Epitope Mapping of Human Monoclonal Antibodies to HLA-B27 By Using Natural and Mutated Antigenic Variants D. Weyl, T. Hansen, Y. Deschamps,K. Hannestad,and A. Toubert ABSTRACT: The epitopes defined by three human monoclonal antibodies (mAbs) (Tr3B6, TrCGI0, TrBH12) against HLA-B27 have been mapped by flow cytometry. For this purpose we used murine transfected cells expressing at their surface hybrid antigens between HLA-B7 and -B27 and, in addition, Epstein-Barr virus cell lines expressing the six HLA-B27 alleles B'2701 to B'2706. The results indicated that the mAbs are domain

specific. TrBHI2 recognizes the first external (alpha-l) domain. Residues critical for the TrBH12 epitope are located in the alpha-1 helix and include the polypepdde stretch 65-76 plus a critical amino acid at pos/don 77. Tr3B6 binds the second external (alpha-2) domain, and one mutation (VALt52 -* GLU 152)destroyed its epkope. TrCGI0 also binds the alpha-2 domain. Human lmmand-

ABBREVIATIONS aa amino acid EBV Epstein-Barr virus FITC fluorescein-isothiocyanate

LCL

ogy 3 1 , 2 7 1 - 2 7 6 (1991)

mAb

lymphoblastoid ceU line monoclonal antibody

INTRODUCTION HLA-B27 antigen is particularly interesting because of its association with ankylosing spondylitis and related disorders [1, 2]. Strong arguments support the hypothesis of a direct involvement of HLA-B27 in disease pathogenesis. In this regard, molecular mimicry between HLA-B27 and bacterial antigens triggering arthritis has been described as a potentially relevant phenomenon [3, 4]. Much advancement has been achieved in defining HLA-B27 antigen by the description of functional subtypes (HLA-B*2701 to B'2706) with different biochemical properties and sequences (for review, see [5]). Site-directed mutagenesis [6] and hybrid gene constructions [7] made it easier to define more precisely the residues involved in the serological and functional rec-

ognition sites of the molecule. Precise mapping of serological epitopes on HLA molecules is difficult to assess with human alloantlsera. For HLA-B27, the study of a panel of monospec/fic alloandsera on HLA-B27 subtypes Epstein-gaff virus (EBV)-transformed lymphoblastoid cell lines (LCLs) led to the definition of some of these epitopes [8]. Several investigators have described human monoclonal antibodies (mAbs) m HLA, and some of them were particularly directed against HLA-B27: Tr3B6, TrCG10, and TrBH12 [9, 10]. In this article we describe their reactivity patterns on routine transfectants expressing at their surface hybrid molecules between the HLA-B7 and -B27 alleles, as well as on EBV cell lines from various B27 subtypes.

From the INSERbl U. 283, H6pital Cochin, Paris Cedex, France (D.W.; Y.D.; A.T.); and the Institute of Medical Biology, Schoolof Medicine, University of Tromso, Norway (T.H.; K.H.). Address reprint requeststo Antoine Toubert, M.D., INSERM U. 283, H#pital Cochin, 27, rue du FgSt-Jacques, 75674 ParisCedex 14, France. Received December 14, 1990; acceptedMarch l, 1991.

MATERIALS AND METHODS

HumanImmunology31, 271-276 (1991) © AmericanSocietyfor Histocompadbilityand Immunog~nedcs,1991

Serological Reagents Human mAbs were produced by fusing EBV-transformed cells from a multlparous woman (HLA-A2,9; 271

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272

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FIGURE 1 Nomenclature of the hybrid moleculestested. Differences between the HLA-B7 (open boxes) and HLAB'2705 (hatched boxes) parental sequences are shown. B7/ B27 and B27/B7 are recombinantsbetween the whole alpha1 and alpha-2 domains. Others are intra-alpha-1 domain recombinantsnamedaccordingm the firstaminoacid position of the recombined sequence. For instance, B7-24 means alpha-1 domain HLA-B7 until position 24 excluded and then HLA-B27. The alpha-2 domainis from HLA-BT. For hybrid gene constructions,see reference [7].

