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The Journal of Immunology, 1998, 161: 2925-2929.
Copyright © 1998 by The American Association of Immunologists

HLA-DR4 (DRB1*0401) Transgenic Mice Expressing an Altered CD4-Binding Site: Specificity and Magnitude of DR4-Restricted T Cell Response1

Shuchong Pan, Theodore Trejo, Julie Hansen, Michele Smart and Chella S. David2

Department of Immunology, Mayo Clinic and Medical School, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-DR4ß(NT) transgene construct in which positions 110 and 139 were altered to resemble endogenous mouse H2 Aß molecules. This construct was introduced into (B10 x SWR) embryos, and DR4ß(NT) transgenic mice were produced. The transgene was transferred into B10.RFB3 (Eß0 E{alpha}p) mice. The transgene-encoded DR4ß molecules paired with endogenous E{alpha} chains to form stable DR4ß/E{alpha} 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-DR4ß(NT) transgenic mice with DR4-restricted peptides induced T cell proliferation in vitro. While the purified T cells from DR4ß(NT) transgenic mice responded strongly to the HA(307–319) presented by M12C3 transfectants expressing altered DR4ß/E{alpha} heterodimers, the response to the same peptides presented by transfectants expressing wild-type DR4ß/E{alpha} 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-DR4ß 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 ß2 domain of MHC class II (4, 5) as a coreceptor to the TCR-{alpha}ß 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 ß2 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-DR4ß was the major contact site with CD4. At the same time, Konig et al. (4) reported that the interaction between CD4 and Aß were diminished when substitutions were made in the same region of ß2 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 ß-chain in which the ß2 domain of HLA-DR molecule was substituted with the ß2 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 ß2 domain of mouse class II molecules was substituted with the human ß2 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 {alpha}2ß2 domain was substituted with the {alpha}2ß2 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 ß2 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 ß-chain was identical to H2-Aß except for an Ala to Val substitution at position 140 (Fig. 1Go). In contrast, H2-Eß had four different amino acid residues at positions 110, 139, 140, and 142. The sequence of HLA-DR4ß(DRB1*0401) was similar to H2-Eß, but Glu at position 110 was substituted by Gln and residue at 142 was the same as H2-Aß. 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 DR4ß 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 DR4ß was more like that of H2-Aß and DQß. HLA-DR4ß(DRB1*0401) transgenic mice with the altered ß2 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.



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  		FIGURE 1. Amino acid sequence comparison of MHC class II molecules between mouse and human.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgene constructions

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-DR4ß(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 DR4ß 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 DR4ß(NT). In mutant DR4ß(NT), the residues Gln at positions 110 and Lys at position 139 were changed to Asn and Thr, respectively. Both wild-type DR4ß and mutant DR4ß(NT) have been confirmed by sequencing. Mutant DR4ß(NT) was subcloned into the pDOI-5 expression vector at the EcoRI site downstream of the H2 E{alpha} promotor and rabbit ß-globulin intron (18). Wild-type DR4ß and DR4ß(NT) were subcloned into the pKCR-7 (19) expression vector separately for later transfection use.

Transgenic mice

The DR4ß(NT)/pDOI-5 construct was double digested with NruI and XbaI to remove the plasmid sequence and microinjected into fertilized eggs from (SWR x 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 DR4ß cDNA as a probe. Founders were intercrossed and backcrossed to B10.RFB3 mice (20), which lack endogenous Eß but express Ea intracytoplasmically.

Cell lines

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 Aßd and Eßd (21). Thus, there is no expression of A or E molecules on M12C3 cell surface. HLA-DR4ß- and DR4ß(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.

RT-PCR

HLA-DR4ß(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.

Flow cytometry

The expression of DR4ß, CD4, and TCR Vß-chains on PBLs of transgenic mice and transfectants were analyzed by flow cytometry using mAbs: L227({alpha}-DRß), 14-4-4s({alpha}-E{alpha}p) GK1.5({alpha}-CD4), HB163({alpha}-Ab), B20.6({alpha}-Vß2), KT4-10({alpha}-Vß4), MR9-8({alpha}-Vß5.1), MR9-4({alpha}-Vß5.1.2), 44-22-1({alpha}-Vß6), TR-310({alpha}-Vß7), KJ-16({alpha}-Vß8.1.2), F23.1({alpha}-Vß8.2), MR10-2({alpha}-Vß9), RR3-15({alpha}-Vß11), 14.2({alpha}-Vß14), and KL23a({alpha}-Vß17). The condition of the FACS analysis has been previously described (22).

Peptide synthesis

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.

T cell proliferation assay

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 x 106) was prepared. Lymphocytes were cultured in 96-well plates at 5 x 105/well in RPMI 1640 medium supplemented with 25 mM HEPES buffer, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 3 x 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.

Purification of T cells

Popliteal, inguinal, and para-aortic lymph node cells from influenza hemagglutinin (HA)3 (307–319) immunized HLA-DR4ß 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 ({alpha}-CD45R) and phycoerythrin-conjugated MAC1 ({alpha}-CD11b) and checked under the fluorescence microscope. The purity of T cells was >99.5%.

