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Immune defence against EBV and EBV-associated disease
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Immune defence against EBV and EBV-associated disease Heather M Long, Graham S Taylor and Alan B Rickinson Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus widespread in the human population and normally contained as an asymptomatic infection by T cell surveillance, nevertheless causes infectious mononucleosis and is strongly linked to several types of human cancer. Here we describe new findings on the range of cellular immune responses induced by EBV infection, on viral strategies to evade those responses and on the links between HLA gene loci and EBVinduced disease. The success of adoptive T cell therapy for EBV-driven post-transplant lymphoproliferative disease is stimulating efforts to target other EBV-associated tumours by immunotherapeutic means, and has reawakened interest in the ultimate intervention strategy, a prophylactic EBV vaccine. Address School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Corresponding author: Rickinson, Alan B ([email protected])
Current Opinion in Immunology 2011, 23:258–264 This review comes from a themed issue on Tumour immunology Edited by Olivera Finn and Cornelis Melief Available online 25th January 2011 0952-7915/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2010.12.014
Introduction Epstein-Barr virus (EBV), a gamma-1 herpesvirus that has co-evolved with our species over millions of years, is found widespread in the human population as a lifelong and largely asymptomatic infection. Acquired orally, the virus replicates in permissive (probably epithelial) cells in the oropharynx but persists as a latent infection of the B cell pool, from which reactivations into lytic cycle continually seed sub-clinical foci of oral shedding. Notwithstanding its benign appearance, EBV has growth-transforming ability and is aetiologically linked to three B cell malignancies, Burkitt Lymphoma (BL), Hodgkin Lymphoma (HL) and post-transplant lymphoproliferative disease (PTLD), to a subset of T and NK cell lymphomas, and to an epithelial tumour, nasopharyngeal carcinoma (NPC). EBV’s dual character, as classical herpesvirus and tumour virus, has long fascinated immunologists [1]. Here we summarise recent progress in that field. Current Opinion in Immunology 2011, 23:258–264
Immune responses to primary and persistent infection Unlike the situation in childhood, around 25% delayed primary infections present as infectious mononucleosis (IM) where the disease symptoms (lymphadenopathy, fever and malaise) are by-products of a highly amplified CD8+ T cell response largely directed against EBV lytic and, to a lesser extent, latent cycle proteins. Using tetramer staining to follow response kinetics over time, recent studies have shown how phenotypic markers such as programmed death 1 (PD-1, transiently up-regulated by antigen stimulation and re-expressed with functional exhaustion) and IL7 receptor-alpha (CD127, transiently down-regulated by antigen stimulation) can distinguish between epitope responses of different sizes [2] and with different long-term fates [3]. There are coincident CD4 responses to EBV in IM but these are much smaller [4] and, as yet, poorly characterised. It is interesting to compare the lytic cycle-specific CD8 response, which is strongly skewed towards immediate early and certain early proteins (i.e. antigens presented by infected cells before immune evasion effects take hold, as described below), and the corresponding CD4 response which is widely spread across immediate early, early and late antigen targets [(5,6, H.M. Long et al., unpublished)]. This supports the view that, while the CD4 response to this B-lymphotropic virus is conventionally cross-primed, the CD8 response is largely driven by direct contact with infected cells. We know very little about the role of innate immune responses early in primary infection; however, three pieces of circumstantial evidence suggest NK cell involvement. First, the recent hints that EBV has NK evasion functions (see below). Second, CD56dim NK cells from human tonsils (likely the first lymphoid tissue exposed to orally transmitted virus) can be primed in vitro by coresident dendritic cells to produce interferon-gamma in amounts capable of delaying EBV-driven B cell transformation [7]. Third, the specific susceptibility to EBV of patients with X-linked lymphoproliferative syndrome (XLP), a rare immunodeficiency that abrogates NK/T cell development and impairs NK cell and T cell interactions with B cells [8]; many patients develop an acute, often fatal, IM-like illness following EBV infection, with massive expansions of infected B cells and reactive T cells. This likely reflects an initial failure of NK (and possibly NK/T) cell controls followed by large but ineffective T cell responses; thus XLP patients do develop EBV-specific CD8+ T cells but they, like XLP NK cells, recognise target B cells very poorly [9]. Infected B cells therefore continue to expand, as do the reactive T www.sciencedirect.com
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cells because, lacking efficient receptor engagement, they do not receive the activation-induced cell death signals that normally regulate response size [10].
