Clin. exp. Immunol. (1990) 82, 16-20
Epitope
mapping of an HLA-B27 monoclonal antibody that reacts with a 35-kD bacterial outer-membrane protein
also
A. TOUBERT, M. HAMACHI*, C. RAFFOUXt, M. S. PARKI & D. T. Y. YU* INSERM U. 283, H6pital Cochin, Paris, France, * Division of Rheumatology, Center for Health Sciences, Los Angeles, CA, USA, t Laboratoire d'Immunologie et d'Histocompatibilit, H6pital St-Louis, Paris, France, and $ Department of Surgery, Center for Health Sciences, Los Angeles, CA, USA (Acceptedfor publication 8 June 1990)
SUMMARY Ye-3 is an HLA-B27-specific murine monoclonal antibody recognizing the heat-modifiable protein of Enterobacteriaceae. Here we used recombinant hybrid molecules between the HLA-B7 and HLAB27 antigens to delineate the epitope recognized by Ye-3. Results of these experiments indicated that the segment of HLA-B*2705 spanning residues 77-81 was critical to the reactive epitope. It is known to be a major serological and functional recognition site of HLA-B*2705 and our data give support for its involvement also in the serological cross-reactivity with bacterial antigens. Keywords HLA-B27 enterobacteria molecular mimicry
protein from the outer membrane of most Gram-negative bacteria (Zhang et al., 1989). By immunizing BALB/c mice with HOM-2, an HLA-B27 (B*2705) positive cell line, we have generated a previously unreported MoAb, Ye-3, of IgM isotype, which reacts with the 35-kD outer membrane component of Enterobacteriaceae. Artificially engineered mutagenized or recombinant molecules have been widely used to study the structure and function of murine and human molecules of the MHC. In the case of HLA-B27, this approach has led to the definition of the major serological (Taurog & El-Zaatari, 1988; Toubert et al., 1988) and functional recognition sites of this molecule (Healy et al., 1989). Such molecules could also define the sequence of HLAB27 involved in the serological cross-reactivity with bacterial antigens. We report here the epitope mapping of the Ye-3 MoAb on HLA-B27 by using transfected cells expressing at their surface recombinant molecules between the HLA-B7 and HLA-B27 alleles.
INTRODUCTION Since its description, the reason for the association between the HLA-B27 antigen and the spondyloarthropathies remains unresolved. The HLA class I molecules are now better clarified. Several subtypes of the HLA-B27 antigen have also been described by using alloreactive (Breuning et al., 1982) or antiviral (Toubert et al., 1984) cytotoxic T cell lines and isoelectric focusing (Choo et al., 1986). Their primary structure has been defined by comparative peptide mapping and protein sequence analysis (reviewed by Rojo et al., 1987). There is no preferential association between any particular HLA-B27 subtype and the disease (Breur-Vriesendorp, Dekker-Saeys & Ivanyi, 1987), neither is there any structural difference between HLA-B27 antigens from a healthy subject and an ankylosing spondylitis patient (Coppin & McDevitt, 1986). One possible approach is to analyse cross-reactive antigens. This cross-reactivity has been suspected as being important because bacteria carrying these cross-reactive proteins can induce reactive arthritis in HLA-B27-positive subjects (Ebringer, Baines & Ptaszynska, 1985; Granfors et al., 1989). Reactivity of Enterobacteriaceae membrane proteins with HLA-B27-specific monoclonal antibodies (MoAbs) have been reported by three groups of investigators (van Bohemen, Grumet & Zanen, 1984; Chen et al., 1987; Raybourne, Bunning & Williams, 1988). Proteins of two molecular weights are positive, one of 35 kD and the other of 19 kD. The 35-kD group has recently been shown to be the heat-modifiable OmpA
MATERIALS AND METHODS Generation of monoclonal antibodies BALB/c mice were immunized with cells from HOM-2, a HLAB27-positive cell line belonging to the B*2705 major subgroup. Generation of MoAbs was as described previously (Chen et al., 1987). Antibodies were screened against the HOM-2 cells using a microcytotoxicity method described previously (Terasaki et al., 1978). One positive hybridoma, Ye-3, was selected. This was subcloned three times and ascites fluids were generated in pristane-injected BALB/c mice. Anti-mouse immunoglobulin antibodies (Litton Bionetics, Kensington, MD) were used to determine the immunoglobulin isotype.
Correspondence: Dr A. Toubert, INSERM U. 283, H6pital Cochin, 27, rue du Fauburg St-Jacques, 75674 Paris Cedex 14, France.
16
Yersinia and HLA-B27 cross-reactivity 24
87
al domain 63 77 82 I I
a2 domain
Serological assays Ye-3 was tested by microcytotoxicity assay (Terasaki et al., 1978) to define its specificity on a panel of 108 HLA-types peripheral blood lymphocytes (PBL) and on the various hybrid transfected cells. Results were expressed as a score of percentages of killed cells: -,0-20%; 1,20-40%; 2,40-60%; 3,60-80%; and 4, 80-100%.
--I
L
B*2705 B7/B27 r B27/B7
B7-24
I
B7-63 I
1
77
1 1
77
F
B7-77 I
M
B7-82 1
Reactivity of YE-3 with bacterial components Growth of bacteria, preparation of Triton X- 100 insoluble membranes, SDS-PAGE, transfer to nitrocellulose membranes and detection of reactivity with antibodies were all as described
previously (Zhang et al., 1989).
