Optimum function of HLA-DR molecules in transgenic mice requires efficient interaction between the class II molecules on APCs and CD4 on T cells. Residues 110 and 139 of the second domain of class II molecules are considered to be critical for recognition of CD4. We generated an HLA-DR4β(NT) transgene construct in which positions 110 and 139 were altered to resemble endogenous mouse H2 Aβ molecules. This construct was introduced into (B10 × SWR) embryos, and DR4β(NT) transgenic mice were produced. The transgene was transferred into B10.RFB3 (Eβ0p) mice. The transgene-encoded DR4β molecules paired with endogenous Eα chains to form stable DR4β/Eα dimers expressed on the cell surface. The hybrid dimers showed similar Ag-binding specificity to HLA-DR4 molecules and positively selected CD4+ T cells in vivo. Immunization of HLA-DR4β(NT) transgenic mice with DR4-restricted peptides induced T cell proliferation in vitro. While the purified T cells from DR4β(NT) transgenic mice responded strongly to the HA(307–319) presented by M12C3 transfectants expressing altered DR4β/Eα heterodimers, the response to the same peptides presented by transfectants expressing wild-type DR4β/Eα molecules was substantially reduced. Taken together, these data confirmed in vitro studies on the importance of these residues in CD4-MHC class II interaction. The altered HLA-DR4β transgenic mice were able to overcome the species barrier and generate efficient HLA-DR4-restricted CD4-specific immune responses. Thus, residues 110 and 139 were critical for the interaction of class II with CD4 T cells during thymic selection as well as peripheral immune responses.

The interaction between CD4 and MHC class II molecules plays a crucial role in both intrathymic selection (1) and peripheral activation of CD4+ T cells (2, 3). During these processes, the transmembrane glycoprotein CD4 binds to the β2 domain of MHC class II (4, 5) as a coreceptor to the TCR-αβ and contributes to differentiation of thymocytes into mature CD4+ T cells (6) and activation of mature T cells in the cellular immune response to Ags presented by MHC class II molecules (7).

The interaction site for CD4 on the MHC class II molecule was mapped to between amino acid 137 and 143 in the β2 domain by two different approaches. Cammarota et al. (5) used soluble HLA-DR4 molecules and rHLA-DR4-derived peptides to bind to immobilized soluble rCD4. They found that the region comprising residues 134 to 148 of HLA-DR4β was the major contact site with CD4. At the same time, Konig et al. (4) reported that the interaction between CD4 and Aβ were diminished when substitutions were made in the same region of β2 domain. Konig et al. (4) showed that position 110 also contributes in the interaction between murine CD4 and human class II. Transfected cell lines expressing exon-shuffled MHC class II β-chain in which the β2 domain of HLA-DR molecule was substituted with the β2 domain of the H2-E molecule resulted in the reduction of human T cell responses (8, 9). These data suggested that a species barrier existed in the recognition of CD4-MHC class II molecules between humans and mice. Similar results were found when the β2 domain of mouse class II molecules was substituted with the human β2 domain, and mouse T cells were used as responder (10). However, one study using H2-A-restricted mouse T cell hybridomas expressing mouse or human CD4 showed that both obtained equivalent responses (11).

Similar controversy was seen in the function of HLA class II molecules in transgenic mice. Mice expressing wild-type DQ(DQ8,DQ6) transgenes in the absence of endogenous class II can interact with CD4 during thymic selection (12) and could generate DQ-restricted T cell responses (13). Meanwhile two studies on HLA-DR4 transgenic mice showed that the species-matched CD4-MHC class II interaction was important (14, 15). In one of them, a chimeric HLA-DR4 molecule in which the α2β2 domain was substituted with the α2β2 from mouse H2-E molecule was used to generate a DR4-restricted T cell response. In the other, human CD4 gene was introduced into HLA-DR4 transgenic mice to obtain DR4-restricted T cell response. In both cases the DR4-restricted T cell response was much lower in frequency and magnitude compared with DQ-restricted T cell response in DQ8.Ab0 transgenic mice.