Viral evasion of immune responses Prompted by findings that antigen presentation to CD8+ T cells falls dramatically during lytic cycle, recent studies [reviewed in [11]] have identified three EBV early lytic proteins with immune evasive effects: BNLF2A binds to the TAP transporter, inhibiting peptide delivery to nascent HLA molecules [12–14], BILF1 binds to HLA I, impairing the export of new complexes and enhancing the lysosomal degradation of existing complexes from the surface [15,16], while BGLF5 reduces new HLA antigen expression as part of its general host shut-off function [17]. The collective effect, lowering surface HLA I levels, alerts lytically infected cells to NK recognition; however, one of the EBV micro-RNAs (miR-BART2) has recently been shown to reduce expression of the NK activating ligand MicB [18] and more NK evasion measures may yet exist. Interestingly EBV also targets the HLA II pathway during lytic cycle, via the immediate early protein, BZLF1, downregulating expression of the class II transactivator CIITA [19]. Among the latent proteins, the latent membrane protein LMP1 actually upregulates HLA I processing yet is itself a poor CD8 T cell target, a property correlating with its ability to self-aggregate [20]. In EBV-transformed B-lymphoblastoid cell lines (LCLs), LMP1 content per individual cell fluctuates because the protein drives both its own synthesis and degradation [21]; parallel fluctuations in antigen processing efficiency means that, at any one time, only a fraction of cells in an LCL culture are visible to CD8+ T cells [22]. Another latent protein, the nuclear antigen EBNA1, has long been a focus of attention because its glycine-alanine repeat (GAr) domain impairs HLA Irestricted presentation of cis-linked epitopes. Recent work suggests that this is achieved by EBNA1 decelerating its own synthesis, either transcriptionally via interference of the nascent GAr-positive protein with ribosome assembly on the mRNA [23,24], or translationally via sub-optimal codon usage within the GAr sequence [25]. Whatever the mechanism, the GAr effect is only partial because, in its natural setting, GAr reduces the presentation of native EBNA1 epitopes by just 30%, similar to its reduction in synthesis rate. This is consistent with the HLA I pathway being fed by newly synthesised antigen, whereas supplying endogenously expressed antigen to the HLA II pathway requires establishment of a mature protein pool [26]. Interestingly, EBNA1 also accesses the HLA II pathway poorly in LCL cells; this is not because of any GArmediated effect but rather the native antigen’s nuclear location, since redirecting expression to the cytoplasm greatly increases EBNA1 CD4 epitope display, with the antigen being delivered into the HLA II processing pathway via autophagy [27]. www.sciencedirect.com
T cell responses, HLA polymorphism and susceptibility to EBV disease The frequency with which T cell-suppressed transplant patients develop PTLD testifies to the importance of T cell surveillance against EBV growth-transforming infections. However, other EBV-associated tumours arising in apparently immunocompetent individuals have more complex aetiologies, involving cellular genetic changes that EBV complements by expressing subsets of latent proteins, typically EBNA1 only in BL, EBNA1 + LMP1 + LMP2 in HL and EBNA1 + LMP2 LMP1 in NPC. Might specific deficiencies in EBV surveillance underpin disease risk in these settings? There are reports of sub-optimal CD4 responses to EBNA1 in BL and HL patients [28,29] and CD8 responses to EBNA1 or LMP2 in NPC patients [30,31], but whether these are a cause or an effect of the disease process is unclear. More interesting is the growing evidence associating HLA polymorphism with disease risk. A seminal paper linking microsatellite markers near the HLA-A locus to increased risk of EBV-positive but not EBV-negative HL [32] has been followed by two larger studies mapping this dose-dependent effect to HLA-A allele identity, with HLA-A*0101 increasing risk (odds ratio >2 per allele dose) and HLA-A*0201 reducing risk (odds ratio = 0.7 per allele dose) [33,34]. This is intriguing since no A*0101restricted epitopes have ever been identified in EBV [1,35], whereas A*0201 presents several LMP2 epitopes [1]. Subsequent work showed that these same microsatellite markers were also associated with an increased risk of IM [36]. However the logical prediction, that A*0101 would be over-represented and A*0201 underrepresented in IM cohorts, has so far not been confirmed [34]. Equally fascinating are recent results of large genome-wide association studies into NPC risk in Chinese populations [37,38]. While a link to the HLA locus has long been suggested in NPC, these genome-wide studies clearly show that markers around the HLA-A gene locus have by far the strongest association with tumour risk. Furthermore, linkage of these markers with HLA identity strengthens the evidence for HLA-A*0207 as a risk allele and for HLA-A*1101 as a protective allele; the latter is particularly interesting since, in Chinese people, most A*1101-restricted responses are directed against a welldefined LMP2 epitope [1].
