Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I-restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8-restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I-restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.     

Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1

The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8(+) cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4(+) T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4(+) T cells, EBNA1 is preferentially recognized. We present evidence that the CD4(+) response may provide a protective role, including interferon gamma secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II-mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4(+) T cell lines to recognize autologous B-LCL-expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4(+) T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4(+) T cell immunity be enhanced to prevent and treat EBV-associated malignancies.     

Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr virus-encoded nuclear antigen 1

Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.     

Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.     

EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) maintains the viral episome in all host cells infected with EBV. Recently, EBNA1 was found to be the main EBV latency antigen for CD4+ T cells and could be recognized in cultures from all donors tested. We now identify a polarized Th1 phenotype and obtain evidence for its presence in vivo. When T cells were stimulated with dendritic cells infected with vaccinia vectors expressing EBNA1, 18 of 19 donors secreted IFN-gamma, whereas only two of 19 secreted IL-4. Magnetic selection was then used to isolate cells from fresh blood based on EBNA1-induced cytokine production. Specific IFN-gamma CD4+ cell lines were established from six of six donors and IL-4 lines from three of six. Only the Th1 lines specifically lysed targets expressing three different sources of EBNA1 protein. When the IgG isotype of EBNA1 plasma Ab's was tested, most specific Ab's were IgG1 and of a high titer, confirming a Th1 response to EBNA1 in vivo. Ab's to other microbial antigens generally were not skewed toward IgG1. Given emerging evidence that Th1 CD4+ T cells have several critical roles in host defense to viral infection and tumors, we propose that EBNA1-specific CD4+ Th1 cells contribute to resistance to EBV and EBV-associated malignancies.     

Identification of a naturally processed HLA-DR-restricted T-helper epitope in Epstein-Barr virus nuclear antigen type 1

Epstein-Barr virus nuclear antigen type 1 (EBNA1), the only viral protein that is unequivocally expressed in all Epstein-Barr virus (EBV)-associated malignant diseases, is essential for viral DNA replication and maintenance of the viral episome in infected cells. A glycine-alanine repeat domain inhibits antigen processing through the ubiquitin-proteasome pathway for presentation on human leukocyte antigen (HLA) class I molecules. EBNA1 is not protected from the HLA class II processing pathway, and CD4+ HLA class II-restricted T cells recognize the antigen. CD4+ T-helper (Th) cells play critical roles in initiating, regulating, and maintaining immune responses against viral infections and tumors, so that inclusion of EBNA1 as a target antigen may improve immunotherapy for EBV-associated cancers. In this study, the authors used the TEPITOPE software program to predict promiscuous class II epitope candidates. After several HLA-DR-restricted peptides were identified by in vitro analysis of the T-cell response to synthetic peptides, a T-cell clone was established that was specific for one of the peptides. Functional studies were performed with this clone. The CD4+ T helper cells specific for the HLA-DR15-restricted peptide EBNA1(482) (AEGLRALLARSHVER) recognized naturally processed EBNA1 protein. This epitope was presented by several HLA-DR alleles, including DR4, DR7, and DR11. The inclusion of the promiscuous, naturally processed EBNA1(482) epitope in vaccine constructs could enhance immune responses against EBV-positive cancers.     

Epstein-barr virus nuclear antigen 1: from immunologically invisible to a promising T cell target

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1)--the one EBV antigen that is expressed in all EBV-associated malignancies--has long been thought to go undetected by the cell-mediated immune system. However, recent studies show that EBNA1 can be presented to both CD4+ and CD8+ T cells, making it a potential new target for immunotherapy of EBV-related cancers.     

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised

We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein- Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen- A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release.     

