A mutation in X-linked inhibitor of apoptosis (G466X) leads to memory inflation of Epstein-Barr virus-specific T cells.

Mutations in the X-linked inhibitor of apoptosis (XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus (EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP (G466X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4(+) CD8(+) population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon (IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAP(G466X) mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease.

Co-infections, inflammation and oncogenesis: future directions for EBV research.

Epstein-Barr virus (EBV) is aetiologically linked to a wide range of human tumours. Some arise as accidents of the virus' lifestyle in its natural niche, the B lymphoid system; these include B-lymphoproliferative disease of the immunocompromised, Hodgkin Lymphoma, Burkitt Lymphoma and particular forms of diffuse large B cell lymphoma. Interestingly, HIV infection increases the incidence of each of these B cell malignancies, though by different degrees and for different reasons. Other EBV-associated tumours arise through rare viral entry into unnatural target tissues; these include all cases of nasal T/NK cell lymphoma and of undifferentiated nasopharyngeal carcinoma plus a small but significant subset of gastric carcinomas, a tumour type more generally associated with chronic Helicobacter pylori infection. Understanding EBV's involvement in the pathogenesis of these different malignancies is an important long-term goal. This article focuses on two overlapping, but relatively neglected, areas of research that could contribute to that goal. The first addresses the mechanisms whereby coincident infections with other pathogens increase the risk of EBV-positive malignancies, and takes as its paradigm the actions of holoendemic malaria and HIV infections as co-factors in Burkitt lymphomagenesis. The second widens the argument to include both infectious and non-infectious sources of chronic inflammation in the pathogenesis of EBV-positive tumours such as T/NK cell lymphoma, nasopharyngeal carcinoma and gastric carcinoma.

Increase in circulating Foxp3+CD4+CD25(high) regulatory T cells in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated disease with high prevalence in Southern Chinese. Using multiparametric flow cytometry, we identified significant expansions of circulating naïve and memory CD4+CD25(high) T cells in 56 NPC patients compared with healthy age- and sex-matched controls. These were regulatory T cells (Treg), as they overexpressed Foxp3 and GITR, and demonstrated enhanced suppressive activities against autologous CD4+CD25- T-cell proliferation in functional studies on five patients. Abundant intraepithelial infiltrations of Treg with very high levels of Foxp3 expression and absence of CCR7 expression were also detected in five primary tumours. Our current study is the first to demonstrate an expansion of functional Treg in the circulation of NPC patients and the presence of infiltrating Treg in the tumour microenvironment. As Treg may play an important role in suppressing antitumour immunity, our findings provide critical insights for clinical management of NPC.

IL-7R alpha versus CCR7 and CD45 as markers of virus-specific CD8+ T cell differentiation: contrasting pictures in blood and tonsillar lymphoid tissue.

In humans, circulating CD8(+) memory T cells to a nonpersistent virus (influenza) lie within CCR7(+)CD45RA(-) central memory, whereas memory to Epstein-Barr virus (EBV) latent, EBV lytic, and cytomegalovirus (CMV) antigens are progressively larger in size and are more biased toward CCR7(-)CD45RA(-) effector memory and CCR7(-)CD45RA(+) terminally differentiated compartments. We found that these populations are also distinguished by progressively lower expression of the interleukin-7 receptor (IL-7R alpha) and by lower IL-7 responsiveness; indeed, percentage IL-7R alpha -positive values showed a tight inverse correlation with population size. However, these relationships among size, differentiation phenotype, and IL-7R alpha status in blood did not hold in tonsillar tissue. In tonsil tissue, although EBV reactivities outnumbered their CMV and influenza counterparts, the distinct CCR7/CD45 isoform signatures of the different virus-specific populations were retained. Moreover, all detectable reactivities showed high levels of IL-7R alpha expression. As a discriminator between different virus-specific populations, IL-7R alpha therefore appears to be more susceptible to tissue location than the classical CCR7/CD45 markers.

CD4+ T-cell responses to Epstein-Barr virus nuclear antigen EBNA1 in Chinese populations are highly focused on novel C-terminal domain-derived epitopes.

Epstein-Barr virus nuclear antigen EBNA1, the one viral protein uniformly expressed in nasopharyngeal carcinoma (NPC), represents a prime target for T-cell-based immunotherapy. However, little is known about the EBNA1 epitopes, particularly CD4 epitopes, presented by HLA alleles in Chinese people, the group at highest risk for NPC. We analyzed the CD4+ T-cell responses to EBNA1 in 78 healthy Chinese donors and found marked focusing on a small number of epitopes in the EBNA1 C-terminal region, including a DP5-restricted epitope that was recognized by almost half of the donors tested and elicited responses able to recognize EBNA1-expressing, DP5-positive target cells.

Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells.

Epstein-Barr virus (EBV), an orally transmitted herpesvirus, efficiently targets B lymphocytes through binding of the viral envelope glycoprotein gp350 to the complement receptor CD21. How the virus accesses epithelial cells is less well understood, because such cells are largely resistant to infection with cell-free virus in vitro. Here, we show that, after binding to primary B cells, most Epstein-Barr virions are not internalized but remain on the B cell surface and from there can transfer efficiently to CD21-negative epithelial cells, increasing epithelial infection by 10(3)- to 10(4)-fold compared with cell-free virus. Transfer infection is associated with the formation of B cell-epithelial conjugates with gp350/CD21 complexes focused at the intercellular synapse; transfer involves the gp85 and gp110 viral glycoproteins but is independent of gp42, the HLA class II ligand that is essential for B cell entry. Therefore, through efficient binding to the B cell surface, EBV has developed a means of simultaneously accessing both lymphoid and epithelial compartments; in particular, infection of pharyngeal epithelium by orally transmitted virus becomes independent of initial virus replication in the B cell system.

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines.

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.

Dual stimulation of Epstein-Barr Virus (EBV)-specific CD4+- and CD8+-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma.

Virus-associated malignancies are potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells. Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, a high-incidence tumor in southern China, expresses a limited set of EBV proteins, including the nuclear antigen EBNA1, an abundant source of HLA class II-restricted CD4(+) T-cell epitopes, and the latent membrane protein LMP2, a source of subdominant CD8(+) T-cell epitopes presented by HLA class I alleles common in the Chinese population. We used appropriately modified gene sequences from a Chinese EBV strain to generate a modified vaccinia virus Ankara recombinant, MVA-EL, expressing the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2. The endogenously expressed fusion protein EL is efficiently processed via the HLA class I pathway, and MVA-EL-infected dendritic cells selectively reactivate LMP2-specific CD8(+) memory T-cell responses from immune donors in vitro. Surprisingly, endogenously expressed EL also directly accesses the HLA class II presentation pathway and, unlike endogenously expressed EBNA1 itself, efficiently reactivates CD4(+) memory T-cell responses in vitro. This unscheduled access to the HLA class II pathway is coincident with EL-mediated redirection of the EBNA1 domain from its native nuclear location to dense cytoplasmic patches. Given its immunogenicity to both CD4(+) and CD8(+) T cells, MVA-EL has potential as a therapeutic vaccine in the context of nasopharyngeal carcinoma.

HLA-A11-restricted epitope polymorphism among Epstein-Barr virus strains in the highly HLA-A11-positive Chinese population: incidence and immunogenicity of variant epitope sequences.

An individual's CD8(+)-cytotoxic-T-lymphocyte (CTL) response to Epstein-Barr virus (EBV) latent cycle antigens focuses on a small number of immunodominant epitopes often presented by just one of the available HLA class I alleles; for example, HLA-A11-positive Caucasians frequently respond to two immunodominant HLA A11 epitopes, IVTDFSVIK (IVT) and AVFDRKSDAK (AVF), within the nuclear antigen EBNA3B. Here, we reexamine the spectrum of EBV strains present in the highly HLA-A11-positive Chinese population for sequence changes in these epitopes relative to the Caucasian type 1 prototype strain B95.8. The IVT epitope was altered in 61 of 64 Chinese type 1 viruses, with four different sequence variants being observed, and the AVF epitope was altered in 46 cases with six different sequence variants; by contrast, all 10 Chinese type 2 viruses retained the prototype 2 epitope sequences. All but one of the type 1 epitope variants were poorly recognized by IVT- or AVF-specific CTLs in pulse-chase assays of peptide-mediated target cell lysis. More importantly, we screened HLA-A11-positive Chinese donors carrying viruses with known epitope mutations for evidence of epitope-specific CTL memory by enzyme-linked immunospot assays: none of the type 1 variants tested, nor the type 2 prototype, appeared to be immunogenic in vivo. The data remain consistent with the possibility that, during virus-host coevolution, pressure from the host CTL-mediated immune response has given A11 epitope-loss viruses a selective advantage.

Latent gene sequencing reveals familial relationships among Chinese Epstein-Barr virus strains and evidence for positive selection of A11 epitope changes.

Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed <2% nucleotide divergence from the B95.8 prototype while type 2 sequences remained even closer to the contemporary African prototype Ag876. However, type 1 isolates could clearly be divided into families based on linked patterns of sequence divergence from B95.8 across all four EBNA loci. Different patterns of IVT and AVF variation were associated with the different type 1 families, and there was additional epitope diversity within families. When the EBNA3 gene sequences of type 1 Chinese strains were subject to computer-based analysis, particular codons within the A11-epitope-coding region were among the few identified as being under positive or diversifying selection pressure. From these results, and the observation that mutant epitopes are consistently nonimmunogenic in vivo, we conclude that the immune selection hypothesis remains viable and worthy of further investigation.