Targeting EBV-positive tumours with LCLstimulated T cell preparations
As recently reviewed [39,40], adoptive transfer of LCLstimulated EBV-specific T cell preparations (EBVCTLs) has proved a remarkably successful therapy for classical PTLD, the lesions of which (like LCLs) are composed of cells expressing the full range of EBV latent proteins plus some cells entering lytic cycle. Overall, among haemopoietic stem cell transplant recipients, allograft donor-derived EBV-CTLs induced complete Current Opinion in Immunology 2011, 23:258–264
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remission in 11/13 PTLD patients, while none of 101 high risk patients treated prophylactically developed PTLD compared to 11% of historic controls [39]. In the solid organ transplant setting, 33 patients with refractory PTLD received third-party EBV-CTLs chosen on a best HLA-match basis; response rates were 52% at 6 months and 12/14 of the patients who achieved complete remission at that time remained disease-free after 4–9 years [40]. Furthermore, despite in vitro evidence that such EBV-CTL preparations include frequent cross-reactivities against allogeneic HLA molecules [41], no cases of graft-versus-host disease have been observed after thirdparty EBV-CTL infusion [40,42,43]. Third-party preparations could therefore become particularly important in transplants using cord blood as a stem cell source [44] since it is difficult to generate virus-specific T cells from EBV-naive cord blood samples in vitro [45]. Because LCL-stimulated effectors tend to be dominated by CD8+ T cells against the EBNA3A,B,C latent proteins, using such effectors against other EBV-positive tumours is problematic since the tumours typically do not express these immunodominant targets [1]. Nevertheless some success has been reported in the context of NPC using this approach [46]. Likewise LCL-stimulated effectors known to be dominated by EBNA3 reactivities induced complete remissions in 5/6 patients with EBNA3-negative HL-like or BL-like tumours [47,48]. Interestingly the more successful EBV-CTL preparations tended to be those with polyclonal T cell receptor usage, implying requirement for a broadly targeted T cell response [47], and with significant numbers of CD4+ T cells [48]. While this may reflect the importance of CD4 help in adoptively transferred preparations, CD4+ T cells may also be acting as effectors in their own right. Thus LCL-stimulated preparations have been shown to contain cytotoxic CD4+ T cells capable of LCL recognition and specific either for EBV latent and lytic proteins [6,49,50] or for cellular antigens whose expression is up-regulated by EBV infection [51].
New methods of generating EBV-specific effectors for tumour therapy Optimising T cell therapies for EBV-positive tumours with limited antigen expression focuses attention on EBNA1 and the LMPs as target antigens. Of these, the richest source of CD8 epitopes, LMP2, is expressed in EBV-positive HL and NPC; furthermore EBV-positive T/NK lymphomas, which express very low levels of fulllength LMP2 transcripts by standard RT-PCR assays, are now known to express an alternative transcript encoding a protein that still contains all known target epitopes [52]. CD4+ T cells are also potential effectors against such tumours with the description of cytotoxic clones against CD4 epitopes in LMP1 [53,54], LMP2 [53] and EBNA1 [55], the latter with proven activity against T/NK lymphoma cells in vitro. Various methods have been designed Current Opinion in Immunology 2011, 23:258–264
to produce effector populations enriched in appropriate specificities, for example by ex vivo selection with EBVspecific pentamers [56] or by in vitro stimulation either with epitope peptides [57,58], with the autologous LCL over-expressing the particular antigen of choice [59], or with an adenovirus encoding EBNA1/LMP1/LMP2 epitope strings [60] or a scrambled EBNA1, LMP1, LMP2 protein sequence [61]. Such chimaeric constructs could also be used to boost relevant antigen-specific responses in the patient by vaccination. Indeed a Modified Vaccinia Ankara (MVA) recombinant expressing the CD4 epitoperich C-terminal fragment of EBNA1 and the full length sequence of LMP2 [62] has just completed Phase I trials in NPC patients with clear evidence of dose-dependent CD4 and CD8 response induction [E.P. Hui et al.; G.S. Taylor et al., unpublished]. An important further development involves genetically manipulating these EBV-specific preparations to render them therapeutically more effective in vivo. Firstly, one aims to maximise the chances of adoptively transferred cells expanding in their new host. One approach being tested in SCID mouse models is to express IL7 receptoralpha (CD127) [63] or IL15 [64] in the cells in order to exploit cytokine circuits that naturally mediate T cell expansion and/or survival. Such manipulations could be combined with prior lymphodepletion of the patient, a procedure designed to make immunological space for