Immunogenetics33: 286-289, 1991

genetics © Springer-Verlag 1991

Two amino acid substitutions at residues 63 and 67 between HLA- B51 and HLA-Bw52 form multiple epitopes recognized by allogeneic T cells

Junji Yamamoto, Masaki Hiraiwa, Hideyuki Hayashi, Manabu Tanabe, Kyoichi Kano, and Masafumi Takiguchi

Department of Immunology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan

Received October 2, 1990; revised version received November 13, 1990

HLA class I antigens are polymorphic glycoprotein ex- pressed on the surface of human somatic cells and are cod- ed by genes in three loci, HI_A-A, -B and -C. These molecules not only function as alloantigens but also pre- sent viral antigens to T cells. The polymorphism of these antigens relates to these functions. More than 70 serological allospecificities of HLA class I antigens are known. HLA-A2 is well studied allospecificity and 10 subtypes of this specificity are known (Ldpez de Castro, 1989). Recent studies of HLA-A2 using site-directed mutagenesis (Hogan et al. 1988, Shimojo et al. 1989) demonstrated that the residues on the c~ helical region of the 0/3/1 and o~ z domains as well as those on the/3 sheet of these domains affect the presentation of influenza virus peptides to T cells. The previous studies (Santos-Aguade et al. 1989) demonstrated that many residues on the 0/ helix and/3 sheet of the 0/1 and o~ 2 domains also affect the allorecognition of T cells. Of the HLA-B antigens, HLA-B27 was well studied and its several residues on the c~ helical region and the 13 sheet of the 0/1 and 0/2 domains were shown to affect allo-recognition of antibody and T cell (L6pez de Casto 1989). However, allodeterminants of other HLA class I antigens remain to be studied.

Our previous studies of HLA-B51 and HLA-Bw52 (Hayashi et al. 1989), HLA-B35 (Ooba et ai. 1989), HLA-B SNA (Sekimata et al. 1990) and HLA-Bw53 (Hayashi et al. 1990) demonstrated that the primary structure of these molecules and HLA-Bw58 are very similar and they might have evolved from the same molecules. HLA-B51 and HLA-Bw52 differ by only two amino acids on the ~ helical region of the c~ 1 domain. HLA-B51 has Ash at residue 63 (Ash 63) and Phe at residue 67 (Phe 67), while HLA-

Address correspondence and offprint requests to: Dr. Masafumi Takiguchi, Department of Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan.

Bw52 has Glu at residue 63 (Glu 63) and Ser at residue 67 (Ser 67). The allospecificities formed by these amino acid substitutions can be discriminated by antibodies, although there are a few monospecific human antisera specific for these antigens. On the other hand, our recent studies (Matsumoto et al. 1990) showed that human T cell clones can definitively discriminate these antigens. Therefore, these amino acid substitutions are thought to affect allorecognition of human T cells.

To investigate which residue affects recognition of HLA-B51 and HLA-Bw52 specific T cells, we generated chimeric genes between HLA-B51 and HLA-Bw52 by ex- change of 1.8 kilobases Bgl II fragments containing 3' of the o/1 domain, the a 2 domain, the 0/3 domain and the transmembrane region between HLA-B51 and HLA-Bw52 genes. The HLA-B51/52 chimeric molecule has Asn 63 and Ser 67, while HLA-B52/51 has Glu 63 and Phe 67. These chimeric genes as well as HLA-B5 (HLA-B51 and HLA-Bw52) genes were transfected into HLA-A, B null Hmy2CIR cells. Clones expressing high levels of these gene products were selected by flow cytometry using W6/32 anti-HLA class I monomorphic mAb. The surface expression of these antigens were confirmed by flow cytometry using TIJ48 HLA-Bw4 specific mAb and com- plement dependent cytotoxicity with human HLA-B5 specific anti-sera (data not shown).

