Journal of Immunological Methods, 151 (1992) 15-26
15
© 1992ElsevierScience PublishersB.V. All rights reserved 0022-1759/92/$05.00
JIM06279
Production of epitope specific monoclonal IgG antibodies to HLA class II molecules by combining in vivo and in vitro immunization Jacob Sten Petersen t and T h o m a s D y r b e r g Hagedorn Research Laboratory, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark
(Received 15 August 1991, revisedreceived29 November1991,accepted 3 SanuaP/1992)
To study the expression of HLA-DQ ,8 chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 ,8 chain sequences. The splenoeytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specifie mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenoeytes, lmmunoblot analysis showed that several of the mAbs cross-reacted with D Q ,8 chain molecules. One mAb to a peptide representing DQw8,8 position [49-60] specifically recognised the DQw8 /3 chain. Three mAbs to a peptide representing DQw8,8 position [39-52] specifically recognised an epitope consisting of Gly-VaI-Tyr in position 45-47, i.e., all DQ,8 alleles except DQw7,8 (position 45-47: Glu-Val-Tyr) and DQw2,8 (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound l~nmphoeytos with the same specificity as found by immunoblotting analysis. Thus, by combining in rive and in vitro immunization we have generated a number of epitope specific monoclonal lgG antibodies that distinguish closely related HLA-DQ ,8 chain alleles in predetermined positions. Key word~. HLA class 11 molecule; Peptide antibody; In vitro immunization;ELISA; Immunoblotting;FACSanalysis
INTRODUCTION The H L A class II immune response gene products are highly polymorphic heterodimers consisting of noncovalently bound a and ,8 chains. Both chains traverse the plasma membrane, are
Correspondence to: J.S. Petersen, Hagedorn Research Laborato~v, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark (Tel.: 45-31-68.08.60; Fax: 45-31-68.05.02). t Jacob Sten Petersen was supporled by the Danish Research Academy. Abbreciation: KLH, keyhole limpel hemocyanin.
glycosylated, and have relative molecular weights of 34 kDa and 29 kDa, respectively (Kaufmaa et al., 1984). Several HLA class iI loci have been identified, and at each Inc~ls multiple allelic forms have been demonstrated (Trowsdale et al., 1985; Gregerson et al., 1986; Bell et al., 1987). This allelic polymorphism resides mainly in the first domain of the /3 chain (Trowsdale et aL, 1985; Gorski et al., 1986; Gregersoa et al., 1986; Bell et al., 1987). The genomic structure, nucleotide sequence and predicted amino acid sequence of HLA elas~ !I molecules have been obtained through molecular cloning and cDNA sequencing (Das et al.,
1983; Larhammar et al., 1983). However, relatively little is known about the relationship between the polymorphic domains and functional properties of the HLA class II molecules, e.g., antigen p~'esentation and T cell recognition. There is an extensive sequence homology between class 11 molecules and in particular between alleles within the same loci. However, only rarely is the particular epitope recognized by the allele specific antibodies (Marsh et al., 1989) characterized. Thus, there is a need for more precisely defined reagents which will permit a more detailed analysis of the HLA gene products. One approach to the development of such reagents has been to generate antibodies to synthetic peptides representing linear sequences of HLA molecules (Church et al., 1983; Deufel et al., 1985; Walker et al., 1985; Choppin et al., 1986; Rojo et al., 1986; Ways et al., 1986; Atar et al., 1989; Petersen et al., 1990). We have recently described the production of several polyelonal antisera which could distinguish the closely related HLA-DQw7 and DQw8 /~ chains based on a single amino acid difference at position 57 (Afar et al., 1989; Petersen et al., 1990). With these antisera, the fl chain molecule could be detected in immunoblotting analysis, but not satisfaetori!y by FACS analysis of intact cells, probably because of high non-specific binding (Petersen et al., 1990). To overcome this problem, we sought to develop mAbs of a predetermined specificity which could be used to detect the D Q molecules on intact cells. To this end, we combined conventional in vivo immunization with in vitro boosting of splenocytes prior to fusion of the cells. This procedure resulted in the generation of IgG mAbs that could distinguish between allelic D Q / 3 chain molecules, both in immunoblotting and FACS analysis.
Materials and methods
Cells Sp2/0-Ag14 myeloma cells and EBV-transformed human B cells (Fig. 1) were cultured in RPMI 1640 (Gibco, Renfrewshire, Scotland) supplemented with 10% fetal calf serum (Sera-lab, Sussex, England), 2 mM L-glutamine (Gibeo), 100
l U / m l penicillin (Gibco), and 100 ixg/ml streptomycin (Gibco) (referred to as RPMI medium).
Synthetic peptides Peptldes of variable lengths representing amino acid sequences between position [39-63] of the fi~t domain of DQw7~ and DQw8j6 (Fig. 1) were synthesized and purified as described (Dyrberg et al., 1990). The peptides used for immt,nization (Fig. 1) were synthesized with an additional carbox'y-terminal eysteine residue and 5 mg of peptide were coupled to 5 mg of keyhole limpet hemocyanin (KLH) using m-maleimido-benzoylN-hydroxy suceinimide as cross-linker (Dyrberg et al., 1990).
Production of monoclonal antibodies For the primary immunization, peptide coupled to KLH was emulsified in Freunds complete adjuvant (Gibco) and injected into multiple intradermal sites on the abdomen of 4-8-week-old female B A L B / c mice (GI. Bomholtgaard, Denmark) (40 ~ g peptide/mouse). For the subsequent immunizations, 40 ttg peptide/mouse was coupled to KLH, emulsified in Freund's incomplete adjuvant (Gibco) and injected at 8 day intervals as described above. Sera from the immunized mice were tested by ELISA, and the mouse with the highest antibody titre was injected intraperitoneany with 40 p.g of unconjugated peptide. 3 days later the spleen was removed and the splenoeytes were either fused immediately or boosted in vitro. The in vitro boosting was performed in 25 em z tissue culture flasks with 7.5 ml conditioned culture medium (DMEM (Gibco) containing 1% non essential amino acid (Gihco), 2 mM L-glutamine, 100 I U / m l penicillin, 100 ,~g/ml streptomycin, 3.5% fetal calf serum and 63% M L C / E L 4 supernatant (Biolnvent International, Lurid, Sweden) at a cell concentration of 6-7 × 106 splenocytes/ ml and with 25 n g / m l of peptide coupled to KLH. After 5 days incubation, the splenocytes were fused with Sp2/0-Agl4 myeloma cells. The in vitro antigen concentration was selected based on preliminary experiments showing that boosting with 25 n g / m l of peptide yielded higher numbers of peptide specific antibodies than 250 n g / m l (data not shown).
Splenocytes and myeloma cells (Sp2/0-Agl4) (ratio 1:2 for in vitro boosted splenocytes and 1 : 4 for splenecytes from only in rive immunized mice) were washed twice in RPMI 1640 and fused by adding 50% v / v polyethylene glycol/RPMI 1640 to the cell pellet over 1 rain. After an
HLA-DQ
8-chain
amine
acid
additional 1 rain the fusion was stopped by adding 2 ml RPM! 1640, followed by 7 ral RPMI 1640 containing 10% FCS. The cells were centrifuged (500 × g for 7 rain), resuspended in RPMI supplemented with HAT (Gibce) and HECS (10% v / v ) (hybridoma growth factor, Costar Europe,
sequences
HLA-DR/DQ 39
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HO104:DR15/DQw6
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DR3/DOw2/POw8~m57
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Immunizin
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peptides:
9
[39-52] [49-60]
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Test peptide:
[44-63]
Fig. 1. The sequence of selected HLA-DQ/] chainsfor the aminoacid residuesat positions[39-63] and the DR3/DQw2, DQw8~l chain mutants(Miehelsenet al., 1990)are shown togetherwith the DQw7 and DQw8peptide sequences used in this study.The peptides used for immunizationwere coupledto carrier protein througha carbox~erminalcysteineresidue.