B7) with the human fusion partner KR4. The production of the Tr3B6 (IgM K), TrBH12 (IgM k), TrCG10 (IgM k) anti-HLA-B27 mAb has been previously described [10]. They were used as crude culture supernatants (Ig concentration 1-3/zg/ml) at 1/10 final dilution. Murine mAbs included anti-HLA monomorphic B9.12.1 or B1.23.2 mAbs as positive controls of HLA expression levels on the transfectants, and anti-I-Ak 10.3.6 mAb as negative control [7]. GS145.2 m,.:rine mAb [11], an IgG1 that reacts to HLA-B27 and weakly to HLA-B7, was kindly provided by Dr. S. Y. Choo (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) and used as 1/1000 dilution of ascites fluid. Cellular Reagents Transfected cells. Murine mastocytoma P815 (HTR) transfected cells expressing various hybrid genes between HLA-B7 and -B27 (B'2705) were previously described [7]. The nomenclature and sequences of these

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hybrids are schematically presented in Fig. 1. Briefly, hybrid genes were constructed by in vivo recombination in E. coli before transfection in P815 (HTR) cells by the calcium phosphate precipitation method and selection in G418 medium (1 mg/ml, Gibco, England). Cells were cloned by limiting dilution and kept in culture in DME containing 10% fetal calf serum and G418 (400 #g/ml). During the course of the experiments, cells were regularly checked for HLA expression with B9.12.1 or B1.23.2 anti-HLA monomorphic mAbs. B7-24 and B763 genes were transfected in a derivative of P815 (HTR) cells expressing the human beta-2 microglobulin (beta-2 m). P815 cells transfected with HLA-B*2705 and expressing the human beta-2 m were obtained from Dr. J. A. Lopez de Castro (Fundacion Jimenez Diaz, Madrid, Spain) to be compared to P815 B27 transfecrants expressing the murine beta-2 m, and gave comparable data.

HLA-B27 subtypes Epstein-Barrvirus lines. Epstein-Barr virus lines belonging to the six HLA-B27 alleles defined at the Tenth International Histocompatibility Workshop were tested and are listed in Table 1. CHI and LH lines were obtained from Dr. S. Y. Choo and the ABO line was obtained from Dr. M. Pla (H6pital St-Louis, Paris, France). Sequence differences among these subtypes are shown in Table 2. Flow Cytometry

Cell preparations. One hundred microliters of the cell suspension (107/ml) were exposed to 100/zl of the anti-

Epitope Mapping of Human HLA-B27 mAbs

TABLE 1

273

HLA typing of EBV-transformed cells expressing different subtypes of HLA-B27 HLA class I

HLA class ll

Name

A

B

C

DR

ABO I.H MI3 A34 A59 CHI ATI A72 A19 A46 A58 A62 LIE 632 920 615

A24,A26 A24, - A1, A28 A2, A9 A24, AI9 A31, A32 A2, All A3, All AI, A11 A2, AI9 A2, AI A2, - A2, All A2, A2 A2, A26 A2, A2

B°2701,BI3 B'2701, B8 B'2702, B8 B'2702, BI8 B°2702,BSI B'2703, BIB B'2704, BI7 B°2704,B5 B'2705, B8 B'2705, B14 B°2705,B8 B°2705,B8 B*2706,B5 B8, B62 BT, B38 B8,--

Cw2,Cw7 Cwl, Cw7 Cw2, Cw7

2, - 5, 5

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1, 3 8, 7,-2,-

RESULTS AND DISCUSSION

1, 4 2, 13 3,--

body to be tested for 43 rain. The second antibody was either fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse 1#3, A, M (Cappel, West Chester, PA) or rabbit anti-human lgM (Miles Yeda Ltd, Weizmann, Rehovot, Israel) sera. W h e n rabbit anti-human was used, this step was followed by incubation with a biotinylated goat anti-rabbit IgG (H + L) (Vector Laboratories, Inc., Burlingame, CA) antibody, then streptavidine-FITC (Immunotech, Marseille, France). In some experiments, human cells were directly stained with a FITC-conjngated sheep anti-human IgM (Paesel G m B H & Co., Frankfurt, Germany). The cells were then fixed for flow cytometry analysis.

