoimmunity the class II HLA DR3-DQ2 haplotype is particularly prominent in that it is linked to an increased risk of insulin-dependent diabetes, thyrogastric autoimmunity, systemic lupus erythematosus, Sjögren’s syndrome, myasthenia gravis, celiac disease, and many other autoimmune conditions (3, 12). The mechanism by which particular haplotypes such as HLA DR3-DQ2 are associated with different autoimmune disorders is not known. Transgenic models have so far relied on single gene transgenic mice (8, 9) or have recreated haplotype combinations by breeding mice to express multiple genes in trans (6, 11, 13). This approach has several drawbacks. First, the DRB3 and DRB4 genes are omitted from consideration despite their importance in many immune responses (15, 16) and the fact that even single chain MHC class II genes can profoundly influence expression of autoimmune disease such as IDDM (17, 18, 19). Second, expression of independent transgenes ignores the possibility that multiple genes in the class II region contribute to disease susceptibility through interactions during positive and negative selection of the T cell repertoire or through aspects of their peripheral coregulation (20, 21). Finally, any role for pseudogenes (e.g., DQB2/A2), intergenic "junk" (22), or endogenous retroviral DNA sequences contained within MHC haplotypes (23) cannot be assessed by a single or multiple independent transgene approach. Therefore, we have derived transgenic mice that express an archetypal HLA DR3-DQ2 autoimmune class II haplotype expressing the linked HLA DRB1*0301-DRB3*0101-DRA*0101-DQB1*0201-DQA1*0501 from a yeast artificial chromosome (YAC) containing the authentic flanking and intervening sequences. In transgenic mice the HLA DR and DQ molecules demonstrate tissue-specific expression, restrict Ag-specific immune responses, and function in protective host immunity, suggesting that these mice will be valuable tools in establishing animal models to unravel the pathogenesis and specificity of MHC-linked autoimmune disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
YAC construction and in vitro transfer

The 320-kb YAC B1D12 (a gift from D. Chaplin, Washington University, St. Louis, MO) was cloned from a cell line with the HLA type DRB1*0301-DRB3*0101-DRA*0101-DQB1*0201-DQA1*0501 (HLA DR3-DQ2) (24). The YAC contains seven HLA class II genes from DRA to DQB2 including flanking and intervening regions as shown in Fig. 1GoA. The YAC was maintained in Saccharomyces cerevisiae AB1380 and retrofitted with the plasmid pUNL13 (a gift from J. T. Lee, Cambridge, MA) containing a neomycin resistance gene. Stable integration of the retrofitted YAC R1B1D12 was achieved in mouse L cells, Chinese hamster ovary (CHO) cells, and the MHC class II-negative mouse B lymphoma line M12.C3 following fusion to YAC-positive yeast spheroplasts (25). After 48 h, G418 selection was applied to the mammalian cells at 0.5 mg/ml and drug-resistant colonies were characterized by flow cytometry and PCR.



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  		FIGURE 1. Regulated expression of the HLA DR3-DQ2 haplotype encoded by the retrofitted YAC R1B1D12. A, The YAC is ~320 kb and contains seven genes from the HLA DR3-DQ2 haplotype from DRA to DQB2, a vector arm on each end, and the neo-resistant gene in the right arm. B, Spheroplast fusion was used to introduce YAC R1B1D12 into mouse L cells (LTA-5, fibroblast-like), CHO cells, and mouse M12.C3 cells (a class II-negative B lymphoma line). Transfectants (+YAC) were stained by indirect immunofluorescence using the mAbs 16.23 (DR + peptide; tightly dotted line), SPV-L3 (DQ; thin line), L243 (DR; thick line), and an isotype control Ab (loosely dotted line). Histograms of the HLA DR3-DQ2 homozygous lymphoblastoid cell line 9022 (DR3-DQ2 BLCL) are shown as a positive control.

 
ES cell transfection

Transfection of C57BL/6 (B6) embryonic stem cells (ES cells) with purified YAC DNA (R1B1D12) was conducted using the cationic lipid DOTAP (Boehringer Mannheim, Mannheim, Germany) as previously described (26). Transfectants were screened by PCR to detect HLA genes and yeast chromosome 3, which comigrates with R1B1D12. Two YAC-transfected ES colonies with intact HLA gene organization were used for microinjection into F1 blastocysts or morula from BALB/c x BALB/B mice. Viable embryos were transferred to the oviducts of pseudopregnant F1 females from CBA x C57BL/6 for development to term. One male chimeric mouse was obtained and crossed to C57BL/6 mice with and without a class II knockout (A{beta}null/null) background. Mice used in experiments were 6–12 wk of age.