Fig. 1. Nomenclature of the hybrid molecules tested. Differences between the HLA-B7 (0) and HLA-B*2705 (-) parental sequences are shown. B7/B27 and B27/B7 are recombinants between the whole a 1 and a2 domains. Others are intra-a l domain recombinants named according to the first amino acid position of the recombined sequence. For instance, B7-24 means al domain HLA-B7 until position 24 excluded and then HLA-B27. The a2 domain is from HLA-B7. For hybrid gene constructions, see Toubert et al. (1988).
Table 1. Amino acid differences between HLA-B7 and HLA-B*2705 antigens in the alpha-1 domain and sequences of the intra-alpha-1 recombinants using the one-letter amino acid code
Amino-acid residues 9
24
32
63
67
70
77
80
82
83
HLA-B7
Y
N
H Y Y Y
S T
Q
HLA-B*2705 B7-24 B7-63 B7-77
Q K
R L
C C Y
S
T T T N
L L L
Y
K K Q Q
S D D D D S
N T
B7-82
E E E N N
Y C
T S S
L L Q Q Q
G R R R R R
Y
17
L
HLA-B*2705 sequences are italicized.
Hybrid gene constructions and transfections The hybrid genes between the HLA-B7 and HLA-B27 alleles were produced by in vivo recombination method as detailed elsewhere (Toubert et al., 1988). The recombinants studied are listed in Fig. I and their amino acid sequences in Table 1. The point of recombination between the homologous sequences was assessed by Sanger dideoxy method. Transfections were carried out in P815 (HTR) murine mastocytoma cell lines by using calcium phosphate DNA precipitation method. Plasmid DNA of the hybrid genes (20 pg) and pAG60 (1 pg) were cotransfected for selection in culture medium containing G418 antibiotic (GIBCO, Paisley, UK). Cells were cloned by limiting dilution and kept in culture in DME containing 10% fetal calf serum (FCS) and G418 (400 ,ug/ml). During the course of the experiments, transfected clones were regularly checked for HLA expression with B9.12.1, an anti-HLA monomorphic MoAb as reported (Toubert et al., 1988). B7-24 and B7-63 genes were transfected in a derivative of P815 (HTR) cells expressing the human fl-2 microglobulin.
RESULTS
Characterization of the Ye-3 The Ye-3 MoAb was determined by immunodiffusion to be an IgM antibody. On testing the panel of PBL from normal individuals, all four HLA-B27-positive cell samples were reactive. Lymphocytes from nine non-HLA-B27 individuals were also positive. The typing of their HLA-B alleles consisted of one of the followings: B38, B39 and B67. Results of Western blot analysis with bacterial membranes Triton X-100 insoluble membranes were prepared from six different Enterobacteriaceae and their reactivity with Ye-3 analysed by Western blot. A component of 35-37 kD was clearly positive. This reactive component was observed with all six bacteria (Fig. 2a). In a separate publication, we have reported that this component did not react with eight other IgM MoAbs that are directed against non-HLA-B27 surface antigens (Zhang et al., 1989). Coomassie blue staining of the membrane proteins showed that this 35-37-kD protein was one of the major proteins of the bacteria membranes (Fig. 2b).
Comparing the reactivity of Ye-3 MoAb among hybrids of HLAB27 and HLA-B7 Ye-3 ascites fluid was tested on the transfectants listed in Fig. 1, using a complement dependent microcytotoxicity assay. Reactivity was tested either directly on the transfectants (Table 2) or by using the transfectants for absorption before testing the residual activity on a panel of HLA-B27 PBL (Table 3). Data obtained in both tests are in agreement. The most striking finding was that the Ye-3 reactivity was totally conserved on B27/B7, B7-24, B763 hybrids but completely lost on the hybrids B7-82 and B7/ B27. The differences between the positive and the negative cells resided in the segment spanning residues 63-81. A second experimental finding was that the B7-77 hybrid was less reactive than the parent protein but retained the reactivity to a large extent. These two sets ofdata indicated that a major epitope was in the segment spanning residues 77-81 of HLA-B*2705.
DISCUSSION Structural knowledge of HLA class I molecules has been advanced through in vitro engineering of mutangenized and hybrid molecules expressed at the cell surface after transfection in eukaryotic cells. This approach was undertaken for HLAB27. Briefly, epitopes recognized by two HLA-B27-specific murine MoAbs, GS 145-2 and TM- 1, were localized at the end of
A. Toubert et al.
18
(a)
( b)
kD 94 67 43
30 14
6 5 4 2 3 MW 6 l 4 5 2 3 Fig. 2. (a) Western blot analysis of the Ye-3 monoclonal antibody with membranes from six different bacteria. Bacteria membranes were isolated and their components separated by SDS-PAGE. After transfer to nitrocellulose membrane, their reactivity was tested with Ye-3 ascites fluid at 1/10 dilution. Lane 1, Yersinia pseudotuberculosis 78; lane 2, Escherichia coli P400; lane 3, Y. enterocolitica 4861; lane 4, Shigellaflexneri 6118; lane 5, Shigella sonnei 59G ATCC 11060; lane 6, Salmonella heidelberg 7736. Technical procedures and control blots with irrelevant IgM antibodies are described in Zhang et al. (1989). (b) SDS-PAGE analysis of bacterial membranes. Membranes from six bacteria were analyzed by SDS-PAGE. The gel was stained with Coomassie blue to visualize the proteins. Bacteria were the same as those shown in (a). Lane designated as MW denotes molecular weight markers.
