Proc. Natl. Acad. Sci. USAVol. 82, pp. 539-542, January 1985Immunology

Human major histocompatibility complex class I antigens: Residues61-83 of the HLA-B7 heavy chain specify an alloreactive site

(synthetic peptide/allospecific)

LESLIE E. WALKER, THERESA A. KETLER, RICHARD A. HOUGHTEN, GREGOR SCHULZ, ALBERTO CHERSI,AND RALPH A. REISFELDDepartment of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037

Communicated by Frank J. Dixon, September 24, 1984

ABSTRACT A chemically synthesized peptide (Asp-Arg-Asn-Thr-Gln-Ile-Tyr-Lys-Ala-Gln-Ala-Gln-Thr-Asp-Arg-Glu-Ser-Leu-Arg-Asn-Leu-Arg-Gly), homologous to residues61-83 of the HLA-B7 heavy chain, induced antibodies thatspecifically recognized the HLA heavy chain-P2-microglob-ulin complex and the free heavy chain of the HLA-B7 antigen.These antibodies specifically immunoprecipitated the HLA-B7(-microglobulin complex solubilized from human lympho-blastoid cells by nonionic detergents and reacted with freeHLA-B7 heavy chains in blots on nitrocellulose. These obser-vations suggest that the antigenic conformation of this regionof the HLA-B7 molecule is independent of the presence of f-microglobulin and that amino acid residues 61-83 mimic analloreactive site expressed by the HLA-B7 antigen.

Human class I histocompatibility antigens are a set of highly-polymorphic integral membrane glycoproteins consisting oftwo noncovalently associated polypeptides ofMr 45,000 andMr 12,000. The smaller component light chain has been iden-tified as the common serum protein f3A-microglobulin, whichlacks allotypic determinants and is encoded on chromosome15, which is outside the major histocompatibility complex(1). The Mr 45,000 heavy chain has been shown to contain allof the structural polymorphism associated with class I anti-gens and is therefore responsible for the serologically de-fined polymorphism of HLA-A, -B, and -C antigens (2). Al-though data obtained from protein amino acid sequencestudies (3) and DNA sequence analyses (4-6) have answeredmany of the questions pertaining to the overall structure andmolecular organization of these antigens, one of the key is-sues that remains to be resolved is the localization of thealloreactive site(s) in the HLA-A, -B heavy chain amino acidsequence. We have attempted to answer this question byproducing antisera to synthetic peptides selected from theamino acid sequence of HLA-B7 antigen (7) followed bytesting the ability of these antisera to bind to the nativeheavy chain-f32-microglobulin complex as well as the isolat-ed heavy chain of class I antigens. Previously, we reportedthe production and characterization of an antibody to one ofthe most hydrophilic peptide regions of HLA-B7 antigen (7).These studies demonstrated that this region of the antigendid not encode for an allorecognition site and the conforma-tion of this region was highly dependent upon the presenceof bound A2-microglobulin.We report here on the characteristics of an antiserum pro-

duced to a synthetic peptide corresponding to residues 61-83of the HLA-B7 antigen heavy chain amino acid sequence,which represents a region of high structural variability whencompared to other class I antigen amino acid sequences (1,8, 9).

MATERIALS AND METHODS

Cell Lines. The human B-lymphoblastoid cell lines used inthis study were cultured in RPMI 1640 medium supplement-ed with 10% fetal calf serum, 2 mM glutamine, and gentamy-cin at 50 ,g/ml.

Isolation of Glycoprotein. Glycoproteins were isolatedfrom the cell line GM3107 or LG-2 by affinity chromatogra-phy on Lens culinaris lectin-Sepharose as described (10).

