Immunogenetics 33: 12-17, 1991 1HllllllllO- genetics

© Springer-Verlag 1991

Generation and characterization of an HLA-DRa-specific monoclonal antibody using L-cell transfectants expressing human and mouse class II major histocompatibility dimers

Donald Palmer, Michael Kevany, Charles Mackworth-Young, Richard Batchelor, Giovanna Lombardi, and Robert Lechler

Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, England

Received June 21, 1990; revised version received September 4, 1990

Abstract. Among the HLA-DR-specific monoclonal an- tibodies (mAbs) that have been characterized previously, there is a marked shortage of DRa chain-specific mAbs which stain unfixed cells. This may result from the high degree of sequence similarity between the ~1 domains of HLA-DR and H-2E leading to a state of cross-tolerance to DRa in H-2-expressing mice. BALB/b (E-negative) mice were immunized with DRl- t rans fec ted mouse L cells. The chain specificity of the resulting DR-specific mAb was determined using a panel of transfectants ex- pressing hybrid mouse/human class II heterodimers. A DRa-specific mAb was generated which was capable of immunoprecipitating DRa~ dimers and inhibiting the an- ti-DR alloresponse of human T-cell clones. The present study demonstrates that, with the selection of a suitable recipient strain, transfectants can be useful in the genera- tion and definition of chain-specific mouse mAbs.

Introduction

Monoclonal antibodies (mAbs) have proved to be valuable tools in the structural and functional analysis of major histocompatibility complex (MHC) molecules (Pierres et al. 1981). MHC-specific class II mAbs with a defined chain specificity and capable of staining cell surface MHC, are vital reagents in monitoring the cell surface ex- pression of individual c~/3 combination following gene transfection. Among the available mAbs specific for human HLA-D gene products there was no known cell surface-staining DRc¢-specific antibody at the outset of this study. In contrast with this deficiency, multiple DRa- specific mAbs have been described which interact with isolated DRc~ chains, or which bind to DR-expressing cels after aldehyde fixation (Guy et al. 1982). One possible

Offprint requests to: R. Lechler.

explanation for this anomaly is the high degree of se- quence similarity between the amino (NH2)-terminal oq domains of HLA-DR (Lee et al. 1982) and H-2E (Benoist et al. 1983), the mouse homologue. This exceeds the ex- tent of sequence similarity that exists between other homologous human and mouse MHC products. As a con- sequence, E-expressing mice are more likely to be tolerant to much of the exposed surface of DRc~. The DRop-specific mAbs that have been produced following immunization with purified protein may well be specific for epitopes in the less conserved c~ 2 or transmembrane portions, which may be less accessible in the native molecule expressed on intact cells. In addition, the absence of an intra-domain disulfide bridge in MHC class II al domains increases the likelihood of conformational alteration when o~ chains are dissociated from their/3-chain partner.

In order to generate cell surface-staining MHC class II-specific antibodies it is advantageous, therefore, to use whole cells as the immunogen, to ensure that the native conformation is preserved. If human cells are used, an- tibodies may be raised against multiple xenogeneic molecules other than the MHC class II chain of interest. Mouse L cells expressing the products of transfected human class II genes have the clear advantage that the ex- pressed HLA-D specificity is the only xenoantigen to which the recipient mouse is exposed. In addition, as shown in this study, L-cell transfectants expressing HLA- D products, either as species-matched or mismatched c~ dimers, are powerful tools in defining the locus specificity and chain specificity of mAbs. Similarily, transfectants expressing haplotype-matched and -mismatched mouse H-2 molecules have proved to be valuable reagents for defining the specificity of mouse alloantibodies (Landais et al. 1986).

As a strategy for raising a DRc~-specific mAb we have used HLA-DRl-expressing L cells to immunize BALB/b mice which do not express E due to a deletion in the Ea

gene (Mathis et al. 1983). The absence of E expression

D. Palmer et al. : Generation and definition of a DRa-specific antibody 13

in these mice should avoid the problem of tolerance to DRop. We have also made use of transfectants expressing hybrid human/mouse MHC class II o~/3 dimers in order to determine the chain specificity of the mAbs generated in this study, as well as of two previously published mAbs.

