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The role of virus-specific CD4+ T cells in the control of Epstein-Barr virus infection
European Journal of Cell Biology 91 (2012) 31–35
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Review
The role of virus-specific CD4+ T cells in the control of Epstein-Barr virus infection Josef Mautner ∗ , Georg W. Bornkamm Clinical Cooperation Group, Pediatric Tumor Immunology, Helmholtz-Zentrum München, Marchioninistr. 25, 81377 Munich, and Children’s Hospital, Technische Universität München, Kölner Platz 1, 80804 Munich, Germany
a r t i c l e
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Article history: Received 31 October 2010 Accepted 22 January 2011 Keywords: Epstein-Barr virus CD4+ T cell Antigen Immunodominance Therapy
a b s t r a c t Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans and has been implicated in the pathogenesis of several human malignancies. Protective immunity against EBV is mediated by T cells, as indicated by an increased incidence of EBV-associated malignancies in immunocompromised patients, and by the successful treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) in transplant recipients by the infusion of polyclonal EBV-specific T cell lines. To implement this treatment modality as a conventional therapeutic option, and to extend this protocol to other EBVassociated diseases, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. To this aim, we studied the poorly defined EBV-specific CD4+ T cell response during acute and chronic infection. © 2011 Elsevier GmbH. All rights reserved.
Introduction Epstein-Barr virus (EBV) is a ubiquitous human ␥-herpesvirus implicated in the pathogenesis of several malignancies of lymphoid and epithelial origin (Kieff and Rickinson, 2007; Kuppers, 2003; Rickinson and Kieff, 2007). Primary infection with EBV usually occurs within the first three years of life by parent-to-child oral transmission in an almost always asymptomatic fashion. Delayed primary infection in adolescence or adulthood may cause the syndrome of infectious mononucleosis (IM). Following oral transmission, the virus replicates in the oropharynx from where it colonizes the host by latently infecting B cells. The reservoir of latently infected B cells can seed foci of virus replication at mucosal sites, and this reactivation of the virus and subsequent re-infection of B lymphocytes allows the virus to persist for life in the infected human host (Kieff and Rickinson, 2007; Kuppers, 2003; Rickinson and Kieff, 2007). In B cells, EBV is able to establish different types of latency characterized by the expression of different sets of viral genes. During the primary phase of B cell infection, as well as in lymphoblastoid cell lines (LCL) generated by infection of B cells with EBV in vitro, the full range of viral latent cycle proteins is expressed that drive the activation and proliferation of the infected cell (Kelly et al., 2009; Kieff and Rickinson, 2007). In vivo, outgrowth of latently infected growth-transformed B cells is curtailed by T cells. The importance of T cell-mediated immune responses in maintaining asymptomatic viral persistence is emphasized by the
∗ Corresponding author at: Children’s Hospital, University of Technology, Kölner Platz 1, D-80804 Munich, Germany. Tel.: +49 89 7099518; fax: +49 89 7099500. E-mail address: [email protected] (J. Mautner). 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.01.007
clinical observation that patients with T cell dysfunction are at risk of developing life-threatening EBV-associated lymphoproliferative disease (Rickinson and Kieff, 2007). In solid organ and hematopoietic stem cell transplant recipients, the incidence of EBV-positive post-transplant lymphoproliferative disease (PTLD) correlates with the degree of the iatrogenically induced immunosuppression. Furthermore, EBV-positive PTLD in transplant recipients has been successfully treated by the infusion of polyclonal EBV-specific T cell lines containing CD4+ and CD8+ T cell components, that were generated by repeated stimulation of peripheral blood T cells with irradiated autologous LCL in vitro (Rooney et al., 1995, 1998). The targets of the EBV-specific cytotoxic CD8+ T cell response have been studied in detail and display a marked hierarchy in immunodominance with epitopes derived from the EBNA3 family of proteins and immediate early as well as early lytic cycle proteins usually inducing the strongest responses across a range of different HLA class I alleles. The same pattern of immunodominance was observed when the EBV latent epitope-specific CD8+ T cell memory was analyzed ex vivo, indicating that the EBV-specific T cell population expanded by LCL stimulation in vitro is an amplification of the in vivo correlate (Khanna and Burrows, 2000; Khanna et al., 1992; Landais et al., 2005b; Murray et al., 1992; Steven et al., 1997; Tan et al., 1999). Specificity of the CD4+ T cells in LCL-stimulated T cell lines Because the corresponding CD4+ T cell response is still poorly defined, we sought to study specificity and function of the EBVspecific CD4+ T cell response in LCL-stimulated T cell cultures. When tested for target-specific cytokine secretion, LCL-stimulated CD4+ T cell lines from healthy virus carriers recognized autologous
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as well as MHC class II-matched EBV-positive, but barely EBVnegative target cells, indicating that these lines were specific for EBV antigens (Adhikary et al., 2007). Of the T cell lines derived from patients with IM, about half displayed EBV-specificity by these criteria while the other half responded similarly against EBV-negative and EBV-positive target cells, indicating that these latter T cell lines predominantly recognized self-antigen(s). All T cell lines derived from virus-negative controls failed to respond against autologous LCL. These results demonstrated that EBV-specific CD4+ T cell memory is efficiently reactivated by LCL stimulation and excluded that de novo priming of EBV-specific CD4+ T cell responses occurred under these in vitro conditions (Adhikary et al., 2007). Surprisingly, except for weak responses against EBNA3C in a few lines, none of the EBV-specific CD4+ T cell lines recognized any of the latent cycle antigens of EBV. Such T cells had been detected in peripheral blood of healthy virus carriers by different groups (Khanna et al., 1995, 1997; Leen et al., 2001; Mautner et al., 2004; Munz et al., 2000), albeit at low frequency (Leen et al., 2001). Therefore, up to 50 restimulations were performed in order to facilitate the expansion of such rare T cell specificities to detectable levels. Again, no reactivity against any of the latent cycle antigens was detected in these late-passage T cell lines. Even the weak responses against EBNA3C had vanished. These results suggested that the LCL-stimulated CD4+ T cell cultures were dominated by CD4+ T cells targeting either lytic cycle antigens of EBV or cellular antigens induced by EBV. To discriminate between these possibilities, a genetically engineered mutant strain of EBV was used, that was still able to infect and growth-transform B cells into so-called miniLCL, but unable to enter the lytic cycle (Adhikary et al., 2006; Kempkes et al., 1995; Moosmann et al., 2002). MiniLCL were barely or not at all recognized by any of the CD4+ T cell lines that had shown EBV-specificity, indicating that these lines recognized lytic cycle proteins of the virus, or cellular proteins induced by viral lytic cycle proteins.