HLA-B51 specific CTL clones were generated from an individual with HLA-A11/A24, Bw52/Bw52, and Cw7/Cw-. Of 19 HLA-B51 specific CTL clones tested, one clone recognized both HLA-B51/52 and HLA-B52/51 chimeric molecules, four clones HLA-B51/52 but not HLA-B52/51 molecules, three clones HLA-B52/51 but not HLA-B51/52 molecules and eleven clones neither HLA-B51/52 nor HLA-B52/51 molecules (Fig. 1 and Table 1). Thus, they revealed four different recognition patterns. Furthermore, five HLA-B51 specific CTL clones from an individual with HLA-A24/A-, B40/B45,

J. Yamamoto et al. : Multiple epitopes on HLA-B5 molecule 287

a b

40 40 40

20 20 20 og

0 2:1 1:1 0.5:1 2:1 1"1 0.5:1 ----- u. o c d o u.i l..iJ a_

60 60 co

40

20 20

2:1 1:1 0.5:1 2:1 1:1 0.5:1

EFFECTOR: TARGET RATIO

Fig. 1 a-d. Recognition of chimeric molecules between HLA-B51 and HLA-Bw52 by HLA-B51 specific CTL clones from an individual with HLA-Bw52. Hmy2CIR cells expressing HLA-B51 ([2), HLA-Bw52 ( 0 ) , HLA-B51/52 ( I ) and HLA-B52/51 ( 0 ) as well as untransfected Hmy2CIR cells were used as target cells. All CTL clones showed less than 10 % specific lysis for untransfected Hmy2CIR cells. Nineteen CTL clones revealed four different recognition patterns, a, 2G4 clone; b, 1DC clone (other three clones showed the same pattern); e, 2B8 clone (other two clones showed the same pattern); d, 2H1 clone (other ten clones showed the same pattern).

C

60

40

20

a b

6o

20

2:1 1:1 0.5:1

d

6O

40

20

2:1 1:1 0.5:1

2:1 1:1 0.5:1

2:1 1:1 0.5:1

EFFECTOR: TARGET RATIO

Fig. 2 a-d . Recognition of chimeric molecules between HLA-B51 and HLA-Bw52 by HLA-Bw52 specific CTL clones from an individual without HLA-B5 CREG. Hmy2CIR cells expressing HLA-B51 ([~), HLA-Bw52 (O), HLA-B51/52 ( I ) , and HLA-B52/51 (O) as well as untransfected Hmy2CIR cells were used as target ceils. All CTL clones showed less than 10 % specific lysis for untransfected Hmy2CIR cells. Five CTL clones revealed four different recognition patterns, a, 10C6 clone; b, 10C38 clone; c, 10C25 clone (other two clones, 10C16 and 10C41, showed the same pattern); d, 10C17 clone.

Table 1. Recognition of the chimeric molecules between HLA-B51 and Bw52 by HLA-B51 specific CTL clones.

H L A - B locus of Target molecules

the effector cells No. of the clones HLA-B51 HLA-B51/52 HLA-B52/51 HLA-Bw52

H L A - B w 5 2 / B w 5 2 1 + * + +

4 + + -

3 + - +

11 + - - H L A - B 4 0 / B 4 5 1 + + +

1 + + -

3 + - -

* Specific lysis of the CTL clones was examined at effector-to-target (E:T) ratio of 2:1, 1 : 1, and 0.5: 1. + , more than 25% specific lysis; - , less than 10% specific lysis at 2:1 E:T ratio.

and Cw3/Cw- revealed three different recognition pat- terns (Table 1). Taken together, these results demonstrated that both Asn 63 and Phe 67 are essential for allorecogni- tion of some HLA-B51 specific CTL clones, but either one is not necessary for allorecognition of other CTL clones. These findings indicate that multiple HLA-B51 specific epitopes are formed by Asn 63 and Phe 67.

Six HLA-Bw52 specific CTL clones were also generated from an individual with HLA-A24/-, B40/B45, and Cw3/Cw- to investigate recognition of the chimeric molecules by HLA-Bw52 specific CTL clones. As shown

in Figure 2, six HLA-Bw52 specific CTL clones revealed four different recognition patterns for the chimeric molecules; The 10C38 clone recognized HLA-B51/52 but not HLA-B52/51 molecules, three other clones (10C25, 10C16, and 10C41) recognized HLA-B52/51 but not HLA-B51/52 molecules and the 10C17 clone neither HLA-B51/52 nor HLA-B52/51 molecules. The 10C6 clone recognized not only HLA-B51/52 but also HLA- B52/51 molecules. Furthermore, recognition of the chimeric molecules by HLA-Bw52 specific CTL clones from an individual with HLA-A2/A2, HLA-B5 l/B51, and

288 J. Yamamoto et al.: Multiple epitopes on HLA-B5 molecule

Table 2. Recognition of the chimeric molecules between HLA-B51 and HLA-Bw52 by HLA-Bw52 specific CTL clones.