expansion that is now being tested in clinical trials in the NPC setting [65]. Secondly, the effector populations need to be able to traffic to the tumour site and resist the immunosuppressive environment that may well exist there. HL provides a useful example in this context, since the malignant Hodgkin–Reed Sternberg (HRS) cells appear to employ multiple immunosuppressive strategies. These range from expression of the PD-1 ligand, which has the capacity to inhibit activated PD1-positive effector cells [66], secretion of the CCL17 and CCL22 chemokines inducing local infiltration of TH2like and regulatory T cells [67], and production of other immunosuppressive cytokines such as IL-10, TGF-b [67], and the immunoregulatory glycan-binding protein, galectin-1 [68], to which EBNA1 and LMP-specific T cells appear selectively susceptible [69]. Several manipulations of adoptively transferred cells are being tested in response to these challenges. For example, one might turn tumour-homing signals to therapeutic advantage by expressing the relevant CCL17/CCL22 receptor CCR4 in the infused cells [70] or overcome local TGF-b-mediated suppression through introduction of a dominant negative TGF-bRII receptor [71]. Finally, there are many situations where T cells are being transferred into patients who are themselves on immunosuppressive therapy, typically involving calcineurin inhibitors such as tacrolimus (FK506) and cyclosporin A (CSA). This has prompted recent studies in which www.sciencedirect.com
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EBV-specific T cell preparations have been genetically manipulated to express a calcineurin mutant protein [72] or used as targets for siRNA knock-down of tacrolimus binding protein (FKBP12) [73], in both cases conferring functional resistance to these immunosuppressive drugs. Note that many of the above genetic modifications have been achieved by retroviral transfer and, as the work moves from the laboratory into the clinic, the issue of safety will become increasingly important. Interestingly, some of the early studies in PTLD therapy actually involved polyclonal EBV-CTL preparations retrovirally transduced with a neomycin transferase gene marker; to date, such cells have been detected in patients up to 105 months after infusion, without adverse effect and without evidence of any neo-marked clone becoming numerically dominant [39].
Vaccination against EBV infection and EBVassociated disease The burden of disease currently linked to EBV infection, namely IM, the various EBV-associated malignancies and possibly also an autoimmune response, multiple sclerosis [74], is fuelling renewed interest in the possibility of a prophylactic vaccine to prevent EBV infection. In recent clinical trials a viral envelope glycoprotein (gp340)-based vaccine, designed primarily to induce neutralising antibody responses, did not protect young EBV-naı¨ve adults from primary infection but did appear to reduce the risk of that leading to IM [75,76]. The challenge now is to incorporate appropriate T cell target antigens into such a vaccine construct, thereby rendering the T cell response able to recognise and destroy the de novo B cell infections upon which establishment of the virus carrier state depends. One of the barriers to such a programme in the past has been the total reliance upon primates as animal models of gamma-1 herpesvirus infection. Very encouragingly, the ability to reconstitute immunodeficient mouse strains with a functional human immune system is now providing new models which not only allow EBV colonisation of the B cell system [77] but also support the induction of EBV-specific CD4+ and CD8+ T cell responses [78,79]. This clears an important obstacle on the road to developing an effective EBV vaccine.
Acknowledgements The authors would like to acknowledge grant funding from the Medical Research Council, Cancer Research UK, Leukaemia & Lymphoma Research and the Gregor MacKay Memorial Fund.
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48. Haque T, Wilkie GM, Jones MM, Higgins CD, Urquhart G, Wingate P, Burns D, McAulay K, Turner M, Bellamy C et al.: Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood 2007, 110:1123-1131. Results of the first trial of adoptive T cell therapy for PTLD using 3rd party donor EBV-CTLs.
63. Vera JF, Hoyos V, Savoldo B, Quintarelli C, Giordano Attianese GM, Leen AM, Liu H, Foster AE, Heslop HE, Rooney CM et al.: Genetic manipulation of tumor-specific cytotoxic T lymphocytes to restore responsiveness to IL-7. Mol Ther 2009, 17:880-888.
49. Adhikary D, Behrends U, Boerschmann H, Pfunder A, Burdach S, Moosmann A, Witter K, Bornkamm GW, Mautner J: Immunodominance of lytic cycle antigens in Epstein-Barr virus-specific CD4+ T cell preparations for therapy. PLoS One 2007, 2:e583.
64. Hoyos V, Savoldo B, Quintarelli C, Mahendravada A, Zhang M, Vera J, Heslop HE, Rooney CM, Brenner MK, Dotti G: Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 2010, 24:1160-1170.