Table 2. Complement-dependent microcytotoxicity assay of Ye-3 monoclonal antibody at various ascites fluid dilutions on P815 untransfected and transfected cells described in Fig. 1.
1/50 1/100 1/200 1/400 1/800 1/1600 1/3200 P815 B*2705
-
-
-
-
-
-
-
4
4
4
4
4
4
-
-
-
-
-
4 4 4 3
4 4 4 3
4 4 4 3
4 4 3 3
3 3 2 3
-
B7-24 B7-63 B7-77
4 4 4 4 3
B7-82
-
-
-
-
-
-
B7/B27 B27/B7
2 1 3 -
Results are expressed as scores of percentages of killed cells:
-,0-20%o; 1, 20-40%0,; 2, 40-60%0,; 3, 60-80%0,; and 4, 80- 1000/o. the first external domain, involving residues 67 (Taurog & ElZaatari, 1988) and 77-80 (Toubert et al., 1988), respectively. Human monospecific alloantisera also recognized sites on the alpha-i domain (Toubert et al., 1988). The dominance of this part of the molecule in serological recognition fits with the data obtained by comparison of the reactivity of various HLA-B27 subtypes with B27M2 MoAb (Vega et al., 1985) and studies of anti-peptide antibodies directed against the 63-84 sequence of HLA-B*2705 (Rojo et al., 1986). It is also consistent with the three-dimensional structure of the HLA-A2 antigen (Bjorkman et al., 1987) if we assume that the overall structure of these molecules is similar. This segment of the molecule is part of an alpha-helix easily accessible to antibodies. The Ye-3 MoAb recognizes HLA-B27, HLA-B38 and HLAB67. Using alloantisera for typing, cross-reactivity among these
has been noticed previously (Park et al., 1988). Our finding that this cross-reactivity could be demonstrated with a single MoAb indicated that these antigens share a similar epitope. Our experiment with the screening panel also showed that the Ye-3 MoAb was not reactive with HLA-B7. This was confirmed by testing HLA-B7-transfected cells and allowed us to use the hybrid transfectants for epitope mapping. All the reactive cells shared the same segment spanning residues 77-81 from HLAB*2705. Hence, this probably constitutes the major epitope for the Ye-3 MoAb. Sequence comparisons between amino acids 78-81 of HLA-B7 and HLA-B*2705 would suggest that residues 77 (S for HLA-B7, D for HLA-B*2705) and/or 80 (N for HLA-B7, T for HLA-B*2705) play a critical role in the antigenic determinant (Table 1). In a previous study (Schwimmbeck, Yu & Oldstone, 1987), a sequence homology between residues 72-77 from HLA-B*2705 and 188-193 from Klebsiellapneumoniae nitrogenase was found. Experimental testing of sera from patients in this study as well as in others (Tsuchiya, Husby & Williams, 1989; Husby et al., 1989) were performed with synthetic peptides 69-84 (Tsuchiya et al., 1989) or 67-84 and 68-83 (Husby et al., 1989) from HLAB*2705. It is therefore possible that residues outside the homologous sequence could be involved in the conformation of the determinant recognized. Interestingly, using large series of overlapping peptides from HLA-B*2705 (residues 65-85) and K. pneumoniae (residues 181-199), it has been demonstrated that two different epitopes are present on HLA-B*2705, one spanning residues 68-75 (epitope I) and another residues 75-82 (epitope II) (Ewing et al., 1989). These data localize epitope I as a major epitope recognized by sera from ankylosing spondylitis patients while murine antisera raised against K. pneumoniae recognized mainly epitope II. This is in agreement with our results obtained with a murine MoAb. Regarding this fact, a
19
Yersinia and HLA-B27 cross-reactivity Table 3. Test of the Ye-3 reactivity on six samples of HLA-B27 peripheral blood lymphocytes (PBL) after twice absorption (45 min at 370C and 15 min at 40C) of the MoAb as 1/50 diluted ascites fluid on the five transfectants (30 x 106 cells each) PBL B27+
1/50 1/100 1/200 1/400 1/800 1/1600 1/3200 1/50 1/100 1/200 1/400 1/800 1/1600 1/3200 Ye-3 absorbed on P815 B27
Ye-3
392 397 398 630 639 649
4 4 4 4 4 4
4 4 4 4 4 4
4 4 3 4 4 4
4 4 2 4 4 4
3 3 2 3 3 3
2 3 1 3 2 1
1 1 1 -
-
-
-
_
_
_
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
I
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2 3 2 2 2 2
1 1 1 1 1 1
-
-
-
-
-
-
1
Ye-3 absorbed on B7-82
Ye-3 absorbed n B-77 392 397 398 630 639 649
-
Ye-3 absorbed on B7-63
Ye-3 absorbed on B7-24 392 397 398 630 639 649
-
4 4 3 4 3 4
4 3 2 4 3 4
2 1 1 3 3
1 2
-
-
-
-
-
-
4 4 4 4 3 4
3 4 4 3 3 3
3 3 4 3 3 3
3 3 3 3 3 3
3 3 3 3 3 3
Microcytotoxicity assay and score as in Table 2.