Abs and flow cytometry

The following mAbs were conjugated with biotin, FITC, or PE: L243 (anti-HLA DR{alpha} chain) (27), SPV-L3 (anti-HLA DQ) (28), 16.23 (anti-DR3/52) (29), Rm5.112 (pan-HLA class II {beta}-chain) (30), ID3 (anti-mCD19), N418 (anti-mCD11c), F4/80 (for macrophages), GK1.5 (anti-mCD4), 56.6.72 (anti-mCD8), 17-3-3 (anti-I-E{beta}b), AF6-120.1 (anti-I-Ab {alpha}-chain). TCR V{beta} mAbs were purchased from BD PharMingen (San Diego, CA). Cells were stained by direct immunofluorescence and analyzed on a FACScan (BD Biosciences, Mountain View, CA) collecting 10,000–20,000 events in the live cell gates. Western blots were conducted as previously described (31).

Salmonella infection

Mice were immunized orally with 0.2 ml of PBS containing 1010 CFU of BRD509 (pTETtac4), the {Delta}aroA, {Delta}aroD attenuated Salmonella mutant SL1344 which expresses the nontoxic COOH-terminal 50-kDa of tetanus toxin. Eight weeks after vaccination, mice were challenged with 107 virulent SL1344 given by the same route, then killed 7 days later. The number of viable bacteria present in the spleens and livers of the mice was determined by serial dilution of tissue homogenates onto Luria-Bertani agar plates, followed by overnight growth at 37°C. Serum taken at 4 wk postvaccination was tested by ELISA for Abs against tetanus toxoid (TT) or Salmonella typhimurium LPS as previously described (32).

Influenza infection and T cell assays

The type A influenza virus Mem 71 (subtype H3N1) was delivered intranasally to penthrane-anesthetized mice as 104.5 PFU of infectious egg-grown virus. Animals were sacrificed on day 6, 8, and 10 after infection and virus titers were measured in the supernatants of lung homogenates (33). For MHC restriction studies peptides heat shock protein (Hsp)3–13 (KTIAYDEEARR) (34) and {alpha}-gliadin 57–73 (QLQPFPQPELPYPQPQS) (35) were synthesized using F-moc chemistry by Chiron (Emeryville, CA). Mice were immunized with 50 µg of synthetic peptide emulsified 1/1 (v/v) in CFA (Difco, Detroit, MI) administered s.c. into the tail base and hind footpad. Lymphocytes from draining lymph nodes (LN) were isolated 7–10 days later and cultured (2 x 105/well) with syngeneic APC (splenocytes) irradiated with 2200 rad (4 x 105/well) in 96-well flat-bottom plates with medium alone, Con A, or synthetic peptides (36). Proliferation of T cells was assayed by their incorporation of [3H]thymidine (1 µCi/well) over 18 h after a 72-h culture period (24 h for Con A). For in vitro blocking studies, culture supernatant containing mAb or 0.5 mg/ml protein A-purified Ab was added to the LN cells (20 µl/well) in the presence of 20 µM peptide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulated expression of HLA DR and DQ gene products in YAC-transfected cell lines

To verify tissue-specific regulation of the DR3-DQ2 gene products, YAC DNA containing a neomycin resistance gene (Fig. 1GoA) was stably transferred into cultured cell lines by spheroplast fusion of YAC+ yeast with mammalian cells from different lineages (Fig. 1GoB). Cell surface expression of DR and DQ was observed on the YAC-transfected murine B lymphoma M12.C3 but not detected on multiple independent clones of the transfected murine fibroblast LTA-5 (L cells) or CHO cells, (Fig. 1GoB). Cell surface expression of DR was greater than for DQ2 similar to the expression pattern on the human DR3-DQ2 homozygous B lymphoblastoid cell line 9022 (Fig. 1GoB). The class II molecules expressed by the murine M12.C3 transfectants were peptide loaded, as demonstrated by the detection of SDS-stable dimers by immunoblots (37) and staining with the peptide-dependent, DR-specific mAb 16.23 (29, 38) (Fig. 1GoB and data not shown). The LTA-5 and CHO transfectants contained integrated copies of HLA DR and DQ genes by PCR (data not shown) and, like the endogenous class II gene products encoded by these cells, surface HLA class II expression was not inducible by exposure to IFN-{gamma} (data not shown). Transfection of class II-negative cell lines with genomic clones of individual MHC class II genes usually results in their surface expression (39). Therefore, we conclude that the differential expression of DR and DQ in the M12.C3 B lymphoma transfectants vs the nonlymphoid L cells and CHO cells reflects physiological regulation of these genes.