Badhoevedorp, The Netherlands) and plated in 96 well tissue culture plates at a concentration of 2-3 × 104 s~)lenocytes/well. Hybridoma supernatants were screened for specific antibodies by ELISA (see below) using the immunizing peptide as solid phase antigen. All hybridoma supernatants that induced a binding five times above background were retested the same day both by a direct and a competition ELISA in the presence of free peptide (100 /*g/ml). Supcrnatants in which binding could be inhibited by more than 50% were defined as positive. Positive hybridomas were cloned twice by limiting dilution in RPMI supplemented with HECS (10% v / v ) and H T (Gibco).
mouse lg standard (Zymed) and the test samples were incubated at room temperature for 2 h; bound lg was quantified with peroxidase-conjugated goat anti-mouse F(ab') z igG (H + L) (Zymed) and the immunoglobulin concentration of the samples was calculated. The dissociation constant ( K D) of the mAbs was determined in a competition ELISA (Friguet et aI., 1985). First, the mAbs were incubated for 15 h in solution with different concentrations of the antigen (in excess) until equilibrium was reached. The preahsorbed samples were then added to microtiter plates coated with the same peptide antigen and the resulting binding measured by an ELISA procedure as described above. The K o was then determined from the following equation:
ELISA
Flat-bottomed, 96 well microtiter plates were coated overnight at room temperature with 50 /*l/well of 5 t~g/ml peptide diluted in 50 mM Na2CO3/NaHCO 3 buffer, pH 9.6. The plates were washed 3 x in PBS with 0.1% Tween 20 before the supernatant, diluted in PBS containing 1% BSA and 0.1% Tween 20 (ELISA dilution buffer) was added a~d incubated for 1.5 h at room temperature. The plates were washed as before and incubated for another 1.5 h at room temperature with pe~'oxidase-conjugated goat anti-mouse F(ab') 2 lgG (H + L) (Zymed, South San Francisco, CA). After washing, substrate (0.4 m g / m l o-phenylenediamine and 0.03% hydrogen peroxide in water) was added. The peroxidase activity was stopped after 30 min by adding 2 M H2SO a and the absorbance measured at 490 nm. The specificity of the binding reaction was confirmed by competition ELISA analysis where free peptide was added together with the mAbs in the first antibody incubation. Irrelevant peptide or no peptide were used as controls. The isotype of selected mAbs was determined in an ELISA using an antigen dependent streptavidin-biotin system (Monoab-Id Sp Mouse Kit, Zymed) according to the manufacturer's instructions. The lg concentration in the culture medium was determined using an antibody capture ELISA assay with micrntiter plates coated with goat anti-mouse IgG (Zymed). Serial dilutions of a
( a(~ × AI)) / ( A o - A ) - an = KD
(Friguet et al., 1985) where a 0 is the total antigen concentration used for preabsorption, A U is the absorbance measured without preabsorption and A the absorbanee measured with preabsorption. Immunoblotting
For immunoblotting analysis, cell membrane proteins were submitted to detergent phase separation as previously described (Atar et al., 1989). The proteins were denatured under reducing conditions, subjected to 10% SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose filters. For immunostaining, nitrocellulose strips were incubated with antibodies, washed, incubated with rabbit-anti mouse IgG F(ab')z conjugated to alkaline-phosphatase (Zymed), washed and finally incubated in substrate. The specificity of the mAbs was confirmed in competition analysis where the primary antibody was incubated together with an excess of free peptide (50 /*g peptide/25 ~ g mAb, using the mAbs at a concentration of 50/zg/ml). FACS analysis
Cells used in FACS analysis were incubated at 3 × l0 s cells/ml in RPMI medium for 24 h before analysis. Preliminary experiments showed
that the mAbs did not bind to living cells, but only to fixed cells. T h e cells were therefore fixed before F A C S analysis: approximately 5 x 106 cells were washed 1 x by centrifugation ( 2 5 0 X g , 7 min) in PBS, 1% BSA and 5 m M N a N 3 ( F A C S buffer), resuspended in 500 ,u.I FACS buffer and 1.5 ml methanol at room temperature was slowly a d d e d drop by drop. A f t e r 2 min, 8 ml FACS buffer w e r e added, the cells washed 3 × in F A C S buffer by centrifugation and resuspended in the same buffer at 2 x 106 e e l l s / m l . Samples of cells (2 × lb"/tube) were incubated for 60 min at 40(: with mAbs diluted in FACS buffer (final concentration: 2 5 / z g / m l ) , washed 1 X by centrifugation in 3 ml F A C S buffer and further incubated for 30 rain at 4°C with 100 t.tl FITC-conjugated F ( a b ' ) 2 rabbit anti-moose l g G (Zymed) diluted ~n F A C S buffer. Finally, the ceils were washed 1 x by eentrifugation, resuspended in 250 /zl F A C S buffer and analysed (FACS-scan, Becton Dickinson, Ca). T h e signal obtained from cells incubated with second antibody alone defined background levels. Competition assays were carried out by adding free peptide together with the mAbs ( 2 5 / z g pept i d e / 5 . 0 / x g mAb, using the mAbs at a concentration of 25 tzg/mI). F A C S buffer and irrelevant peptide were used as positive controls.
Results
Production of peptide specific monoclonal antibodies Six out of a total of 30 immunized mice were selected for fusion studies based on a reactivity with the immunizing peptide in ELISA. T h e peptide antibody titer in serum from mice selected for in vitro boosting did not differ from that of mice from which splenoeytes were fused directly after in vivo immunization (data not shown). All the fusions resulted in multiple growing hybridomas ( 3 . 7 - 5 6 x 103 h y b r i d o m a s / s p l e e n ) (Table I). N o peptide specific mAbs were isolated from the first three fusions (Table I, mice 1, 2, and ~,~ using splenocytes from only in vivo immunized mice. T o enhance the e f f i c i e n ~ of antibody production, the in vivo immunization was combined with in vitro boosting of the splenocytes before fusion (Table I, mice 3a, and 4 - 6 ) . Peptide specific mAbs were isolated from all fusions using the in vitro boosted splenocytes. T o directly compare in vivo with in v i v o / v i t r o immunization, splenocytes from one mouse (Table 1, mouse 3), immunized in vivo, were divided in two equal parts. O n e was fused immediately and the other
TABLE I HLA-DQ .8 CHAIN MONOCLONAL PEPTIDE ANTIBODIES Mouse/antigen
Immunization
Hybridore#s/well
no. of wells
Positivehybridomas DQw7 DQw8
DQw7/DQw8
(1) DQw7/8.0[49-60] /2) DQw8,8[49-60] (3) DQw7 ~[49-60]
In viva In rive (a) In vlvo (bl In rive/vitro In vivo/vitro In rive/vitro In rive/vitro
4.7 4.4 4.2 2,6 3.2 3.1 2.9
i,152 1,152 576 576 1,152 1,152 1,152
0 0 0 10 {~ II II
0 0 0 2 8 2 2
(4) DQw8 #[49-60] (5) DQw8 .0[39-52] (6) DQw8 #[39-52]
0 O 0 O 4t 3 3
Splcnocytes from mice 1-6 immunized in rive (]-3a) or in rive combined ~;th an in vitro boosting (3b-61 (see materials and methnds section). Hybridomas/well are indicated as an average of 200 wells, no. of wells arc the total number of wells used to seed the fused spleen cells. Hybridomas were defined as positive based on ELISA react[vity and indicated as follows. Mice 1-4:DQw7 binding to HLA-DQw7 0[44-63], DQw8 binding to HLA-DQw8 B[44-63], DQw7/DQw8 binding to HLADQw7 and DQw8 /][44- 53]. Mice 5-6:DQw7 binding to HLA-DQw7 .0139-52]¢ys,DQw8 binding to HLA-DQw8 ~39-52]cys. DOw7/DQw8 binding to HLA-DQw7 and DOw8 fl[39-52]cys.
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Fig. 2. Bindingof mAb 2H3 to HLA-DQw8•[39-52]cys in immunoblotso[ EBV-transformed cells. The mAb was tested out a concentration o[ 25 ~g/ml. -, direct binding. +, the mAbwas co-incubated with HLA-DOw8/3139-52]cysat a concentrationof 50 ~g/ml. A: DR3/3, DQw2/2; B: DR8/8, DQw4/4; C: DRI6/16, DOwS/5; D: DRI5/15, DQw6/6; E: DR4/4, DQw7/7; F: DR4/4, DQwS/8. The 2B5 and 1A1 mAbsto HLA-DQw813139-52]cysshowed the same bindingspecificityas 2H3 (not shown).
was boosted in vitro. Only fusion of the in vitro boosted splenocytes resulted in peptide specific mAbs, even though the number of hybridomas was approximately 60% higher fol[owing in vivo immunization only, i.e., 2.5 × 103 compared to 1.6 × 103 in 500 wells.
Characterization of mAbs to HLA-DQ/3 position 149-601 Peptide specificity. The peptide specificity of the mAhs was analysed by ELISA using peptides representing position [44-63] of DQwT/3 or DQw8/3 as antigens (Fig. 1). These peptides differ only at position 57 within the immunizing sequence.
Thefusion of splenocytes immunized in vivo/iu vitro with DQw7 fl[49-60]cys (Table I, mouse 3) resulted in 12 peptide specific mAbs, ten bound only to DQw7 /3[44-63] and two bound both DQw7 and DQw8 /3[44-63]. 49 hybridomas producing peptide specific mAbs were isolated from splenocytes immunized in vivo/in vitro with DQw8 /3[49-60] (Table I, mouse 4), 41 bound only DQw8 /3[44-63] and 8 bound both DQw7 and DQw8/3[44-63]. The isotype and dissociation constants (KD) were determined for 15 hybridomas which had been cloned twice by limiting dilution. All mAbs were IgGl,k except for two which were IgM,k. The K D of five selected IgG1 mAbs was 3.1-5.4
U14= 29e3¢JOI~51%FLI~F|u
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Fig. 3. FACS analysis of methanol fixed EBV-transformed cells. The shaded curves show bindingof mAb 21"]3to DOw8 /][39-52]eys at a concentration of 25 ttg/ml. The solidblack line and open curve show bindingof 21-13after co-incubationwith HLA.DQw8/~[39-52]cys at a concentration of 200 tLg/ml.The pale greyline and open curveshowhingingof cellsincubatedwith second antibody only (goat anti-mouse IgG H+L). A: DR3/3, DOw2/2; B: DR8/8, DQw4/4; C: DRI6/16, DOwS/5; D: DRIS/IS, DQwfi/6;E: DP,4/4, DQw7/7; F: DR4/4, DQwS/8 (see Fig. 1). The 2B5 and 1A1 mAbsto HLA-DQwg/3139-52]cys showed the same bindingspecificityas 2H3 (not shown).
x 10 -5 M -~, whereas the two IgM mAbs displayed a lower affinity, i.e., 1.1-1.4 x 10 -3 M 1.