Based on previous mapping results, we aligned the amino acid sequences of the β2 domain of HLA-DQ8(DQB1*0302) and HLA-DR4(DRB1*0401) with the corresponding H2-A and H2-E molecules in mice. By comparing the amino acid sequences in these regions, we found that HLA-DQ8 β-chain was identical to H2-Aβ except for an Ala to Val substitution at position 140 (Fig. 1). In contrast, H2-Eβ had four different amino acid residues at positions 110, 139, 140, and 142. The sequence of HLA-DR4β(DRB1*0401) was similar to H2-Eβ, but Glu at position 110 was substituted by Gln and residue at 142 was the same as H2-Aβ. We hypothesized that residues 110 and 139 may be the key to better interaction of DR molecules with CD4. To test this hypothesis, we introduced two substitutions into the DR4β gene construct. The Gln at position 110 and Lys at position 139 were substituted by Asn and Thr, respectively. Thus, the sequence of the CD4-binding region in the altered DR4β was more like that of H2-Aβ and DQβ. HLA-DR4β(DRB1*0401) transgenic mice with the altered β2 gene were generated. By using these mice, the molecular basis of the interaction between mouse CD4 and human DR4 molecules was studied both in vitro and in vivo.

FIGURE 1.

Amino acid sequence comparison of MHC class II molecules between mouse and human.

FIGURE 1.

Amino acid sequence comparison of MHC class II molecules between mouse and human.

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Human DRB1*0401 gene cDNA was generated by the RT-PCR method using mRNA isolated from the Priess cell line (16). The specific site-directed mutagenesis was conducted by using the overlap PCR method (17). Briefly, the 5′-end and 3′-end complementory oligo primers of HLA-DR4β(DRB1*0401) cDNA were synthesized as DR4b-5′ (5′-CCGGAATTCATGGTGTGTCTGAAGTTC-3′) and DR4b-3′ (5′-GTGGAATTCTCAGCTCAGGAATCCTG-3′). Two pairs of the mutagenic oligos were synthesized as follows: A1 (5′-CTGAATCACCACAACCTCCTGGTC-3′), A2 (5′-GACCAGGAGGTTGTGGTGATTCAG-3′), B1 (5′-AGTAGTCTCTTCCTGGCCGTTCCG-3′), and B2 (5′-CGGAACGGCCAGGAAGAGACTACT-3′). The bold letters indicate mutant sites. The first two fragments were generated by PCR using DR4b-5′(+A2) and DR4b-3′(+A1) as primers and DR4β cDNA as a template. The two fragments were purified and overlap extension was used to generate the full length gene containing the mutation at position 110. Using this mutant gene as a template, DR4b-5′(+B2) and DR4b-3′(+B1) were used as the primers to generate double mutant DR4β(NT). In mutant DR4β(NT), the residues Gln at positions 110 and Lys at position 139 were changed to Asn and Thr, respectively. Both wild-type DR4β and mutant DR4β(NT) have been confirmed by sequencing. Mutant DR4β(NT) was subcloned into the pDOI-5 expression vector at the EcoRI site downstream of the H2 Eα promotor and rabbit β-globulin intron (18). Wild-type DR4β and DR4β(NT) were subcloned into the pKCR-7 (19) expression vector separately for later transfection use.

The DR4β(NT)/pDOI-5 construct was double digested with NruI and XbaI to remove the plasmid sequence and microinjected into fertilized eggs from (SWR × B10)F1 mice. Viable embryos were reimplanted into the oviducts of pseudopregnant foster mothers. Mice carrying the transgene were identified by Southern blot analysis using DR4β cDNA as a probe. Founders were intercrossed and backcrossed to B10.RFB3 mice (20), which lack endogenous Eβ but express Ea intracytoplasmically.

The M12C3 cell line (gift from Dr. David Mckean, Mayo Clinic, Rochester, MN) was grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 0.1 mM 2-ME and buffered to pH 7.3 with 10 mM HEPES. M12C3 cells express the H2d MHC haplotype with mutated Aβd and Eβd (21). Thus, there is no expression of A or E molecules on M12C3 cell surface. HLA-DR4β- and DR4β(NT)-pKCR-7 gene constructs were cotransfected with pMC1neo poly(A) (Stratagene, La Jolla, CA) into M12C3 cells separately by electroporation using a gene pulser (Bio-Rad, Richmond, CA). The transfected cells were cultured in select medium containing 1 mg/ml G418 for 2 wk. Stable clones DR4b-9 and DR4bNT-2 were chosen by flow cytometry analysis (data not shown) and maintained in the culture medium described above.