Synthesis of Peptide. The sequence of the peptide was se-lected from the HLA-B7 sequence reported by Orr et al.(11). The peptide that was chosen (Asp-Arg-Asn-Thr-Gln-Ile-Tyr-Lys-Ala-Gln-Ala-Gln-Thr-Asp-Arg-Glu-Ser-Leu-Arg-Asn-Leu-Arg-Gly) contained an amino acid sequencewith a decidedly high local hydrophificity, as determined bythe method of Hopp and Woods, and also is hypervariablewhen compared to other class I antigen heavy chain se-quences (12, 13). The peptide was synthesized by the solid-phase method of Merrifield (14, 15) using a Beckman model990B peptide synthesizer. A cysteine was added to the finalresidue at the carboxyl terminus to facilitate coupling to key-hole limpet hemocyanin (KLH). The composition of the pep-tide was confirmed by amino acid analysis.Coupling of Peptide to Carrier Protein. The peptide was

coupled to KLH as described by Green et al. (16) with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (17)through the cysteine at its carboxyl terminus. Briefly, 425 p.lofMBS at 5 mg/ml in dimethylformamide was slowly addedto 20 mg ofKLH in 1 ml of 0.01 M sodium phosphate buffer(pH 7.2). After 30 min at room temperature, unreacted MBSwas removed by gel filtration at 40C on a 25 x 0.7 cm Sepha-dex G-25 column equilibrated with 0.5 M sodium phosphatebuffer (pH 6.0). The peptide (3 mg) dissolved in 1 ml of 0.1 Msodium phosphate buffer (pH 7.2) was mixed with 3 mg ofKLH-MBS. The reaction mixture was adjusted to pH 7.2and stirred for 3 hr at room temperature. After dialysis over-night against 2 liters of phosphate-buffered saline (pH 7.2),the peptide-KLH conjugate was stored at -20'C.

Production of Antiserum HLA-B761-83. After an initial in-tramuscular injection of 200 pxg of peptide-KLH conjugatein complete Freund's adjuvant, rabbits received four weeklysubcutaneous injections of 100 ttg of peptide-KLH in in-complete adjuvant. Animals were bled 7-10 days after thelast injection. Pooled serum was then purified by affinitychromatography on peptide 61-83 conjugated to cyanogenbromide-activated Sepharose. The peptide was conjugatedat -0.5 mg/ml and elution was by equilibration with 0.1 Mglycine HCl buffer (pH 2.5).

Electrophoretic Transfer Blotting Analysis. Glycoproteins(5 ,ug per lane) isolated from the cell line GM3107 or LG-2were mixed with 100 ,ud of 0.068 M Tris HCl (pH 6.8) con-

Abbreviations: KLH, keyhole limpet hemocyanin; MBS, m-male-imidobenzoyl-N-hydroxysuccinimide ester.

539

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540 Immunology: Walker et a!.

taining 2% NaDodSO4 and 5% (vol/vol) 2-mercaptoethanoland heated for 2 min at 100°C.

Electrophoretic Transfer of Proteins. Free HLA-A, -Bheavy chains, after separation from p2-microglobulin by Na-DodSO4/PAGE (18), were electrophoretically transferred tonitrocellulose sheets as described by Towbin et al. (19). Toreduce nonspecific binding of the antiserum, the nitrocellu-lose sheets were first incubated for 1 hr with 0.01 M Tris HClbuffer (pH 7.4) containing 0.5 M NaCl and 3% (wt/vol) bo-vine serum albumin. The blots were then incubated at roomtemperature for 2 hr with either 10 ml of rabbit antiserum,diluted 1:4 in phosphate-buffered saline, containing 0.1% bo-vine serum albumin and 0.2% Tween 20 or with 10 ml ofmonoclonal antibody (5 ,g/ml) in the same buffer. After fivewashes with phosphate-buffered saline containing 0.2%Tween 20 and 0.1% ovalbumin, the blots were incubated for1 hr with 10 ml of a 1:100 dilution of staphylococcal proteinA conjugated to horseradish peroxidase (Sigma). After anadditional five washes with the same buffer, the locations ofthe bound antibodies were visualized by incubation at roomtemperature with 10 ml of 0.01 M Tris HCl (pH 7.4) contain-ing 0.8 mM o-dianisidine dihydrochloride and 0.001% H202.The reaction was stopped by washing with water containing0.02% sodium azide.