Binding to HLA-DQ ~ chain molecules in immunoblot analysis. None of the 12 mAbs against HLA-DQw7 ~[49-60] bound in immunoblot analysis of EBV-transformed lympbocytes. In contrast, 3 of the 49 mAbs against DQw8/3149-60]cys (2A12, 6B12, and 8D12) bound to a 29 kDa protein in immunoblots of DR4/DQw8 positive cells, identical in size to the band recognized by a monoelonal antibody to H L A - D R / D Q /3 chain molecules (data not shown). To test the allele specificity of the antibodies, the three mAbs were also tested for binding to D R 3 / D Q w 2 ,
DR8/DQw4, DRI5/DQw5, DR15/DQw6, and DR4/DQw7 homozygous cells. One mAb, 6B12 did not bind to the /3 chain molecule of any of these cells; 2A12 recognized the DR3/DQw2 /3 chain molecule, and 8D12 showed a much broader reactivity binding to DR8/DQw4, DR1/DOwS, and DR4/DQw7 positive cells. The sequence specific binding of the mAbs was confirmed by competition experiments: co-incubation with the DQw8 peptide in all cases blocked the binding completely to the 29 kDa protein, whereas the DQw7 peptide only blocked the binding for 8D12 (not shown). None of the DQw8/~[49-60]~.3's mAbs bound
1"-[~ Ol4B;~'4tB~9~14xFklxFluorescence
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Fig. 4. FACSanalysisof methanol fixedEBV-transformedtransrect with either nativeor mutant HLA-DOw8/3chain. The shaded curve showbindingof mAh 2H3 to DQw8j~[39-52]cysat a concentration of 25/~g/ml.The solidblack line and open curveshow bindingof 2H3 after ¢o-incubalionwith HLA-DQw$ #[39-52]cys in a concentration of 2{10p.g/ml. The pale grey line and open curve show bindingof cells incubated with second antibodyonly(goat anti-mouse IgG H+L). A: DR3/DQw2; B: DR3/DQw2 transfected with DQw8Bm45~ chain; C: DI~/DQw2 transfected with nativeDQw8 ~ chain; D: DR3/DQw2 transteeted with DOw8/3m57fl chain(Fig. 1). in immunoblot analysis of peripheral blood lymphocytes or in FACS analysis to living or fixed cells.
Characterization of mAbs to HLA-DQ/3 position 139-521 Peptide specificity. The two fusions with splenocytes from mice immunized irr vivo/in vitro with DQw8 /3139-52]cys (Table i, mice 5 and 6) each resulted in five peptide specific mAbs. Five of these were specific for DQw8/3139-52]cys and five bound both DQw7 and DQw8 ,B[39-52]cys. Analysis of five selected hybridomas, cloned twice by limiting dilution, demonstrated that all were of the IgG1,K isotype with a KD between 1 . 7 × 1 0 - 5 - 1 . 5 × 1 0 -6 M-t.
Binding to HLA-DQ fl chain molecules in immunoblot analysis. Three of the ten mAbs (2H3, 2B5, and 1A1) specific for DQw8 /3139-52]eys recognised the /3 chain molecule on DQw8, DQw4, DQw5, and DQw6 positive cells, but not on DQw2 and DQw7 positive cells (Fig. 2). To confirm the sequence specificity of the bindir.g, the three mAbs were incubated with an excess of the DQw8 /3139-52]cys peptide and tested for binding to DQw4, DQw5, DQw6, and DQw8 positive cells. In all cases the DQw8 peptide blocked the binding completely (Fig. 2). The specificity of these mAbs was further analysed by testing their binding to DR3/DQw2 homozygous cells transfeeted with site directed DQw8/3 gene mutants (Fig. 1) (Kwok et al., 1989). The 2H3,
2B5 and 1AI mAbs all recognis~.d the/3 chain on the DQwg~ and DOwg/3m57 mutant cells (results not shown), the mutant DQw8~m57 encoding a molecule identical to DQw8/~ except that Ala at position 57 was replaced by Asp corresponding to the residue in DQw7fl in that position. In contrast, none of the mAbs recognised the ~ ehain molecule on the non-transfected cells or on the DQw8flm45 mutant cells, where the residue at position 45 of DQwgfl (Gly) has been replaced by the corresponding residue from DQw7/~ (Glu). Binding to intact cells: FACS analysis None of the ten DQw8 /3139-52]cys mAbs bound to living cells in FACS analysis. Since anti-peptide antibodies recognise sequential rather than conformational eptiopes, the lymphocytes were fixed to induce a partial denaturation of the membrane proteins. Following this procedure, the three mAbs that recognised the DQ /~ chain in immunoblot analysis now clearly bound to intact cells confirming the immunoblot results: the mAbs bound to DR8/DQw4, DR1/DQw5, D R I 5 / D Q w 6 and DR4/DQw8 positive cells but not to DR3/DQw2 and DR4/DQw7 positive cells (Fig. 3). The binding was displaced completely by incubation with an excess of fre~ DQw8 /3139-52]cys peptide (Fig. 3). Furthern, ..:, the mAbs bound with the same specificity as observed in immunoblot analysis to the cells ex-
A
Discussion Here we report the production of epitope specific mAbs that distinguish HLA-DQ fl chain alleles based on single amino acid substitutions at either position 45 or 57 in the first, variable domain. Two important points emerge from these results: firstly, they demonstrate that it is possible to make useful mAbs of a predetermined specificity down to the level of a single amino acid. Such mAbs may be valuable tools in the serological analysis of closely related HLA class II alleles.
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pressing the different DOw8 /3 chain mutants, i.e., binding to the DOwg/3 and DOw8,gm57 mutant cells, but no binding to the mutant DQw8/~m45 and the non-transfected DR3/ DQw2 cell line. The mAbs were also tested for binding to PBL from a DR2/DR3, DQwl/DQw2 positive individual in a dual immunofluorescence analysis using FITC-conjugated HLA-D specific mAb (Dakopatts) and a phycoerythrin conjugated rat anti-mouse K for the detection of the DQw8 /3139-52]cys mAbs (Fig. 5). The results showed that the HLA-D positive cells were specifically stained by the DQw8 /3139-52]cys mAbs; co-incubation with DQw8 fl[39-52]cys pcptide reduced the binding to background levels (Fig. 5).
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i --->
Fig. 5. Bindingof mAb 2H3 to HLA-DQw8 /3139-52]cysin FACSanalysisof methanol fixedperipheral blood lymphocytes.The mAb 2H3 was tested at a concentration of 25/tg/ml. A: FLI. mAb to HLA-DR/DQ/3 chains(Dakopatts, Glostrup.Denmark); FL2, mAb 2H3 to HLA-DQw8 /3139-52]cys. B: FLI, mAb to HLA-DR/DQ fl chain; FL2, 2143co-incubated with HLA-DOw8 /3139-52]cys at a concentration of 200 /xg/ml. The 2B5 and IAI mAbs tu HLA-DQw8 /3[39 52]cysshowed the same binding specificityas 2H3(not shown).