HLA-DR4β(NT) transgenic mice and negative littermates were sacrificed and fresh tissues were removed and immediately frozen in liquid nitrogen. Total RNA of livers, hearts, thymi, and spleens were isolated using RNeasy Kit (Qiagen, Santa Clarita, CA). RT-PCR was performed according to the instruction of the manufacturer (Boehringer Mannheim, Indianapolis, IN). After the first strand of cDNA was synthesized, DR4b-5′ and DR4b-3′ oligos described above were used as primers in the following PCR. The final products were analyzed on 1.2% argarose gel.

The expression of DR4β, CD4, and TCR Vβ-chains on PBLs of transgenic mice and transfectants were analyzed by flow cytometry using mAbs: L227(α-DRβ), 14-4-4s(α-Eαp) GK1.5(α-CD4), HB163(α-Ab), B20.6(α-Vβ2), KT4-10(α-Vβ4), MR9-8(α-Vβ5.1), MR9-4(α-Vβ5.1.2), 44-22-1(α-Vβ6), TR-310(α-Vβ7), KJ-16(α-Vβ8.1.2), F23.1(α-Vβ8.2), MR10-2(α-Vβ9), RR3-15(α-Vβ11), 14.2(α-Vβ14), and KL23a(α-Vβ17). The condition of the FACS analysis has been previously described (22).

Peptides were synthesized by the Peptide Core Facility at the Mayo Foundation using an automated 430A peptide synthesizer (Applied Biosystems, Foster City, CA) and were purified by HPLC. Amino acid composition was confirmed by N-terminal sequencing using Edman’s method.

Mice were immunized with 100 μg of peptide emulsified in a saline solution and CFA, and T cell proliferation assay was performed (23). Briefly, draining lymph nodes were removed from the mice at 7 days after immunization and a single cell suspension (5 × 106) was prepared. Lymphocytes were cultured in 96-well plates at 5 × 105/well in RPMI 1640 medium supplemented with 25 mM HEPES buffer, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 3 × 10−5 M 2-ME, 1 mM sodium pyruvate, 5% (v/v) horse serum (HyClone, Logan, UT), and 2% (v/v) of TCM serum extender (Celox, Hopkins, MN). Cells were challenged with 100 μl of medium (negative control), Con A (2 μg/ml, positive control), or immunizing peptide (2, 10, and 50 μg/ml) at 37°C for 48 h. Eighteen hours before the termination, 10 μl of a 180 μCi/ml solution of [3H]thymidine was added to each well. Cells were harvested onto filter paper disks and incorporation of [3H]thymidine was determined by liquid scintillation counting. Results were expressed as the mean cpm of triplicate cultures. For the inhibition experiment, 20 μl (5 μg Ab) of culture supernatant containing mAb GK1.5 or L227 was added to the cells challenged in vitro with peptides at 50 μg/ml.

Popliteal, inguinal, and para-aortic lymph node cells from influenza hemagglutinin (HA)3 (307–319) immunized HLA-DR4β transgenic mice were removed at day 10 after immunization. T cells were isolated by using Dynabeads M-450 Thy-1.2 (Dynal, Lake Success, NY) according to the manufacturer’s instruction. The isolated cells were stained by FITC-conjugated B220 (α-CD45R) and phycoerythrin-conjugated MAC1 (α-CD11b) and checked under the fluorescence microscope. The purity of T cells was >99.5%.

M12C3, DR4b-9, and DR4bNT-2 cell lines were used as APCs to test HLA-restricted T cell response. Purified T cells (2 × 105) plus 5 × 105 irradiated (9000 rad) APCs of each cell line were cultured in 0.2 ml of culture medium per well in the presence of HA (307–319) Ag peptide. The T cell proliferation assay was conducted as described above.