Indirect Immunoprecipitation. Lymphoblastoid cell lineswere intrinsically labeled with [3H]leucine, extracted withnonionic detergent, and centrifuged to remove particulatematerials as described (20). The extracts were then allowedto react with protein A-Sepharose conjugated with eitherantiserum HLA-B76183, normal rabbit IgG, or monoclonalantibody W6/32, which is directed to a structure common toall HLA-A, -B, and -C antigens. The indirect immunopre-cipitates were analyzed by NaDodSO4/PAGE (18) followedby fluorography (20) to visualize the separated components.Antibody-Dependent Cellular Cytotoxicity Assay. Anti-

body-dependent cellular cytotoxicity assays were performedessentially as described (21). Briefly, the cell lines to be usedas targets GM3107 (HLA-A3,3; B7,7) and LG-2 (HLA-2,2;B27,27) were labeled with 51Cr by incubating 1 x 106 cells inRPMI 1640 media containing 10%o fetal calf serum with 100,uCi (1 Ci - 37 GBq) of 5tCr for 1 hr at 37°C. The labeled cellswere washed, resuspended in media, and aliquoted into 96-well tissue culture plates at 1 x 104 cells per well. The cells

HLA-B7 +

Proc. NatL Acad. Sci. USA 82 (1985)

Table 1. Reactivity of antiserum HLA6183 with phenotypicallydistinct cell lines

% class ICell line Phenotype antigen bound

GM3107 HLA-A3,3; B7,7 50GM3161 HLA-A3,3; B7,7 55GM1857 HLA-A9,11; B7,W15 26GM2707 HLA-A9,2; B18,7 16LG-2 HLA-A2,2; B27,27 0GM3098 HLA-A2,2; B17,17 5GM3103 HLA-A9,9; B14,14 0GM3163 HLA-A30,30; B13,13 0GM3160 HLA-A2,2; B35,12 0GM3164 HLA-A2,24; B27 0.5

The above cell lines were intrinsically labeled with [3H]leucineand extracted with nonionic detergent (20). The radiolabeled cellsupernatants were then allowed to react with protein A-Sepharoseconjugated with either antiserum HLA6143, normal rabbit immu-noglobulin, monoclonal antibody W6/32, or cell culture supernatantfrom the murine myeloma P3X63Ag8. The % class I antigen boundwas calculated as follows: % class I antigen = (cpm antiserumHLA6183 - cpm rabbit IgG)/(cpm W6/32 - cpm P3X63Ag8).Typically for those cell lines that reacted with antiserum HLA6183,14,000-50,000 cpm were specifically bound. Cell lines that did notreact with the antiserum specifically bound 0-300 cpm and the cellline GM3098 (5%) specifically bound 2400 cpm.

were then incubated with 1 14g of antibody or control IgG for1 hr at room temperature and washed by centrifugation toremove unbound antibody. Purified human mononuclearcells were then added at a target:effector ratio of 1:50. Fol-lowing centrifugation, cell lysis was monitored by assaying100 ,ul of supernatant in an LKB y counter.