slightly different recognition of bacterial cross-reactive epitopes in human and murine species could be possible. It should be emphasized that we used here hybrid HLA molecules whose structure and expression are similar to the parental class I antigens but possess discrete parts of the HLAB27 sequence. Thus, it is highly possible that the cross-reactive epitope is present on the native conformation of the HLA-B27 molecule. This is also in agreement with in situ findings on synovial tissues (Husby et al., 1989). Recent reports concern the bacterial counterpart which would be a candidate to trigger the immune phenomenon. The 35-kD bacterial envelope component has been characterized as the OmpA protein of Enterobacteriaceae (Zhang et al., 1989). The high homology of this protein among the various strains of bacteria tested could explain the multiple reactivities observed (Fig. 2). However, there is no correlation between OmpA expression or Ye-3 reactivity and the arthritogenic potential of these strains. Another important finding was the identification by Stieglitz, Fosmire & Lipsky (1989) of a plasmid, pHS-2, common to arthritogenic strains of Shigella and whose sequence carries five consecutive amino acids identical to residues 71-75 of HLA-B*2705 in an open reading frame of 22 amino acids. Regulation of expression of such plasmid sequences in HLAB27 individuals would be an important consideration.
Taken together, these data support the hypothesis of a crossreactivity between HLA-B27 and epitopes expressed by Gramnegative bacteria. Several reports are in agreement to map the HLA-B27 residues involved in a structurally dominant part of the molecule. By using HLA-B27 recombinant molecules, we were able to confirm this fact on integral HLA class I antigens.
ACKNOWLEDGMENTS We are indebted to Dr J. Charreire and to Professor B. Amor for helpful discussions, to N. Bazely (H6pital Cochin) and C. Dehay (H6pital StLouis) for technical assistance and to J. Decaix for help in the preparation of the manuscript. This work was supported in part by the Nora Eccles Trendwell Foundation and the Association de Recherches sur la Polyarthrite.
REFERENCES BWORKMAN, P.J., SAPER, M.A., SAMRAOUI, B., BENNETT, W.S., STROMINGER, J.L. & WILEY, D.C. (1987) Structure of the human class I histocompatibility antigen, HLA-A2. Nature, 329, 506. BREUNING, M.H., LUCAS, C.J., BREUR, B.S., ENGELSMA, M.Y., DE LANGE, C.G., DEKKER, A.J., BIDDISON, W.E. & IVANYI, P. (1982) Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and their role in self-recognition. Hum. Immunol. 5, 259. BREUR-VRIESENDORP, B.S., DEKKER-SAEYS, A.J. & IVANYI, P. (1987)
20
A. Toubert et al.
Distribution of HLA-B27 subtypes in patients with ankylosing spondylitis: the disease is associated with a common determinant of the various B27 molecules. Ann. rheum. Dis. 46, 353. CHEN, J.H., KONO, D.H., ZHOU, Y., PARK, M.S., OLDSTONE, M.M.B.A. & Yu, D.T.Y. (1987) A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27. J. Immunol. 139, 3003. CHOO, S.Y., ANTONELLI, P., NISPEROS, B., NEPOM, G.T. & HANSEN, J.A. (1986) Six variants of HLA-B27 identified by isoelectric focusing. Immunogenetics, 23, 24. COPPIN, H.L. & McDEVITT, H.O. (1986) Absence of polymorphism between HLA-B27 genomic exon sequences isolated from normal donors and ankylosing spondylitis patients. J. Immunol. 137, 2168. EBRINGER, A., BAINES, M. & PTASZYNSKA, T. (1985) Spondyloarthritis, uveitis, HLA-B27 and Klebsiella. Immunol. Rev. 86, 101. EWING, E., EBRINGER, R., TRIBBICK, G. & GEYSEN, M. (1989) Antibody activity in ankylosing spondylitis sera to two sites on HLA-B27 at the MHC groove region (within sequence 65-85) and to a Klebsiella pneumoniae nitrogenase peptide (within sequence 181-199). Arthritis Rheum. 32, S22 (Abstract). GRANFORS, K., JALKANEN, S., VON ESSEN, R., LAHESMAA-RANTALA, R., ISOMAKI, O., PEKKOLA-HEINO, K., MERILAHTI-PALO, R., SAARIO, R., ISOMAKI, H. & TOIVANEN, A. (1989) Yersinia antigens in synovialfluid cells from patients with reactive arthritis. N. Engl. J. Med. 320, 216. HEALY, F., TOUBERT, A., GOMARD, E., JORDAN, B.R. & LEVY, J.P. (1989) Delineation of determinants on HLA-B7 and HLA-B27 that are necessary for cytolytic T cell recognition by using inter- and intradomain recombinants. J. Immunol. 143, 2357. HUSBY, G., TSUCHIYA, N., SCHWIMMBECK, P.L., KEAT, A., PAHLE, J.A., OLDSTONE, M.B.A. & WILLIAMS, R.C. (1989) Cross-reactive epitope with Klebsiella pneumoniae nitrogenase in articular tissue of HLAB27 + patients with ankylosing spondylitis. Arthritis Rheum. 32,437. PARK, M.S., TERASAKI, P.I., BARBETTI, A., HAHN, H. & CECKA, J.M. (1988) Significance of the HLA molecular structure to transplantation. In Clinical Transplant (ed. by P. I. Terasaki) p. 301. UCLA Press, Los Angeles. RAYBOURNE, R.B., BUNNING, V.K. & WILLIAMS, K.M. (1988) Reaction of anti-HLA-B monoclonal antibodies with envelope proteins of Shigella species. Evidence for molecular mimicry in the spondyloarthropathies. J. Immunol. 140, 3489. Rojo, S., APARICIO, P., HANSEN, J.A., CHOO, S.Y. & LOPEZ DE CASTRO, J.A. (1987) Structural analysis of an HLA-B27 functional variant, B27d, detected in American Blacks. J. Immunol. 139, 3396.