Materials and methods

Cell lines. The transfectants used in this study are described in Table 1. The parental mouse L cell (DAP.3) and all the transfectants were maintained in culture in Dulbecco's modified Eagle medium (Flow Laboratories, McClean, West Virginia) supplemented with 10% fetal calf serum (FCS), 2 mM L-glntamine, and 10 gg/ml gentamicin. Selec- tive drugs for the transfected cell lines were also added to the culture medium as appropriate. The L cells were subcultured following tryp- sinization once weekly. The homozygous DR7-expressing, Epstein-Barr virus (EBV)-transformed B-cell line Mann (Bodmer et al. 1984) was cultured in RPMI-1640 supplemented with 10% FCS, 2 mM L- glutamine, and antibiotics. The nonsecreting mouse myeloma Ag8, used as the fusion partner for hybridoma generation, was cultured in RPMI-1640 with 10% FCS and antibiotics.

mAbs. The mAbs used in this study were 14.4.4S (anti-Ea; Ozato et al. 1980), M5/114 (anti-A/3 b'a and anti-E~d; Bhattacharya et al. 1981), 10.2.16 (anti-A~k; Oi et al. 1978), L243 (anti-DR; Lampson and Levy 1980), Genox 3.3 (anti-DQwl; Brodsky et al. 1980), SG465 (anti-DP, DQ, DR; Goyert and Silver 1983), and HB.50 (anti-mouse class I; Ozato et al. 1980). They were used as cell-free hybridoma supernatants for flow cytometric analysis and at the concentration indicated for inhibition experiments.

Flow microfluorimetric analysis. For flow microfluorimetric analysis, 5 × 105 cells were incubated with 100 Ixl of the indicated mAb, or with medium alone for the negative control, at 4 °C for 30 min. After washing

Table 1. A list of cell lines used in this study.

Cell line Property Reference

Human T-cell clones G l l anti-DR1, Dwl L9 anti-DR1, Dwl

Lombardi et al. 1989 G. Lombardi (unpublished data)

Human EBV-transformed B cell Mann DR7 Bodmer et al. 1984 Daudi HLA class I-negative Klein et al. 1968

Human fibroblast M1 HLA class II-negative Mentzer et al. 1986

Transfected mouse L cells (DAP.3) DAP.3 untransfected Lechler et al. 1988 DR1 Lechler et al. 1988 E~dEc~ R. Lechler

(unpublished data) E~kE~ Lechler et al. 1988 E~dDRc~ Lechler et al. 1990 Et3kDRa Lechler et al. 1990 DRI~Ec~ Lechler et al. 1988 A~kAc~ k Lechler et al. 1990 Aj3kDRa Lechler et al. 1990

twice in phosphate-buffered saline with 2.5 % FCS, the cells were in- cubated for a further 30 min at 4 °C with 100 ~tl of a 1 : 50 dilution of fluoresceinated sheep anti-mouse Ig (Amersham, Amersham, UK). After two additional washes, stained ceils were analyzed using the EPICS Profile Flow Cytometer (Coulter Electronics, Luton, UK).

T-cell proliferation assay. Two anti-DR1 alloreactive human T-cell clones, G11 and L9, were raised as previously described (Lombardi et al. 1989a). For the proliferative assay, T cells (104 cells/well) were cultured in the presence of mitomycin C-treated DR1 L cells (5 × 104 cells/well) in flat-bottomed microtiter plates in a total volume of 200 p.1. A range of concentrations of mAb supernatant were added to the cultures, as indicated in the figure legends. After 2 days, 1 ~tCi/well of 3H-thymidine ([3H]-TdR) was added and the cultures harvested onto glass fibre filters. Proliferation was measured as [3H]-TdR incor- poration as determined by liquid scintillation spectroscopy. The results are expressed as cpm + / - standard deviation for triplicate cultures.