APC lines for analyzing HLA-restricted T cell response

M12C3, DR4b-9, and DR4bNT-2 cell lines were used as APCs to test HLA-restricted T cell response. Purified T cells (2 x 105) plus 5 x 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation and characterization of DR4ß(NT)/B10.RFB3 transgenic mice

Transgene-positive offspring were identified by Southern hybridization to DR4ß cDNA probes. The founder, which was backcrossed to B10.RFB3 (Eß0 E{alpha}p), had five integrated transgenes (data not shown). The introduction of DR4ß(NT) transgenes onto the B10.RFB3 strain enables the DR4ß chains to pair with endogenous E{alpha} chains. After four to five backcrosses to B10.RFB3 mice, the PBL from DR4ß(NT)/B10.RFB3 transgenic mice and negative littermates were analyzed for surface expression of the DR4 mutant molecule by FACS. Figure 2GoA shows that a subpopulation (about 40% of total cell population) from transgenic mice was stained by mAbs L227 ({alpha}-DR4ß) and 14-4-4s ({alpha}-E{alpha}) 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. 2GoB). 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 DR4ß(NT) transgenes were found in the thymus and spleen from transgene-positive mice, but not in the liver and heart (Fig. 3Go).



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  		FIGURE 2. Analysis of HLA-DR4ß expression in the transgenic HLA-DR4ß(NT)+/B10.RFB3 and HLA-DR4ßNT-/B10.RFB3 mice. A, PBL from HLA-DR4ß(NT)+/B10.RFB3 and HLA-DR4ßNT-/B10.RFB3 mice were analyzed by flow cytometry for surface expression of the molecules HLA-DR4ß(NT)/E{alpha}. B, Dot plots of two-color flow cytometry analysis of HLA-DR4ß(NT) and B220 cell-surface expression on PBL from HLA-DR4ß(NT)+/B10.RFB3 and HLA-DR4ßNT-/B10.RFB3 mice.

 


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  		FIGURE 3. Detection of tissue distribution of DR4ß(NT) gene transcripts in the HLA-DR4ß(NT)+/B10.RFB3 and HLA-DR4ßNT-/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.

 
Hybrid DR4ß(NT)/E{alpha} molecules influence T cell development in transgenic mice.

It has been shown that MHC class II molecules influenced the development of CD4+ T cells in the thymus. To determine whether HLA-DR4ß transgenes effected the T cell repertoire, PBLs were analyzed for the coexpression of murine CD4 and a number of TCR Vß-chains (Table IGo). In transgenic mice, T cells coexpressing CD4 and Vß5.1.2, Vß7, and Vß11 were reduced about fivefold compared with nontransgenic littermates.


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  		Table I. TCR Vß expression in CD4+ T cells

 
DR4ßNT/E{alpha} hybrid molecules can serve as restriction elements in transgenic mice

To study the function of DR4ß(NT)/E{alpha} hybrid molecules in transgenic mice, the Ag-binding specificity of DR4ß(NT)/E{alpha} 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. 4Go). More DR4-restricted peptides were tested later (Table IIGo). All tested peptides elicited immune responses in transgenic mice but not in nontransgenic littermates and these responses could be inhibited by mAb L227 ({alpha}-DR) and GK1.5 ({alpha}-CD4) (Table IIGo). The mAb 10.2.16 ({alpha}-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 DR4ß(NT)/E{alpha} 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 DR4ß(NT)/E{alpha}, but helped produce a more efficient interaction between human class II and mouse CD4 molecules.



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  		FIGURE 4. T cell proliferation to antigenic peptides in HLA-DR4ßNT 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 {delta}cpm plus SD from two mice per group assayed individually. Background counts of T cells and medium were <4500 {delta}cpm.

 

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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

 
Mouse CD4 T cells recognize DR4ß(NT)/Ea but not DR4ß/Ea molecules

Although T cell proliferative response in transgenic mice implied that mouse CD4 molecules could recognize the altered DR4ß ß2 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 5Go 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):DR4ß/E{alpha} suggested an inefficient interaction between mouse CD4 and DR4ß/E{alpha} 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 Eß) or Gln and Lys (in DR4ß) were in those positions, the binding affinity decreased and the T cell response was weak or abolished.



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  		FIGURE 5. T cell responses to HA (307–319) presented by altered and wild-type DR4ß/E{alpha} hybrid molecules. A total of 2 x 105 purified T cells from draining lymph nodes of HLA-DR4ß(NT) transgenic mice, which had been immunized with HA (307–319), were cultured with 5 x 104 irradiated M12C3, DR4b-9, and DR4bNT-2 cells in the presence of HA (307–319) at a concentration of 50 µg/ml.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 DR4ß(NT) transgenic mice we have generated differ from the wild-type DR4ß in only two amino acids (110 and 139) in the ß2 domain. Since these two residues do not take part in binding of peptides or interaction with the TCRs, the function of DR4ß transgene in the transgenic mice should resemble the human immune response.

The DR4ß(NT) molecules pair with E{alpha} and fold into a proper conformation and expressed on the cells surface in a tissue-specific manner due to the E{alpha} promotor upstream of DR4ß(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 DR4ß ß2 domain effectively and that the DR4ß(NT)/E{alpha} 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 DR{alpha} into a mouse lacking endogenous class II molecules (Aß0). 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 Vß segments (26). Since DR4ß(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 DR4ß(NT)/E{alpha} molecule presents the superantigens coded by the Mtv genes to immature T cells, resulting in the deletions of Vß5.1.2-, Vß7-, and Vß11-expressing CD4+ T cells, similar to the DR{alpha}/Eß 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).


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


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

2 Address correspondence and reprint requests to Dr. Chella S. David, Department of Immunology, Mayo Clinic and Medical School, Rochester, MN 55905. E-mail address: Back

3 Abbreviation used in this paper: HA, hemagglutinin Back

Received for publication April 10, 1998. Accepted for publication May 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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