Rojo, S., LOPEZ DE CASTRO, J.A., APARICIO, P., VAN SEVENTER, G. & BRAGADO, R. (1986) HLA-B27 antigenicity: antibodies against the chemically synthesized 63-84 peptide from HLA-B27. 1 display alloantigenic specificity and discriminate among HLA-B27 subtypes. J. Immunol. 137, 904. SCHWIMMBECK, P.L., Yu, D.T.Y. & OLDSTONE, M.B.A. (1987) Autoantibodies to HLA-B27 in the sera of HLA-B27 patients with ankylosing spondylitis and Reiter's syndrome. Molecular mimicry with Klebsiella pneumoniae as potential mechanism of autoimmune disease. J. exp. Med. 166, 173. STIEGLITZ, H., FosMIRE, S. & LIPSKY, P. (1989) Identification of a 2-Md plasmid from Shigella flexneri associated with reactive arthritis. Arthritis Rheum. 32, 937. TAUROG, J.D. & EL-ZAATARI, F.A.K. (1988) In vitro mutagenesis of HLA-B27. Substitution of an unpaired cysteine residue in the alpha- 1 domain causes loss of antibody-defined epitopes. J. cdin. Invest. 82, 987. TERASAKI, P.I., BERNOCO, D., PARK, M.S., OZTURK, G. & IWAKI, Y. (1978) Microdroplet testing for HLA-A, -B, -C and -D antigens. Am. J. cdin. Pathol. 69, 103. TOUBERT, A., GOMARD, E., GRUMET, F.C., AMOR, B., MULLER, J.Y. & LEVY, J.P. (1984) Identification of several functional subgroups of HLA-B27 by restriction of the activity of antiviral T killer lymphocytes. Immunogenetics, 20, 513. TOUBERT, A., RAFFOUX, C., BORETTO, J., SIRE, J., SODOYER, R., THURAU, S.R., AMOR, B., COLOMBANI, J., LEMONNIER, F.A. & JORDAN, B.R. (1988) Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants. J. Immunol. 141, 2503. TsUCHIYA, N., HUSBY, G. & WILLIAMs, R.C. (1989) Studies of humoral and cell-mediated immunity to peptides shared by HLA-27.1 and Klebsiella pneumoniae nitrogenase in ankylosing spondylitis. Clin. exp. Immunol. 76, 354. VAN BOHEMEN, C.G., GRUMET, F.C. & ZANEN, H.C. (1984) Identification of HLA-B27 Ml and M2 cross-reactive antigens in Klebsiella, Shigella and Yersinia. Immunology, 52, 607. VEGA, M.A., EZQUERRA, A., Rojo, S., APARICIO, P., BRAGADO, R. & LOPEZ DE CASTRO, J. (1985) Structural analysis of an HLA-B27 functional variant: identification of residues that contribute to the specificity of recognition by cytolytic T lymphocytes. Proc. nati Acad. Sci. USA, 82, 7394. ZHANG, J.J., HAMACHI, M., HAMACHI, T., ZHAO, Y.P. & Yu, D.T.Y. (1989) The bacterial outer membrane protein which reacts with antiHLA-B27 antibodies is the OmpA protein. J. Immunol. 143, 2955.
Epitope
mapping of an HLA-B27 monoclonal antibody that reacts with a 35-kD bacterial outer-membrane protein
also
A. TOUBERT, M. HAMACHI*, C. RAFFOUXt, M. S. PARKI & D. T. Y. YU* INSERM U. 283, H6pital Cochin, Paris, France, * Division of Rheumatology, Center for Health Sciences, Los Angeles, CA, USA, t Laboratoire d'Immunologie et d'Histocompatibilit, H6pital St-Louis, Paris, France, and $ Department of Surgery, Center for Health Sciences, Los Angeles, CA, USA (Acceptedfor publication 8 June 1990)
SUMMARY Ye-3 is an HLA-B27-specific murine monoclonal antibody recognizing the heat-modifiable protein of Enterobacteriaceae. Here we used recombinant hybrid molecules between the HLA-B7 and HLAB27 antigens to delineate the epitope recognized by Ye-3. Results of these experiments indicated that the segment of HLA-B*2705 spanning residues 77-81 was critical to the reactive epitope. It is known to be a major serological and functional recognition site of HLA-B*2705 and our data give support for its involvement also in the serological cross-reactivity with bacterial antigens. Keywords HLA-B27 enterobacteria molecular mimicry
protein from the outer membrane of most Gram-negative bacteria (Zhang et al., 1989). By immunizing BALB/c mice with HOM-2, an HLA-B27 (B*2705) positive cell line, we have generated a previously unreported MoAb, Ye-3, of IgM isotype, which reacts with the 35-kD outer membrane component of Enterobacteriaceae. Artificially engineered mutagenized or recombinant molecules have been widely used to study the structure and function of murine and human molecules of the MHC. In the case of HLA-B27, this approach has led to the definition of the major serological (Taurog & El-Zaatari, 1988; Toubert et al., 1988) and functional recognition sites of this molecule (Healy et al., 1989). Such molecules could also define the sequence of HLAB27 involved in the serological cross-reactivity with bacterial antigens. We report here the epitope mapping of the Ye-3 MoAb on HLA-B27 by using transfected cells expressing at their surface recombinant molecules between the HLA-B7 and HLA-B27 alleles.