Transgene-positive offspring were identified by Southern hybridization to DR4β cDNA probes. The founder, which was backcrossed to B10.RFB3 (Eβ0p), had five integrated transgenes (data not shown). The introduction of DR4β(NT) transgenes onto the B10.RFB3 strain enables the DR4β chains to pair with endogenous Eα chains. After four to five backcrosses to B10.RFB3 mice, the PBL from DR4β(NT)/B10.RFB3 transgenic mice and negative littermates were analyzed for surface expression of the DR4 mutant molecule by FACS. Figure 2,A shows that a subpopulation (about 40% of total cell population) from transgenic mice was stained by mAbs L227 (α-DR4β) and 14-4-4s (α-Eα) but no staining of cells was seen in negative littermates. The result of two-color FACS analysis showed that most of the B220-positive cells were stained by DR-specific Ab L227 (Fig. 2,B). To test whether the transgenes were transcribed in a tissue-specific manner, the RNA isolated from different tissues were analyzed by RT-PCR. The transcription of DR4β(NT) transgenes were found in the thymus and spleen from transgene-positive mice, but not in the liver and heart (Fig. 3).

FIGURE 2.

Analysis of HLA-DR4β expression in the transgenic HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice. A, PBL from HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice were analyzed by flow cytometry for surface expression of the molecules HLA-DR4β(NT)/Eα. B, Dot plots of two-color flow cytometry analysis of HLA-DR4β(NT) and B220 cell-surface expression on PBL from HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice.

FIGURE 2.

Analysis of HLA-DR4β expression in the transgenic HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice. A, PBL from HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice were analyzed by flow cytometry for surface expression of the molecules HLA-DR4β(NT)/Eα. B, Dot plots of two-color flow cytometry analysis of HLA-DR4β(NT) and B220 cell-surface expression on PBL from HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice.

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FIGURE 3.

Detection of tissue distribution of DR4β(NT) gene transcripts in the HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice by RT-PCR. Lanes 1 and 5 are liver, lanes 2 and 6 are hearts, lanes 3 and 7 are spleen, lanes 4 and 8 are thymi, and lane 9 is PCR product from transgene construct as positive control.

FIGURE 3.

Detection of tissue distribution of DR4β(NT) gene transcripts in the HLA-DR4β(NT)+/B10.RFB3 and HLA-DR4βNT/B10.RFB3 mice by RT-PCR. Lanes 1 and 5 are liver, lanes 2 and 6 are hearts, lanes 3 and 7 are spleen, lanes 4 and 8 are thymi, and lane 9 is PCR product from transgene construct as positive control.

Close modal

It has been shown that MHC class II molecules influenced the development of CD4+ T cells in the thymus. To determine whether HLA-DR4β transgenes effected the T cell repertoire, PBLs were analyzed for the coexpression of murine CD4 and a number of TCR Vβ-chains (Table I). In transgenic mice, T cells coexpressing CD4 and Vβ5.1.2, Vβ7, and Vβ11 were reduced about fivefold compared with nontransgenic littermates.

Table I.

TCR Vβ expression in CD4+ T cells

VβTransgenic (%)Nontransgenic (%)
 5.37 4.42 
10.46 10.15 
5.1 0.84 0.32 
5.1.2 0.91a 4.84 
10.37 11.57 
0.11 0.51 
8.1.2 23.9 24.28 
8.1.2.3 29.24 34.27 
8.2 15.53 20.42 
3.47 1.74 
11 0.19 1.2  
14 7.55 7.3 
17 0.89 0.95 
VβTransgenic (%)Nontransgenic (%)
 5.37 4.42 
10.46 10.15 
5.1 0.84 0.32 
5.1.2 0.91a 4.84 
10.37 11.57 
0.11 0.51 
8.1.2 23.9 24.28 
8.1.2.3 29.24 34.27 
8.2 15.53 20.42 
3.47 1.74 
11 0.19 1.2  
14 7.55 7.3 
17 0.89 0.95 
a

Bold type indicates TCR Vβ showing deletion.