Flow cytoraetry atlalysis. Flow cytometry analysis was performed with a fluorescence-activated cell analyzer

TABLE 2

Sequence differences in HLA-B27 subtypes B ' 2 7 0 1 to B ' 2 7 0 6 Residues

Alleles

59

74

B'2701 TYR TYR B'2702 - ASP B'2703 HIS ASP B°2704 - ASP B°2705 m ASP B'2706 - ASP

77

80

81

114

116

(FACscan, Becton Dickinson, Mountain View, CA) equipped with an argon laser. Dead cells were eliminated from the analysis on forward and side light scatter. Background fluorescence was determined with control samples using unrelated isotype-matched antibody (10.3.6) for murine antisera assays and samples without the first antibody for human antibody tests. The Kolmogorov-Smirnov two-sample test, which is a standard nonparametric statistical test, has been used to compare histograms [12]. T h e D-value is defined as the maximum difference between the cumulative area-normalized experimental and control histograms.

152

ASN THR ALA HIS ASP VAL -lie ASP -LEU SER -LEU GLU ASP ~ LEU SER -LEU ASP TYR GLU

Dashesdenote residues identicalto that in B'2701 taken as reference.

The human mAbs were first tested on P815 untransfected cells to ascertain the absence o f nonspecific binding on these cells (data not shown), and then on P815 transfected with HLA-B7 or HLA-B*2705. As expected from the typing of the donor (HLA-A2,9; B7) who gave lymphocytes for the fusion and production of the mAbs, Tr3B6, T r C G I 0 , and TrBH12 reacted on HLA-B27 and not on HLA-B7 transfected cells, which allowed testing them on the hybrid transfectants. A problem that arises when testing transfected cells is that the expression levels of the transfected gene products could differ from one cell line to another or among experiments. To minimize that point, we used FACscan analysis to check the expression level of the transfectants with BI.23.2, an HLA-specific monomorphic murlne mAb, and we tested simultaneously in the same experiment the three human mAbs on each llne. W e could thus rule out that differences in binding of these mAbs could be due to differences in gene expression levels. Tr3B6 As seen by comparisons of binding on the transfectants (Fig. 2), the second external domain of the B27 molecule, alpha-2, is necessary as no binding occurred on B27 B7. The part of this domain from amino acid (an) 177 to the end is not a major part o f this epitope as the hybrid B27-177 was well recognized. Additional data were obtained with EBV lines from various B27 subtypes (Fig. 3): substitutions at residue 59 (B'2703) or at residues 77, 80, and 81 (t3"2702) did not affect recognition, and these different lines were recognized as well as the major B ' 2 7 0 5 subtype. By contrast, VAL t~2 seems important because LCLs LIE expressing B ' 2 7 0 6 (GLUt52), and A71, A72 expressing B ' 2 7 0 4 (GLU t52) dicl not react with Tr3B6. These data combined emphasize the influence o f the alpha-2 domain preceding residue 177, as shown specifically for the residue at position 152, in the formation of the Tr3B6 epitopo. However,

274

D. Weyl et al.

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Contrary to Tr3B6 and TrCG10, TrBH12 is mostly reactive on the alpha-1 domain, as shown by its reactivity with B27 B7 and B7 B27. On this domain, aa stretch 63-77 i~ critical, as seen by comparison of reactivity between B7-63 and B7-77 hybrids. The involvement of this part of the molecule is further evidenced by the reactivity on the EBV lines. B'2701 and B'2702 expressing lines are nonreactive. The sequence of B'2705 (ASP TM ASP 77 T H R s° LEU sl) differs at three positions from B'2701 (TYR TMASN 77 ALAsl) and from B'2702 (ASN 77 ILEs° ALA81), as shown in Table 2. As LEU sl

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FIGURE 2 Analysis of Tr3B6, TrCGI0, and TrBH12 mAbs on the murine transfectants. D values characterize the specific staining of the mAbs as defined in Materials and Methods.