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells.

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function.

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.

Epstein-Barr virus-specific cytotoxic T lymphocyte responses in the blood and tumor site of Hodgkin's disease patients: implications for a T-cell-based therapy.

Approximately 40% of Hodgkin's disease (HD) cases carry EBV in the malignant Hodgkin-Reed Sternberg (H-RS) cells, with expression of viral latent membrane proteins (LMPs) 1 and 2. These viral proteins are targets for CTLs in healthy EBV carriers, and their expression in EBV-associated HD raises the possibility of targeting them for a CTL-based immunotherapy. Here we characterize the CTL response to EBV latent antigens in both the blood and tumor-infiltrating lymphocytes of HD patients using two approaches: (a) in vitro reactivation of CTLs by stimulation with the autologous EBV-transformed lymphoblastoid cell line; and (b) an enzyme-linked immunospot assay to quantify frequencies of CTLs specific for known LMP1/2 epitopes. We detected EBV-specific CTLs in blood and biopsy samples from both EBV-negative and EBV-positive HD patients. However, as in healthy EBV carriers, LMP-specific CTL precursors occurred only at low frequency in the blood of HD patients, and with the exception of one EBV-negative HD case, were undetectable in the tumor. These data give rise to two considerations: (a) they may explain why EBV-positive tumor cells persist in the presence of an existing EBV-specific immune response; and (b) they provide a rationale for selectively boosting/eliciting LMP-specific CTL responses as a therapy for EBV-positive HD.

Memory T cells constitute a subset of the human CD8+CD45RA+ pool with distinct phenotypic and migratory characteristics.

Using HLA class I-viral epitope tetramers to monitor herpes virus-specific CD8(+) T cell responses in humans, we have shown that a significant fraction of responding cells revert from a CD45RO(+) to a CD45RA(+) state after priming. All tetramer-binding CD45RA(+) cells, regardless of epitope specificity, expressed a phenotype LFA-1(high)CCR7(low) that was stable for at least 10 years in infectious mononucleosis patients and indefinitely in asymptomatic carriers. CD8(+)CD45RA(+)LFA-1(high) cells were not present in cord blood but in adults account for up to 50% of CD8(+)CD45RA(+) cells. These CD45RA(+)LFA-1(high) cells have significantly shorter telomeres than CD45RA(+)LFA-1(low) cells, suggesting that the latter represent a naive population, while the former are memory cells. CD45RA(+) memory cells are a stable population of noncycling cells, but on stimulation they are potent producers of IFN-gamma, while naive CD8(+) cells produce only IL-2. The chemokine receptor profile and migratory potential of CD45RA(+) memory cells is very similar to CD45RO(+) cells but different to naive CD8 cells. In accord with this, CD45RA(+) memory cells were significantly underrepresented in lymph nodes, but account for virtually all CD8(+)CD45RA(+) T cells in peripheral tissues of the same individuals.

Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes.