50. Taylor GS, Long HM, Haigh TA, Larsen M, Brooks J, Rickinson AB: 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 2006, 177:3746-3756.
65. Louis CU, Straathof K, Bollard CM, Gerken C, Huls MH, Gresik MV, Wu MF, Weiss HL, Gee AP, Brenner MK et al.: Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients. Blood 2009, 113:2442-2450. First paper indicating that prior lymphodepletion enhances in vivo expansion of EBV-CTLs adoptively transferred in a non-stem cell transplant patient setting.
51. Long HM, Zuo J, Leese AM, Gudgeon NH, Jia H, Taylor GS, Rickinson AB: CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus-transformed B cells. Blood 2009, 114:807-815. 52. Fox CP, Haigh TA, Taylor GS, Long HM, Lee SP, Shannon-Lowe C, O’Connor S, Bollard CM, Iqbal J, Chan WC et al.: A novel latent membrane 2 transcript expressed in Epstein-Barr viruspositive NK- and T-cell lymphoproliferative disease encodes a target for cellular immunotherapy. Blood 2010, 116:3695-3704. 53. Haigh TA, Lin X, Jia H, Hui EP, Chan AT, Rickinson AB, Taylor GS: EBV latent membrane proteins (LMPs) 1 and 2 as immunotherapeutic targets: LMP-specific CD4+ cytotoxic T cell recognition of EBV-transformed B cell lines. J Immunol 2008, 180:1643-1654. 54. Kobayashi H, Nagato T, Takahara M, Sato K, Kimura S, Aoki N, Azumi M, Tateno M, Harabuchi Y, Celis E: Induction of EBVlatent membrane protein 1-specific MHC class II-restricted Tcell responses against natural killer lymphoma cells. Cancer Res 2008, 68:901-908. 55. Demachi-Okamura A, Ito Y, Akatsuka Y, Tsujimura K, Morishima Y, Takahashi T, Kuzushima K: Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells. Cancer Sci 2008, 99:1633-1642. 56. Uhlin M, Okas M, Gertow J, Uzunel M, Brismar TB, Mattsson J: A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation. Cancer Immunol Immunother 2010, 59:473-477. 57. Jones K, Nourse JP, Morrison L, Nguyen-Van D, Moss DJ, Burrows SR, Gandhi MK: Expansion of EBNA1-specific effector T cells in posttransplantation lymphoproliferative disorders. Blood 2010, 116:2245-2252.
66. Yamamoto R, Nishikori M, Kitawaki T, Sakai T, Hishizawa M, Tashima M, Kondo T, Ohmori K, Kurata M, Hayashi T et al.: PD-1PD-1 ligand interaction contributes to immunosuppressive microenvironment of Hodgkin lymphoma. Blood 2008, 111:3220-3224. 67. Aldinucci D, Gloghini A, Pinto A, De Filippi R, Carbone A: The classical Hodgkin’s lymphoma microenvironment and its role in promoting tumour growth and immune escape. J Pathol 2010, 221:248-263. 68. Gandhi MK, Wilkie GM, Dua U, Mollee PN, Grimmett K, Williams T, Whitaker N, Gill D, Crawford DH: Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders. Am J Transplant 2007, 7:1293-1299. 69. Smith C, Beagley L, Khanna R: Acquisition of polyfunctionality by Epstein-Barr virus-specific CD8+ T cells correlates with increased resistance to galectin-1-mediated suppression. J Virol 2009, 83:6192-6198. 70. Di Stasi A, De Angelis B, Rooney CM, Zhang L, Mahendravada A, Foster AE, Heslop HE, Brenner MK, Dotti G, Savoldo B: T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood 2009, 113:6392-6402. 71. Foster AE, Dotti G, Lu A, Khalil M, Brenner MK, Heslop HE, Rooney CM, Bollard CM: Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor. J Immunother 2008, 31:500-505.
58. Moosmann A, Bigalke I, Tischer J, Schirrmann L, Kasten J, Tippmer S, Leeping M, Prevalsek D, Jaeger G, Ledderose G et al.: Effective and long-term control of EBV PTLD after transfer of peptide-selected T cells. Blood 2010, 115:2960-2970.
72. Brewin J, Mancao C, Straathof K, Karlsson H, Samarasinghe S, Amrolia PJ, Pule M: Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease. Blood 2009, 114:4792-4803.
59. Bollard CM, Gottschalk S, Leen AM, Weiss H, Straathof KC, Carrum G, Khalil M, Wu MF, Huls MH, Chang CC et al.: Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and Tlymphocyte transfer. Blood 2007, 110:2838-2845.