RESULTSAntiserum to the HLA-B7 synthetic peptide (Asp-Arg-Asn-Thr-Gln-Ile-Tyr-Lys-Ala-Gln-Ala-Gln-Thr-Asp-Arg-Glu-Ser-Leu-Arg-Asn-Leu-Arg-Gly), designated antiserumHLA-B76143, following purification on peptide 61-83 conju-gated to Sepharose, was tested for its ability to bind to apanel of extracts from lymphoblastoid cells that had beenintrinsically radiolabeled with [3H]leucine. The cpm boundby the antiserum HLA-B76143 peptide antibody, after sub-

+ +

GM3107 LG-2 GM3163 GM3161 GM1857 GM3160 GM3098

CHAIN -[

CHAIN -E

- -~~~-.: . _

a*,._-- _

.468o~w-om-u '

(m N CN m~ NY NY m NY n CY (n CYD m G m G m OD n OD mn OD ( D (

I IN Icw N NI N I N

ANTI0B

%f'0 0ANT IBODY %o 39 %O 3t wo v 39 %O 3 %O x % 39

z

I-z

P-

z

P-

zI-z

I-z z

FIG. 1. Indirect immunoprecipitation patterns of antiserum HLA6183 and W6/32 against B-lymphoid cell lines. The cell lines were intrinsi-cally radiolabeled with [3H]leucine, extracted with 2% Renex-30 in 0.01 M Tris/0.15 M sodium chloride/0.001 M phenylmethylsulfonyl chlo-ride, pH 8.5, and allowed to react with either antiserum HLA6183 or W6/32 bound to protein A-Sepharose. The bound antigens were separatedby NaDodSO4/PAGE and the components were visualized by fluorography. The Mr 45,000 molecule is shown. The positions of the intrinsicallylabeled light (L) and heavy (H) chains of immunoglobulin produced by the cell lines and whether the cell lines are serologically positive forHLA-B7 antigens are indicated.

Ig H

Ig L

Proc. NatL Acad ScL USA 82 (1985) 541

traction of background binding of normal rabbit IgG, werecompared to those obtained with the monoclonal antibodyW6/32 (22), following subtraction of background binding ofthe murine myeloma P3X63 supernatant. Monoclonal anti-body W6/32 has been shown to bind to all HLA-A, -B, and-C antigens, and its reactivity therefore represents the totalamount of HLA-A, -B, and -C antigens es-esent. As is shownin Table 1, only those cell lines that serologically typed asHLA-B7 positive were capable of reacting with the anti-pep-tide antibody. Cross-reactions with the antiserum HLA-B76143 were not observed with extracts containing class Iantigens such as HLA-B27, which would be expected to behighly cross-reactive, based on serotyping analyses. Howev-er, all extracts were reactive with anti-HLA-B76183 that didserologically express HLA-B7 antigen when tested by indi-rect immunoprecipitation against a number of intrinsicallylabeled B-lymphoid cell lines.

Fig. 1 depicts patterns obtained by NaDodSO4/PAGE ofthese indirect immunoprecipitates. For comparative pur-poses, Fig. 1 indicates whether the cell line types serologi-cally positive for HLA-B7 antigen and it also depicts the pat-terns of class I antigens that are precipitated by the monoclo-nal antibody W6/32. It is apparent that all cell lines testedthat contain HLA-B7 antigen (GM3107, GM3161, andGM1857) contain a Mr 45,000 molecule that has the samemobility as the heavy chain of class I antigens reactive withmonoclonal antibody W6/32. In contrast, those cell linesthat are not serologically positive for HLA-B7 antigen (LG-2, GM3163, GM3160, and GM3098) did not react with antise-rum HLA-B76143. The appearance of two closely spacedcomponents with approximate Mr 45,000 present in the pat-tern of GM3107 in Fig. 1 does not necessarily indicate that aseparation of molecules with HLA-B7 and HLA-A2 specific-ities was achieved. It is likely that each component containsa mixture of these two molecules when monoclonal antibodyW6/32 was used in the reaction. When the anti-peptide anti-b6dy HLA-B76143 was employed, it is likely that each of thetwo components contained HLA-B7 molecules since thesame two component patterns were observed with monoclo-nal antibody Q6/64, which does not react with HLA-A2 (7).Some of the indirect immunoprecipitatiorn patterns also re-veal the heavy and light chains of immunoglobulins pro-duced by the intrinsically labeled cell lines. These immu-noglobulin chains are present since they bind to the proteinA-Sepharose that was used for the indirect immunoprecipita-tion and thus they do not specifically react with the antibod-ies employed in the experiments.To determine whether the antigenic conformation of the