A bacterial expression cloning approach for direct antigen identification The EBV genome contains approximately 100 putative open reading frames, but neither the precise number nor the identity of all genes expressed during lytic replication is known (Kieff and Rickinson, 2007). To assess whether the EBV-specific CD4+ T cells recognized lytic cycle antigens, a method previously developed to rapidly map epitopes within proteins, was adapted to facilitate antigen identification within the whole viral genome (Milosevic et al., 2005). This so-called direct antigen identification (DANI) method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria which are subsequently fed to MHC class II+ antigen presenting cells and probed with antigen-specific T cells (Milosevic et al., 2006). EBV DNA was digested with frequently cutting restriction enzymes, the resulting short DNA fragments ligated into the expression vector mix, and the resulting EBV library plated at 60 cfu/well. With an average insert size of 82 bp, and an EBV plasmid size of 182 kb, a single 96-well plate covered the whole EBV genome approximately 2.5 times. The bacterial suspensions were added to autologous miniLCL which were subsequently probed with the EBV-specific T cells. Bacterial pools recognized by the T cells were plated on agar plates and single bacterial colonies tested in the same way. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of LCL-stimulated CD4+ T cells, demonstrating that unknown viral antigens are efficiently identified with this method and that lytic cycle antigens are the main targets of the EBV-specific CD4+ T cell response (Milosevic et al., 2006).
Table 1 Antigens recognized by LCL-stimulated CD4+ T cell lines. Donor
Dominant antigens
Subdominant antigens
IM1 IM2 IM5 GB JM DA MS SM MA TK
BcLF1a BXLF2a BNRF1a BNRF1a BALF2b BVRF2a BALF4a BALF2b , BNRF1a BMRF1b , BNRF1a BORF1a
BFRF3a , BXLF2a BDLF1b , BNRF1a BALF2b BXRF1a , BORF1a , BDLF1a , BBRF3a BDLF1a , BXRF1a , BALF4a BNRF1a , BCRF1a , BORF1a , EBNA3Cc EBNA3Cc BMRF1b Nd Nd
The antigens recognized by the EBV-reactive CD4+ cell lines from three patients with IM and seven healthy virus carriers were identified by using PBMC or miniLCL pulsed with single latent or lytic cycle proteins of EBV as targets. Responses against dominant antigens were maintained up to fifty restimulations, while responses against subdominant antigens were detected at early passages of the T cell lines only. No responses were detected against the immediate early antigens BZLF1 and BRLF1. Nd, not determined. a Late lytic cycle antigens. b Early lytic cycle proteins. c Latent cycle antigens.
Immunodominance of virion antigens Specificity as well as V chain analyses revealed that even after repeated rounds of stimulation in vitro, LCL-stimulated CD4+ T cell lines were still oligoclonal and recognized more than one antigen. To follow the evolution of the lines in vitro in more detail, all latent cycle proteins and 50 different lytic cycle genes of EBV, including the immediate early antigens BZLF1 and BRLF1, 23 early antigens, and 25 late antigens, were recombinantly expressed and purified. Autologous APC were pulsed with the various proteins and probed with the LCL-stimulated T cell lines after different passages. These experiments revealed that all T cell lines recognized at least one of the lytic cycle antigens tested (Adhikary et al., 2007). Except for the lytic cycle proteins BDLF1, BMRF1, BCRF1, BVRF2, and BALF2, all of these lytic cycle antigens were derived from virion proteins (Table 1). Moreover, all lines targeted at least one virion antigen. With the notable exception of the tegument protein BNRF1, which was recognized by six of the ten T cell lines tested, diverse sets of antigens were targeted. Moreover, T cell clones established from T cell lines stimulated more than 40 times often expressed different TCR-V chains and recognized more than one antigen. Thus, most LCL-stimulated CD4+ T cell lines from EBV-positive individuals had remained oligoclonal even beyond a year and a half in culture. These results suggested that the virus-specific CD4+ T cell response is not focused on one or a few immunodominant antigens, but is directed against a broad set of antigens, mostly derived from the group of structural proteins of EBV. The weak and transient responses against EBNA3C detected in two of the T cell lines established from healthy virus carriers implied that T cells specific for latent cycle antigens expanded under these in vitro culture conditions, albeit less efficiently than lytic cycle antigen-specific CD4+ T cells. However, when peripheral blood CD4+ T cells from healthy virus carriers were stimulated with miniLCL, which fail to express lytic cycle proteins, the resulting T cell lines recognized predominantly autoantigens and only sporadically latent cycle antigens of EBV, even after more than 25 passages (Adhikary et al., 2007). This was unexpected because all latent cycle proteins are expressed in LCL, and CD4+ T cells specific for latent antigens have been consistently detected in the peripheral blood of EBV-seropositive donors (Rickinson and Kieff, 2007). Thus, the targets of the LCL-stimulated CD4+ T cell response display a marked hierarchy in immunodominance: lytic cycle (structural) antigens > autoantigens > latent cycle antigens.
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Fig. 1. Efficient presentation of virion antigens following receptor-mediated uptake. EBV infection of B cells is initiated by the binding of the viral glycoprotein BLLF1 to CD21 on B cells. Following envelope fusion with the endosomal or the cell membrane, the nucleocapsid is released into the cytoplasm and the viral DNA eventually delivered into the cell nucleus. Proteins in the viral envelope are retained in the endosomal compartment, degraded, and peptide fragments loaded on MHC class II molecules for recognition by CD4+ T cells.