Creation of HLA DR3-DQ2 haplotype-transgenic mice by transfection of ES cells with YAC DNA

Purified YAC DNA was transfected into C57BL/6 mouse ES cells, producing two ES clones in which the relevant HLA loci had integrated into the ES genome without detectable deletion or rearrangement and which lacked yeast chromosome 3 (data not shown). Multiple blastocyst injections were performed with the transfected ES cells, leading to the generation of a single founder line (1 of 50 pups) which was expanded by breeding with C57BL/6 mice bearing a class II knockout background, I-A{beta}null/null (A{beta}0/0) (40). The YAC transgene was transmitted as an autosomal haplotype segregating independently of the endogenous mouse MHC on chromosome 17. YAC-transgenic animals bred normally and there was no detectable phenotype of the YAC+ animals compared with nontransgenic littermates. The DR-DQ-containing YAC transgene was bred to homozygosity, indicating nonlethal insertion of the 320-kb YAC DNA into the mouse genome.

Regulated expression of HLA DR and DQ in YAC-transgenic mice

To examine the regulation of class II expression in YAC-transgenic mice, cell surface expression of DR and DQ molecules was examined on splenocytes from five different genotypes (Fig. 2Go). For comparison, PBMCs from a HLA DR3-DQ2 homozygous blood donor were also stained for DR and DQ expression and the B cell marker CD19 (Fig. 2GoB). HLA DR and DQ expression was evident on transgenic mouse B cells (Fig. 2Go, A and C), dendritic cells (Fig. 2Go, C and D), and a proportion of macrophages (data not shown) as expected. Several observations attest to the proper regulation of DR-DQ expression in the transgenic animals. First, HLA class II expression was higher on mouse dendritic cells (CD11c+) compared with B cells as was the case for endogenous H-2b mouse class II molecules (Fig. 2GoC). Nonetheless, HLA class II molecules were up-regulated in activated B cells from YAC-transgenic mice (data not shown). Second, in humans, but not in mice, class II molecules are expressed on activated T cells (41). Accordingly, expression of DR and DQ was not detectable on mouse T cells (Fig. 3GoA) even after in vitro stimulation with the T cell mitogen Con A (data not shown). Last, transgenic mouse B cells expressed higher levels of DR than DQ as seen on human LCL 9022, the murine M12.C3 transfectants (Fig. 1GoB), and human B peripheral blood B cells (Fig. 2GoB), reflecting the differential expression of these gene products in human cells (42). B cells from transgenic mice expressed heterogeneous levels of DR and DQ molecules and at lower levels than observed in peripheral blood B cells from healthy blood donors (compare A and B in Fig. 2Go). The expression of human class II molecules on mouse B cells was also lower than observed for endogenous I-Ab, perhaps indicating competition for peptide Ag. Although A{beta}null/null mice lack I-E molecules, they synthesize I-E{beta} chains potentially capable of forming interspecies DR{alpha}/I-E{beta} heterodimers. Spleen cells from YAC/A{beta}null/null mice reacted with the I-E{beta} chain-specific mAb 17-3-3, indicating surface expression of interspecies DR/I-E dimers at levels ~10-fold lower than HLA DR expression in the transgenic animals (Fig. 2GoA, lower panel). Breeding of YAC-transgenic mice to complete class II knockout mice (43) lacking all I-A and I-E genes resulted in similar levels of HLA DR and DQ expression to that seen on the A{beta}null/null background, except that I-E{beta} determinants were not detected in these mice (data not shown).