region of the HLA-B7 antigens defined by the residues 61-83required interaction with f32-microglobulin, antiserum HLA-B76143 was tested by electrophoretic transfer blotting analy-sis against partially purified glycoprotein extracts of B-lym-phoblastoid cells expressing either HLA-B7 (GM3107) orHLA-B27 (LG-2). When such extracts were dissociated byNaDodSO4/PAGE and electrophoretically transferred to ni-trocellulose paper and then allowed to react with antiserumHLA-B76183, reactivity was detected only with the heavychain of HLA-B7 and not with that of the HLA-B27 antigen(Fig. 2). The presence of HLA heavy chain in the immunob-lots of both antigens is verified by the reactivity with mono-clonal antibody Q1/28.Although antiserum HLA-B76183 binds significantly only

to the HLA-B7 antigen, a possibility remained that this reac-tivity may be directed to an intracellular HLA-B7 antigenprecursor, especially since all of our studies utilized deter-gent-solubilized cell extracts. To ascertain whether the re-gion of the HLA-B7 heavy chain, comprised by its residues61-83, is indeed expressed on the lymphoid cell surface, celllines LG2 (HLA-A2,2; B27,27) and GM3107 (HLA-A3,3;B7,7) were labeled with 51Cr, following which we deter-

GM3107 LG-2

45 ---{

F-':t cot

zM0 lra(

z

FIG. 2. Electrophoretic transfer blotting analysis of antiserumHLA61483. Glycoproteins prepared from the cell lines GM3107 (HLA-A3,3; B7,7) and LG-2 (HLA-A2,2; B27,27) were separated by Na-DodSO4/PAGE and transferred to nitrocellulose paper. The blotswere then incubated for 1 hr with anti-HLA61-3 or monoclonal anti-body Q1/28. Following washing, the nitrocellulose strips were incu-bated with protein A conjugated to horseradish peroxidase and thereactive bands were visualized with o-diansidine dihydrochloridedissolved in 0.01 M Tris HCI/0.001% H202, pH 7.4. The Mr 45,000molecule is shown.

mined the ability of antiserum HLA-B76183 to mediate lysisin the presence of complement. As shown in Fig. 3, onlyGM3107 cells bearing the HLA-B7 haplotype were lysed byantiserum HLA-B761 83 whereas the monomorphic monoclo-nal antibody W6/32 was reactive with both GM3107 andLG2 cells. The HLA-B7 antigen detected by antiserumHLA-B761 83 is therefore most likely expressed on the sur-face of the plasma cell membrane and does not represent anintracellular precursor.

DISCUSSIONThe results presented in this manuscript demonstrate that asynthetic peptide homologous to residues 61-83 of the HLA-B7 antigen heavy chain is immunogenic and elicits antibod-ies that specifically react with HLA-B7 antigen. Conse-quently, this synthetic peptide appears to mimic an alloreac-tive site expressed by the HLA-B7 antigen. The uniquenessof this peptide region to the HLA-B7 molecule is apparentfrom our data, indicating that antibodies to this peptide onlybind to or indirectly immunoprecipitate antigen from thosecell lines that according to tissue typing are known to sero-logically express HLA-B7. The specificity of the anti-pep-tide antibody for HLA-B7 is further indicated by lack of re-activity with cell line LG-2, expressing HLA-B27, which isknown to cross-react with HLA-B7 by tissue typing.