Immunodominance of structural antigens based on receptor-mediated antigen uptake of virions Given that usually only about 1% of cells in an LCL culture spontaneously become permissive for lytic replication and express lytic cycle proteins, while all express latent cycle proteins, this immunodominance of structural antigens was surprising. First insights into the factors and mechanisms dictating this pattern of immunodominance were obtained when different EBVspecific CD4+ T cell clones were assayed for target cell recognition. Although all T cell clones recognized their cognate peptide antigens with similar affinity, only CD4+ T cells specific for glycoproteins, but not for the transcription factor BZLF1, recognized autologous as well as HLA-matched allogeneic EBV-infected target cells (Adhikary et al., 2006). Since miniLCL were not recognized, T cell recognition was probably dependent on sporadic lytic replication occurring in a low percentage of cells in culture. However, when the percentage of cells in an LCL culture that were recognized by the T cells was determined, a substantially higher number was obtained than those positive for lytic cycle proteins in immunofluorescence studies. Co-culture experiments of HLA-mismatched LCL and HLAmatched miniLCL indicated that the antigen was transferred from one cell type to the other. Several lines of evidence suggested that the antigen transferred between cells was derived from released virions. First, miniLCL pulsed with purified virus preparations were recognized by the T cells, demonstrating that virions can serve as the source of the antigen. Because miniLCL pulsed with heatinactivated virus were still recognized, T cell recognition did not depend on productive infection of the target cells. Second, the antigen was most efficiently transferred to B cells and this transfer was blocked by BLLF1- and CD21-specific monoclonal antibodies, implying that antigen uptake is receptor-mediated. Third, the failure of BZLF1-specific T cells to recognize LCL, and the failure of virion-specific T cells to recognize autologous dendritic cells
(DC) co-cultured with HLA-mismatched LCL demonstrated that the amount of antigen released by dead or dying cells was not sufficient for T cell detection (Adhikary et al., 2006). Besides structural proteins, some LCL-stimulated CD4+ T cells had also targeted lytic cycle proteins that are not constituents of virions. In the case of BMRF1, presentation involved intercellular antigen transfer, probably by release of protein from lytically infected cells and uptake as exogenous protein by neighboring cells as described previously for other EBV proteins (Landais et al., 2005a; Taylor et al., 2006). Why these but not other lytic cycle proteins such as BZLF1 are efficiently transferred between cells is not known, but might reflect quantitative differences in protein expression levels, protein stability and/or the timing of expression later in the lytic cycle which might facilitate release from cells. Likewise, the impaired expansion of latent cycle antigen-specific CD4+ T cells in LCL-stimulated culture might also be the consequence of inefficient presentation of epitopes derived from latent cycle proteins on MHC class II. The observation that mainly B cells, whether primary or EBVinfected, presented virion proteins most efficiently implicated a protective role of such T cells in controlling the spread of infection. Importantly, virion-specific CD4+ T cells recognized target cells pulsed with virus supernatant before the EBV genome has circularized and before EBNA2, the protein essential for primary B cell growth-transformation, is expressed. EBV infection of B cells is initiated by BLLF1 adsorption to CD21 on the B cell plasma membrane, followed by viral endocytosis and envelope fusion with the endosomal or cell membrane and nucleocapsid exocytosis into the cytoplasm (Hutt-Fletcher, 2007). During this process of virus uncoating, proteins of the viral envelope are probably retained at the cell membrane, as indicated by the higher percentage of cells positive for BLLF1 than for BZLF1 in immunofluorescence experiments (Lee et al., 1993). Endosomal and cell membrane proteins efficiently access the MHC class II processing and loading
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compartment, thereby allowing CD4+ T cells specific for envelope antigens to detect EBV-infected cells before viral latency is established. This receptor-mediated virion uptake and subsequent presentation of virion protein-derived peptides on MHC class II turned out to be extremely efficient. BALF4- and BLLF1-specific T cells were able to recognize B cells incubated with less than one EBV genome equivalent per cell (Adhikary et al., 2006, 2008). Thus, virion proteins retained in endosomes or the cell surface during virus uncoating label newly EBV-infected B cells for immune attack by CD4+ T cells specific for virion proteins (Fig. 1). A potential role of virion antigen-specific CD4+ T cells in the control of EBV infection Importantly, virion antigen-specific CD4+ T cells proved to be cytolytic and able to prevent the outgrowth of primary B cells infected with EBV in vitro. Thus, by diminishing the pool of EBVinfected B cells, virion antigen-specific CD4+ T cells might aid in controlling EBV infection in vivo. In fact, recent results from preclinical models indicate that EBVspecific CD4+ T cells are effective against PTLD-like tumors even in the absence of CD8+ T cells, and that the CD4+ and CD8+ T cell components of LCL-stimulated T cell preparations are equally efficacious in controlling PTLD in mice (Merlo et al., 2010, and unpublished results). Furthermore, an important role of CD4+ T cells in establishing antiviral immunity has been inferred from clinical experience; low numbers of endogenous CD4+ T cells have been identified as an important risk factor for the development of EBV-associated diseases in immunosuppressed patients (Sebelin-Wulf et al., 2007), and patients with PTLD showed better clinical responses in a phase II trial when the infused LCL-stimulated T cell lines contained higher proportions of CD4+ T cells (Haque et al., 2007). Finally, in a patient with PTLD showing complete and stable remission after infusion of EBV peptides-selected T lymphocytes, CD4+ T cells specific for a peptide derived from the virion protein BNRF1 had expanded significantly, suggesting that these T cells contributed to the antitumoral immune response in vivo (Moosmann et al., 2010). Collectively, these findings suggest that EBV-specific CD4+ and CD8+ T cells complement each other by targeting different phases of the virus’ life cycle. Standardized and efficient expansion of virion-specific CD4+ T cells using virus-like particles For unknown reasons, the CD4/CD8 ratio in LCL-stimulated T cell preparations can vary from 2:98 to 98:2 (Smith et al., 1995). Because EBV-specific CD4+ T cells in LCL-stimulated T cell preparations are almost exclusively directed against structural antigens of the virus (Adhikary et al., 2007), which are efficiently presented on MHC II following receptor-mediated uptake of released viral particles, we investigated which factors compromised the expansion of EBV-specific CD4+ T cells in LCL-stimulated T-cell preparations. These experiments showed that spontaneous virus production by LCL and, hence, the presentation of viral antigens, varies intraand interindividually. Virus production is further impaired by acyclovir treatment of LCL, which is performed in most currently applied clinical protocols to suppress virus production in stimulator LCL to minimize residual infectious viral particles within adoptively transferred T cells. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4+ T cells. All CD4+ T cell lines that were repeatedly stimulated with LCL grown in FCSsupplemented media eventually showed FCS-reactivity, even lines established from patients with acute IM who are expected to have
