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.
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 ...
Group I Burkitt lymphoma (BL) lines retaining the original BL tumor cell phenotype are unable to present endogenously expressed antigens to HLA class I-restricted cytotoxic T cells (CTL) but can be recognized if the relevant HLA class I/peptide epitope complex is reconstituted at the cell surface by exogenous addition of synthetic target peptide. Endogenous antigen-processing function is restored in BL lines that have undergone Epstein-Barr virus (EBV)-induced drift in culture to the group III phenotype typically displayed by EBV-transformed lymphoblastoid cell lines (LCL) of normal B cell origin. We compared group I versus group III cells for their expression of proteasome components, transporter proteins and HLA-class I antigens, all of which are thought to be involved in the endogenous antigen processing pathway. By Western blot analysis, there were not consistent differences in the low molecular mass protein subunits of proteasomes (lmp)-2, lmp-7 and delta, although the mb-1 proteasome subunit was regularly present at higher levels in group I BL lines relative to group III lines or LCL. By contrast there were marked differences in the expression of peptide transporter-associated proteins (Tap), with down-regulation of Tap-1 and Tap-2 in 8/8 and 7/8 group I BL lines, respectively. Surface levels of HLA class I antigens were also consistently lower in group I cells; this was not associated with an intracellular accumulation of free HLA heavy chains, such as is seen in the Tap-deficient T2 processing-mutant line, but instead reflected a reduced rate of HLA class I synthesis in group I cells. Analysis of EBV gene transfectants of the B lymphoma lines BJAB and BL41 showed that the virus-encoded latent membrane protein-1 (LMP1), which is one of several EBV antigens expressed in group III but not in group I cells, was uniquely able to up-regulate expression both of the Tap proteins and HLA class I. Furthermore, this was accompanied by a restoration of antigen-processing function as measured by the ability of these cells to present an endogenously expressed viral antigen to CTL. These effects of LMP1 were similar to those induced in the same cell lines by interferon-gamma treatment. The results implicate both Tap and HLA class I expression as factors limiting the antigen-processing function of BL cells, and suggest that the accessibility of other EBV-associated malignancies to CTL surveillance may be critically dependent upon their LMP1 status.
SummaryThe importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of EpsteinBarr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-[3 clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8 + individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8 + EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TC1L-[3 sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-[3 repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed longterm preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TC1L-[3 clonotypes and antigen-specific TCR-VJ3 family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TC1L-[3 expansions were present at the clonotype level, but not at the TC1L-V[3 family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TC1L stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.
The identification of MHC-restricted and tumour-specific cytotoxic T lymphocytes (CTLs) provides strong evidence in support of T cell-mediated immune surveillance against human tumour cells. These CTLs recognize short peptides derived from tumour-associated antigens in conjunction with class I molecules expressed on tumour cells. In contrast to these observations there are now numerous examples to suggest that a number of tumours escape this CTL-mediated control either by down-regulating accessory molecules or by blocking the intracellular processing of tumour-specific antigens. Recently a number of tumour cell lines have been identified which display a transcriptional deficiency of transporters associated with antigen processing (also referred to as TAP). The Epstein-Barr virus (EBV)-associated tumour, Burkitt's lymphoma (BL), is a classic example in this category. In the present study we have restored class I-restricted antigen processing in a BL cell line by transfecting a minigene expression vector encoding a CTL epitope derived from EBV linked to an endoplasmic reticulum translocation signal sequence. These minigene transfected BL cells were not only susceptible to lysis by virus-specific CTL but were also capable of efficiently activating an antigen-specific CTL response. Interestingly, the immunogenicity of these BL cells was not affected by the significantly down-regulated expression of adhesion molecules such as LFA1 alpha, LFA1 beta and LFA3. These findings suggest that resistance of tumour cells to CTL-mediated immune control can be reversed if the relevant peptide epitopes are appropriately presented on the cell surface.
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