HLA-B locus of Target molecules

the effector cells No. of the clones HLA-B51 HLA-B51/52 HLA-B52/51 HLA-Bw52

HLA-B51/B 51

HLA-B40/B45

2 - * + + + 2 - + - + 1 - - - + 1 - + + + 1 - + - + 3 - - + + 1 - - - +

* Specific lysis of the CTL clones was examined at effector-to-target (E:T) ratio of 2:1, 1 : 1, and 0.5 : 1. + , more than 25 % specific lysis; - , less than 10 % specific lysis at 2:1 E:T ratio.

Cw-/Cw- was investigated. These clones also revealed three different patterns of recognition (Table 2). These results demonstrated that Glu 63 and Ser 67 form multiple epitopes of HLA-Bw52 recognized by T cells. Thus, the present study demonstrated that a single substitution not only at residue 63 but also at residue 67 affects the recogni- tion of some HLA-B51 or HLA-Bw52 specific CTL clones. These indicate that both residues are involved in formation of HLA-B51 or HLA-Bw52 allodeterminants which T ceils recognize.

Various studies (Buerstedde et al. 1989; Pierres et al. 1989) suggested that alloreactive T ceils recognize MHC- bound peptides from cytoplasmic proteins. The X-ray crystallographic studies of HLA-A2 molecule (Bjorkman et al. 1987) has provided the evidence that HLA class I molecules bind peptide fragments from cytoplasmic pro- teins within the antigen binding groove. Furthermore, re- cent studies (Heath et al. 1989) directly demonstrated that alloreactive T cells recognize a peptide consisting of 10 to 15 amino acids. Thus, as alloreactive T cells recognize MHC molecules binding peptides, allorecognition of T cells is influenced by the change of TCR or peptide bin- ding to MHC molecules by amino acid substitutions on MHC molecules. As the residues 63 and 67 are thought to be peptide binding sites by the X-ray crystallographic studies of HLA-A2 molecules, the substitutions at these residues may affect the binding of peptides to HLA-B5 molecules.

Previous studies (Santos-Aguade et al. 1989; Hogan et al. t989) showed that there is a large repertoire of allogeneic T cells specific for HLA-A2. If HLA-peptide complex forms the allodeterminants which T cells recognize and various peptides but not a single peptide are able to bind one HLA molecule, the large repertoire of allogeneic T cells might be induced by stimulation of various peptide-HLA complexes. The present study show- ed that HLA-B51 or HLA-Bw52 specific CTL clones reveal four different recognition patterns for the chimeric molecules between HLA-B51 and HLA-Bw52. These suggest that the residues 63 and 67 may play an important

role in binding of peptides to HLA-B5 molecules and that each HLA-B51 and HLA-Bw52 molecules may be able to bind at least four different peptides, although one can not completely rule out the possibility that T cell receptor recognizes four different epitopes formed by one allopep- tide and HLA-B51 or Bw52 molecule and that these epitopes are affected by the substitution at residues 63 and 67. The present study also showed that HLA-B51 or HLA- Bw52 specific CTL clones not only from an individual without HLA-B5 but also from that with HLA-B5 reveal- ed three or four different specificities. This indicates that a large allogeneic T cell repertoire is generated even from the individual with HLA-B5.

In the present study, we demonstrate that multiple epitopes of HLA-B51 or HLA-Bw52 are formed by two amino acid substitutions at residues 63 and 67, and suggest that HLA-B51 or HLA-Bw52 specific T cells recognize at least four different allopeptides. Further studies of HLA-B5 binding allopeptides might provide the evidence supporting the concepts that multiple allodeterminants on one HLA molecule are formed by the binding of various allopeptides to the HLA molecule.

Acknowledgments. We thank Dr. C. Miiller for gift of the TU48 monoclonal antibody. Ms. Sachiko Karaki and Ms. Ai Kariyone for technical assistance and Ms. Mashu Noda for secretarial assistance. This work was supported by a grant from the Aid for Scientific Research, of the Ministry of Education, Culture and Science, Japan and also by a grant from the Ryoichi Naito Foundation of Medical Research.

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