a strong antiviral T cell response (Adhikary et al., 2008). Given that more than 90% of the adult population is EBV-seropositive and carries virus-neutralizing antibodies that diminish virion uptake, supplementing media with human serum may result in reduced presentation of virion antigens. These findings indicated that addition of excess amounts of EBV particles may antagonize the inhibitory effect of human serum and compensate for differences in virus production by different LCLs, and thereby facilitate to establish uniform and standardized stimulation conditions. Because incubation of stimulator cells with wild-type EBV would pose an incalculable health risk to patients, the possibility of using genome-deficient Epstein-Barr virus-like particles (EB-VLP) was explored. EB-VLP produced by human cells in serum-free media are readily available in large quantities and transfer structural antigens as efficiently as wild-type EBV. Instead of LCL, PBMC pulsed with EB-VLP were used as stimulators because PBMC do not produce virus and are immediately available. This strategy facilitated the specific and rapid expansion of EBV-specific CD4+ T cells and, thus, might contribute to the development of standardized protocols for the generation of T cell lines with improved clinical efficacy (Adhikary et al., 2008). Conclusions The reconstitution of EBV-specific immunity in transplant patients by the adoptive transfer of polyclonal virus-specific T cell lines has provided important proof of principle for immunotherapy of EBV-associated tumors, and for cancer immunotherapy in general (Foster and Rooney, 2006; Gattinoni et al., 2006; Ho et al., 2003; Tey et al., 2006). Given the significant burden of EBV-associated tumors worldwide, important future goals of this adoptive T cell therapy are the introduction into mainstream clinical practice and the extension to EBV-associated tumor entities other than PTLD (Gottschalk et al., 2005; Tey et al., 2006). Thus, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. A better understanding of the critical immune effector cells and the relevant immune targets may eventually expedite the preparation of T cell lines and improve the clinical effectiveness of this form of immunotherapy and, hence, the long term survival of patients. References Adhikary, D., Behrends, U., Boerschmann, H., Pfunder, A., Burdach, S., Moosmann, A., Witter, K., Bornkamm, G.W., Mautner, J., 2007. Immunodominance of lytic cycle antigens in Epstein-Barr virus-specific CD4+ T cell preparations for therapy. PLoS One 2, e583. Adhikary, D., Behrends, U., Feederle, R., Delecluse, H.J., Mautner, J., 2008. Standardized and highly efficient expansion of Epstein-Barr virus-specific CD4+ T cells by using virus-like particles. J. Virol. 82, 3903–3911. Adhikary, D., Behrends, U., Moosmann, A., Witter, K., Bornkamm, G.W., Mautner, J., 2006. Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins. J. Exp. Med. 203, 995–1006. Foster, A.E., Rooney, C.M., 2006. Improving T cell therapy for cancer. Expert Opin. Biol. Ther. 6, 215–229. Gattinoni, L., Powell Jr., D.J., Rosenberg, S.A., Restifo, N.P., 2006. Adoptive immunotherapy for cancer: building on success. Nat. Rev. Immunol. 6, 383–393. Gottschalk, S., Heslop, H.E., Rooney, C.M., 2005. Adoptive immunotherapy for EBVassociated malignancies. Leuk. Lymphoma 46, 1–10. Haque, T., Wilkie, G.M., Jones, M.M., Higgins, C.D., Urquhart, G., Wingate, P., Burns, D., McAulay, K., Turner, M., Bellamy, C., et al., 2007. Allogeneic cytotoxic T cell therapy for EBV-positive post transplant lymphoproliferative disease: results of a phase II multicentre clinical trial. Blood 110, 1123–1131. Ho, W.Y., Blattman, J.N., Dossett, M.L., Yee, C., Greenberg, P.D., 2003. Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction. Cancer Cell 3, 431–437. Hutt-Fletcher, L.M., 2007. Epstein-Barr virus entry. J. Virol. 81, 7825–7832. Kelly, G.L., Long, H.M., Stylianou, J., Thomas, W.A., Leese, A., Bell, A.I., Bornkamm, G.W., Mautner, J., Rickinson, A.B., Rowe, M., 2009. An Epstein-Barr virus antiapoptotic protein constitutively expressed in transformed cells and implicated in Burkitt lymphomagenesis: the Wp/BHRF1 link. PLoS Pathog. 5, e1000341.
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Milosevic, S., Behrends, U., Christoph, H., Mautner, J., 2005. Direct mapping of MHC class II epitopes. J. Immunol. Methods 306, 28–39. Moosmann, A., Bigalke, I., Tischer, J., Schirrmann, L., Kasten, J., Tippmer, S., Leeping, M., Prevalsek, D., Jaeger, G., Ledderose, G., et al., 2010. Effective and longterm control of EBV PTLD after transfer of peptide-selected T cells. Blood 115, 2960–2970. Moosmann, A., Khan, N., Cobbold, M., Zentz, C., Delecluse, H.J., Hollweck, G., Hislop, A.D., Blake, N.W., Croom-Carter, D., Wollenberg, B., et al., 2002. B cells immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells. Blood 100, 1755–1764. Munz, C., Bickham, K.L., Subklewe, M., Tsang, M.L., Chahroudi, A., Kurilla, M.G., Zhang, D., O’Donnell, M., Steinman, R.M., 2000. Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1. J. Exp. Med. 191, 1649–1660. Murray, R.J., Kurilla, M.G., Brooks, J.M., Thomas, W.A., Rowe, M., Kieff, E., Rickinson, A.B., 1992. 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. J. Exp. Med. 176, 157–168. Rickinson, A.B., Kieff, E., 2007. Epstein-Barr virus. In: Knipe, D.M., Howley, P.M. (Eds.), Field’s Virology. Lippincott Williams & Wilkins, Philadelphia, pp. 2575–2627. Rooney, C.M., Smith, C.A., Ng, C.Y., Loftin, S., Li, C., Krance, R.A., Brenner, M.K., Heslop, H.E., 1995. Use of gene-modified virus-specific T lymphocytes to control EpsteinBarr-virus-related lymphoproliferation. Lancet 345, 9–13. Rooney, C.M., Smith, C.A., Ng, C.Y., Loftin, S.K., Sixbey, J.W., Gan, Y., Srivastava, D.K., Bowman, L.C., Krance, R.A., Brenner, M.K., et al., 1998. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92, 1549–1555. Sebelin-Wulf, K., Nguyen, T.D., Oertel, S., Papp-Vary, M., Trappe, R.U., Schulzki, A., Pezzutto, A., Riess, H., Subklewe, M., 2007. Quantitative analysis of EBV-specific CD4/CD8 T cell numbers, absolute CD4/CD8 T cell numbers and EBV load in solid organ transplant recipients with PLTD. Transpl. Immunol. 