INTRODUCTION Since its description, the reason for the association between the HLA-B27 antigen and the spondyloarthropathies remains unresolved. The HLA class I molecules are now better clarified. Several subtypes of the HLA-B27 antigen have also been described by using alloreactive (Breuning et al., 1982) or antiviral (Toubert et al., 1984) cytotoxic T cell lines and isoelectric focusing (Choo et al., 1986). Their primary structure has been defined by comparative peptide mapping and protein sequence analysis (reviewed by Rojo et al., 1987). There is no preferential association between any particular HLA-B27 subtype and the disease (Breur-Vriesendorp, Dekker-Saeys & Ivanyi, 1987), neither is there any structural difference between HLA-B27 antigens from a healthy subject and an ankylosing spondylitis patient (Coppin & McDevitt, 1986). One possible approach is to analyse cross-reactive antigens. This cross-reactivity has been suspected as being important because bacteria carrying these cross-reactive proteins can induce reactive arthritis in HLA-B27-positive subjects (Ebringer, Baines & Ptaszynska, 1985; Granfors et al., 1989). Reactivity of Enterobacteriaceae membrane proteins with HLA-B27-specific monoclonal antibodies (MoAbs) have been reported by three groups of investigators (van Bohemen, Grumet & Zanen, 1984; Chen et al., 1987; Raybourne, Bunning & Williams, 1988). Proteins of two molecular weights are positive, one of 35 kD and the other of 19 kD. The 35-kD group has recently been shown to be the heat-modifiable OmpA
MATERIALS AND METHODS Generation of monoclonal antibodies BALB/c mice were immunized with cells from HOM-2, a HLAB27-positive cell line belonging to the B*2705 major subgroup. Generation of MoAbs was as described previously (Chen et al., 1987). Antibodies were screened against the HOM-2 cells using a microcytotoxicity method described previously (Terasaki et al., 1978). One positive hybridoma, Ye-3, was selected. This was subcloned three times and ascites fluids were generated in pristane-injected BALB/c mice. Anti-mouse immunoglobulin antibodies (Litton Bionetics, Kensington, MD) were used to determine the immunoglobulin isotype.
Correspondence: Dr A. Toubert, INSERM U. 283, H6pital Cochin, 27, rue du Fauburg St-Jacques, 75674 Paris Cedex 14, France.
16
Yersinia and HLA-B27 cross-reactivity 24
87
al domain 63 77 82 I I
a2 domain
Serological assays Ye-3 was tested by microcytotoxicity assay (Terasaki et al., 1978) to define its specificity on a panel of 108 HLA-types peripheral blood lymphocytes (PBL) and on the various hybrid transfected cells. Results were expressed as a score of percentages of killed cells: -,0-20%; 1,20-40%; 2,40-60%; 3,60-80%; and 4, 80-100%.
--I
L
B*2705 B7/B27 r B27/B7
B7-24
I
B7-63 I
1
77
1 1
77
F
B7-77 I
M
B7-82 1
Reactivity of YE-3 with bacterial components Growth of bacteria, preparation of Triton X- 100 insoluble membranes, SDS-PAGE, transfer to nitrocellulose membranes and detection of reactivity with antibodies were all as described
previously (Zhang et al., 1989).
Fig. 1. Nomenclature of the hybrid molecules tested. Differences between the HLA-B7 (0) and HLA-B*2705 (-) parental sequences are shown. B7/B27 and B27/B7 are recombinants between the whole a 1 and a2 domains. Others are intra-a l domain recombinants named according to the first amino acid position of the recombined sequence. For instance, B7-24 means al domain HLA-B7 until position 24 excluded and then HLA-B27. The a2 domain is from HLA-B7. For hybrid gene constructions, see Toubert et al. (1988).
Table 1. Amino acid differences between HLA-B7 and HLA-B*2705 antigens in the alpha-1 domain and sequences of the intra-alpha-1 recombinants using the one-letter amino acid code
Amino-acid residues 9
24
32
63
67
70
77
80
82
83
HLA-B7
Y
N
H Y Y Y
S T
Q
HLA-B*2705 B7-24 B7-63 B7-77
Q K
R L
C C Y
S
T T T N
L L L
Y
K K Q Q
S D D D D S
N T
B7-82
E E E N N
Y C
T S S
L L Q Q Q
G R R R R R
Y
17
L
HLA-B*2705 sequences are italicized.
Hybrid gene constructions and transfections The hybrid genes between the HLA-B7 and HLA-B27 alleles were produced by in vivo recombination method as detailed elsewhere (Toubert et al., 1988). The recombinants studied are listed in Fig. I and their amino acid sequences in Table 1. The point of recombination between the homologous sequences was assessed by Sanger dideoxy method. Transfections were carried out in P815 (HTR) murine mastocytoma cell lines by using calcium phosphate DNA precipitation method. Plasmid DNA of the hybrid genes (20 pg) and pAG60 (1 pg) were cotransfected for selection in culture medium containing G418 antibiotic (GIBCO, Paisley, UK). Cells were cloned by limiting dilution and kept in culture in DME containing 10% fetal calf serum (FCS) and G418 (400 ,ug/ml). During the course of the experiments, transfected clones were regularly checked for HLA expression with B9.12.1, an anti-HLA monomorphic MoAb as reported (Toubert et al., 1988). B7-24 and B7-63 genes were transfected in a derivative of P815 (HTR) cells expressing the human fl-2 microglobulin.