To study the function of DR4β(NT)/Eα hybrid molecules in transgenic mice, the Ag-binding specificity of DR4β(NT)/Eα and the HLA-DR4-restricted T cell response were analyzed. The DR4-binding peptide HA (307–319) (24), MBP (84–106) (14), and GAD65 (274–286) (25) emulsified in CFA were used to immunize transgenic and nontransgenic mice, respectively. Seven days later, lymphocytes from draining lymph nodes were isolated and challenged with the same peptide in vitro. T cell proliferative responses specific to HA (307–319), MBP (84–106), and GAD65 (274–286) were observed only in transgenic mice (Fig. 4). More DR4-restricted peptides were tested later (Table II). All tested peptides elicited immune responses in transgenic mice but not in nontransgenic littermates and these responses could be inhibited by mAb L227 (α-DR) and GK1.5 (α-CD4) (Table II). The mAb 10.2.16 (α-Af) had no effect on these responses (data not shown). The peptide GAD65 (339–351) did not bind to DR4 molecule (25) and the insulin peptide (44–68) did not fit the DR4-binding motif on the basis of computer analysis. These two peptides failed to induce T cell response both in transgenic and nontransgenic mice. These results indicated that DR4β(NT)/Eα hybrid molecules had similar binding specificity to DR4 molecules and were capable of presenting these peptides to murine T cells to generate immune responses. The two altered amino acid residues did not effect the peptide binding site of DR4β(NT)/Eα, but helped produce a more efficient interaction between human class II and mouse CD4 molecules.

FIGURE 4.

T cell proliferation to antigenic peptides in HLA-DR4βNT transgenic mice. Responses of popliteal lymph node cells were analyzed in mice which have been immunized with HA (307–319) (A), MBP (84–106), (B), and GAD65 (274–286) (C). Results are shown as the mean δcpm plus SD from two mice per group assayed individually. Background counts of T cells and medium were <4500 δcpm.

FIGURE 4.

T cell proliferation to antigenic peptides in HLA-DR4βNT transgenic mice. Responses of popliteal lymph node cells were analyzed in mice which have been immunized with HA (307–319) (A), MBP (84–106), (B), and GAD65 (274–286) (C). Results are shown as the mean δcpm plus SD from two mice per group assayed individually. Background counts of T cells and medium were <4500 δcpm.

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Table II.

In vitro T cell proliferation of HLA-DR4/B10.RFB3 transgenic mice to selected DR4-specific peptide in the presence or absence of anti-CD4(GK1.5) or −DR4(L227) mAb

PeptideThymidine Incorporation (Δ cpm × 1000)
Peptide onlyPeptide + GK1.5Peptide + L227
HA (307–319) 63.8 ± 5.2 0.6 ± 0.2 12.1 ± 0.2 
MBP (87–106) 47.3 ± 6.5 0.5 ± 0.2 14.1 ± 0.3 
mPLP (175–192) 51.6 ± 6.3 0.5 ± 0.3 12.9 ± 0.2 
GAD65 (274–286) 45.8 ± 3.5 0.4 ± 0.2 8.0 ± 0.4 
GAD67 (114–133) 49.2 ± 4.9 0.8 ± 0.3 10.5 ± 0.6 
Insulin (1–24) 43.5 ± 5.4 0.6 ± 0.2 14.5 ± 0.5 
PeptideThymidine Incorporation (Δ cpm × 1000)
Peptide onlyPeptide + GK1.5Peptide + L227
HA (307–319) 63.8 ± 5.2 0.6 ± 0.2 12.1 ± 0.2 
MBP (87–106) 47.3 ± 6.5 0.5 ± 0.2 14.1 ± 0.3 
mPLP (175–192) 51.6 ± 6.3 0.5 ± 0.3 12.9 ± 0.2 
GAD65 (274–286) 45.8 ± 3.5 0.4 ± 0.2 8.0 ± 0.4 
GAD67 (114–133) 49.2 ± 4.9 0.8 ± 0.3 10.5 ± 0.6 
Insulin (1–24) 43.5 ± 5.4 0.6 ± 0.2 14.5 ± 0.5 

Although T cell proliferative response in transgenic mice implied that mouse CD4 molecules could recognize the altered DR4β β2 domain, we further confirmed this by using selected transfectant clones as APCs. T cells isolated from mice immunized with HA (307–319) peptide were tested against a panel of APCs. Figure 5 shows the responses of T cells specific for HA (307–319) presented by different APC clones. The DR4b(NT)-2 cell line elicited a strong T cell response compared with that of the DR4b-9 cell line. No T cell response was elicited by M12C3 cell lines. The absence of proliferative response of T cells for HA (307–319):DR4β/Eα suggested an inefficient interaction between mouse CD4 and DR4β/Eα molecules. These data showed that mouse CD4 molecules could recognize and interact efficiently with altered DR4 molecules to induce DR4-restricted T cell response in vivo. The amino acids Asn at position 110 and Thr at 139 were critical for mouse CD4 binding. When Glu and Lys (in Eβ) or Gln and Lys (in DR4β) were in those positions, the binding affinity decreased and the T cell response was weak or abolished.