17, 203–210. Smith, C.A., Ng, C.Y., Heslop, H.E., Holladay, M.S., Richardson, S., Turner, E.V., Loftin, S.K., Li, C., Brenner, M.K., Rooney, C.M., 1995. Production of genetically modified Epstein-Barr virus-specific cytotoxic T cells for adoptive transfer to patients at high risk of EBV-associated lymphoproliferative disease. J. Hematother. 4, 73–79. Steven, N.M., Annels, N.E., Kumar, A., Leese, A.M., Kurilla, M.G., Rickinson, A.B., 1997. Immediate early and early lytic cycle proteins are frequent targets of the EpsteinBarr virus-induced cytotoxic T cell response. J. Exp. Med. 185, 1605–1617. Tan, L.C., Gudgeon, N., Annels, N.E., Hansasuta, P., O’Callaghan, C.A., Rowland-Jones, S., McMichael, A.J., Rickinson, A.B., Callan, M.F., 1999. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J. Immunol. 162, 1827–1835. Taylor, G.S., Long, H.M., Haigh, T.A., Larsen, M., Brooks, J., Rickinson, A.B., 2006. A role for intercellular antigen transfer in the recognition of EBV-transformed B cell lines by EBV nuclear antigen-specific CD4+ T cells. J. Immunol. 177, 3746–3756. Tey, S.K., Bollard, C.M., Heslop, H.E., 2006. Adoptive T-cell transfer in cancer immunotherapy. Immunol. Cell Biol. 84, 281–289.
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European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb
Review
The role of virus-specific CD4+ T cells in the control of Epstein-Barr virus infection Josef Mautner ∗ , Georg W. Bornkamm Clinical Cooperation Group, Pediatric Tumor Immunology, Helmholtz-Zentrum München, Marchioninistr. 25, 81377 Munich, and Children’s Hospital, Technische Universität München, Kölner Platz 1, 80804 Munich, Germany
a r t i c l e
i n f o
Article history: Received 31 October 2010 Accepted 22 January 2011 Keywords: Epstein-Barr virus CD4+ T cell Antigen Immunodominance Therapy
a b s t r a c t Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans and has been implicated in the pathogenesis of several human malignancies. Protective immunity against EBV is mediated by T cells, as indicated by an increased incidence of EBV-associated malignancies in immunocompromised patients, and by the successful treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) in transplant recipients by the infusion of polyclonal EBV-specific T cell lines. To implement this treatment modality as a conventional therapeutic option, and to extend this protocol to other EBVassociated diseases, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. To this aim, we studied the poorly defined EBV-specific CD4+ T cell response during acute and chronic infection. © 2011 Elsevier GmbH. All rights reserved.
Introduction Epstein-Barr virus (EBV) is a ubiquitous human ␥-herpesvirus implicated in the pathogenesis of several malignancies of lymphoid and epithelial origin (Kieff and Rickinson, 2007; Kuppers, 2003; Rickinson and Kieff, 2007). Primary infection with EBV usually occurs within the first three years of life by parent-to-child oral transmission in an almost always asymptomatic fashion. Delayed primary infection in adolescence or adulthood may cause the syndrome of infectious mononucleosis (IM). Following oral transmission, the virus replicates in the oropharynx from where it colonizes the host by latently infecting B cells. The reservoir of latently infected B cells can seed foci of virus replication at mucosal sites, and this reactivation of the virus and subsequent re-infection of B lymphocytes allows the virus to persist for life in the infected human host (Kieff and Rickinson, 2007; Kuppers, 2003; Rickinson and Kieff, 2007). In B cells, EBV is able to establish different types of latency characterized by the expression of different sets of viral genes. During the primary phase of B cell infection, as well as in lymphoblastoid cell lines (LCL) generated by infection of B cells with EBV in vitro, the full range of viral latent cycle proteins is expressed that drive the activation and proliferation of the infected cell (Kelly et al., 2009; Kieff and Rickinson, 2007). In vivo, outgrowth of latently infected growth-transformed B cells is curtailed by T cells. The importance of T cell-mediated immune responses in maintaining asymptomatic viral persistence is emphasized by the
∗ Corresponding author at: Children’s Hospital, University of Technology, Kölner Platz 1, D-80804 Munich, Germany. Tel.: +49 89 7099518; fax: +49 89 7099500. E-mail address: [email protected] (J. Mautner). 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.01.007
clinical observation that patients with T cell dysfunction are at risk of developing life-threatening EBV-associated lymphoproliferative disease (Rickinson and Kieff, 2007). In solid organ and hematopoietic stem cell transplant recipients, the incidence of EBV-positive post-transplant lymphoproliferative disease (PTLD) correlates with the degree of the iatrogenically induced immunosuppression. Furthermore, EBV-positive PTLD in transplant recipients has been successfully treated by the infusion of polyclonal EBV-specific T cell lines containing CD4+ and CD8+ T cell components, that were generated by repeated stimulation of peripheral blood T cells with irradiated autologous LCL in vitro (Rooney et al., 1995, 1998). The targets of the EBV-specific cytotoxic CD8+ T cell response have been studied in detail and display a marked hierarchy in immunodominance with epitopes derived from the EBNA3 family of proteins and immediate early as well as early lytic cycle proteins usually inducing the strongest responses across a range of different HLA class I alleles. The same pattern of immunodominance was observed when the EBV latent epitope-specific CD8+ T cell memory was analyzed ex vivo, indicating that the EBV-specific T cell population expanded by LCL stimulation in vitro is an amplification of the in vivo correlate (Khanna and Burrows, 2000; Khanna et al., 1992; Landais et al., 2005b; Murray et al., 1992; Steven et al., 1997; Tan et al., 1999). Specificity of the CD4+ T cells in LCL-stimulated T cell lines Because the corresponding CD4+ T cell response is still poorly defined, we sought to study specificity and function of the EBVspecific CD4+ T cell response in LCL-stimulated T cell cultures. When tested for target-specific cytokine secretion, LCL-stimulated CD4+ T cell lines from healthy virus carriers recognized autologous