FIGURE 3 Analysis of Tr3B6, TrCG10, and TrBH12 mAbs on EBV LCLs from various HLA-B27 subtypes. Results are expressed as in Fig. 2. HLA typing of the LCI.~is given in Table 1. Ear ,nes

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there are arguments for a minor role of alpha-l, as B7 B27-177 was less reactive than B27-177 hybrid. The influence of the first domain on the reactivity of Tr3B6 could not be more precisely evaluated with the intraalpha-1 recombinants because they all possess a B7 alpha-2 domain and were therefore unreactive. TrCG10 As for Tr3B6, the recognition pattern of TrCG10 involves the second domain of the molecule; however, Tr3B6 and TrCG10 epitopes are not similar. When tested on the transfectants, B7 B27-177 and B27-177 were less reactive than B7 B27 and B27, respectively, suggesting that the sequence from 177 to the end of the alpha-2 domain, contrary to Tr3B6, influences the reactivitT of T r C G ! 0 mAb. Another point is that, unlike Tr3B6, two LCLs from the B'2704 subtype and the B'2706 LCL LIE were recognized as positive, which implies that the mutation GLU instead of VAL (B'2705) at residue 152 is less important for TrCG10

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Epitope Mapping of Human HLA-B27 mAbs

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275

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recognized by GS145.2 is more complex and does not involve the alpha-1 domain only because hybrid B7B27 was also positive. These data were confirmed by radioimmunoassay as in Toubert et al. [7] (data not shown). Tested on EBV lines, all subtypes were recognized, contrary to TrBH12, which was unreactive on B'2701 and B'2702 LCLs. The two epitopes are therefore clearly different. CONCLUSION

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FIGURE 4 Analysis ofGS145.2 mAb on the murine transfectants and EBV LCLs. Cells and results are expressed as in Figs. 2 and 3. ALAs~ is a conservative substitution, the ASP 77 --~ ASN 77 mutation is the most likely cause for the epitope loss. Lines from the other subtypes (B'2703, B'2704, B'2705, and B'2706) are still recognized with a high reactivity. Particularly, B'2703 different from B'2705 at residue 59 (HIS instead of TYR) was well recognized. B'2704 and B'2706 cell lines were also reactive, suggesting that the change at position 77 is not sufficient by itself to abrogate recognition. The nature of the substitution at residue 77 in the B'2704 and B'2706 subtypes (SER) or B'2702 subtype (ASN), instead of ASP in B'2705, could also be taken into account in the different reactivities observed with TrBH12. The fact that HLA-Bw47, which is also recognized by TrBH12 [10], is the only other HLA-B sequence described having ASP at position 77 [13] is another argument for the importance of this residue. GS145.2 Because the epitope recognized by GS145.2 routine mAb was reported to involve residue 67 [6~ and could therefore be similar to TrBH12, we tested this mAb on the transfectants and EBV lines by using the same protocols as for the human mAbs. Results are presented on Fig. 4. The involvement of residues 63-77 of alphaol domain is apparent from the loss of reactivity of B7o77 compared with B7-63 hybrid. However, the epitope

The HLA-B27 system appears as an especially good candidate for comparisons of murioe and human mAbs recognition as somc murine HLA-B27 specific mAbs have already been mapped on the molecule: 77-81 aa sequence at the end of the alpha-1 domain has been involved in B27-M2 [14] and T M I [7] recognition sites. As mentioned above, GS145.2 mAb has been shown to be influenced by substitutions at residue 67 [6], the size of the substituted a being of primary importance [15]. P56-I mAb was found to be negative on B'2704 and B'2706 subtypes and therefore dependent on residue 152 [16]. Finally, HLA-BT, B27 cross-reactive mAbs ME1 and GSP5.3 are affected by changes in the 6 3 - 7 0 sequence and at residues 69 and 71 [17]. On the whole, most murine mAbs were mapped on the alpha-1 domain. The most comparable reactivities between human and murine mAbs are those of Tr3B6 with p56.1 mAb and of TrBH12 with B27.M2 mAb, whereas TrCG10 reactivity is unique. The epitopes defined in previous studies with human alloantisera on B27 subtypes LCLs [8, 16] are also different from those defined here. This work puts emphasis on the wide range of serological epitopes on the HLA-B27 molecule as more specific sera and specifically designed cells can be tested to characterize them. These reagents of human or/gin could also be of interest to test the cross-reactivity between HLA-B27 and bacterial antigens putatively involved in ankylosing spondylitis pathogenesis. ACKNOWLEDGMENTS