Immunoprecipitation of MHC class 11 molecules. Two human cell l ines-Daudi, a B cell which lacks MHC class I expression and M1, a fibroblast which lacks class II expression-were surface-labeled with 12Slodine (2 × 106 cells for each labeling) and lysed in 1% digitonin (Sigma, London, UK). Immunoprecipitation was performed using a con- ventional protocol (Hudson and Hay 1989). Briefly, after pre-clearing with an irrelevant mouse mAb (OKT3) and staphylococcal A-linked Sepharose 4B beads (Pharmacia, Uppsala, Sweden), the lysates were incubated with 50 ~tl of hybridoma supernatant containing the test an- tibody. W6/32 (anti-HLA class I; Barnstaple et al. 1978) and L243 (anti- HLA-DR; Lampson and Levy 1980) were used as control mAbs. The lysates were then incubated with staphylococcal A-linked beads. The beads were pelletted by centrifugation, washed, and boiled in a reducing buffer. The supernatant containing the released protein was then elec- trophoresed on a 12 % sodium dodecyl sulfate (SDS) polyacrylamide gel, dried, and autoradiographed.

Results

Generation of DR-specific mAbs following immunization with DRl-expressing L cells. BALB/b mice carrying the E-negative H-2 b haplotype were primed intraperitoneally with 1 × 107 L-cell (H-2 ~) transfectants expressing high levels of cell surface HLA-DR1. After 4 weeks the mice were boosted in the same way with 2 × 106 DR1 L cells and their spleen cells fused with the Ag8 myeloma line 48 h later. Supernatants from 70 wells with growing hybridomas were screened initially on EBV transformed human B cells using a cell surface enzyme-linked im- munosorbent assay (ELISA). The 20 supernatants that gave positive results in the ELISA were tested by flow cytometric analysis on the DR1 L cells, and three were found to produce surface fluorescence. The cells from one of these positive wells were subcloned by limiting dilution and one subclone, 7-4.1, was selected for further study.

The fluorescence profiles generated with 7-4.1 on the untransfected L-cell line DAP.3, DR1 L cells, and an EBV-transformed human B-cell line are shown in Figure 1. As can be seen, 7-4.1 stained the two DR-expressing cells, but not DAP.3. The level of fluorescence was similar to that obtained with the DR-specific mAb L243.

14 D. Palmer et a]. : Generation and definition of a DRc~-specific antibody

B LCL

I i

DRo:DR113

I i

DAP-3

I I 101 10 2 10 3

Relative fluorescence intensity

Fig. 1. The 7-4. I mAb produces HLA-DR-dependent cell surface stain- ing. Flow cytometric profiles of a human EBV-transformed B-cell line (B-LCL), a DRl-expressing L-celt transfeetant (DRII3:DRc0, and the untransfected L-cell line (DAP.3) are shown. Background fluorescence following incubation with the fiuorescein isothiocyanate-conjugated an- ti-mouse immunoglobulin alone is portrayed by the dotted line, staining with L243 is represented by the dashed line, and with 7-4.1 by the solid l i n e .

Mapping the specificity of 7-4.1 for DRo~ using transfec- rants expressing hybrid human/mouse class H dimers. In order to determine the chain specificity of the 7-4.1 mAb, a series of transfected L cells expressing species-mis- matched human/mouse MHC class II c~B dimers was used. The derivation of these transfectants is described elsewhere (Lechler et al. 1990). As displayed in Figure 2, 7-4.1 stained the E/3aDR~-expressing cells, but not the DR1/3Ea or the E/3aEc~ transfectant. No staining of cells expressing native A molecules was observed (data not shown). These results demonstrate that 7-4.1 is specific for the DRc~ chain.

The L243 and SG465 mAbs are ~ chain-specific. The previously published mAbs L243 (anti-DR) and SG465, which reacts with all human class II products, were tested on the panel of human/mouse hybrid transfectants. As can be seen in Figure 3, L243 gives exactly the same pattern of staining as the 7-4.1 antibody produced in this study, and appears to recognize an epitope on the DRa chain. The SG465 mAb, which recognizes as conserved determi- nant on all human class II molecules, also appears to be

chain-specific. As for 7-4.1 and L243, SG465 stains cells expressing E/3dDRc~ but not DR1/3Ec~ (Fig. 3). It

E

©

I

DRo~DRlp / Eo~DR1,5

I I L I I I

DRc~E,Sd l Eo~Epd

I t I I ; I 101 10 2 10 3 101 1 2 10 3

Relative fluorescence intensity

Fig. 2. The 7-4.1 mAb gives a DR~-dependent pattern of cell surface staining. Flow cytometric profiles of L-cell transfectants expressing the MHC class II dimers as indicated on each panel are displayed. Background staining is shown as a dotted line, and staining with the 7-4.1 mAb as a solid line.

gives an additional reaction on cells expressing ABkAa k, but did not stain Ad-expressing transfectants.