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as well as MHC class II-matched EBV-positive, but barely EBVnegative target cells, indicating that these lines were specific for EBV antigens (Adhikary et al., 2007). Of the T cell lines derived from patients with IM, about half displayed EBV-specificity by these criteria while the other half responded similarly against EBV-negative and EBV-positive target cells, indicating that these latter T cell lines predominantly recognized self-antigen(s). All T cell lines derived from virus-negative controls failed to respond against autologous LCL. These results demonstrated that EBV-specific CD4+ T cell memory is efficiently reactivated by LCL stimulation and excluded that de novo priming of EBV-specific CD4+ T cell responses occurred under these in vitro conditions (Adhikary et al., 2007). Surprisingly, except for weak responses against EBNA3C in a few lines, none of the EBV-specific CD4+ T cell lines recognized any of the latent cycle antigens of EBV. Such T cells had been detected in peripheral blood of healthy virus carriers by different groups (Khanna et al., 1995, 1997; Leen et al., 2001; Mautner et al., 2004; Munz et al., 2000), albeit at low frequency (Leen et al., 2001). Therefore, up to 50 restimulations were performed in order to facilitate the expansion of such rare T cell specificities to detectable levels. Again, no reactivity against any of the latent cycle antigens was detected in these late-passage T cell lines. Even the weak responses against EBNA3C had vanished. These results suggested that the LCL-stimulated CD4+ T cell cultures were dominated by CD4+ T cells targeting either lytic cycle antigens of EBV or cellular antigens induced by EBV. To discriminate between these possibilities, a genetically engineered mutant strain of EBV was used, that was still able to infect and growth-transform B cells into so-called miniLCL, but unable to enter the lytic cycle (Adhikary et al., 2006; Kempkes et al., 1995; Moosmann et al., 2002). MiniLCL were barely or not at all recognized by any of the CD4+ T cell lines that had shown EBV-specificity, indicating that these lines recognized lytic cycle proteins of the virus, or cellular proteins induced by viral lytic cycle proteins.
A bacterial expression cloning approach for direct antigen identification The EBV genome contains approximately 100 putative open reading frames, but neither the precise number nor the identity of all genes expressed during lytic replication is known (Kieff and Rickinson, 2007). To assess whether the EBV-specific CD4+ T cells recognized lytic cycle antigens, a method previously developed to rapidly map epitopes within proteins, was adapted to facilitate antigen identification within the whole viral genome (Milosevic et al., 2005). This so-called direct antigen identification (DANI) method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria which are subsequently fed to MHC class II+ antigen presenting cells and probed with antigen-specific T cells (Milosevic et al., 2006). EBV DNA was digested with frequently cutting restriction enzymes, the resulting short DNA fragments ligated into the expression vector mix, and the resulting EBV library plated at 60 cfu/well. With an average insert size of 82 bp, and an EBV plasmid size of 182 kb, a single 96-well plate covered the whole EBV genome approximately 2.5 times. The bacterial suspensions were added to autologous miniLCL which were subsequently probed with the EBV-specific T cells. Bacterial pools recognized by the T cells were plated on agar plates and single bacterial colonies tested in the same way. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of LCL-stimulated CD4+ T cells, demonstrating that unknown viral antigens are efficiently identified with this method and that lytic cycle antigens are the main targets of the EBV-specific CD4+ T cell response (Milosevic et al., 2006).
Table 1 Antigens recognized by LCL-stimulated CD4+ T cell lines. Donor
Dominant antigens
Subdominant antigens
IM1 IM2 IM5 GB JM DA MS SM MA TK
BcLF1a BXLF2a BNRF1a BNRF1a BALF2b BVRF2a BALF4a BALF2b , BNRF1a BMRF1b , BNRF1a BORF1a
BFRF3a , BXLF2a BDLF1b , BNRF1a BALF2b BXRF1a , BORF1a , BDLF1a , BBRF3a BDLF1a , BXRF1a , BALF4a BNRF1a , BCRF1a , BORF1a , EBNA3Cc EBNA3Cc BMRF1b Nd Nd
The antigens recognized by the EBV-reactive CD4+ cell lines from three patients with IM and seven healthy virus carriers were identified by using PBMC or miniLCL pulsed with single latent or lytic cycle proteins of EBV as targets. Responses against dominant antigens were maintained up to fifty restimulations, while responses against subdominant antigens were detected at early passages of the T cell lines only. No responses were detected against the immediate early antigens BZLF1 and BRLF1. Nd, not determined. a Late lytic cycle antigens. b Early lytic cycle proteins. c Latent cycle antigens.
Immunodominance of virion antigens Specificity as well as V chain analyses revealed that even after repeated rounds of stimulation in vitro, LCL-stimulated CD4+ T cell lines were still oligoclonal and recognized more than one antigen. To follow the evolution of the lines in vitro in more detail, all latent cycle proteins and 50 different lytic cycle genes of EBV, including the immediate early antigens BZLF1 and BRLF1, 23 early antigens, and 25 late antigens, were recombinantly expressed and purified. Autologous APC were pulsed with the various proteins and probed with the LCL-stimulated T cell lines after different passages. These experiments revealed that all T cell lines recognized at least one of the lytic cycle antigens tested (Adhikary et al., 2007). Except for the lytic cycle proteins BDLF1, BMRF1, BCRF1, BVRF2, and BALF2, all of these lytic cycle antigens were derived from virion proteins (Table 1). Moreover, all lines targeted at least one virion antigen. With the notable exception of the tegument protein BNRF1, which was recognized by six of the ten T cell lines tested, diverse sets of antigens were targeted. Moreover, T cell clones established from T cell lines stimulated more than 40 times often expressed different TCR-V chains and recognized more than one antigen. Thus, most LCL-stimulated CD4+ T cell lines from EBV-positive individuals had remained oligoclonal even beyond a year and a half in culture. These results suggested that the virus-specific CD4+ T cell response is not focused on one or a few immunodominant antigens, but is directed against a broad set of antigens, mostly derived from the group of structural proteins of EBV. The weak and transient responses against EBNA3C detected in two of the T cell lines established from healthy virus carriers implied that T cells specific for latent cycle antigens expanded under these in vitro culture conditions, albeit less efficiently than lytic cycle antigen-specific CD4+ T cells. However, when peripheral blood CD4+ T cells from healthy virus carriers were stimulated with miniLCL, which fail to express lytic cycle proteins, the resulting T cell lines recognized predominantly autoantigens and only sporadically latent cycle antigens of EBV, even after more than 25 passages (Adhikary et al., 2007). This was unexpected because all latent cycle proteins are expressed in LCL, and CD4+ T cells specific for latent antigens have been consistently detected in the peripheral blood of EBV-seropositive donors (Rickinson and Kieff, 2007). Thus, the targets of the LCL-stimulated CD4+ T cell response display a marked hierarchy in immunodominance: lytic cycle (structural) antigens > autoantigens > latent cycle antigens.