We are indebted to Dr. J. Charreire for helpful discussions, Dr. A. Kahan for help in FACscan analysis, N. Bazdy for excellent technical assistance, and J. Decaix for the preparation of this manuscript. This work was supported in part by the Association de Recherches sat la Polyarthr/te (ARP). D. Weyl was supported by a grant from the Fondation pour la Recherche M~dicale (FRM). REFERENCES 1. Brewerton DA, Caffrev M, Hart FD, James DCO, Nicholls A, Smrrock RD: Ankylosing spondylitis and HL-A27. Lancet I:904, 1973.

276

2. Schlosstein L, Terasaki PI, Bluestone R, Pearson CM: High association of an HLA antigen W27, with ankylosing spondylitis. N EnglJ Med 288:704, 1973. 3. Ebringer A, Balnes M, Ptaszynska T: Spondyloarthritis, uveitis, HLA-B27 and Klebsiella. Immunol Rev 86:101, 1985. 4. Yu DTY, Choo SY, Schaak T: Molecular mimicry in HLA-B27 related arthritis. Ann Intern Med 111:581, 1989. 5. Lopez de Castro JA: FILA-B27 and HLA-A2 subtypes: Structure, evolution and function. Immunol Today 10:239, 1989. 6. Tautog JD, EI-Zaatari FAK: In vitro mutagenesis of HLA-B27 substitution of an unpaired cysteine residue in the alpha-1 domain causes loss of antibody-defined epitopes. J Clin Invest 82:987, 1988. 7. Toubert A, Raffoux C, BorettoJ, Sire J, Sodoyer R, Thurau SR, Amor B, Colombani J, Lemonnier FA, Jordan BR: Epkope mapping of HLA-B27 and HLA-B7 antigens using intradomain recombinants. J Immunol 141:2503, 1988. 8. De Waal LP, Krom FEJM, Breur-Vriesendorp BS, Engelfriet CP, Lopez de Castro JA, Ivanyi P: Conventional alloantisera can recognize the same HLA-B27 polymorphism as detected by cytotoxic T lymphocytes. Hum Immuno120:265, 1987. 9. Hansen T, Hannestad K: Simple rosette assay for HLAB27 typing of whole blood samples. Tissue Antigens 30:198, 1987. 10. Hansen T, Braille A, Hannestad K: Four cytotoxic hu-

D. Weyl et al.

man hybridoma antibodies that react with HLA-B27. Tissue Antigens 32:267, 1988. 11. Johnston J, Garbutt J, Nelson KA: A monoclonal antibody specific for HLA-B27 (abstract). Hum Immunol 14:134, 1985. 12. Young IT: Proof without prejudice: Use of the Kolmogorov-Smirnov test for the analysis of histograms from flow systems and other sources. J Histochem Cytochem 25:935, 1977. 13. Pohla H, Kuon W, Tabaczewski P, Doerner C, Weiss E: Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles. Immunogenetics 29:297, 1989. 14. Vega MA, Ezquerra A, Rojo S, Aparicio P, Bragado R, Lopez de Castro J: Structural analysis of an HLA=B27 functional variant: Identification of residues that contribute to the specificity of recognition by cytolytic T lymphocytes. Proc Natl Acad Sci USA 82:7394, 1985. 15. EI-Zaatari F, Sams KC, Taurog JD: In vitro mutagenesis of HLA-B27. Aminoacid substitutions at position 67 disrupt anti-B27 monoclonal antibody binding in direct relation to the size of the substituted side chain. J Immunol 144:1512, 1990. 16. Choo SY, St. John T, Orr FIT, Hansen JA: Molecular analysis of the variant alloantigen HLA-B27d (HLAB'2703) identifies a unique single aminoacid substitution. Hum Immunol 21:209, 1988. 17. Tautog JD, EI.Zaatari F, Tang JP: Mapping of HLA-B27 antigenic determinants by site-directed mutagenesis (abstract). Arthritis Rheum 33:S26, 1990.