73. De Angelis B, Dotti G, Quintarelli C, Huye LE, Zhang L, Zhang M, Pane F, Heslop HE, Brenner MK, Rooney CM et al.: Generation of Epstein-Barr-virus-specific cytotoxic T lymphocytes resistant to the immunosuppressive drug tacrolimus (FK506). Blood 2009.
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74. Levin LI, Munger KL, O’Reilly EJ, Falk KI, Ascherio A: Primary infection with the Epstein-Barr virus and risk of multiple sclerosis. Ann Neurol 2010, 67:824-830. The most recent in a series of papers providing strong epidemiological evidence of a link between EBV infection and MS risk. 75. Moutschen M, Leonard P, Sokal EM, Smets F, Haumont M, Mazzu P, Bollen A, Denamur F, Peeters P, Dubin G et al.: Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults. Vaccine 2007, 25:4697-4705. 76. Sokal EM, Hoppenbrouwers K, Vandermeulen C, Moutschen M, Leonard P, Moreels A, Haumont M, Bollen A, Smets F, Denis M: Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults. J Infect Dis 2007, 196:1749-1753. 77. Cocco M, Bellan C, Tussiwand R, Corti D, Traggiai E, Lazzi S, Mannucci S, Bronz L, Palummo N, Ginanneschi C et al.:
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CD34+ cord blood cell-transplanted Rag2S/S gamma(c)S/S mice as a model for Epstein-Barr virus infection. Am J Pathol 2008, 173:1369-1378. 78. Strowig T, Gurer C, Ploss A, Liu YF, Arrey F, Sashihara J, Koo G, Rice CM, Young JW, Chadburn A et al.: Priming of protective T cell responses against virus-induced tumors in mice with human immune system components. J Exp Med 2009, 206:1423-1434. This paper along with Ref. [79] highlighting the potential to study human T cell responses to experimental EBV infection in humanised mouse models. 79. Shultz LD, Saito Y, Najima Y, Tanaka S, Ochi T, Tomizawa M, Doi T, Sone A, Suzuki N, Fujiwara H et al.: Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice. Proc Natl Acad Sci U S A 2010, 107:13022-13027. See annotation to Ref. [78].
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Immune defence against EBV and EBV-associated disease Heather M Long, Graham S Taylor and Alan B Rickinson Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus widespread in the human population and normally contained as an asymptomatic infection by T cell surveillance, nevertheless causes infectious mononucleosis and is strongly linked to several types of human cancer. Here we describe new findings on the range of cellular immune responses induced by EBV infection, on viral strategies to evade those responses and on the links between HLA gene loci and EBVinduced disease. The success of adoptive T cell therapy for EBV-driven post-transplant lymphoproliferative disease is stimulating efforts to target other EBV-associated tumours by immunotherapeutic means, and has reawakened interest in the ultimate intervention strategy, a prophylactic EBV vaccine. Address School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Corresponding author: Rickinson, Alan B ([email protected])
Current Opinion in Immunology 2011, 23:258–264 This review comes from a themed issue on Tumour immunology Edited by Olivera Finn and Cornelis Melief Available online 25th January 2011 0952-7915/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2010.12.014
Introduction Epstein-Barr virus (EBV), a gamma-1 herpesvirus that has co-evolved with our species over millions of years, is found widespread in the human population as a lifelong and largely asymptomatic infection. Acquired orally, the virus replicates in permissive (probably epithelial) cells in the oropharynx but persists as a latent infection of the B cell pool, from which reactivations into lytic cycle continually seed sub-clinical foci of oral shedding. Notwithstanding its benign appearance, EBV has growth-transforming ability and is aetiologically linked to three B cell malignancies, Burkitt Lymphoma (BL), Hodgkin Lymphoma (HL) and post-transplant lymphoproliferative disease (PTLD), to a subset of T and NK cell lymphomas, and to an epithelial tumour, nasopharyngeal carcinoma (NPC). EBV’s dual character, as classical herpesvirus and tumour virus, has long fascinated immunologists [1]. Here we summarise recent progress in that field. Current Opinion in Immunology 2011, 23:258–264
Immune responses to primary and persistent infection Unlike the situation in childhood, around 25% delayed primary infections present as infectious mononucleosis (IM) where the disease symptoms (lymphadenopathy, fever and malaise) are by-products of a highly amplified CD8+ T cell response largely directed against EBV lytic and, to a lesser extent, latent cycle proteins. Using tetramer staining to follow response kinetics over time, recent studies have shown how phenotypic markers such as programmed death 1 (PD-1, transiently up-regulated by antigen stimulation and re-expressed with functional exhaustion) and IL7 receptor-alpha (CD127, transiently down-regulated by antigen stimulation) can distinguish between epitope responses of different sizes [2] and with different long-term fates [3]. There are coincident CD4 responses to EBV in IM but these are much smaller [4] and, as yet, poorly characterised. It is interesting to compare the lytic cycle-specific CD8 response, which is strongly skewed towards immediate early and certain early proteins (i.e. antigens presented by infected cells before immune evasion effects take hold, as described below), and the corresponding CD4 response which is widely spread across immediate early, early and late antigen targets [(5,6, H.M. Long et al., unpublished)]. This supports the view that, while the CD4 response to this B-lymphotropic virus is conventionally cross-primed, the CD8 response is largely driven by direct contact with infected cells. We know very little about the role of innate immune responses early in primary infection; however, three pieces of circumstantial evidence suggest NK cell involvement. First, the recent hints that EBV has NK evasion functions (see below). Second, CD56dim NK cells from human tonsils (likely the first lymphoid tissue exposed to orally transmitted virus) can be primed in vitro by coresident dendritic cells to produce interferon-gamma in amounts capable of delaying EBV-driven B cell transformation [7]. Third, the specific susceptibility to EBV of patients with X-linked lymphoproliferative syndrome (XLP), a rare immunodeficiency that abrogates NK/T cell development and impairs NK cell and T cell interactions with B cells [8]; many patients develop an acute, often fatal, IM-like illness following EBV infection, with massive expansions of infected B cells and reactive T cells. This likely reflects an initial failure of NK (and possibly NK/T) cell controls followed by large but ineffective T cell responses; thus XLP patients do develop EBV-specific CD8+ T cells but they, like XLP NK cells, recognise target B cells very poorly [9]. Infected B cells therefore continue to expand, as do the reactive T www.sciencedirect.com
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cells because, lacking efficient receptor engagement, they do not receive the activation-induced cell death signals that normally regulate response size [10].
Viral evasion of immune responses Prompted by findings that antigen presentation to CD8+ T cells falls dramatically during lytic cycle, recent studies [reviewed in [11]] have identified three EBV early lytic proteins with immune evasive effects: BNLF2A binds to the TAP transporter, inhibiting peptide delivery to nascent HLA molecules [12–14], BILF1 binds to HLA I, impairing the export of new complexes and enhancing the lysosomal degradation of existing complexes from the surface [15,16], while BGLF5 reduces new HLA antigen expression as part of its general host shut-off function [17]. The collective effect, lowering surface HLA I levels, alerts lytically infected cells to NK recognition; however, one of the EBV micro-RNAs (miR-BART2) has recently been shown to reduce expression of the NK activating ligand MicB [18] and more NK evasion measures may yet exist. Interestingly EBV also targets the HLA II pathway during lytic cycle, via the immediate early protein, BZLF1, downregulating expression of the class II transactivator CIITA [19]. Among the latent proteins, the latent membrane protein LMP1 actually upregulates HLA I processing yet is itself a poor CD8 T cell target, a property correlating with its ability to self-aggregate [20]. In EBV-transformed B-lymphoblastoid cell lines (LCLs), LMP1 content per individual cell fluctuates because the protein drives both its own synthesis and degradation [21]; parallel fluctuations in antigen processing efficiency means that, at any one time, only a fraction of cells in an LCL culture are visible to CD8+ T cells [22]. Another latent protein, the nuclear antigen EBNA1, has long been a focus of attention because its glycine-alanine repeat (GAr) domain impairs HLA Irestricted presentation of cis-linked epitopes. Recent work suggests that this is achieved by EBNA1 decelerating its own synthesis, either transcriptionally via interference of the nascent GAr-positive protein with ribosome assembly on the mRNA [23,24], or translationally via sub-optimal codon usage within the GAr sequence [25]. Whatever the mechanism, the GAr effect is only partial because, in its natural setting, GAr reduces the presentation of native EBNA1 epitopes by just 30%, similar to its reduction in synthesis rate. This is consistent with the HLA I pathway being fed by newly synthesised antigen, whereas supplying endogenously expressed antigen to the HLA II pathway requires establishment of a mature protein pool [26]. Interestingly, EBNA1 also accesses the HLA II pathway poorly in LCL cells; this is not because of any GArmediated effect but rather the native antigen’s nuclear location, since redirecting expression to the cytoplasm greatly increases EBNA1 CD4 epitope display, with the antigen being delivered into the HLA II processing pathway via autophagy [27]. www.sciencedirect.com