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  		FIGURE 2. Regulated surface expression of HLA DR and DQ molecules on B lymphocytes and dendritic cells from YAC-transgenic mice. A, Spleen cells from mice with the indicated genotypes were double-stained with mAbs specific for murine B cells (CD19) and class II determinants as follows: Rm5.112 (pan-HLA class II {beta}-chain), L243 (DR {alpha}-chain),SPV.L3 (DQ), AF6-120.1 (I-Ab {alpha}-chain), and 17-3-3 (I-E{beta} chain). B, PBMCs from a HLA DR3-DQ2 homozygous blood donor were double-stained with directly conjugated mAbs recognizing the human B cell marker CD19 and Rm5.112 (pan-HLA class II {beta}-chain), L243 (DR, {alpha}-chain) or SPV.L3 (DQ). C, Histograms showing the relative surface expression of MHC class II on B cells (CD19 gated) and dendritic cells (CD11c gated) in B6+/null and YAC+/+A{beta}null/null mice. D, Spleen cells from mice with the indicated genotypes were double-stained for the dendritic cell marker CD11c and either Rm5.112 (pan-HLA class II {beta}-chain) or AF6-120.1 (I-Ab {alpha}-chain). The percentage of double-staining cells (dendritic cells) is shown in the upper right quadrant.

 


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  		FIGURE 3. Functional expression of HLA class II molecules by splenic APC but not T cells in YAC-transgenic mice. A, Double staining of CD4 (GK1.5, left panel) and CD8 T cells (56.6.72, right panel) for HLA class II expression (mAb Rm5.112). B, Human class II molecules are expressed as SDS-stable dimers in YAC-transgenic mice. Immunoblotting of spleen cell lysates with mAb Rm5.112 (pan-class II {beta}-chain). Lysates from mice of the indicated genotypes were either boiled for 2 min (b) or were loaded unboiled (u) in sample buffer. The positive control (+ve control) cell lysate is from the DR3-DQ2 homozygous BLCL 9022. Molecular mass markers are shown and the arrow indicates SDS-stable dimers. Monomers migrate at 28–31 kDa.

 
Peptide loading of class II molecules requires productive interactions with H-2M and H-2O molecules (44, 45). Therefore, we determined whether the HLA class II molecules expressed in mouse cells formed stable dimers in SDS, indicating their assembly with peptide Ag. Western blots of splenic cell lysates demonstrated the presence of stable human class II molecules in the YAC-transgenic mice, and these were converted to monomeric chains upon boiling (Fig. 3GoB). Thus, human class II molecules load with peptide Ag in mouse cells.

Positive and negative selection of CD4 T cells in YAC-transgenic mice

To determine whether the introduction of the DR3-DQ2 transgenes restored the CD4 T lymphocyte compartment in the class II knockout A{beta}null/null mice, T cell subpopulations were enumerated in thymocytes, splenocytes, and peripheral blood cells from transgenic and control mice (Fig. 4Go). The A{beta}null/null mice lacked significant numbers of CD4+ T cells; however, in YAC-transgenic heterozygotes (YAC+/nullA{beta}null/null), splenic (Fig. 4GoA, upper panel) and blood (Fig. 4GoB) CD4 T cell numbers were restored to approximately one-third of those in B6+/null mice, and, in homozygotes (YAC+/+A{beta}null/null), CD4 T cell numbers were restored to two-thirds normal. The lower number of CD4+ T lymphocytes in YAC-transgenic animals (YAC+/nullA{beta}null/null and YAC+/+A{beta}null/null) compared with B6+/null mice almost certainly reflects inefficient interaction between murine CD4 and human class II molecules (46). The percentage of single positive CD4+ thymocytes did not significantly differ between YAC-transgenic heterozygotes (YAC+/nullA{beta}null/null) and homozygotes (YAC+/+A{beta}null/null) (Fig. 4GoA, lower panel), perhaps implying more efficient export of CD4+ T lymphocytes in the homozygous animals. The repertoire of T cells selected by HLA class II molecules was diverse in that the proportion of T cells using V{beta} 1–4, 6–9, 10, and 13 was similar in transgenic and nontransgenic B6 mice. Some V{beta}-bearing subfamilies (such as V{beta}8 family) appeared overrepresented in CD4 T cells from YAC+ mice (Fig. 4GoC), perhaps reflecting deletion of V{beta}5-, V{beta}11-, and V{beta}12-bearing T cells due to endogenous expression of mouse mammary tumor virus superantigens (47). A similar deletion pattern occurred in the CD8 T cell compartment of YAC+ mice, although V{beta}5-bearing CD8 T cells were barely detectable even in normal B6 mice. Finally, histology of the thymus and spleen from YAC-transgenic mice are all normal (data not shown).