Surprisingly, the region comprised by amino acid residues61-83 of the HLA-B7 heavy chain is apparently conforma-

Immunology: Walker et aL

Proc. NatL AcadJ Sci USA 82 (1985)

40

30

~20

10

Anti- W6/32 IgG Anti- W6/32 IgGpeptide peptide

FIG. 3. Antibody-dependent cellular cytoxocity of anti-HLA61-83- (A) GM3107 cells (HLA-A3,3; B7,7) used as targets; (B) LG-2cells (HLA-A2,2; B27,27) used as targets. Following radiolabeling ofthe cell lines with 51Cr, the cells were allowed to react with eitherantiserum HLA6183, monoclonal antibody W6/32, or control IgGfor 1 hr and were washed to removed unbound antibody. Effectorcells, purified human mononuclear cells, were added at a target:ef-fector ratio of 1:50. Cell lysis was monitored by assaying 100 ,Al ofeach supernatant in an LKB y counter. The % lysis of target cellswas calculated as follows: % lysis = [(measured cpm - backgroundcpm)/(maximal cpm - background cpm)] x 100. In this system,100% cell lysis-i.e., maximal cpm-was equivalent to 19,100 cpmand background cell lysis was 1820 cpm.

tionally independent of the presence of bound 32-microglob-ulin since antiserum HLA-B76183 that reacts with the pep-tide as well as the native HLA-B7-p32-microglobulin complexby indirect immunoprecipitation also reacts with the isolatedHLA-B7 heavy chain when tested by blotting analysis. Thisfinding contrasts with that in our previous report describingan antibody to another synthetic peptide of HLA-B7 (resi-dues 39-50). The conformation of this very hydrophilic pep-tide region was greatly affected by the presence of bound f3A-microglobulin since antisera to this peptide could only reactwith the free peptide and the isolated HLA-B7 heavy chain.To date, only one other antibody-i.e., monoclonal antibodyQ1/28-demonstrated the ability to react with both the na-tive HLA heavy chain-f32-microglobulin complex and theheavy chain devoid of f32-microglobulin (23). Since monoclo-nal antibody Q1/28 is monomorphic and therefore does notreact with the peptide region of the HLA-B7 heavy chaindescribed in this study, there appear to be at least two sitesin the HLA heavy chain that are conformationally indepen-dent of f2-microglobulin. The total number of such sites isprobably small since no other monoclonal antibodies toHLA-A, -B, and -C antigens have been reported to reactwith both forms of the class I antigen heavy chain. This con-tention is strengthened by the observed lack of reactivity ofthe heavy chain-32-microglobulin complex with two rabbitantisera produced to other synthetic peptides of the HLA-B7heavy chain, representing residues 168-186 (Leu-Arg-Tyr-Leu-Gln-Asn- Gly - Lys -Asp- Lys - Leu-Glu-Arg-Ala-Asp-Pro-Pro-Lys) and residues 215-232 (Leu-Thr-Trp-Gln-Arg-Asp-Gly-Glu-Asp-Asn-Thr-Gln-Asp-Thr-Glu-Leu-Val-Glu),respectively (unpublished observations). The significance ofthe conformational changes induced by the binding of f-

microglobulin upon the heavy chain of class I antigens is notentirely clear at this point, however, our data suggest thatthese changes may have relatively little effect on the presen-

tation of a peptide region of an HLA heavy chain that may

contribute considerably towards the serological polymor-phism of class I histocompatibility antigens.

In conclusion, chemically synthesized peptides of theHLA-B7 heavy chain were shown to be capable of generat-ing antiserum that is allospecific and to be of considerableuse in mapping important antigenic regions of this molecule.It will be of considerable interest to produce additional anti-sera to equivalent regions ofHLA-A, -B antigens with differ-ent phenotypes to ascertain whether the findings reportedhere can be generalized for class I histocompatibility anti-gens.

We thank Ms. Karen Truesdell for her expert technical assistanceand Ms. Bonnie Pratt Filiault for her excellent secretarial assistance.This work was supported by U.S. Public Health Service Grant AI10180 from the National Institutes of Health. This is Scripps Publi-cation no. IMM-3621.

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542 Immunology: Walker et aL