The reaction patterns of 7-4.1, L243, and SG465 are summarized in Table 2. The 31.1 (DeKretser et al. 1982) DR/3-specific mAb is included for comparison.

The 7.4.1 mAb immunoprecipitated a cell surface DR heterodimer from a Daudi cell lysate. In order to confirm that the 7-4.1 mAb binds to HLA class II molecules, an immunoprecipitation experiment was performed (Fig. 4). The Daudi cell line, which lacks HLA class I expression, and the M1 human fibroblast, which lacks HLA class II

DRc~DR1,5 Ec~DR1 [3

0 2 1'0 3 101 1'0 2 1'0 3 DRaEI 3d Ao:kA ~, k

O

101 1'0 2 110 3 1() 1

Relative fluorescence intensity

1'0 2 1'0 3

Fig. 3. The patterns of staining of transfected L ceils expressing mouse/human MHC class II heterodimers indicate that the mAbs L243 and SG465 are both directed against an c~-chain epitope. Flow cytometric profiles of L-cell transfectants expressing the MHC class II dimers as indicated on each panel are shown. Background staining is represented as a dotted line, staining with L243 as a dashed line, and with SG465 as a solid line.

D. Palmer et al.: Generation and definition of a DRc~-specific antibody 15

Table 2. Patterns of staining of transfected cell lines by the 7-4.1, L243, and SG465 mAbs.

mAb MHC class II molecule expressed on transfected cell line

DRADRB DRAEb EaDRB EaEb DRAAb AaAb

7-4.1 + + - - + - L243 + + - - + - SG465 + + - - + +

used. Full inhibition of the response of G 11 was seen when the culture contained 20% 7-4.1 supernatant; 5% L243 supernatant was required to cause the same degree of in- hibition for this clone. No inhibition was seen with the negative control mAb, Genox 3.53 (anti-DQwl). The 7-4.1 supernatant was only weakly inhibitory of the response of the T-cell clones to DRl-expressing human B-cell lines, compared to the effects of the L243 mAb.

Discussion

Following immunization of BALB/b, E-negative mice with DRl-expressing mouse L-cell transfectants, a DRc~- specific mAb, 7-4.1, was produced which stained unfixed cells. The chain specificities of this and two previously

Fig. 4. The 7-4.1 mAb immunoprecipitates a heterodimer with the same reIative mass as HLA-DR. An autoradiograph of a reducing SDS- polyacrylamide gel electrophoresis is shown. Lysates from surface- iodinated Daudi cells (left panel) and from M1 cells (right panel) were immunoprecipitated using the mAbs indicated at the head of each lane. The migration of relative mass markers is shown.

expression, were used. The HLA class I-specific mAb W6/32 immunoprecipitated bands of 45 000 and 12 000 from the M1 line and not from Daudi and L243, the HLA- DR-specific mAb, brought down bands of 32 000 and 28 000 from the Daudi lysate and not from M1. The 7-4.1 mAb precipitated bands of the same relative mass as L243, confirming its specificity for HLA-D region products.

The 7-4.1 mAb inhibits the proliferation of alloreactive anti-DR1 human T-cell clones. The effect of the 7-4.1, DRc~-specific mAb on the proliferative response of two anti-DR1 human T-cell clones, L9 and G11, was com- pared to the inhibition caused by the DR-specific mAb L243 (Fig 5). The clones were co-cultured with the DRl-expressing L-cell transfectant to which they were known to respond. For the L9 clone, the level of inhibition with 7-4.1 and L243 mAbs was comparable at all doses

1 2

8

10 20 30 40

30 b)

10-

i I I

0 10 20 30 40

~t 1 mAb/well

Fig. 5. The 7-4.1 mAb inbhibits the proliferative response of two human alloreactive T-cell clones. The T-cell clones Gl l (a) and L9 (b) (104 cells per well) wre co-c~tured with mitomycin C-treated DRl-express- ing L-cell transfeetants (5 x 104 cells per well) in the presence of in- creasing concentrations of 7-4.1 (O), L243 (C)), or Genox 3.1 ([7), HLA-DQ-specific mAb supernatants for 72 h. Results are recorded as cpm +-standard deviation.