FIGURE 5.

T cell responses to HA (307–319) presented by altered and wild-type DR4β/Eα hybrid molecules. A total of 2 × 105 purified T cells from draining lymph nodes of HLA-DR4β(NT) transgenic mice, which had been immunized with HA (307–319), were cultured with 5 × 104 irradiated M12C3, DR4b-9, and DR4bNT-2 cells in the presence of HA (307–319) at a concentration of 50 μg/ml.

FIGURE 5.

T cell responses to HA (307–319) presented by altered and wild-type DR4β/Eα hybrid molecules. A total of 2 × 105 purified T cells from draining lymph nodes of HLA-DR4β(NT) transgenic mice, which had been immunized with HA (307–319), were cultured with 5 × 104 irradiated M12C3, DR4b-9, and DR4bNT-2 cells in the presence of HA (307–319) at a concentration of 50 μg/ml.

Close modal

Generation of functional HLA-DR transgenic mice are critical for future in-depth studies on the function of HLA-DR molecules in vivo and generation of viable HLA-DR-restricted disease models. Attempts to generate functional HLA-DR4 transgenic mice have been difficult in our laboratory (our unpublished observations) and others (15), mainly due to an inability of mouse CD4 to interact efficiently with human HLA-DR molecules. Currently available DR4 transgenic mice either suffer from inadequate interaction with mouse CD4 T cells or expression of chimeric human/mouse hybrid molecules in which the unique role of human HLA-DR molecules cannot be determined. The DR4β(NT) transgenic mice we have generated differ from the wild-type DR4β in only two amino acids (110 and 139) in the β2 domain. Since these two residues do not take part in binding of peptides or interaction with the TCRs, the function of DR4β transgene in the transgenic mice should resemble the human immune response.

The DR4β(NT) molecules pair with Eα and fold into a proper conformation and expressed on the cells surface in a tissue-specific manner due to the Eα promotor upstream of DR4β(NT). These molecules bind a panel of DR4-specific peptides and present them to T cells to generate DR4-restricted T cell response. This demonstrates that the mouse CD4 can interact with mutated DR4β β2 domain effectively and that the DR4β(NT)/Eα molecule can positively/negatively select T cells expressing specific TCR to shape the mouse T cell repertoire. We are currently introducing this transgene along with the DRα into a mouse lacking endogenous class II molecules (Aβ0). Such a transgenic mouse will be valuable in the study of the immune response and disease association of the DR gene in the absence of endogenous mouse class II genes.

Mouse mammary tumor virus-encoded superantigens (Mtv) play an important role in the shaping of T cell repertoire. The MHC class II molecules present these superantigens to the immature T cells in the thymus and mediate clonal deletion of maturing T cells bearing certain Vβ segments (26). Since DR4β(NT).B10.RFB3 transgenic mice have the C57BL/10 background genes they should express the Mtv 7, 8, 9, 14, and 17 genes (27, 28). The DR4β(NT)/Eα molecule presents the superantigens coded by the Mtv genes to immature T cells, resulting in the deletions of Vβ5.1.2-, Vβ7-, and Vβ11-expressing CD4+ T cells, similar to the DRα/Eβ transgenic mice (29).

We are currently generating a double transgenic mouse containing an altered DR4 gene along with the HLA-DQ8 gene to simulate the human HLA haplotype DQA1*0301/DQB1*0302/DRA*0101/DRB1*0401. This haplotype is linked to many human autoimmune diseases and thus can have potential value in those studies. Furthermore, our findings confirm the in vitro studies showing the importance of residues 110 and 139 on the class II second domain for optimum interaction with the CD4 T cells. Similar strategy can be used to generate functional HLA-DR transgenic mice with DR genes involved in other human autoimmune diseases such as diabetes (DR3) and multiple sclerosis (DR2).

We thank Dr. D. Mathis (Strasbourg, France) for the pDOI-5 vector.

1

This work was supported by National Institutes of Health Grant CA 24473.

3

Abbreviation used in this paper: HA, hemagglutinin

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