Secondly, we show that in vitro boosting of splenocytes from in vivo immunized mice may be helpful in the generation of mAbs against small peptide antigens. Thus, the amino acid sequence of the peptide to be used as an antigen may be selected by criteria other than predicted antigenicity. In this study it was not possible to isolate any pcptide specific mAbs from mice receiving in vivo immunization alone, although the three fusions made all resulted in large numbers of hybridomas (Table I). In vitro immunization using unprimed mouse spleen cells has previously been shown to result in antibodies to antigens which are normally not immunogenic (Van Ness et al., 1984; Borrebaeck, ]980; Borrebaeck et al., 1986). However, such mAbs are predominantly IgM and of low affinity (Borrebacck, 1986; De Boer et al., 1987), e.g., in one study (De Boer et al., 1987) 90% of the mAbs against human thyroglobulin generated by in vitro immunization were of the IgM isotype. By combining in vivo immunization with in vitro boosting of the splenocytes before fusion, we have isolated a large number of relatively high affinity peptide specific mAbs of the IgG isotype. These results suggest that during the in vitro boosting, antigen specific B ceils primed in vivo may be preferentially expanded. Most antibody epitopes in proteins are believed to be discontinuous and conformationally dependent (Van Regenmortel et al., 1988). Thus peptides may mimic only part of an epitope and therefore often induce limited crossreactivity to the native antigen (Van Regenmortel, 1989). Accordingly, peptide antibodies tend to react much better with a denatured form of the protein. These points are exemplified in the present study: of the six mAbs which reacted with the denatured HLA-DQw8 ,8 chain protein in immunoblutting, none reacted with the HLA class II molecules on living cells in FACS analysis. However, after cells were fixed in methanol, which induces a partial denaturation, the three mAbs to DQw8 /3[3952]eys bound to both EBV-transformed cells and PBL with the same specificity as observed in immunoblotting analysis (Figs. 3-5). Three mAbs to HLA-DQw8 fl[39-52]cys (2B5, 2H3, and 1A1) reacted selectively with D Q ,8 chain molecules with a Gly-VaI-Tyr segment in
position [45-47], i.e., DQw4//, DQw5fl, DQw6.8, and DQwg.8 hut not DQw7.8 (Glu in position 45) or DQw2fl (Glu-Phe in position 46-47). The specificity of mAbs binding was confirmed using two different techniques: (1) in competition experiments the immunizing pepfides, but not irrelevant peptides, inhibited binding of the mAbs completely in both immunoblotting and FACS analysis (Figs. 2 and 3) and (2) analysis of D R 3 / D Q w 2 positive cells transfectod with native or mutated DQw8/3 chain genes coding for DQw7 /3 chain amino acid substitutions in position 45 and 57 (Michelsen et al., 1990) showed a binding pattern that correlated with the binding to cells homozygous for the DQw7 or DQw8 .8 chain allele (Fig. 4). These results clearly demonstrate that the 29 kDa protein recognized is the DQw8 chain and not an unrelated protein. The three mAbs to DQw8 .8139-52]cys (2B5, 2H3, and 1A1) bound to approximately 10% of the cells from peripheral blood lymphocytes from a DQwl positive control. The proportion of cells that were stained corresponded well to the expected value of HLA class lI positive cells, i.e., B and activated T cells. Experiments with double fluorescence showed that all cells stained with a HLA-D specific mAb (Dakopatts) were also recognised by the mAbs to DQw8 .8139-52]cys (Fig. 5). In contrast, none of the mAbs to DQw8/3[4960]cys bound to intact cells fixed in methanol. Two observations may explain the discrepancy between the binding patterns observed in immunoblotting and FACS analysis. First, the fact that the mAbs to DQw8 fl[49-60]cys are of a lower affinity than the mAbs to DQw8 ,8[3952]cys, may result in a lower potential for binding. The position 45 epitope is more hydrophilic than the position 57 epitope, which may explain the difference in affinity between the two groups of mAbs. Second, in a recent study (Michelsen et al., 1990) of the fine specificity of DQw7 or DQw8 ,8 chain specific mAbs using site directed mutagenesis of the DQw8 ,8-chain, it was demonstrated that the amino acid in position 45 is critical for generating the serologic epitopes characterizing the DQw7 and DQw8 .8 chain molecules. Taken together, these results indicate that the epitope around position 57 may not be
exposed to an tib o d y b in d in g , even in fixed cells, M a n y a u t o i m m u n e diseases have b e e n s hown to b e associated with specific H L A a n t i g e n s ( S v e j g a a r d et al., 1983; T i w a r i et al. 1985; M i c h e l s e n et al., 1987, 1990; T o d d et al., 1987; H o r n et al., 1988). In type 1 diabetic p a t i e n t s the frequency of t h e H L A - D R 4 associated D Q w 8 /3 c h a i n allele is significantly increased c o m p a r e d t o t h e b a c k g r o u n d p o p u l a t i o n ( M i c h e l s e n et al., 1987, 1990; T o d d et al., 1987; H o r n et al., 1988). T h e o t h e r D R 4 associated D Q /3 c ha i n allele, D Q w T , is negatively associated with type 1 diab e t e s a n d differs from the D Q w 8 / 3 c ha i n only by f o u r a m i n o acid substitutions at positions 13, 26, 45, a n d 57 w i t h i n the first d o m a i n ( T o d d et al., 1987). A t p r e s e n t it is n o t k n o w n h o w these two closely r e l a t e d alleles c o n f e r susceptibility or resistance t o the disease, b u t the w h o l e m o l e c u l e is believed to b e involved r a t h e r t h a n a single a m i n o acid. E p i t o p e specific m A b s to the di a be t e s assoc i a t e d D Q p ch ain m o l e c u l e s m a y be useful tools for s t u d y i ng t h e expression o f the s e alleles in a u t o i m m u n i t y . T h e r e f o r e , to test the utility of the H L A - D Q / 3 ch ain reactive m A b s , they w e r e tested in i m m u n o b l o t analysis o n a p a n e l of l ymphobl a s told ceils f r o m D Q w 8 positive type 1 di abe t i c p a t i e n t s a n d D O w 7 o r D Q w 8 positive healthy controls. T h e m A b s specific for the e p i t o p e s at p o s i t i o n s 45 a n d 57 s h o w e d conclusive b i n d i n g p a t t e r n s c o n f i r m i n g that the cells w e r e e i t h e r D Q w 7 o r D Q w 8 positive. T h u s , e p i t o p e specific m A b s to H L A class 1I m o l e c u l e s m a y prove to be useful for serologic analysis of H L A alleles associated w i t h a u t o i m m u n i t y .
Acknowledgements W e are i n d e b t e d to Else Jost J e n s e n for expert technical assistance a n d to Harts Kofod for synthesizing the peptides. T h e cells carrying the D O w 8 / 3 m u t a n t m o l e c u l e s were kindly d o n a t e d by W i l l i a m Kwok, V i r g i n i a M a s o n R e s e a r c h Center, Seattle, W A .
References Atar, D., Dvrberg, T., Michelsen, B., Karlsen, A., Kofod, H., Molvig J. and Lernmark, .~. (1989) Site-specific antibodies
distinguish single amino acid substitutions in position 57 in H L A - D O beta-chain alleles associated with insulin-dependent diabetes. J. ImmunoL 143, 533-538,
Bell, J.I, Denney, Jr., D., Foster, L, Bell, T,, Todd, J,A. and McDeviU, H.O. (1987) ABelic variation in the DR subregion of the human major hislocompafibifty complex, proc. Nad. Acad. 8ci. USA 84, b234-6238, Borrebaeck, C.A.K. (1986) In vitro immunization fc~ ~roduclion of murine and human monoclonal antibodies: Present status, Trends. Biotechnol. 4, 147-153. Borrebacck, C.A.K. and Moiler, S.A. (1980) In vitro immunization. Effect of growth and differentiation factors on antigen-specific B cell activation and production of monoclonal antibodies 1o autologous antigens and weak immunogens. J. ImmunoL 136, 3710-3716. Choppin, J., Metzger, J.J., Bouillot, M,. Briand, J.P.].. Connan, F., Van Regcnmortel M.H. and Levy J.P. (1986) Recognition of HLA class 1 molecules by antisera directed to synthetic peptides corresponding to different regions of the HLA-B7 heavy chain. J. ImmunoL 136, 1738-1744. Church, W.R., Walker, L,E,, Houghten, R.A. and Reisfdd, R.A. (1983) Anfi-HLA antibodies of predetermined specificity: a chemically synthized peptide induces antibodies specific for HLA-A. B heavy chain. Proc. Natl. Acad. 8ci. USA 80, 255-258. Das, H.K., Lawrance, S.K. and Weissmann, S.M. (1983) Structure and nucleotide sequence of the heavy chain gene of HLA-DR. Proc. Natl, Acad. Sd. USA 80. 3543-3547. De Boer, M.. Ossendorp, F.A,, Bruning, P.F. and Tager, J.M. (1987) Direct evidence for a primary immune response of routine B-lymphocyles after in vitro immunization of dissociated splenoc'/tes. Hyhridoma 6, 253-258. Deufek T., Grove, A., Kofod, H. and Lernmark. A. (1~5) Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain. FEBS Len. 189, 329-337. Dyrberg, T. and Kofod, H. (1990) Synthetic peplides as antigens. In: OIdstone, M.B.A. (Ed.), Animal Virus Pathogenesis: A Practical Approach. Oxford University Press. Oxford, p. 163. Friguet, B., Chaffone, A.F., Djavadi-Ohaniance. L. and Goldberg, M.E. (1985) Measurement of the true affinity conslant in solution of antigen-'~ntifiody complexes hy enzyme-linked immunosorbeni assay. J. Immunol. Methods 77, 305-319. Gorski, J. and Mach, B. (1986) Polymorphism of human la antigens: gene conversation between two DR beta loci results in a new FILA-/DR specificity. Nature 322, 67-70. Gregerson, P.K., Moriuchi, T., Karr. R.W., Obata. F., Moriuehi, J., Maccari, L, Goldberg, D., Winchester, R.J. and Silver, J. (1986) Polymorphism of HI.A-DR beta chains in DR-4. DR-7 and DR-9 fiaplotypes implications for the mechanisms of allelle variation. Proc. Natl. Acad. Set. USA 83, 9149-9153. Horn, G.T., Bugawan, T.L., Long, C.M. and Erlich, H.A. (1988) Allelic sequence variation of the IILA-DQ loci: relationship to serology and to insufin-independent diabetes susceptibility, proc. Nail. Acad. Set. USA 85, 60126016.