CD8+ T cell subsets of cytotoxic T lymphocytes induced by Epstein-Barr virus infection in infectious mononucleosis

Mononuclear peripheral blood lymphocytes (PBL) from patient with infectious mononucleosis (IM) were tested in a 51Cr-release assay for cytotoxicity against autologous and allogeneic lymphoblastoid cell line (LCL), or Epstein-Barr virus (EBV)-genome positive and negative cell line. In acute phase, PBL lyse an autologous LCL as well as allogeneic LCL (Wa cells). High levels of cytotoxicity were observed in the combinations between effector and target cells sharing HLA-Class 1 product. EBV-genome positive Daudi and Raji cells which lack HLA-Class 1 antigen and have mismactched HLA-Class 1 antigen, respectively showed resistance to killing. EBV-genome negative tumor cells except NK sensitive K562 cells were not killed by IM lymphocytes. However, the IM lymphocytes without atypical form in convalescent phase failed to show killing activity against autologous and allogeneic LCL. These findings suggest that cell surface membrane antigen structure on EBV-infected LCL may be able to explain the recognition and triggering of lysis of target cells by HLA-Class 1 restricted cytotoxic T cells (CTL) from acute IM. Phenotypic analysis of PBL with atypical form from IM was made by two-color flow cytometry. The data demonstrate that CD8+ T cells quantitatively represent the major population of lymphocytes expanded during acute IM. Furthermore, approximately 70% of these CD8+ T cells express HLA-DR on these surface, suggesting that they have undergone activation. However, IL 2R (CD25 antigen) expression was not significantly elevated on activated T cells. The salient profile on cytofluorographs of an acute IM was the increased number of CD3+CD19-, CD8+CD11b-, CD8+CD28+ and CD8+S6F1+ cells. However, CD3-CD19+, CD8+CD11b+, CD8+S6F1-, CD4+Leu8- and CD25+HLA-DR+ antigens were little expressed. Increased number of CD8+CD11b-, CD8+CD28+ and CD8+S6F1+ cells, which are regarded as CTL were reduced according to the improvement of the clinical symptoms and laboratory findings. These results together with HLA typing analysis suggested a possibility HLA-Class 1 restriction of the CTL with surface phenotype of CD8+CD11b-, CD8+CD28+, and CD8+S6F1+.     

Critical role of EBNA1-specific CD4+ T cells in the control of mouse Burkitt lymphoma in vivo

CD4+ T cells play important roles in orchestrating host immune responses against cancer and infectious diseases. Although EBV-encoded nuclear antigen 1-specific (EBNA1-specific) CD4+ T cells have been implicated in controlling the growth of EBV-associated tumors such as Burkitt lymphoma (BL) in vitro, direct evidence for their in vivo function remains elusive due to the lack of an appropriate experimental BL model. Here, we describe the development of a mouse EBNA1-expressing BL tumor model and the identification of 2 novel MHC H-2I-A(b)-restricted T cell epitopes derived from EBNA1. Using our murine BL tumor model and the relevant peptides, we show that vaccination of mice with EBNA1 peptide-loaded DCs can elicit CD4+ T cell responses. These EBNA1-specific CD4+ T cells recognized peptide-pulsed targets as well as EBNA1-expressing tumor cells and were necessary and sufficient for suppressing tumor growth in vivo. By contrast, EBNA1 peptide-reactive CD8+ T cells failed to recognize tumor cells and did not contribute to protective immunity. These studies represent what we believe to be the first demonstration that EBNA1-specific CD4+ T cells can suppress tumor growth in vivo, which suggests that CD4+ T cells play an important role in generating protective immunity against EBV-associated cancer.     

