SummaryTo examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-cx and -/3 chains from HLA-B8-restricted, CD8 + cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by dones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of c~ and/3 chain rearrangements suggests that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved/3 chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

Summal~There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type I and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA I as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the downregulation of latent antigens can be reversed.

SummaryTwo unusual characteristics ofthe memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate selftolerance and T cell receptor (TCR) plasticity in humans . First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D . L. Doolan, A. Suhrbier, D . J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp . Med. 180:2335-2340 . Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B*4402 on uninfected cells (Burrows, S. R., R. Khanna, J . M. Burrows, and D . J. Moss. 1994. J. Exp . Med . 179 :1155-1161 . No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self-reactive T cells in humans . A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions . In addition, a significant public TCR component was identified in which several distinct cx/R rearrangements are shared by CTL clones from a number of unrelated HLA B8+ , B*4402 + donors . The striking dominance ofpublic TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination . Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues . Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NHZ-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402 . Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination ofTCR-binding residues .T cells that express the aR TCR heterodimer recognize immunogenic peptides presented by self-MHC molecules. TCR diversity arises during T cell development in the thymus by rearrangement of variable (TCRAV and TCRBV), diversity (TCRBD), and joining (TCRAJ and TCRBJ) gene segments, as well as N region diversity at the junctional regions (1, 2). The hypervariable complementarity determining region 3 (CDR3)' spans the junctional regions and interacts directly with peptide epitopes (3) . The 'Abbreviations used in this paper: CDR3, complementarity determining region 3; CTLp, CTL precursor; LCL, lymphoblastoid cell line ; LDA, limiting dilution analysis .S. R. Burrows and S. L. Silins contributed equally to this work . mature T cell repertoire expresses only a small proportion ...

Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis and is associated with Burkitt's lymphoma and nasopharyngeal carcinoma. The virus is harboured for life in all previously infected individuals and is apparently controlled by a population of EBV-specific memory T lymphocytes, specifically activated to recognize the functionally defined lymphocyte-detected membrane antigen. Two types (A and B) of EBV have been identified that show DNA sequence divergence within the BamH1 WYH region of the genome encoding the transformation-associated antigen, Epstein-Barr nuclear antigen 2 (EBNA 2) (ref. 4). To define the function of EBNA 2 in T-cell recognition, we have compared the ability of EBV-specific cytotoxic T-cell clones to distinguish between autologous B lymphocytes transformed by A- or B-type virus. We have now isolated both CD4 and CD8 cytotoxic T-cell clones that recognize autologous A-type but not B-type transformed lymphoblastoid cell lines, thus providing the first evidence that EBV-specific T-cell recognition can be mediated by EBNA 2. As this antigen is not expressed in Burkitt's lymphoma, this finding explains the failure of EBV-specific T-cell surveillance to eliminate the tumour.

All individuals previously infected with Epstein-Barr virus (EBV) harbor life-long a population of virus-infected B cells whose number appears to be controlled by EBVspecific, HLA-restricted (class I and class II) CTLs (CD4 and CD8) that recognize a functionally defined lymphocyte-determined membrane antigen, LYDMA (1, 2). There are two recognized types of EBV (A and B) that show sequence divergence within the reading frames encoding EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 (3, 4) and differences in the molecular weight of these proteins (4, 5).Our general approach to identifying CTL epitopes has been to isolate EBVspecific T cell clones that recognize autologous A-type transformant but fail to lyse the corresponding B-type transformant (6). The present report defines the first target epitope recognized by EBVspecific CTLs. Our approach to identifying this epitope has been to screen selected peptide sequences from the EBNA proteins using specific CTLs and the autologous B-type transformant.Volume 171 January 1990 [345][346][347][348][349] Materials and Methods Brief Definitive ReportCellLines. EBV transformed cell lines (LCLs) were established either spontaneously (i.e., by transformation with endogenous virus) or by the addition ofexogenous virus (7) . A spontaneous LCL from the EBV seropositive donor LC was established using cyclosporin A (8) . The type of endogenous virus (A or B) in the spontaneous cell lines was determined using Southern analysis (6) and was used to define donor LC as an A-type donor.Cell lines were established using exogenous A-or B-type virus by infecting B lymphocytes from an EBV seropositive donor Lc, with virus recovered from two A-type cell lines, IARC-BL74 (8) and IARC-BL36, (4) and two B-type cell lines, Ag876 (3) and L4 (4) . Southern and Western analyses were used to confirm the presence of A-or B-type virus in the respective LC transformants (designated LC/BL74, LC/BL36, LC/Ag876, and LC/L4) as previously described (6) .CTL Activation and 5/ Cr-release Assay. Lymphocytes from donor LC (HLA Al, B8, B18) were cocultivated with the irradiated autologous A-type LCL, LC/BL74 for 3 d and the activated T cells were cloned in agar and expanded in the presence of IL-2 and specific stimulating cells (2) . T cell clones were screened in the standard 5 'Cr release assay (E/T ratio of 10 :1) against a panel of cell lines including autologous and allogeneic A-and B-type LCLs .Peptide Selection. To identify the CTL epitope expressed on the A-type transformants but not on the autologous B-type transformants, a series of peptides (20-25 amino acids) deduced from the known sequences of the A-type EBNAs 2, 3, 4, 5, and 6 proteins were synthesized (9) and screened on the autologous B-type transformant LC/Ag876 . Peptides selected were