Kaufman, J.F., Auffray C., Korman A,J., Shakelford D.A. and Strominger, J. (1984) The class It molecules of the human and murine major histocompatibility complex. Cell 36, 1-14. Kwok, W.W., Lotshaw, C., Milncr, E.C. Knitter-Jack, N. and Nepom, G.T. (1989) Mutational analysis of the HLADQ3.2 insulin-dependent diabetes mellitus susceptibilily gene. Proc. Natl. Acad. Sci. USA 86, 1027-30. Larhammar, D., Hyldig-Nielscn, J.J., Se~vcnius, B. Andersson, G.. Rask, L. and Pelerson, P.A. (1983) Exon-intron organization and complete nudeotide sequence of a human major histocompalibility antigen DC beta gene. Proc. Natl. Acad. Sci. USA 80, 7313-7317. March, S.G and Bodmer, J.G. (1989) HLA-DR and -DQ epitopes and monoclonal antibody specificity, lmmunol. Today 10, 305-312. Michelsea. B. and Lernmark, A. (1987) Molecular cloning of a polymorphic DNA endonuelease fragment associates insulin-dependent diabetes mellitus with HLA-DQ. J. Clin. Invest. 79, 1144-1152. Michelsen. B., Dyrberg, T., Vissing, H., Serup, P. and Lernmark, A. (1990) HLA-DQ and -DX genes in insulin-dependent diabetes mellitus. Curt. Top. Microbiol. Immunol. 164, 57-68. Pctcrsen, J.S., Atar, D., Karlsen, A., Kofod, H. and Dyrberg, T. (1990} Characterization of the fine specificity of peptide antibodies to HLA-DQ beta-chain molecules. Biomed. Biochem. Acta 49, 1223-1232. RoD, S.. Lopez de Castro, J.A., Aparieio, P., Bragado R. and Van-Seventer, G. (1986) HLA-B27 antigenieity: antibodies ag", the chemically synthesized 63-84 peptide from HLA-B27.1 display alloantigenic specificity and discriminate among HLA-B27 subtypes. J. lmmunol. 137, 904-910.
Svejgaard A . Platz, P. and Ryder, L.P. (1983) H L A and disease 1982 - a survey. Immunol. Rev. 70, 193-218. Tiwari, J.L. and Terasaki, P.I. (1985) H L A and Disease Association. Springer-Verlag, New York. Todd, J.A., Bell, J.I. and McDevitl, H.O. (1987) HLA-DQ beta gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329, 599-604. Trowsdale, J., Young, J.A., Kelly, A.P., Austin, P.J.. Carson, S,, Meunier, H., So, A., Erlich, H.A., Spielsman, R.S. and Bodmer, L et al. (1985) Structure, sequence and polymorphism in the HLA-D region. [ramunoL Rcv. 85, 5-43. Van Ness, J., Laemmli, U.IC and Pettijohn, D.E. (1984) Immunization in vitro and production of monoclonal antibodies specific to insoluble and weakly immunogenic proteins. Prec. Natl. Acad. Sci. USA 81, 7897-7901. Van Regenmortel, M.H.V. (1989) Structural and functional approaches to the study of protein antigenicity, lmmunoL Today 10, 266-272. Van Regenmortel, M.H.V. and Daney, D.M.G. (1988) An assessment of prediction methods for locating continuous epitopes in proteins. ImmunoL Lett. 17, 95-108. Walker, L.E., Ketler, T.A. Houghten, R.A., Schulz, G., Cbersi, A, and Reisf¢ld, R.A. (1985) Human major histocompatibility complex class I antigens: residues 61-83 of the HLAB7 heavy chain specify an alloreactive site. Prec. Natl. Acad. Sci. USA 82, 539-542. Ways, I.P., Rothbard, J.B. and Parham, P. (1986) Amino acid residues 56 to 69 of HLA-A2 specify an antigenic determinant shared by HLA-A2 and HLA-B17. J. ImmunoL 137, 217 222.
15
© 1992ElsevierScience PublishersB.V. All rights reserved 0022-1759/92/$05.00
JIM06279
Production of epitope specific monoclonal IgG antibodies to HLA class II molecules by combining in vivo and in vitro immunization Jacob Sten Petersen t and T h o m a s D y r b e r g Hagedorn Research Laboratory, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark
(Received 15 August 1991, revisedreceived29 November1991,accepted 3 SanuaP/1992)
To study the expression of HLA-DQ ,8 chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 ,8 chain sequences. The splenoeytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specifie mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenoeytes, lmmunoblot analysis showed that several of the mAbs cross-reacted with D Q ,8 chain molecules. One mAb to a peptide representing DQw8,8 position [49-60] specifically recognised the DQw8 /3 chain. Three mAbs to a peptide representing DQw8,8 position [39-52] specifically recognised an epitope consisting of Gly-VaI-Tyr in position 45-47, i.e., all DQ,8 alleles except DQw7,8 (position 45-47: Glu-Val-Tyr) and DQw2,8 (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound l~nmphoeytos with the same specificity as found by immunoblotting analysis. Thus, by combining in rive and in vitro immunization we have generated a number of epitope specific monoclonal lgG antibodies that distinguish closely related HLA-DQ ,8 chain alleles in predetermined positions. Key word~. HLA class 11 molecule; Peptide antibody; In vitro immunization;ELISA; Immunoblotting;FACSanalysis
INTRODUCTION The H L A class II immune response gene products are highly polymorphic heterodimers consisting of noncovalently bound a and ,8 chains. Both chains traverse the plasma membrane, are
Correspondence to: J.S. Petersen, Hagedorn Research Laborato~v, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark (Tel.: 45-31-68.08.60; Fax: 45-31-68.05.02). t Jacob Sten Petersen was supporled by the Danish Research Academy. Abbreciation: KLH, keyhole limpel hemocyanin.
glycosylated, and have relative molecular weights of 34 kDa and 29 kDa, respectively (Kaufmaa et al., 1984). Several HLA class iI loci have been identified, and at each Inc~ls multiple allelic forms have been demonstrated (Trowsdale et al., 1985; Gregerson et al., 1986; Bell et al., 1987). This allelic polymorphism resides mainly in the first domain of the /3 chain (Trowsdale et aL, 1985; Gorski et al., 1986; Gregersoa et al., 1986; Bell et al., 1987). The genomic structure, nucleotide sequence and predicted amino acid sequence of HLA elas~ !I molecules have been obtained through molecular cloning and cDNA sequencing (Das et al.,
1983; Larhammar et al., 1983). However, relatively little is known about the relationship between the polymorphic domains and functional properties of the HLA class II molecules, e.g., antigen p~'esentation and T cell recognition. There is an extensive sequence homology between class 11 molecules and in particular between alleles within the same loci. However, only rarely is the particular epitope recognized by the allele specific antibodies (Marsh et al., 1989) characterized. Thus, there is a need for more precisely defined reagents which will permit a more detailed analysis of the HLA gene products. One approach to the development of such reagents has been to generate antibodies to synthetic peptides representing linear sequences of HLA molecules (Church et al., 1983; Deufel et al., 1985; Walker et al., 1985; Choppin et al., 1986; Rojo et al., 1986; Ways et al., 1986; Atar et al., 1989; Petersen et al., 1990). We have recently described the production of several polyelonal antisera which could distinguish the closely related HLA-DQw7 and DQw8 /~ chains based on a single amino acid difference at position 57 (Afar et al., 1989; Petersen et al., 1990). With these antisera, the fl chain molecule could be detected in immunoblotting analysis, but not satisfaetori!y by FACS analysis of intact cells, probably because of high non-specific binding (Petersen et al., 1990). To overcome this problem, we sought to develop mAbs of a predetermined specificity which could be used to detect the D Q molecules on intact cells. To this end, we combined conventional in vivo immunization with in vitro boosting of splenocytes prior to fusion of the cells. This procedure resulted in the generation of IgG mAbs that could distinguish between allelic D Q / 3 chain molecules, both in immunoblotting and FACS analysis.
Materials and methods
Cells Sp2/0-Ag14 myeloma cells and EBV-transformed human B cells (Fig. 1) were cultured in RPMI 1640 (Gibco, Renfrewshire, Scotland) supplemented with 10% fetal calf serum (Sera-lab, Sussex, England), 2 mM L-glutamine (Gibeo), 100
l U / m l penicillin (Gibco), and 100 ixg/ml streptomycin (Gibco) (referred to as RPMI medium).
Synthetic peptides Peptldes of variable lengths representing amino acid sequences between position [39-63] of the fi~t domain of DQw7~ and DQw8j6 (Fig. 1) were synthesized and purified as described (Dyrberg et al., 1990). The peptides used for immt,nization (Fig. 1) were synthesized with an additional carbox'y-terminal eysteine residue and 5 mg of peptide were coupled to 5 mg of keyhole limpet hemocyanin (KLH) using m-maleimido-benzoylN-hydroxy suceinimide as cross-linker (Dyrberg et al., 1990).
Production of monoclonal antibodies For the primary immunization, peptide coupled to KLH was emulsified in Freunds complete adjuvant (Gibco) and injected into multiple intradermal sites on the abdomen of 4-8-week-old female B A L B / c mice (GI. Bomholtgaard, Denmark) (40 ~ g peptide/mouse). For the subsequent immunizations, 40 ttg peptide/mouse was coupled to KLH, emulsified in Freund's incomplete adjuvant (Gibco) and injected at 8 day intervals as described above. Sera from the immunized mice were tested by ELISA, and the mouse with the highest antibody titre was injected intraperitoneany with 40 p.g of unconjugated peptide. 3 days later the spleen was removed and the splenoeytes were either fused immediately or boosted in vitro. The in vitro boosting was performed in 25 em z tissue culture flasks with 7.5 ml conditioned culture medium (DMEM (Gibco) containing 1% non essential amino acid (Gihco), 2 mM L-glutamine, 100 I U / m l penicillin, 100 ,~g/ml streptomycin, 3.5% fetal calf serum and 63% M L C / E L 4 supernatant (Biolnvent International, Lurid, Sweden) at a cell concentration of 6-7 × 106 splenocytes/ ml and with 25 n g / m l of peptide coupled to KLH. After 5 days incubation, the splenocytes were fused with Sp2/0-Agl4 myeloma cells. The in vitro antigen concentration was selected based on preliminary experiments showing that boosting with 25 n g / m l of peptide yielded higher numbers of peptide specific antibodies than 250 n g / m l (data not shown).