RESULTS
Characterization of the Ye-3 The Ye-3 MoAb was determined by immunodiffusion to be an IgM antibody. On testing the panel of PBL from normal individuals, all four HLA-B27-positive cell samples were reactive. Lymphocytes from nine non-HLA-B27 individuals were also positive. The typing of their HLA-B alleles consisted of one of the followings: B38, B39 and B67. Results of Western blot analysis with bacterial membranes Triton X-100 insoluble membranes were prepared from six different Enterobacteriaceae and their reactivity with Ye-3 analysed by Western blot. A component of 35-37 kD was clearly positive. This reactive component was observed with all six bacteria (Fig. 2a). In a separate publication, we have reported that this component did not react with eight other IgM MoAbs that are directed against non-HLA-B27 surface antigens (Zhang et al., 1989). Coomassie blue staining of the membrane proteins showed that this 35-37-kD protein was one of the major proteins of the bacteria membranes (Fig. 2b).
Comparing the reactivity of Ye-3 MoAb among hybrids of HLAB27 and HLA-B7 Ye-3 ascites fluid was tested on the transfectants listed in Fig. 1, using a complement dependent microcytotoxicity assay. Reactivity was tested either directly on the transfectants (Table 2) or by using the transfectants for absorption before testing the residual activity on a panel of HLA-B27 PBL (Table 3). Data obtained in both tests are in agreement. The most striking finding was that the Ye-3 reactivity was totally conserved on B27/B7, B7-24, B763 hybrids but completely lost on the hybrids B7-82 and B7/ B27. The differences between the positive and the negative cells resided in the segment spanning residues 63-81. A second experimental finding was that the B7-77 hybrid was less reactive than the parent protein but retained the reactivity to a large extent. These two sets ofdata indicated that a major epitope was in the segment spanning residues 77-81 of HLA-B*2705.
DISCUSSION Structural knowledge of HLA class I molecules has been advanced through in vitro engineering of mutangenized and hybrid molecules expressed at the cell surface after transfection in eukaryotic cells. This approach was undertaken for HLAB27. Briefly, epitopes recognized by two HLA-B27-specific murine MoAbs, GS 145-2 and TM- 1, were localized at the end of
A. Toubert et al.
18
(a)
( b)
kD 94 67 43
30 14
6 5 4 2 3 MW 6 l 4 5 2 3 Fig. 2. (a) Western blot analysis of the Ye-3 monoclonal antibody with membranes from six different bacteria. Bacteria membranes were isolated and their components separated by SDS-PAGE. After transfer to nitrocellulose membrane, their reactivity was tested with Ye-3 ascites fluid at 1/10 dilution. Lane 1, Yersinia pseudotuberculosis 78; lane 2, Escherichia coli P400; lane 3, Y. enterocolitica 4861; lane 4, Shigellaflexneri 6118; lane 5, Shigella sonnei 59G ATCC 11060; lane 6, Salmonella heidelberg 7736. Technical procedures and control blots with irrelevant IgM antibodies are described in Zhang et al. (1989). (b) SDS-PAGE analysis of bacterial membranes. Membranes from six bacteria were analyzed by SDS-PAGE. The gel was stained with Coomassie blue to visualize the proteins. Bacteria were the same as those shown in (a). Lane designated as MW denotes molecular weight markers.
Table 2. Complement-dependent microcytotoxicity assay of Ye-3 monoclonal antibody at various ascites fluid dilutions on P815 untransfected and transfected cells described in Fig. 1.