INTRODUCTION: Epstein-Barr virus (EBV) is transmitted orally, replicates in the oropharynx and establishes life-long latency in human B lymphocytes. T-cell responses to latent and lytic/replicative cycle proteins are readily detectable in peripheral blood from healthy EBV-seropositive individuals. EBV has also been detected within synovial tissue, and T-cell responses to EBV lytic proteins have been reported in synovial fluid from a patient with rheumatoid arthritis (RA). This raises the question regarding whether T cells specific for certain viruses might be present at high frequencies within synovial fluid and whether such T cells might be activated or able to secrete cytokines. If so, they might play a 'bystander' role in the pathogenesis of inflammatory joint disease. OBJECTIVES: To quantify and characterize T cells that are specific for epitopes from EBV, cytomegalovirus (CMV) and influenza in peripheral blood and synovial fluid from patients with arthritis. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from patients with inflammatory arthritis (including those with RA, osteoarthritis, psoriatic arthritis and reactive arthritis). Samples from human leucocyte antigen (HLA)-A2-positive donors were stained with fluorescent-labelled tetramers of HLA-A2 complexed with the GLCTLVAML peptide epitope from the EBV lytic cycle protein BMLF1, the GILGFVFTL peptide epitope from the influenza A matrix protein, or the NLVPMVATV epitope from the CMV pp65 protein. Samples from HLA-B8-positive donors were stained with fluorescent-labelled tetramers of HLA-B8 complexed with the RAKFKQLL peptide epitope from the EBV lytic protein BZLF1 or the FLRGRAYGL peptide epitope from the EBV latent protein EBNA3A. All samples were costained with an antibody specific for CD8. CD4+ T cells were not analyzed. Selected samples were costained with antibodies specific for cell-surface glycoproteins, in order to determine the phenotype of the T cells within the joint and the periphery. Functional assays to detect release of IFN- or tumour necrosis factor (TNF)- were also performed on some samples. RESULTS: The first group of 15 patients included 10 patients with RA, one patient with reactive arthritis, one patient with psoriatic arthritis and three patients with osteoarthritis. Of these, 11 were HLA-A2 positive and five were HLA-B8 positive. We used HLA-peptide tetrameric complexes to analyze the frequency of EBV-specific T cells in PBMCs and SFMCs (Figs 1 and 2). Clear enrichment of CD8+ T cells specific for epitopes from the EBV lytic cycle proteins was seen within synovial fluid from almost all donors studied, including patients with psoriatic arthritis and osteoarthritis and those with RA. In donor RhA6, 9.5% of CD8+ SFMCs were specific for the HLA-A2 restricted GLCTLVAML epitope, compared with 0.5% of CD8+ PBMCs. Likewise in a donor with osteoarthritis (NR4), 15.5% of CD8+ SFMCs were specific for the HLA-B8-restricted RAKFKQLL epitope, compared with 0.4% of CD8+ PBMCs. In contrast, we did not find enrichment of T cells specific for the HLA-B8-restricted FLRGRAYGL epitope (from the latent protein EBNA3A) within SFMCs compared with PBMCs in any donors. In selected individuals we performed ELISpot assays to detect IFN- secreted by SFMCs and PBMCs after a short incubation in vitro with peptide epitopes from EBV lytic proteins. These assays confirmed enrichment of T cells specific for epitopes from EBV lytic proteins within synovial fluid and showed that subpopulations of these cells were able to secrete proinflammatory cytokines after short-term stimulation. We used a HLA-A2/GILGFVFTL tetramer to stain PBMCs and SFMCs from six HLA-A2-positive patients. The proportion of T cells specific for this influenza epitope was low (<0.2%) in all donors studied, and we did not find any enrichment within SFMCs. We had access to SFMCs only from a second group of four HLA-A2-positive patients with RA. A tetramer of HLA-A2 complexed to the NLVPMVATV epitope from the CMV pp65 protein reacted with subpopulations of CD8+ SFMCs in all four donors, with frequencies of 0.2, 0.5, 2.3 and 13.9%. SFMCs from all four donors secreted TNF after short-term incubation with COS cells transfected with HLA-A2 and pp65 complementary DNA. We analyzed the phenotype of virus-specific cells within PBMCs and SFMCs in three donors. The SFMC virus-specific T cells were more highly activated than those in PBMCs, as evidenced by expression of high levels of CD69 and HLA-DR. A greater proportion of SFMCs were CD38+, CD62L low, CD45RO bright, CD45RA dim, CD57+ and CD28- when compared with PBMCs. DISCUSSION: This work shows that T cells specific for certain epitopes from viral proteins are present at very high frequencies (up to 15.5% of CD8+ T cells) within SFMCs taken from patients with inflammatory joint disease. This enrichment does not reflect a generalized enrichment for the 'memory pool' of T cells; we did not find enrichment of T cells specific for the GILGFVFTL epitope from influenza A or for the FLRGRAYGL epitope from the EBV latent protein EBNA3A, whereas we found clear enrichment of T cells specific for the GLCTLVAML epitope from the EBV lytic protein BMLF1 and for the RAKFKQLL epitope from the EBV lytic protein BZLF1. The enrichment might reflect preferential recruitment of subpopulations of virus-specific T cells, perhaps based on expression of selectins, chemokine receptors or integrins. Alternatively, T cells specific for certain viral epitopes may be stimulated to proliferate within the joint, by viral antigens themselves or by cross-reactive self-antigens. Finally, it is theoretically possible that subpopulations of T cells within the joint are preferentially protected from apoptotic cell death. Whatever the explanation, the virus-specific T cells are present at high frequency, are activated and are able to secrete proinflammatory cytokines. They could potentially interact with synoviocytes and contribute to the maintenance of inflammation within joints in many different forms of inflammatory arthritis.

Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype.

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.

Changing patterns of dominant TCR usage with maturation of an EBV-specific cytotoxic T cell response.

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.

Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation.

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines.

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.

T-cell memory: lessons from Epstein-Barr virus infection in man.

Epstein-Barr virus offers an ideal opportunity to follow the human T-cell response to a virus infection over time from its acute primary phase, as seen in infectious mononucleosis patients, into the memory phase that accompanies life-long virus persistence. Here we review recent evidence on the development and maturation of cytotoxic T-cell memory using this viral system.