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Fig. 1. Efficient presentation of virion antigens following receptor-mediated uptake. EBV infection of B cells is initiated by the binding of the viral glycoprotein BLLF1 to CD21 on B cells. Following envelope fusion with the endosomal or the cell membrane, the nucleocapsid is released into the cytoplasm and the viral DNA eventually delivered into the cell nucleus. Proteins in the viral envelope are retained in the endosomal compartment, degraded, and peptide fragments loaded on MHC class II molecules for recognition by CD4+ T cells.
Immunodominance of structural antigens based on receptor-mediated antigen uptake of virions Given that usually only about 1% of cells in an LCL culture spontaneously become permissive for lytic replication and express lytic cycle proteins, while all express latent cycle proteins, this immunodominance of structural antigens was surprising. First insights into the factors and mechanisms dictating this pattern of immunodominance were obtained when different EBVspecific CD4+ T cell clones were assayed for target cell recognition. Although all T cell clones recognized their cognate peptide antigens with similar affinity, only CD4+ T cells specific for glycoproteins, but not for the transcription factor BZLF1, recognized autologous as well as HLA-matched allogeneic EBV-infected target cells (Adhikary et al., 2006). Since miniLCL were not recognized, T cell recognition was probably dependent on sporadic lytic replication occurring in a low percentage of cells in culture. However, when the percentage of cells in an LCL culture that were recognized by the T cells was determined, a substantially higher number was obtained than those positive for lytic cycle proteins in immunofluorescence studies. Co-culture experiments of HLA-mismatched LCL and HLAmatched miniLCL indicated that the antigen was transferred from one cell type to the other. Several lines of evidence suggested that the antigen transferred between cells was derived from released virions. First, miniLCL pulsed with purified virus preparations were recognized by the T cells, demonstrating that virions can serve as the source of the antigen. Because miniLCL pulsed with heatinactivated virus were still recognized, T cell recognition did not depend on productive infection of the target cells. Second, the antigen was most efficiently transferred to B cells and this transfer was blocked by BLLF1- and CD21-specific monoclonal antibodies, implying that antigen uptake is receptor-mediated. Third, the failure of BZLF1-specific T cells to recognize LCL, and the failure of virion-specific T cells to recognize autologous dendritic cells
(DC) co-cultured with HLA-mismatched LCL demonstrated that the amount of antigen released by dead or dying cells was not sufficient for T cell detection (Adhikary et al., 2006). Besides structural proteins, some LCL-stimulated CD4+ T cells had also targeted lytic cycle proteins that are not constituents of virions. In the case of BMRF1, presentation involved intercellular antigen transfer, probably by release of protein from lytically infected cells and uptake as exogenous protein by neighboring cells as described previously for other EBV proteins (Landais et al., 2005a; Taylor et al., 2006). Why these but not other lytic cycle proteins such as BZLF1 are efficiently transferred between cells is not known, but might reflect quantitative differences in protein expression levels, protein stability and/or the timing of expression later in the lytic cycle which might facilitate release from cells. Likewise, the impaired expansion of latent cycle antigen-specific CD4+ T cells in LCL-stimulated culture might also be the consequence of inefficient presentation of epitopes derived from latent cycle proteins on MHC class II. The observation that mainly B cells, whether primary or EBVinfected, presented virion proteins most efficiently implicated a protective role of such T cells in controlling the spread of infection. Importantly, virion-specific CD4+ T cells recognized target cells pulsed with virus supernatant before the EBV genome has circularized and before EBNA2, the protein essential for primary B cell growth-transformation, is expressed. EBV infection of B cells is initiated by BLLF1 adsorption to CD21 on the B cell plasma membrane, followed by viral endocytosis and envelope fusion with the endosomal or cell membrane and nucleocapsid exocytosis into the cytoplasm (Hutt-Fletcher, 2007). During this process of virus uncoating, proteins of the viral envelope are probably retained at the cell membrane, as indicated by the higher percentage of cells positive for BLLF1 than for BZLF1 in immunofluorescence experiments (Lee et al., 1993). Endosomal and cell membrane proteins efficiently access the MHC class II processing and loading
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compartment, thereby allowing CD4+ T cells specific for envelope antigens to detect EBV-infected cells before viral latency is established. This receptor-mediated virion uptake and subsequent presentation of virion protein-derived peptides on MHC class II turned out to be extremely efficient. BALF4- and BLLF1-specific T cells were able to recognize B cells incubated with less than one EBV genome equivalent per cell (Adhikary et al., 2006, 2008). Thus, virion proteins retained in endosomes or the cell surface during virus uncoating label newly EBV-infected B cells for immune attack by CD4+ T cells specific for virion proteins (Fig. 1). A potential role of virion antigen-specific CD4+ T cells in the control of EBV infection Importantly, virion antigen-specific CD4+ T cells proved to be cytolytic and able to prevent the outgrowth of primary B cells infected with EBV in vitro. Thus, by diminishing the pool of EBVinfected B cells, virion antigen-specific CD4+ T cells might aid in controlling EBV infection in vivo. In fact, recent results from preclinical models indicate that EBVspecific CD4+ T cells are effective against PTLD-like tumors even in the absence of CD8+ T cells, and that the CD4+ and CD8+ T cell components of LCL-stimulated T cell preparations are equally efficacious in controlling PTLD in mice (Merlo et al., 2010, and unpublished results). Furthermore, an important role of CD4+ T cells in establishing antiviral immunity has been inferred from clinical experience; low numbers of