Changing patterns of dominant TCR usage with maturation of an EBV-specific cytotoxic T cell response.
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Infection with EBV provides a unique opportunity to follow the human CD8(+) T cell response to a persistent, genetically stable agent from the primary phase, as seen in infectious mononucleosis (IM) patients, into long-term memory. This study focuses on the response to an immunodominant HLA-A2.01-restricted epitope, GLCTLVAML, from the EBV-lytic cycle Ag BMLF1. TCR analysis of the highly amplified primary response to this epitope revealed markedly oligoclonal receptor usage among in vitro-derived clones, with similar clonotypes dominant in all three IM patients studied. Direct staining of IM T cell preparations with the A2.01/GLCTLVAML tetramer linked this oligoclonal epitope-specific response with appropriate Vbeta subset expansions in the patients' blood. These patients were studied again >2 years later, at which time TCR analysis of in vitro-reactivated clones suggested that rare clonotypes within the primary response had now come to dominate memory. Five additional A2. 01-positive IM patients were studied prospectively for Vbeta subset representation within primary and memory epitope-specific populations as identified by tetramer staining. In each case, the primary response contained large Vbeta2, Vbeta16, or Vbeta22 components, and in three of five cases the originally dominant Vbeta was represented very poorly, if at all, in memory. We conclude 1) that an EBV epitope-specific primary response large enough to account for up to 10% CD8(+) T cells in IM blood may nevertheless be dominated by just a few highly expanded clonotypes, and 2) that with persistent viral challenge such dominant T cell clonotypes may be lost and replaced by others in memory. (+info)
Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype.
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We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4(+) and CD8(+) T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8(+). The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8(+) T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool. (+info)
EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency.
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Infection of humans with Epstein-Barr virus (EBV) may cause infectious mononucleosis (IM). Analysis of single EBV-infected cells from tonsils of IM patients for rearranged immunoglobulin genes revealed two strategies of EBV for rapid and massive spread in the B cell compartment: the direct infection of many naive as well as memory and/or germinal center B cells and the expansion of the latter cells to large clones. In IM, the generation of virus-harboring memory B cells from naive B cells passing through a germinal center reaction likely plays no role. Members of clones can show distinct morphologies and likely also EBV gene expression patterns, and this ability implies a mechanism by which EBV-harboring cells can evade immune surveillance and establish a pool of persisting EBV-infected B cells. (+info)
CD8(+) T-cell selection, function, and death in the primary immune response in vivo.
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The primary immune response to Epstein Barr virus (EBV) is characterized by striking proliferation of EBV-specific CD8(+) T cells. In this study we have investigated the clonal composition and functional properties of the cells mediating this primary response and have analyzed the mechanisms that control the downregulation of the primary response and the selection of memory cells. We show that massively expanded T-cell clones often dominate the primary antigen-specific T-cell response. Despite the enormous extent of expansion, the virus-specific T cells express high levels of intracellular perforin and are potently cytotoxic. They are, however, functionally heterogeneous in their ability to secrete proinflammatory cytokines, with subpopulations of the antigen-specific T cells being hyporesponsive. The primary response is closely regulated, and the majority of cells are programmed to die via a cytokine-rescuable pathway, leaving only small populations of memory T cells surviving. Comparison of the clonal composition of primary and memory responses in vivo shows that the clones that dominate the primary response are relatively heavily culled during the downregulation of the primary response and the establishment of T-cell memory. (+info)
Analysis of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene and promoter in Hodgkin's disease isolates: selection against EBV variants with mutations in the LMP-1 promoter ATF-1/CREB-1 binding site.