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  		FIGURE 4. Positive and negative selection of CD4+ T cells in HLA DR3-DQ2-transgenic mice. A, Splenic (upper panel) and thymic (lower panel) T cells from mice with the indicated genotypes were double-stained with mAb GK1.5 (CD4) and mAb 56.6.72 (CD8). The percentage of single positive CD4 T cells as a proportion of all gated lymphocytes is shown in the lower right quadrant of each panel. B, The average proportion of CD4 T cells was determined in tail blood lymphocytes from B6+/null (n = 3), YAC+/nullB6+/null (n = 8), YAC+/+A{beta}null/null (n = 5), YAC+/nullA{beta}null/null (n = 12), and A{beta}null/null (n = 8) animals. C, Selection of a diverse range of V{beta} families in the CD4 T cells of DR3-DQ2-transgenic mice. Splenic T cells from B6+/null (filled bar), YAC+/nullB6+/null (hatched bar), and YAC+/nullA{beta}null/null (open bar) were double-stained for either CD4 (upper panel) or CD8 (lower panel) and the indicated anti-V{beta} mAbs. The percentage of the total CD4 or CD8 T cell population staining with each of these mAbs is shown on the vertical axis.

 
HLA DR3-DQ2-transgenic mice develop protective immunity following infectious challenge

To determine the immune function of the HLA DR3-DQ2-transgenic mice, animals of different genotypes were challenged with Salmonella (a model of CD4 T cell-dependent immunity) (48) or influenza virus (a model in which viral clearance is mediated predominantly by CD8 T cells) (49).

Vaccination of mice with attenuated S. typhimurium conferred protection against infection with the wild-type bacterium in normal B6 (B6+/+), B6.A{beta}+/null (B6+/0), YAC+/nullA{beta}+/null (YAC+/0B6+/0), and YAC+/nullA{beta}null/null (YAC+/0A{beta}0/0) mice, but not in A{beta}null/null (A{beta}0/0) mice (Fig. 5GoA). Accordingly, bacterial counts in spleens of vaccinated and unvaccinated A{beta}null/null mice were not significantly different (p < 0.64), whereas vaccination of YAC+/nullA{beta}null/null, B6+/+, B6+/null, and YAC+/nullB6+/null mice resulted in significantly lower bacterial counts than in A{beta}null/null mice (p < 0.0027). Salmonella clearance from the liver was similar to the spleen (data not shown). The Ag-specific Ab titer against LPS and TT was also estimated in mice after vaccination with attenuated Salmonella, which expresses the COOH fragment of tetanus toxin. The titer of specific serum Ig to LPS in YAC+/nullA{beta}null/null mice was comparable in control B6+/+, B6+/null, and YAC+/nullB6+/null mice but was significantly higher than that from A{beta}null/null mice (p < 0.0046) (Fig. 5GoB). The YAC+/nullA{beta}null/null mice were less efficient at generating specific Ig against TT, in that Ab titers were lower than those in B6+/+ (p < 0.006), B6+/null (p < 0.001), and YAC+/nullB6+/null (p < 0.01) mice and only marginally higher than those in A{beta}null/null mice (p < 0.062) (Fig. 5GoC). This could reflect limitations of the CD4 T cell immunity in DR3-DQ2 YAC-transgenic mice.



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  		FIGURE 5. Protective immunity to S. typhimurium and influenza virus in HLA DR3-DQ2-transgenic mice. A, Mice of the indicated genotypes were immunized intragastrically with 1010 live attenuated S. typhimurium (BRD509, aroA-, aroD-). Eight weeks after immunization, these mice (Vacc +; hatched bar) were challenged with 107 virulent S. typhimurium SL1344. Unimmunized (Vacc -; open bar), genotype-matched mice were included as controls. At day 6 postchallenge, mice were sacrificed and the bacterial count in the spleen and liver was determined. The histogram shows the mean count ± SE, and values for individual mouse spleens (•). Four weeks after initial immunization with attenuated Salmonella mice were bled and serum Abs specific for marker Ags LPS (a component of Gram-negative bacteria) (B) and TT (produced by the Salmonella) (C) were determined by ELISA. Error bar represents the SD of the mean. Values for individual mice are shown (• and {triangleup}). D, Protective immunity to influenza virus infection in DR3-DQ2 A{beta}null/null-transgenic mice. Mice with the indicated genotypes were inoculated intranasally with influenza virus and their lungs were removed at days 6, 8, and 10 postinfection. Virus titers in the lungs of individual mice were measured in a plaque-forming assay. Each circle represents the virus titer of individual mice and the line represents the average titer of each group.