Eradication of Epstein-Barr virus episome and associated inhibition of infected tumor cell growth by adenovirus vector-mediated transduction of dominant-negative EBNA1.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1), a latent viral protein consistently expressed in infected proliferating cells, is essentially required in trans to maintain EBV episomes in cells. We constructed a mutant (mt) EBNA1 and examined whether it exerted dominant-negative effects on maintenance of the viral episome thereby leading to abrogation of EBV-infected tumor cell growth. Using lymphocyte and epithelial cell lines converted with neomycin-resistant recombinant EBV (rEBV) as models, adenovirus vector-mediated transduction of mtEBNA1, but not LacZ, brought about rapid and striking reductions in rEBV-derived wild-type EBNA1 levels and viral genomic loads in converted lines of three major viral latencies. This outcome was further validated at the single-cell level by cellular loss of G418 resistance and viral signals in situ. The mtEBNA1 transduction significantly impaired growth of naturally EBV-harboring Burkitt lymphoma cells in vitro and in vivo, largely in association with the eradication of viral episomes. Expression of mtEBNA1 per se caused no detectable cytotoxicity in EBV-uninfected cells. These results indicate that mtEBNA1 can act as a dominant-negative effector that efficiently impedes the EBV-dependent malignant phenotypes in cells regardless of viral latency or tissue origin. The mutant will afford an additional therapeutic strategy specifically targeting EBV-associated malignancies.     

Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans by latently infecting B cells, with occasional cycles of reactivation, virus production, and reinfection. Protective immunity against EBV is mediated by T cells, but the role of EBV-specific T helper (Th) cells is still poorly defined. Here, we study the Th response to the EBV lytic cycle proteins BLLF1 (gp350/220), BALF4 (gp110), and BZLF1 and show that glycoprotein-specific Th cells recognize EBV-positive cells directly; surprisingly, a much higher percentage of target cells than those expressing lytic cycle proteins were recognized. Antigen is efficiently transferred to bystander B cells by receptor-mediated uptake of released virions, resulting in recognition of target cells incubated with <1 virion/cell. T cell recognition does not require productive infection and occurs early after virus entry before latency is established. Glycoprotein-specific Th cells are cytolytic and inhibit proliferation of lymphoblastoid cell lines (LCL) and the outgrowth of LCL after infection of primary B cells with EBV. These results establish a novel role for glycoprotein-specific Th cells in the control of EBV infection and identify virion proteins as important immune targets. These findings have implications for the treatment of diseases associated with EBV and potentially other coated viruses infecting MHC class II-positive cells.     

Cell-cell adhesion mediated by CD8 and human histocompatibility leukocyte antigen G, a nonclassical major histocompatibility complex class 1 molecule on cytotrophoblasts

The lymphocyte differentiation marker CD8 acts as a coreceptor with the T cell receptor (TCR) during recognition of peptide presented by major histocompatibility complex (MHC) class I molecules. The functions of CD8 in the TCR complex are thought to be signaling through the association of CD8 with the protein tyrosine kinase p56lck and adhesion to MHC class I through the alpha 3 domain. While the ability of the CD8 alpha/alpha homodimer to bind to classical MHC class I molecules has been shown, it is unclear whether CD8 can also bind nonclassical molecules. Of particular interest is human histocompatibility leukocyte antigen (HLA)-G which is expressed on placental cytotrophoblast cells. These cells do not express HLA-A, -B and -C molecules. In this report, we demonstrate that CD8 can bind to HLA-G. It is possible, therefore, that a cell bearing CD8 may interact with HLA-G-expressing cells.     

Requirement for CD8-major histocompatibility complex class I interaction in positive and negative selection of developing T cells.

The interaction of the T cell surface glycoprotein CD8 with major histocompatibility complex (MHC) class I molecules on target cells is required for effective T cell activation. Mutations in the alpha 3 domain of the MHC class I molecule can disrupt binding to CD8, yet leave antigen presentation unaffected. Here we show that such a mutation can interfere with positive and negative selection of T cells bearing T cell receptors (TCRs) that interact specifically with the mutant class I molecule. Autoreactive T cells in male mice expressing a transgenic TCR specific for the male antigen H-Y and H-2Db were not deleted in the context of a transgenic Db molecule bearing a mutation at residue 227. Similarly, CD8+ cells were not positively selected in female mice expressing both the TCR and mutant class I transgenes. Endogenous MHC class I molecules were competent to bind CD8, but were unable to rescue the defect, indicating a requirement for coordinate recognition of antigen/MHC by a complex of the TCR and CD8 coreceptor for both positive and negative selection of thymocytes.     

Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies.

Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins.     

EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-gamma and IL-2

Symptomatic primary Epstein-Barr virus (EBV) infection and elevated humoral immune responses to EBV are associated with an increased risk of developing multiple sclerosis (MS). We explored mechanisms leading to this change in EBV-specific immunity in untreated patients with MS and healthy virus carriers matched for MS-associated HLA alleles. MS patients showed selective increase of T cell responses to the EBV nuclear antigen 1 (EBNA1), the most consistently recognized EBV-derived CD4⁺ T cell antigen in healthy virus carriers, but not to other EBV-encoded proteins. In contrast, influenza and human cytomegalovirus–specific immune control was unchanged in MS. The enhanced response to EBNA1 was mediated by an expanded reservoir of EBNA1-specific central memory CD4⁺ T helper 1 (Th1) precursors and Th1 (but not Th17) polarized effector memory cells. In addition, EBNA1-specific T cells recognized myelin antigens more frequently than other autoantigens that are not associated with MS. Myelin cross-reactive T cells produced IFN-γ, but differed from EBNA1-monospecific cells in their capability to produce interleukin-2, indicative of a polyfunctional phenotype as found in controlled chronic viral infections. Our data support the concept that clonally expanded EBNA1-specific CD4⁺ T cells potentially contribute to the development of MS by cross-recognition of myelin antigens.     

Molecular size variation of EBNA is determined by the EB viral genome

The size classes of the Epstein-Barr virus(EBV)-induced nuclear antigen (EB-NA) have been determined in EBV-carrying sublines of two originally EBV-negative Burkitt lymphomas, converted in vitro by two different EBV substrains, and in somatic hybrids between two EBV-carrying cell lines or between an EBV-positive and an EBV-negative line. Partially purified EBNA components were detected by Western blotting and subsequent exposure to antiserum/alkaline-phosphatase-coupled protein A complexes. Converted Ramos and BJAB sublines contained the main EBNA components characteristic for the virus donor strain. The EBNA components of the Ramos and BJAB cells converted by P3HR-1 virus were similar to each other and to the components of the P3HR-1 donor cell, whereas the EBNA of the cells converted by B95-8 virus resembled the B95-8 donor line. It was concluded that the size variation of EBNA is determined by the viral genome. The parental lines of the somatic hybrids studied (Raji, P3HR-1, Namalwa and Daudi) contained EBNA components of different molecular weight (MW) classes. The higher MW forms ranged from 70K to 80K. Several distinctive lower MW components also were present. The somatic hybrids expressed the main EBNA components of both parental lines. Hybrids between EBV-carrying and EBV-genome-negative lines contained the characteristic EBNA component of the EBV-positive parent. It was concluded that the size variation of EBNA is under strict genetic control which prevails in the hybrids in a codominant fashion.     

Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency

CD8 molecules expressed on the surface of a subset of T cells participate in the selection of class I MHC antigen-restricted T cells in the thymus, and in MHC-restricted immune responses of mature class I MHC antigen-restricted T cells. Here we describe an immune-deficient patient with lack of CD8+ peripheral blood cells. The patient presented with Pneumocystis carinii pneumonia and was unable to reject an allogeneic skin graft, but had normal primary and secondary antibody responses. Examination of the patient's thymus revealed that the loss of CD8+ cells occurred during intrathymic differentiation: the patient's immature cortical thymocytes included both CD4+ and CD8+ cells while the mature medullary cells expressed the CD4 but not the CD8 protein on their surface. Northern blot and polymerase chain reaction analyses revealed the presence of CD8 alpha and beta mRNA in the patient's thymus but not in the peripheral blood. Both class I MHC antigen expression and the expressed TCR V beta repertoire are normal in this patient. These data are consistent with an impaired selection of CD8+ cells in the patient's thymus and support the role of the CD8 surface protein in thymic selection previously characterized in genetically manipulated and inbred mice.