1/50 1/100 1/200 1/400 1/800 1/1600 1/3200 P815 B*2705
-
-
-
-
-
-
-
4
4
4
4
4
4
-
-
-
-
-
4 4 4 3
4 4 4 3
4 4 4 3
4 4 3 3
3 3 2 3
-
B7-24 B7-63 B7-77
4 4 4 4 3
B7-82
-
-
-
-
-
-
B7/B27 B27/B7
2 1 3 -
Results are expressed as scores of percentages of killed cells:
-,0-20%o; 1, 20-40%0,; 2, 40-60%0,; 3, 60-80%0,; and 4, 80- 1000/o. the first external domain, involving residues 67 (Taurog & ElZaatari, 1988) and 77-80 (Toubert et al., 1988), respectively. Human monospecific alloantisera also recognized sites on the alpha-i domain (Toubert et al., 1988). The dominance of this part of the molecule in serological recognition fits with the data obtained by comparison of the reactivity of various HLA-B27 subtypes with B27M2 MoAb (Vega et al., 1985) and studies of anti-peptide antibodies directed against the 63-84 sequence of HLA-B*2705 (Rojo et al., 1986). It is also consistent with the three-dimensional structure of the HLA-A2 antigen (Bjorkman et al., 1987) if we assume that the overall structure of these molecules is similar. This segment of the molecule is part of an alpha-helix easily accessible to antibodies. The Ye-3 MoAb recognizes HLA-B27, HLA-B38 and HLAB67. Using alloantisera for typing, cross-reactivity among these
has been noticed previously (Park et al., 1988). Our finding that this cross-reactivity could be demonstrated with a single MoAb indicated that these antigens share a similar epitope. Our experiment with the screening panel also showed that the Ye-3 MoAb was not reactive with HLA-B7. This was confirmed by testing HLA-B7-transfected cells and allowed us to use the hybrid transfectants for epitope mapping. All the reactive cells shared the same segment spanning residues 77-81 from HLAB*2705. Hence, this probably constitutes the major epitope for the Ye-3 MoAb. Sequence comparisons between amino acids 78-81 of HLA-B7 and HLA-B*2705 would suggest that residues 77 (S for HLA-B7, D for HLA-B*2705) and/or 80 (N for HLA-B7, T for HLA-B*2705) play a critical role in the antigenic determinant (Table 1). In a previous study (Schwimmbeck, Yu & Oldstone, 1987), a sequence homology between residues 72-77 from HLA-B*2705 and 188-193 from Klebsiellapneumoniae nitrogenase was found. Experimental testing of sera from patients in this study as well as in others (Tsuchiya, Husby & Williams, 1989; Husby et al., 1989) were performed with synthetic peptides 69-84 (Tsuchiya et al., 1989) or 67-84 and 68-83 (Husby et al., 1989) from HLAB*2705. It is therefore possible that residues outside the homologous sequence could be involved in the conformation of the determinant recognized. Interestingly, using large series of overlapping peptides from HLA-B*2705 (residues 65-85) and K. pneumoniae (residues 181-199), it has been demonstrated that two different epitopes are present on HLA-B*2705, one spanning residues 68-75 (epitope I) and another residues 75-82 (epitope II) (Ewing et al., 1989). These data localize epitope I as a major epitope recognized by sera from ankylosing spondylitis patients while murine antisera raised against K. pneumoniae recognized mainly epitope II. This is in agreement with our results obtained with a murine MoAb. Regarding this fact, a
19
Yersinia and HLA-B27 cross-reactivity Table 3. Test of the Ye-3 reactivity on six samples of HLA-B27 peripheral blood lymphocytes (PBL) after twice absorption (45 min at 370C and 15 min at 40C) of the MoAb as 1/50 diluted ascites fluid on the five transfectants (30 x 106 cells each) PBL B27+
1/50 1/100 1/200 1/400 1/800 1/1600 1/3200 1/50 1/100 1/200 1/400 1/800 1/1600 1/3200 Ye-3 absorbed on P815 B27
Ye-3
392 397 398 630 639 649
4 4 4 4 4 4
4 4 4 4 4 4
4 4 3 4 4 4
4 4 2 4 4 4
3 3 2 3 3 3
2 3 1 3 2 1
1 1 1 -
-
-
-
_
_
_
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
I
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2 3 2 2 2 2
1 1 1 1 1 1
-
-
-
-
-
-
1
Ye-3 absorbed on B7-82
Ye-3 absorbed n B-77 392 397 398 630 639 649
-
Ye-3 absorbed on B7-63
Ye-3 absorbed on B7-24 392 397 398 630 639 649
-
4 4 3 4 3 4
4 3 2 4 3 4
2 1 1 3 3
1 2
-
-
-
-
-
-
4 4 4 4 3 4
3 4 4 3 3 3
3 3 4 3 3 3
3 3 3 3 3 3
3 3 3 3 3 3
Microcytotoxicity assay and score as in Table 2.
slightly different recognition of bacterial cross-reactive epitopes in human and murine species could be possible. It should be emphasized that we used here hybrid HLA molecules whose structure and expression are similar to the parental class I antigens but possess discrete parts of the HLAB27 sequence. Thus, it is highly possible that the cross-reactive epitope is present on the native conformation of the HLA-B27 molecule. This is also in agreement with in situ findings on synovial tissues (Husby et al., 1989). Recent reports concern the bacterial counterpart which would be a candidate to trigger the immune phenomenon. The 35-kD bacterial envelope component has been characterized as the OmpA protein of Enterobacteriaceae (Zhang et al., 1989). The high homology of this protein among the various strains of bacteria tested could explain the multiple reactivities observed (Fig. 2). However, there is no correlation between OmpA expression or Ye-3 reactivity and the arthritogenic potential of these strains. Another important finding was the identification by Stieglitz, Fosmire & Lipsky (1989) of a plasmid, pHS-2, common to arthritogenic strains of Shigella and whose sequence carries five consecutive amino acids identical to residues 71-75 of HLA-B*2705 in an open reading frame of 22 amino acids. Regulation of expression of such plasmid sequences in HLAB27 individuals would be an important consideration.
Taken together, these data support the hypothesis of a crossreactivity between HLA-B27 and epitopes expressed by Gramnegative bacteria. Several reports are in agreement to map the HLA-B27 residues involved in a structurally dominant part of the molecule. By using HLA-B27 recombinant molecules, we were able to confirm this fact on integral HLA class I antigens.
ACKNOWLEDGMENTS We are indebted to Dr J. Charreire and to Professor B. Amor for helpful discussions, to N. Bazely (H6pital Cochin) and C. Dehay (H6pital StLouis) for technical assistance and to J. Decaix for help in the preparation of the manuscript. This work was supported in part by the Nora Eccles Trendwell Foundation and the Association de Recherches sur la Polyarthrite.
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