 
After infection with influenza virus all mice had high virus titers in their lungs at day 6 (Fig. 5GoD). By day 8, B6+/null, YAC+/nullB6+/null, and YAC+/nullA{beta}null/null mice cleared the virus, indicating that the CD8 compartment in YAC+/nullA{beta}null/null was functional. Half (two of four) of the A{beta}null/null mice showed a modest delay in clearance of the influenza virus with detectable virus in their lungs on day 8; however, all the A{beta}null/null mice had cleared the virus by day 10. The slight delay in viral clearance by class II-deficient (A{beta}null/null) mice compared with the DR3-DQ2 transgenic mice (YAC+/nullA{beta}null/null) may reflect a Th cell contribution in the YAC-transgenic mice. This result is compatible with the findings in CD4 T cell-depleted mice (50), which also show a slight delay in clearance of virus in the absence of CD4 T cells, which are necessary for the expansion of CD8 T cells (51) and for the development of T-dependent Ab responses (52).

HLA DR- and DQ-restricted T cell responses in YAC-transgenic mice

To demonstrate HLA DR- and DQ-restricted immunity in YAC-transgenic mice animals were immunized with the DR3-restricted mycobacterial Hsp3–13 peptide (34) or the DQ2-restricted {alpha}-gliadin peptide 57–73 (35). LN cells were tested for Ag-specific proliferation in vitro 7–10 days later (Fig. 6Go). T cells from both B6 mice and DR3-DQ2-transgenic animals responded to the {alpha}-gliadin peptide, with a greater response seen in the transgenic animals (Fig. 6GoA). Proliferation of {alpha}-gliadin-specific T cells from transgenic mice could be blocked with SPV-L3 (anti-DQ) but not by L243 (anti-DR{alpha}), suggesting intact DQ2-restricted responses in transgenic mice (Fig. 6GoB). The DR3-DQ2-transgenic mice responded in a dose-dependent manner to the Hsp3–13 peptide, while T cells from B6 mice did not react to this peptide (Fig. 6GoC). Furthermore, proliferation of Hsp3–13-specific T cells could be blocked by mAb L243 (anti-DR{alpha}) and Rm5.112 (pan-human class II {beta}) but remained unaffected by mAb SPV-L3 (anti-DQ) or 17.3.3 (anti I-E{beta}) (Fig. 6GoD). Normal C57BL/6 mice, but not YAC-transgenic mice lacking I-Ab molecules (YAC+/nullA{beta}null/null), responded to OVA323–339 following immunization with this peptide in CFA (Fig. 6GoE). Because Freund’s adjuvant contains mycobacterial Hsps we also tested whether T cells from these YAC+/nullA{beta}null/null mice were primed to Hsp65kDa Ag that contains the Hsp3–13 determinant. As shown in Fig. 6GoE, YAC-transgenic mice responded specifically to the MT65kDa Hsp3–13 peptide, indicating natural presentation of this determinant by HLA DR molecules.