Splenocytes and myeloma cells (Sp2/0-Agl4) (ratio 1:2 for in vitro boosted splenocytes and 1 : 4 for splenecytes from only in rive immunized mice) were washed twice in RPMI 1640 and fused by adding 50% v / v polyethylene glycol/RPMI 1640 to the cell pellet over 1 rain. After an
HLA-DQ
8-chain
amine
acid
additional 1 rain the fusion was stopped by adding 2 ml RPM! 1640, followed by 7 ral RPMI 1640 containing 10% FCS. The cells were centrifuged (500 × g for 7 rain), resuspended in RPMI supplemented with HAT (Gibce) and HECS (10% v / v ) (hybridoma growth factor, Costar Europe,
sequences
HLA-DR/DQ 39
45
BM-92:DR4/DQw8
R
DEU:
. . . . .
DR4/DQW7
F
D
S
V
G
V
50 Y
E
R
A
V
T
57
P
60
L G P P A A E Y W N S
. . . . . . . . . . .
D
. . . . . .
HO104:DR15/DQw6
.
.
.
.
.
.
.
.
.
.
.
.
.
Q
-
R
CAL:
DR16/DQw5
.
.
.
.
.
.
.
.
.
.
.
.
.
Q
-
R-
OLGA:
DR8/DQw4
.
.
.
.
.
.
.
.
.
.
.
.
.
R
L
D
. . . . . .
COX:
DR3/DQw2
. . . . . .
E
F
. . . .
L
-
-
L
-
A
. . . . . .
. . . . . .
E
F
. . . .
L
-
-
L
-
A
. . . . . .
DR3/DQw2/
....
DR3/DQw2/DQwS~
.
DR3/DQw2/DQw8Bm45
. . . . .
DR3/DOw2/POw8~m57
.
.
.
.
.
.
.
.
.
.
.
.
Immunizin
.
.
.
.
.
.
.
.
.
.
.
.
peptides:
9
[39-52] [49-60]
.
.
.
.
.
. E
. .
. .
. .
. .
. .
.
. . .
.
. . .
. . .
. . .
. . .
. . . . . . . . V
. .
. .
. . . . . .
. .
. .
D
. .
. .
. .
.
. . . . . . .
.
. . . . . . . . . . . . .
Test peptide:
[44-63]
Fig. 1. The sequence of selected HLA-DQ/] chainsfor the aminoacid residuesat positions[39-63] and the DR3/DQw2, DQw8~l chain mutants(Miehelsenet al., 1990)are shown togetherwith the DQw7 and DQw8peptide sequences used in this study.The peptides used for immunizationwere coupledto carrier protein througha carbox~erminalcysteineresidue.
Badhoevedorp, The Netherlands) and plated in 96 well tissue culture plates at a concentration of 2-3 × 104 s~)lenocytes/well. Hybridoma supernatants were screened for specific antibodies by ELISA (see below) using the immunizing peptide as solid phase antigen. All hybridoma supernatants that induced a binding five times above background were retested the same day both by a direct and a competition ELISA in the presence of free peptide (100 /*g/ml). Supcrnatants in which binding could be inhibited by more than 50% were defined as positive. Positive hybridomas were cloned twice by limiting dilution in RPMI supplemented with HECS (10% v / v ) and H T (Gibco).
mouse lg standard (Zymed) and the test samples were incubated at room temperature for 2 h; bound lg was quantified with peroxidase-conjugated goat anti-mouse F(ab') z igG (H + L) (Zymed) and the immunoglobulin concentration of the samples was calculated. The dissociation constant ( K D) of the mAbs was determined in a competition ELISA (Friguet et aI., 1985). First, the mAbs were incubated for 15 h in solution with different concentrations of the antigen (in excess) until equilibrium was reached. The preahsorbed samples were then added to microtiter plates coated with the same peptide antigen and the resulting binding measured by an ELISA procedure as described above. The K o was then determined from the following equation:
ELISA
Flat-bottomed, 96 well microtiter plates were coated overnight at room temperature with 50 /*l/well of 5 t~g/ml peptide diluted in 50 mM Na2CO3/NaHCO 3 buffer, pH 9.6. The plates were washed 3 x in PBS with 0.1% Tween 20 before the supernatant, diluted in PBS containing 1% BSA and 0.1% Tween 20 (ELISA dilution buffer) was added a~d incubated for 1.5 h at room temperature. The plates were washed as before and incubated for another 1.5 h at room temperature with pe~'oxidase-conjugated goat anti-mouse F(ab') 2 lgG (H + L) (Zymed, South San Francisco, CA). After washing, substrate (0.4 m g / m l o-phenylenediamine and 0.03% hydrogen peroxide in water) was added. The peroxidase activity was stopped after 30 min by adding 2 M H2SO a and the absorbance measured at 490 nm. The specificity of the binding reaction was confirmed by competition ELISA analysis where free peptide was added together with the mAbs in the first antibody incubation. Irrelevant peptide or no peptide were used as controls. The isotype of selected mAbs was determined in an ELISA using an antigen dependent streptavidin-biotin system (Monoab-Id Sp Mouse Kit, Zymed) according to the manufacturer's instructions. The lg concentration in the culture medium was determined using an antibody capture ELISA assay with micrntiter plates coated with goat anti-mouse IgG (Zymed). Serial dilutions of a
( a(~ × AI)) / ( A o - A ) - an = KD
(Friguet et al., 1985) where a 0 is the total antigen concentration used for preabsorption, A U is the absorbance measured without preabsorption and A the absorbanee measured with preabsorption. Immunoblotting
For immunoblotting analysis, cell membrane proteins were submitted to detergent phase separation as previously described (Atar et al., 1989). The proteins were denatured under reducing conditions, subjected to 10% SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose filters. For immunostaining, nitrocellulose strips were incubated with antibodies, washed, incubated with rabbit-anti mouse IgG F(ab')z conjugated to alkaline-phosphatase (Zymed), washed and finally incubated in substrate. The specificity of the mAbs was confirmed in competition analysis where the primary antibody was incubated together with an excess of free peptide (50 /*g peptide/25 ~ g mAb, using the mAbs at a concentration of 50/zg/ml). FACS analysis
Cells used in FACS analysis were incubated at 3 × l0 s cells/ml in RPMI medium for 24 h before analysis. Preliminary experiments showed
that the mAbs did not bind to living cells, but only to fixed cells. T h e cells were therefore fixed before F A C S analysis: approximately 5 x 106 cells were washed 1 x by centrifugation ( 2 5 0 X g , 7 min) in PBS, 1% BSA and 5 m M N a N 3 ( F A C S buffer), resuspended in 500 ,u.I FACS buffer and 1.5 ml methanol at room temperature was slowly a d d e d drop by drop. A f t e r 2 min, 8 ml FACS buffer w e r e added, the cells washed 3 × in F A C S buffer by centrifugation and resuspended in the same buffer at 2 x 106 e e l l s / m l . Samples of cells (2 × lb"/tube) were incubated for 60 min at 40(: with mAbs diluted in FACS buffer (final concentration: 2 5 / z g / m l ) , washed 1 X by centrifugation in 3 ml F A C S buffer and further incubated for 30 rain at 4°C with 100 t.tl FITC-conjugated F ( a b ' ) 2 rabbit anti-moose l g G (Zymed) diluted ~n F A C S buffer. Finally, the ceils were washed 1 x by eentrifugation, resuspended in 250 /zl F A C S buffer and analysed (FACS-scan, Becton Dickinson, Ca). T h e signal obtained from cells incubated with second antibody alone defined background levels. Competition assays were carried out by adding free peptide together with the mAbs ( 2 5 / z g pept i d e / 5 . 0 / x g mAb, using the mAbs at a concentration of 25 tzg/mI). F A C S buffer and irrelevant peptide were used as positive controls.