16 D. Palmer et al.: Generation and definition of a DRc~-specific antibody

45 55 65 75 85

DR(~ TVWRLEEF GRFASFEAQGALANIAVDKANLE IMTKRSNYTP I TN

I-E~ -I . . . . . . AK . . . . . . . . . . . . . . . . . . . . DY-KE---N--DA-

Fig. 6. Amino acid sequence comparison of the carboxy-terminal halves of the DR and E c h domains. The single-letter amino acid code is used. The sequence of Ec~ is only shown when it differs from DRc~. The sequence of DRc~ was from Lee and co-workers (1982) and that of Ec~ from Benoist and co-workers (1983).

published mAbs were defined using a panel of transfec- tants expressing human and mouse hybrid class II molecules. The DRop-specific mAb was able to im- munoprecipitate cell surface-labeled DRc~/3 dimers and to inhibit the proliferative responses of two alloreactive, anti-DR1, human T-cell clones.

None of the previously described DRop-specific mAbs has been shown to stain intact, unfixed cells. There are two possible ways to account for this. The first is that many of these antibodies were generated using purified DRot chains as the immunogen (Guy et al. 1982). Unlike the/31 domains of class II molecules, o~ 1 domains to not have an internal disulfide bond. As a consequence, oq domains are likely to be conformationally plastic and native conformation may be destroyed by denaturation of a/3 dimers. Analysis of mAb-defined epitopes on mouse Acq domains when co-expressed with haplotype-mis- matched A/3 chains indicates that al domain conformat- ion is susceptible to alteration even in the context of an assembled c~/3 dimer (Braunstein and Germain 1987). Several examples of A~ 1 epitope loss due to haplotype- mismatched pairing have been seen, and two examples of the appearance of an epitope imposed by pairing with a mismatched /3 chain have been described. Thus, im- munization with denatured DRo¢ chains might not be ex- pected to generate antibodies which recognize the molecule in its native conformation.

A second explanation for the shortage of cell surface- staining DRa-specific mAbs is tolerance in E-expressing mice to many DRc~ epitopes due to sequence similarity between DRa I and Eai domains. Not only do the two se- quences share 80 % similarity, but the predicted locations of conserved regions in the al domain three-dimensional structure may further increase the extent of cross- tolerance. Based on the hypothetical model of class II structure (Brown et al. 1988) that was derived from the crystal structure of HLA-A2 (Bjorkman et al. 1987), there is a region of sequence identity comprising a contiguous stretch of 20 amino acids, from residues 55 to 74, which is predicted to form a major part of the c h domain c~- helix. The identity of amino acids at the positions which are predicted to be on the upper face of the two c~-helices, and available for antibody binding, is of particular relevance. The sequences of the carboxy-terminal halves of these domains are compared in Figure 6. Given that

the epitopes recognised by the majority of anti-MHC class II mAbs map to the c~-helical portion of the amino-ter- minal domains, this similarity is likely to reduce greatly the inlmunogenicity of DRot in E-expressing mice. Fur- ther functional evidence for the similarity of the exposed surfaces of these two domains is provided by the inter- changeability of DRcz and Ea in the restriction element for two DRl-restricted influenza-specific human T-cell clones (Lechler et al. 1988) and in the stimulator molecules for two alloreactive T-cell clones (Lombardi et al. 1989b).

The strategy of immunizing BALB/b mice, which fail to express E, was successful in that several surface-stain- ing DRc¢-specific mAbs were produced. However, ironically these antibodies did not stain Eel-expressing cells, which raises the possibility that epitope(s) they recognize may also be immunogenic in E-expressing mice.

In conclusion, mouse cells expressing individual MHC class II molecules have clear advantages as specific ilnmunogens, and as screening reagents. The transfectants used here which express only one human class II polypep- tide paired to a mouse partner are particularly powerful in defining the chain specificity of class II-specific mAbs. Antibodies with a defined chain specificity, in turn, pro- vide a very useful means of detecting cell surface isotype- mismatched dimers following gene transfection.

Acknowledgments. We wish to thank Sanya Goyert for generously pro- viding the SG465 antibody, and Anthony Warrens for critical reading of the manuscript.

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