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  		FIGURE 6. HLA DR- and DQ-restricted Ag-specific T cell responses in YAC-transgenic mice. C57BL/6 and YAC+/null/A{beta}null/null mice were immunized in the tail and hind footpad with 50 µg of the DQ2-restricted wheat {alpha}-gliadin peptide 57–73 (QLQPFPQPELPYPQPQS) (A and B), the DR3-restricted mycobacterial peptide, Hsp3–13 (KTIAYDEEARR) (C and D), or the I-A{beta}b-restricted OVA peptide OVA332–339 (E) emulsified 1/1 (v/v) in CFA. Peptide-specific T cell proliferation was assayed by [3H]thymidine incorporation (1 µCi/well) of regional LN T cells stimulated 7–10 days later with Ag-loaded and irradiated syngeneic APC. Ab blocking studies confirmed HLA restriction of the responses in B and D where 20 µl per well of culture supernatant containing the indicated mAb or 0.5 mg/ml protein A-purified mAb was added to the LN cells in the presence of 20 µM peptide. E, The recall response to the OVA332–339 and MT65kDa Hsp3–13 peptides was assayed 8 days after priming B6 and YAC+/nullA{beta}null/null mice with OVA332–339 in CFA. Actual cpm for YAC+/nullA{beta}null/null mice T cells stimulated with MT65kDa Hsp3–13 were 7970 (with peptide) and 447 (without peptide). MHCI (DQ binder) and P25 (I-Ab binder) are negative controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of HLA-transgenic mouse models have been developed to study autoimmunity, and these have proven valuable in evaluating the role of individual HLA genes in disease susceptibility (8, 9, 53, 54, 55). However, there are no HLA-transgenic mouse models that express multiple linked genes from common autoimmune-prone human haplotypes. There is considerable evidence that haplotypic combinations of genes within the MHC play an important role in autoimmunity (4, 12). Haplotypes potentially contribute to disease phenotype through the sum of the interactions between individual gene products and their coregulation. These interactions cannot easily be studied in multiplex transgenic animals expressing unlinked single HLA genes in trans. For example, introduction of a DR4 transgene onto DQ8-transgenic NOD mice significantly reduces the incidence of IDDM in these animals (13), but the DR4-DQ8 haplotype also expresses the DRB4 (DR53) class II gene product that is largely ignored in this context and is known to present autoantigenic peptides (16). To this end it is important to develop more physiological animal models of MHC-linked disease in which haplotypes of the HLA class II region function properly in transgenic mice. The transgenic mice described in this study express a YAC containing a 320-kb region of the HLA DR3-DQ2 haplotype. Evaluation of HLA DR3-DQ2 haplotype-transgenic mice indicates regulated HLA gene expression, T cell selection, and functional Ag presentation mediated by the DR and DQ genes encoded within the YAC transgene. Histological examination of tissues in YAC-transgenic mice, including the pancreatic {beta}-islets, does not reveal any evidence of spontaneous pathology in these animals (data not shown).

Previous single chain DR{alpha}- or DQ{beta}-transgenic systems have demonstrated interspecies pairing between IE/DR or IA/DQ (56, 57). However, in transgenic mice expressing DR{alpha}{beta} or DQ{alpha}{beta} heterodimers, these become dominant and serve as the main restriction element for T cell responses (46). We have detected the presence of interspecies pairing between DR{alpha} and I-E{beta} but not between DQ{beta} and I-A in the DR3-DQ2-transgenic animals. We cannot rule out that the DR{alpha}/I-E{beta} molecules account for some of the T cell selection and peripheral immune responses in YAC-transgenic mice. However, we believe this is likely to be a minor role because I-E{beta} was expressed at approximately one-tenth the level of HLA DR determinants, and the MMTV-mediated negative selection of T cells bearing V{beta} 5, 10, and 11 was far more efficient than previously observed when I-E is expressed alone (~60% efficient) (58). Moreover, our preliminary findings in complete class II knockout mice (MHCII{Delta}/{Delta}) (43) expressing the DR3-DQ2 haplotype indicate that these mice select a similar number of CD4+ T cells with an equally diverse V{beta} usage, as observed in mice expressing the DR3-DQ2 haplotype on the A{beta}null/null background. The incomplete restoration of CD4+ T cell numbers in the peripheral blood of DR3-DQ2 haplotype-transgenic mice (33–66% normal) probably reflects inefficient interaction between HLA class II molecules and murine CD4 (46). For this reason we are currently introducing human CD4 onto the background of the DR3-DQ2-transgenic mice.

This study is the first published demonstration that significant components of human HLA haplotypes of class II genes can be functionally expressed in transgenic mice. This observation should greatly facilitate experimental dissection of the role of these genes in autoimmunity and permit development of humanized animal models of autoimmune disease.


   Acknowledgments
 
We thank David Chaplin, Jeannie Lee, Graeme Russ, Anthony D’Apice, Louise Barnett, and scientists of the Victorian Transplantation and Immunogenetics Service and the Australian Red Cross Blood Service (Victoria, Australia) for gifts of reagents and for technical assistance.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia, the Juvenile Diabetes Research Foundation, and the Arthritis Foundation of Australia. Back

2 Address correspondence and reprint requests to Dr. James McCluskey, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail address: jamesm1{at}unimelb.edu.au Back

3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; YAC, yeast artificial chromosome; CHO, Chinese hamster ovary; LTA, L cell; ES cell, embryonic stem cell; LN, lymph node; Hsp, heat shock protein. Back

Received for publication September 25, 2001. Accepted for publication January 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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