Results
Production of peptide specific monoclonal antibodies Six out of a total of 30 immunized mice were selected for fusion studies based on a reactivity with the immunizing peptide in ELISA. T h e peptide antibody titer in serum from mice selected for in vitro boosting did not differ from that of mice from which splenoeytes were fused directly after in vivo immunization (data not shown). All the fusions resulted in multiple growing hybridomas ( 3 . 7 - 5 6 x 103 h y b r i d o m a s / s p l e e n ) (Table I). N o peptide specific mAbs were isolated from the first three fusions (Table I, mice 1, 2, and ~,~ using splenocytes from only in vivo immunized mice. T o enhance the e f f i c i e n ~ of antibody production, the in vivo immunization was combined with in vitro boosting of the splenocytes before fusion (Table I, mice 3a, and 4 - 6 ) . Peptide specific mAbs were isolated from all fusions using the in vitro boosted splenocytes. T o directly compare in vivo with in v i v o / v i t r o immunization, splenocytes from one mouse (Table 1, mouse 3), immunized in vivo, were divided in two equal parts. O n e was fused immediately and the other
TABLE I HLA-DQ .8 CHAIN MONOCLONAL PEPTIDE ANTIBODIES Mouse/antigen
Immunization
Hybridore#s/well
no. of wells
Positivehybridomas DQw7 DQw8
DQw7/DQw8
(1) DQw7/8.0[49-60] /2) DQw8,8[49-60] (3) DQw7 ~[49-60]
In viva In rive (a) In vlvo (bl In rive/vitro In vivo/vitro In rive/vitro In rive/vitro
4.7 4.4 4.2 2,6 3.2 3.1 2.9
i,152 1,152 576 576 1,152 1,152 1,152
0 0 0 10 {~ II II
0 0 0 2 8 2 2
(4) DQw8 #[49-60] (5) DQw8 .0[39-52] (6) DQw8 #[39-52]
0 O 0 O 4t 3 3
Splcnocytes from mice 1-6 immunized in rive (]-3a) or in rive combined ~;th an in vitro boosting (3b-61 (see materials and methnds section). Hybridomas/well are indicated as an average of 200 wells, no. of wells arc the total number of wells used to seed the fused spleen cells. Hybridomas were defined as positive based on ELISA react[vity and indicated as follows. Mice 1-4:DQw7 binding to HLA-DQw7 0[44-63], DQw8 binding to HLA-DQw8 B[44-63], DQw7/DQw8 binding to HLADQw7 and DQw8 /][44- 53]. Mice 5-6:DQw7 binding to HLA-DQw7 .0139-52]¢ys,DQw8 binding to HLA-DQw8 ~39-52]cys. DOw7/DQw8 binding to HLA-DQw7 and DOw8 fl[39-52]cys.
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Fig. 2. Bindingof mAb 2H3 to HLA-DQw8•[39-52]cys in immunoblotso[ EBV-transformed cells. The mAb was tested out a concentration o[ 25 ~g/ml. -, direct binding. +, the mAbwas co-incubated with HLA-DOw8/3139-52]cysat a concentrationof 50 ~g/ml. A: DR3/3, DQw2/2; B: DR8/8, DQw4/4; C: DRI6/16, DOwS/5; D: DRI5/15, DQw6/6; E: DR4/4, DQw7/7; F: DR4/4, DQwS/8. The 2B5 and 1A1 mAbsto HLA-DQw813139-52]cysshowed the same bindingspecificityas 2H3 (not shown).
was boosted in vitro. Only fusion of the in vitro boosted splenocytes resulted in peptide specific mAbs, even though the number of hybridomas was approximately 60% higher fol[owing in vivo immunization only, i.e., 2.5 × 103 compared to 1.6 × 103 in 500 wells.
Characterization of mAbs to HLA-DQ/3 position 149-601 Peptide specificity. The peptide specificity of the mAhs was analysed by ELISA using peptides representing position [44-63] of DQwT/3 or DQw8/3 as antigens (Fig. 1). These peptides differ only at position 57 within the immunizing sequence.
Thefusion of splenocytes immunized in vivo/iu vitro with DQw7 fl[49-60]cys (Table I, mouse 3) resulted in 12 peptide specific mAbs, ten bound only to DQw7 /3[44-63] and two bound both DQw7 and DQw8 /3[44-63]. 49 hybridomas producing peptide specific mAbs were isolated from splenocytes immunized in vivo/in vitro with DQw8 /3[49-60] (Table I, mouse 4), 41 bound only DQw8 /3[44-63] and 8 bound both DQw7 and DQw8/3[44-63]. The isotype and dissociation constants (KD) were determined for 15 hybridomas which had been cloned twice by limiting dilution. All mAbs were IgGl,k except for two which were IgM,k. The K D of five selected IgG1 mAbs was 3.1-5.4
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Fig. 3. FACS analysis of methanol fixed EBV-transformed cells. The shaded curves show bindingof mAb 21"]3to DOw8 /][39-52]eys at a concentration of 25 ttg/ml. The solidblack line and open curve show bindingof 21-13after co-incubationwith HLA.DQw8/~[39-52]cys at a concentration of 200 tLg/ml.The pale greyline and open curveshowhingingof cellsincubatedwith second antibody only (goat anti-mouse IgG H+L). A: DR3/3, DOw2/2; B: DR8/8, DQw4/4; C: DRI6/16, DOwS/5; D: DRIS/IS, DQwfi/6;E: DP,4/4, DQw7/7; F: DR4/4, DQwS/8 (see Fig. 1). The 2B5 and 1A1 mAbsto HLA-DQwg/3139-52]cys showed the same bindingspecificityas 2H3 (not shown).
x 10 -5 M -~, whereas the two IgM mAbs displayed a lower affinity, i.e., 1.1-1.4 x 10 -3 M 1.
Binding to HLA-DQ ~ chain molecules in immunoblot analysis. None of the 12 mAbs against HLA-DQw7 ~[49-60] bound in immunoblot analysis of EBV-transformed lympbocytes. In contrast, 3 of the 49 mAbs against DQw8/3149-60]cys (2A12, 6B12, and 8D12) bound to a 29 kDa protein in immunoblots of DR4/DQw8 positive cells, identical in size to the band recognized by a monoelonal antibody to H L A - D R / D Q /3 chain molecules (data not shown). To test the allele specificity of the antibodies, the three mAbs were also tested for binding to D R 3 / D Q w 2 ,
DR8/DQw4, DRI5/DQw5, DR15/DQw6, and DR4/DQw7 homozygous cells. One mAb, 6B12 did not bind to the /3 chain molecule of any of these cells; 2A12 recognized the DR3/DQw2 /3 chain molecule, and 8D12 showed a much broader reactivity binding to DR8/DQw4, DR1/DOwS, and DR4/DQw7 positive cells. The sequence specific binding of the mAbs was confirmed by competition experiments: co-incubation with the DQw8 peptide in all cases blocked the binding completely to the 29 kDa protein, whereas the DQw7 peptide only blocked the binding for 8D12 (not shown). None of the DQw8/~[49-60]~.3's mAbs bound
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Fig. 4. FACSanalysisof methanol fixedEBV-transformedtransrect with either nativeor mutant HLA-DOw8/3chain. The shaded curve showbindingof mAh 2H3 to DQw8j~[39-52]cysat a concentration of 25/~g/ml.The solidblack line and open curveshow bindingof 2H3 after ¢o-incubalionwith HLA-DQw$ #[39-52]cys in a concentration of 2{10p.g/ml. The pale grey line and open curve show bindingof cells incubated with second antibodyonly(goat anti-mouse IgG H+L). A: DR3/DQw2; B: DR3/DQw2 transfected with DQw8Bm45~ chain; C: DI~/DQw2 transfected with nativeDQw8 ~ chain; D: DR3/DQw2 transteeted with DOw8/3m57fl chain(Fig. 1). in immunoblot analysis of peripheral blood lymphocytes or in FACS analysis to living or fixed cells.
Characterization of mAbs to HLA-DQ/3 position 139-521 Peptide specificity. The two fusions with splenocytes from mice immunized irr vivo/in vitro with DQw8 /3139-52]cys (Table i, mice 5 and 6) each resulted in five peptide specific mAbs. Five of these were specific for DQw8/3139-52]cys and five bound both DQw7 and DQw8 ,B[39-52]cys. Analysis of five selected hybridomas, cloned twice by limiting dilution, demonstrated that all were of the IgG1,K isotype with a KD between 1 . 7 × 1 0 - 5 - 1 . 5 × 1 0 -6 M-t.
Binding to HLA-DQ fl chain molecules in immunoblot analysis. Three of the ten mAbs (2H3, 2B5, and 1A1) specific for DQw8 /3139-52]eys recognised the /3 chain molecule on DQw8, DQw4, DQw5, and DQw6 positive cells, but not on DQw2 and DQw7 positive cells (Fig. 2). To confirm the sequence specificity of the bindir.g, the three mAbs were incubated with an excess of the DQw8 /3139-52]cys peptide and tested for binding to DQw4, DQw5, DQw6, and DQw8 positive cells. In all cases the DQw8 peptide blocked the binding completely (Fig. 2). The specificity of these mAbs was further analysed by testing their binding to DR3/DQw2 homozygous cells transfeeted with site directed DQw8/3 gene mutants (Fig. 1) (Kwok et al., 1989). The 2H3,
2B5 and 1AI mAbs all recognis~.d the/3 chain on the DQwg~ and DOwg/3m57 mutant cells (results not shown), the mutant DQw8~m57 encoding a molecule identical to DQw8/~ except that Ala at position 57 was replaced by Asp corresponding to the residue in DQw7fl in that position. In contrast, none of the mAbs recognised the ~ ehain molecule on the non-transfected cells or on the DQw8flm45 mutant cells, where the residue at position 45 of DQwgfl (Gly) has been replaced by the corresponding residue from DQw7/~ (Glu). Binding to intact cells: FACS analysis None of the ten DQw8 /3139-52]cys mAbs bound to living cells in FACS analysis. Since anti-peptide antibodies recognise sequential rather than conformational eptiopes, the lymphocytes were fixed to induce a partial denaturation of the membrane proteins. Following this procedure, the three mAbs that recognised the DQ /~ chain in immunoblot analysis now clearly bound to intact cells confirming the immunoblot results: the mAbs bound to DR8/DQw4, DR1/DQw5, D R I 5 / D Q w 6 and DR4/DQw8 positive cells but not to DR3/DQw2 and DR4/DQw7 positive cells (Fig. 3). The binding was displaced completely by incubation with an excess of fre~ DQw8 /3139-52]cys peptide (Fig. 3). Furthern, ..:, the mAbs bound with the same specificity as observed in immunoblot analysis to the cells ex-
A
Discussion Here we report the production of epitope specific mAbs that distinguish HLA-DQ fl chain alleles based on single amino acid substitutions at either position 45 or 57 in the first, variable domain. Two important points emerge from these results: firstly, they demonstrate that it is possible to make useful mAbs of a predetermined specificity down to the level of a single amino acid. Such mAbs may be valuable tools in the serological analysis of closely related HLA class II alleles.