endogenous CD4+ T cells have been identified as an important risk factor for the development of EBV-associated diseases in immunosuppressed patients (Sebelin-Wulf et al., 2007), and patients with PTLD showed better clinical responses in a phase II trial when the infused LCL-stimulated T cell lines contained higher proportions of CD4+ T cells (Haque et al., 2007). Finally, in a patient with PTLD showing complete and stable remission after infusion of EBV peptides-selected T lymphocytes, CD4+ T cells specific for a peptide derived from the virion protein BNRF1 had expanded significantly, suggesting that these T cells contributed to the antitumoral immune response in vivo (Moosmann et al., 2010). Collectively, these findings suggest that EBV-specific CD4+ and CD8+ T cells complement each other by targeting different phases of the virus’ life cycle. Standardized and efficient expansion of virion-specific CD4+ T cells using virus-like particles For unknown reasons, the CD4/CD8 ratio in LCL-stimulated T cell preparations can vary from 2:98 to 98:2 (Smith et al., 1995). Because EBV-specific CD4+ T cells in LCL-stimulated T cell preparations are almost exclusively directed against structural antigens of the virus (Adhikary et al., 2007), which are efficiently presented on MHC II following receptor-mediated uptake of released viral particles, we investigated which factors compromised the expansion of EBV-specific CD4+ T cells in LCL-stimulated T-cell preparations. These experiments showed that spontaneous virus production by LCL and, hence, the presentation of viral antigens, varies intraand interindividually. Virus production is further impaired by acyclovir treatment of LCL, which is performed in most currently applied clinical protocols to suppress virus production in stimulator LCL to minimize residual infectious viral particles within adoptively transferred T cells. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4+ T cells. All CD4+ T cell lines that were repeatedly stimulated with LCL grown in FCSsupplemented media eventually showed FCS-reactivity, even lines established from patients with acute IM who are expected to have
a strong antiviral T cell response (Adhikary et al., 2008). Given that more than 90% of the adult population is EBV-seropositive and carries virus-neutralizing antibodies that diminish virion uptake, supplementing media with human serum may result in reduced presentation of virion antigens. These findings indicated that addition of excess amounts of EBV particles may antagonize the inhibitory effect of human serum and compensate for differences in virus production by different LCLs, and thereby facilitate to establish uniform and standardized stimulation conditions. Because incubation of stimulator cells with wild-type EBV would pose an incalculable health risk to patients, the possibility of using genome-deficient Epstein-Barr virus-like particles (EB-VLP) was explored. EB-VLP produced by human cells in serum-free media are readily available in large quantities and transfer structural antigens as efficiently as wild-type EBV. Instead of LCL, PBMC pulsed with EB-VLP were used as stimulators because PBMC do not produce virus and are immediately available. This strategy facilitated the specific and rapid expansion of EBV-specific CD4+ T cells and, thus, might contribute to the development of standardized protocols for the generation of T cell lines with improved clinical efficacy (Adhikary et al., 2008). Conclusions The reconstitution of EBV-specific immunity in transplant patients by the adoptive transfer of polyclonal virus-specific T cell lines has provided important proof of principle for immunotherapy of EBV-associated tumors, and for cancer immunotherapy in general (Foster and Rooney, 2006; Gattinoni et al., 2006; Ho et al., 2003; Tey et al., 2006). Given the significant burden of EBV-associated tumors worldwide, important future goals of this adoptive T cell therapy are the introduction into mainstream clinical practice and the extension to EBV-associated tumor entities other than PTLD (Gottschalk et al., 2005; Tey et al., 2006). Thus, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. A better understanding of the critical immune effector cells and the relevant immune targets may eventually expedite the preparation of T cell lines and improve the clinical effectiveness of this form of immunotherapy and, hence, the long term survival of patients. References Adhikary, D., Behrends, U., Boerschmann, H., Pfunder, A., Burdach, S., Moosmann, A., Witter, K., Bornkamm, G.W., Mautner, J., 2007. Immunodominance of lytic cycle antigens in Epstein-Barr virus-specific CD4+ T cell preparations for therapy. PLoS One 2, e583. Adhikary, D., Behrends, U., Feederle, R., Delecluse, H.J., Mautner, J., 2008. Standardized and highly efficient expansion of Epstein-Barr virus-specific CD4+ T cells by using virus-like particles. J. Virol. 82, 3903–3911. Adhikary, D., Behrends, U., Moosmann, A., Witter, K., Bornkamm, G.W., Mautner, J., 2006. Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins. J. Exp. Med. 203, 995–1006. Foster, A.E., Rooney, C.M., 2006. Improving T cell therapy for cancer. Expert Opin. Biol. Ther. 6, 215–229. Gattinoni, L., Powell Jr., D.J., Rosenberg, S.A., Restifo, N.P., 2006. Adoptive immunotherapy for cancer: building on success. Nat. Rev. Immunol. 6, 383–393. Gottschalk, S., Heslop, H.E., Rooney, C.M., 2005. Adoptive immunotherapy for EBVassociated malignancies. Leuk. Lymphoma 46, 1–10. Haque, T., Wilkie, G.M., Jones, M.M., Higgins, C.D., Urquhart, G., Wingate, P., Burns, D., McAulay, K., Turner, M., Bellamy, C., et al., 2007. Allogeneic cytotoxic T cell therapy for EBV-positive post transplant lymphoproliferative disease: results of a phase II multicentre clinical trial. Blood 110, 1123–1131. Ho, W.Y., Blattman, J.N., Dossett, M.L., Yee, C., Greenberg, P.D., 2003. Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction. Cancer Cell 3, 431–437. Hutt-Fletcher, L.M., 2007. Epstein-Barr virus entry. J. Virol. 81, 7825–7832. Kelly, G.L., Long, H.M., Stylianou, J., Thomas, W.A., Leese, A., Bell, A.I., Bornkamm, G.W., Mautner, J., Rickinson, A.B., Rowe, M., 2009. An Epstein-Barr virus antiapoptotic protein constitutively expressed in transformed cells and implicated in Burkitt lymphomagenesis: the Wp/BHRF1 link. PLoS Pathog. 5, e1000341.
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