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AIMS: To study the distribution of Epstein-Barr virus (EBV) variants containing mutations in the latent membrane protein 1 (LMP-1) oncogene and promoter in EBV associated Hodgkin's disease and infectious mononucleosis compared with previous findings in asymptomatic EBV carriers. METHODS: Sequence analysis of the EBV LMP-1 promoter and gene in isolates from Danish patients with Hodgkin's disease (n = 61) and infectious mononucleosis (n = 10). RESULTS: Viruses (previously designated group D) that contain two mutations in the activating transcription factor/cAMP response element (ATF/CRE) in the LMP-1 promoter, which are known to decrease promoter activity greatly, were significantly less frequent in Hodgkin's disease than in both infectious mononucleosis (p = 0.0081) and asymptomatic EBV carriers (p = 0.0084). In some cases, the LMP-1 gene contained mutations in a recently identified cytotoxic T cell (CTL) epitope. Most viral isolates contained mutations shown to increase nuclear factor kappa B (NF-kappa B) activation and had one of two newly identified C-terminal activation regions 3 (CTAR-3) deleted. The exon 1 Xho-I restriction site in the LMP-1 gene could be lost through a range of different mutations. CONCLUSIONS: These findings indicate selection pressure against EBV strains with weak LMP-1 promoter activity in Hodgkin's disease and thus provide further strong circumstantial evidence for the pathogenic role of EBV (and LMP-1) in this disease. Mutation of the CTL epitope suggests immune selection of EBV strains. Many EBV isolates contain functionally important mutations in the LMP-1 gene. Loss of the Xho-I restriction site should not be used as a marker of specific LMP-1 variants. (+info)
A 17-year-old boy with juvenile rheumatoid arthritis presented with jaundice, confusion, hemolytic anemia, thrombocytopenia, and acute renal failure secondary to titer-confirmed acute Epstein-Barr virus (EBV). Renal biopsy specimen revealed interstitial nephritis with an inflammatory infiltrate composed of cytotoxic/suppressor T cells, and interstitial mononuclear cell nuclei expressed EBV encoded RNA-1 (EBER-1) mRNA. Methylprednisolone treatment resulted in rapid improvement. (+info)
CD11b expression identifies CD8+CD28+ T lymphocytes with phenotype and function of both naive/memory and effector cells.
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A previously unreported CD8(+)CD28(+)CD11b(+) T cell subset occurs in healthy individuals and expands in patients suffering from primary viral infections. In functional terms, these cells share the features of naive/memory CD8(+)CD28(+)CD11b(-) and terminally differentiated effector CD8(+)CD28(-)CD11b(+) subpopulations. Like CD28(-) cells, CD28(+)CD11b(+) lymphocytes have the ability to produce IFN-gamma, to express perforin granules in vivo, and to exert a potent cytolytic activity. Moreover, these cells can respond to chemotactic stimuli and can efficiently cross the endothelial barrier. In contrast, like their CD11b(-) counterpart, they still produce IL-2 and retain the ability to proliferate following mitogenic stimuli. The same CD28(+)CD11b(+) subpopulation detected in vivo could be generated by culturing naive CD28(+)CD11b(-) cells in the presence of mitogenic stimuli following the acquisition of a CD45RO(+) memory phenotype. Considering both phenotypic and functional properties, we argue that this subset may therefore constitute an intermediate phenotype in the process of CD8(+) T cell differentiation and that the CD11b marker expression can distinguish between memory- and effector-type T cells in the human CD8(+)CD28(+) T cell subset. (+info)
The flow cytometric analysis of telomere length in antigen-specific CD8+ T cells during acute Epstein-Barr virus infection.
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Acute infectious mononucleosis (AIM) induced by Epstein-Barr virus (EBV) infection is characterized by extensive expansion of antigen-specific CD8+ T cells. One potential consequence of this considerable proliferative activity is telomere shortening, which predisposes the EBV-specific cells to replicative senescence. To investigate this, a method was developed that enables the simultaneous identification of EBV specificity of the CD8+ T cells, using major histocompatibility complex (MHC) class I/peptide complexes, together with telomere length, which is determined by fluorescence in situ hybridization. Despite the considerable expansion, CD8+ EBV-specific T cells in patients with AIM maintain their telomere length relative to CD8+ T cells in normal individuals and relative to CD4+ T cells within the patients themselves and this is associated with the induction of the enzyme telomerase. In 4 patients who were studied up to 12 months after resolution of AIM, telomere lengths of EBV-specific CD8+ T cells were unchanged in 3 but shortened in one individual, who was studied only 5 months after initial onset of infection. Substantial telomere shortening in EBV-specific CD8+ T cells was observed in 3 patients who were studied between 15 months and 14 years after recovery from AIM. Thus, although telomerase activation may preserve the replicative potential of EBV-specific cells in AIM and after initial stages of disease resolution, the capacity of these cells to up-regulate this enzyme after restimulation by the persisting virus may dictate the extent of telomere maintenance in the memory CD8+ T-cell pool over time. (+info)