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pressing the different DOw8 /3 chain mutants, i.e., binding to the DOwg/3 and DOw8,gm57 mutant cells, but no binding to the mutant DQw8/~m45 and the non-transfected DR3/ DQw2 cell line. The mAbs were also tested for binding to PBL from a DR2/DR3, DQwl/DQw2 positive individual in a dual immunofluorescence analysis using FITC-conjugated HLA-D specific mAb (Dakopatts) and a phycoerythrin conjugated rat anti-mouse K for the detection of the DQw8 /3139-52]cys mAbs (Fig. 5). The results showed that the HLA-D positive cells were specifically stained by the DQw8 /3139-52]cys mAbs; co-incubation with DQw8 fl[39-52]cys pcptide reduced the binding to background levels (Fig. 5).
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Fig. 5. Bindingof mAb 2H3 to HLA-DQw8 /3139-52]cysin FACSanalysisof methanol fixedperipheral blood lymphocytes.The mAb 2H3 was tested at a concentration of 25/tg/ml. A: FLI. mAb to HLA-DR/DQ/3 chains(Dakopatts, Glostrup.Denmark); FL2, mAb 2H3 to HLA-DQw8 /3139-52]cys. B: FLI, mAb to HLA-DR/DQ fl chain; FL2, 2143co-incubated with HLA-DOw8 /3139-52]cys at a concentration of 200 /xg/ml. The 2B5 and IAI mAbs tu HLA-DQw8 /3[39 52]cysshowed the same binding specificityas 2H3(not shown).
Secondly, we show that in vitro boosting of splenocytes from in vivo immunized mice may be helpful in the generation of mAbs against small peptide antigens. Thus, the amino acid sequence of the peptide to be used as an antigen may be selected by criteria other than predicted antigenicity. In this study it was not possible to isolate any pcptide specific mAbs from mice receiving in vivo immunization alone, although the three fusions made all resulted in large numbers of hybridomas (Table I). In vitro immunization using unprimed mouse spleen cells has previously been shown to result in antibodies to antigens which are normally not immunogenic (Van Ness et al., 1984; Borrebaeck, ]980; Borrebaeck et al., 1986). However, such mAbs are predominantly IgM and of low affinity (Borrebacck, 1986; De Boer et al., 1987), e.g., in one study (De Boer et al., 1987) 90% of the mAbs against human thyroglobulin generated by in vitro immunization were of the IgM isotype. By combining in vivo immunization with in vitro boosting of the splenocytes before fusion, we have isolated a large number of relatively high affinity peptide specific mAbs of the IgG isotype. These results suggest that during the in vitro boosting, antigen specific B ceils primed in vivo may be preferentially expanded. Most antibody epitopes in proteins are believed to be discontinuous and conformationally dependent (Van Regenmortel et al., 1988). Thus peptides may mimic only part of an epitope and therefore often induce limited crossreactivity to the native antigen (Van Regenmortel, 1989). Accordingly, peptide antibodies tend to react much better with a denatured form of the protein. These points are exemplified in the present study: of the six mAbs which reacted with the denatured HLA-DQw8 ,8 chain protein in immunoblutting, none reacted with the HLA class II molecules on living cells in FACS analysis. However, after cells were fixed in methanol, which induces a partial denaturation, the three mAbs to DQw8 /3[3952]eys bound to both EBV-transformed cells and PBL with the same specificity as observed in immunoblotting analysis (Figs. 3-5). Three mAbs to HLA-DQw8 fl[39-52]cys (2B5, 2H3, and 1A1) reacted selectively with D Q ,8 chain molecules with a Gly-VaI-Tyr segment in
position [45-47], i.e., DQw4//, DQw5fl, DQw6.8, and DQwg.8 hut not DQw7.8 (Glu in position 45) or DQw2fl (Glu-Phe in position 46-47). The specificity of mAbs binding was confirmed using two different techniques: (1) in competition experiments the immunizing pepfides, but not irrelevant peptides, inhibited binding of the mAbs completely in both immunoblotting and FACS analysis (Figs. 2 and 3) and (2) analysis of D R 3 / D Q w 2 positive cells transfectod with native or mutated DQw8/3 chain genes coding for DQw7 /3 chain amino acid substitutions in position 45 and 57 (Michelsen et al., 1990) showed a binding pattern that correlated with the binding to cells homozygous for the DQw7 or DQw8 .8 chain allele (Fig. 4). These results clearly demonstrate that the 29 kDa protein recognized is the DQw8 chain and not an unrelated protein. The three mAbs to DQw8 .8139-52]cys (2B5, 2H3, and 1A1) bound to approximately 10% of the cells from peripheral blood lymphocytes from a DQwl positive control. The proportion of cells that were stained corresponded well to the expected value of HLA class lI positive cells, i.e., B and activated T cells. Experiments with double fluorescence showed that all cells stained with a HLA-D specific mAb (Dakopatts) were also recognised by the mAbs to DQw8 .8139-52]cys (Fig. 5). In contrast, none of the mAbs to DQw8/3[4960]cys bound to intact cells fixed in methanol. Two observations may explain the discrepancy between the binding patterns observed in immunoblotting and FACS analysis. First, the fact that the mAbs to DQw8 fl[49-60]cys are of a lower affinity than the mAbs to DQw8 ,8[3952]cys, may result in a lower potential for binding. The position 45 epitope is more hydrophilic than the position 57 epitope, which may explain the difference in affinity between the two groups of mAbs. Second, in a recent study (Michelsen et al., 1990) of the fine specificity of DQw7 or DQw8 ,8 chain specific mAbs using site directed mutagenesis of the DQw8 ,8-chain, it was demonstrated that the amino acid in position 45 is critical for generating the serologic epitopes characterizing the DQw7 and DQw8 .8 chain molecules. Taken together, these results indicate that the epitope around position 57 may not be
exposed to an tib o d y b in d in g , even in fixed cells, M a n y a u t o i m m u n e diseases have b e e n s hown to b e associated with specific H L A a n t i g e n s ( S v e j g a a r d et al., 1983; T i w a r i et al. 1985; M i c h e l s e n et al., 1987, 1990; T o d d et al., 1987; H o r n et al., 1988). In type 1 diabetic p a t i e n t s the frequency of t h e H L A - D R 4 associated D Q w 8 /3 c h a i n allele is significantly increased c o m p a r e d t o t h e b a c k g r o u n d p o p u l a t i o n ( M i c h e l s e n et al., 1987, 1990; T o d d et al., 1987; H o r n et al., 1988). T h e o t h e r D R 4 associated D Q /3 c ha i n allele, D Q w T , is negatively associated with type 1 diab e t e s a n d differs from the D Q w 8 / 3 c ha i n only by f o u r a m i n o acid substitutions at positions 13, 26, 45, a n d 57 w i t h i n the first d o m a i n ( T o d d et al., 1987). A t p r e s e n t it is n o t k n o w n h o w these two closely r e l a t e d alleles c o n f e r susceptibility or resistance t o the disease, b u t the w h o l e m o l e c u l e is believed to b e involved r a t h e r t h a n a single a m i n o acid. E p i t o p e specific m A b s to the di a be t e s assoc i a t e d D Q p ch ain m o l e c u l e s m a y be useful tools for s t u d y i ng t h e expression o f the s e alleles in a u t o i m m u n i t y . T h e r e f o r e , to test the utility of the H L A - D Q / 3 ch ain reactive m A b s , they w e r e tested in i m m u n o b l o t analysis o n a p a n e l of l ymphobl a s told ceils f r o m D Q w 8 positive type 1 di abe t i c p a t i e n t s a n d D O w 7 o r D Q w 8 positive healthy controls. T h e m A b s specific for the e p i t o p e s at p o s i t i o n s 45 a n d 57 s h o w e d conclusive b i n d i n g p a t t e r n s c o n f i r m i n g that the cells w e r e e i t h e r D Q w 7 o r D Q w 8 positive. T h u s , e p i t o p e specific m A b s to H L A class 1I m o l e c u l e s m a y prove to be useful for serologic analysis of H L A alleles associated w i t h a u t o i m m u n i t y .
Acknowledgements W e are i n d e b t e d to Else Jost J e n s e n for expert technical assistance a n d to Harts Kofod for synthesizing the peptides. T h e cells carrying the D O w 8 / 3 m u t a n t m o l e c u l e s were kindly d o n a t e d by W i l l i a m Kwok, V i r g i n i a M a s o n R e s e a r c h Center, Seattle, W A .
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distinguish single amino acid substitutions in position 57 in H L A - D O beta-chain alleles associated with insulin-dependent diabetes. J. ImmunoL 143, 533-538,
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