T cell responses, HLA polymorphism and susceptibility to EBV disease The frequency with which T cell-suppressed transplant patients develop PTLD testifies to the importance of T cell surveillance against EBV growth-transforming infections. However, other EBV-associated tumours arising in apparently immunocompetent individuals have more complex aetiologies, involving cellular genetic changes that EBV complements by expressing subsets of latent proteins, typically EBNA1 only in BL, EBNA1 + LMP1 + LMP2 in HL and EBNA1 + LMP2 LMP1 in NPC. Might specific deficiencies in EBV surveillance underpin disease risk in these settings? There are reports of sub-optimal CD4 responses to EBNA1 in BL and HL patients [28,29] and CD8 responses to EBNA1 or LMP2 in NPC patients [30,31], but whether these are a cause or an effect of the disease process is unclear. More interesting is the growing evidence associating HLA polymorphism with disease risk. A seminal paper linking microsatellite markers near the HLA-A locus to increased risk of EBV-positive but not EBV-negative HL [32] has been followed by two larger studies mapping this dose-dependent effect to HLA-A allele identity, with HLA-A*0101 increasing risk (odds ratio >2 per allele dose) and HLA-A*0201 reducing risk (odds ratio = 0.7 per allele dose) [33,34]. This is intriguing since no A*0101restricted epitopes have ever been identified in EBV [1,35], whereas A*0201 presents several LMP2 epitopes [1]. Subsequent work showed that these same microsatellite markers were also associated with an increased risk of IM [36]. However the logical prediction, that A*0101 would be over-represented and A*0201 underrepresented in IM cohorts, has so far not been confirmed [34]. Equally fascinating are recent results of large genome-wide association studies into NPC risk in Chinese populations [37,38]. While a link to the HLA locus has long been suggested in NPC, these genome-wide studies clearly show that markers around the HLA-A gene locus have by far the strongest association with tumour risk. Furthermore, linkage of these markers with HLA identity strengthens the evidence for HLA-A*0207 as a risk allele and for HLA-A*1101 as a protective allele; the latter is particularly interesting since, in Chinese people, most A*1101-restricted responses are directed against a welldefined LMP2 epitope [1].
Targeting EBV-positive tumours with LCLstimulated T cell preparations
As recently reviewed [39,40], adoptive transfer of LCLstimulated EBV-specific T cell preparations (EBVCTLs) has proved a remarkably successful therapy for classical PTLD, the lesions of which (like LCLs) are composed of cells expressing the full range of EBV latent proteins plus some cells entering lytic cycle. Overall, among haemopoietic stem cell transplant recipients, allograft donor-derived EBV-CTLs induced complete Current Opinion in Immunology 2011, 23:258–264
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remission in 11/13 PTLD patients, while none of 101 high risk patients treated prophylactically developed PTLD compared to 11% of historic controls [39]. In the solid organ transplant setting, 33 patients with refractory PTLD received third-party EBV-CTLs chosen on a best HLA-match basis; response rates were 52% at 6 months and 12/14 of the patients who achieved complete remission at that time remained disease-free after 4–9 years [40]. Furthermore, despite in vitro evidence that such EBV-CTL preparations include frequent cross-reactivities against allogeneic HLA molecules [41], no cases of graft-versus-host disease have been observed after thirdparty EBV-CTL infusion [40,42,43]. Third-party preparations could therefore become particularly important in transplants using cord blood as a stem cell source [44] since it is difficult to generate virus-specific T cells from EBV-naive cord blood samples in vitro [45]. Because LCL-stimulated effectors tend to be dominated by CD8+ T cells against the EBNA3A,B,C latent proteins, using such effectors against other EBV-positive tumours is problematic since the tumours typically do not express these immunodominant targets [1]. Nevertheless some success has been reported in the context of NPC using this approach [46]. Likewise LCL-stimulated effectors known to be dominated by EBNA3 reactivities induced complete remissions in 5/6 patients with EBNA3-negative HL-like or BL-like tumours [47,48]. Interestingly the more successful EBV-CTL preparations tended to be those with polyclonal T cell receptor usage, implying requirement for a broadly targeted T cell response [47], and with significant numbers of CD4+ T cells [48]. While this may reflect the importance of CD4 help in adoptively transferred preparations, CD4+ T cells may also be acting as effectors in their own right. Thus LCL-stimulated preparations have been shown to contain cytotoxic CD4+ T cells capable of LCL recognition and specific either for EBV latent and lytic proteins [6,49,50] or for cellular antigens whose expression is up-regulated by EBV infection [51].
New methods of generating EBV-specific effectors for tumour therapy Optimising T cell therapies for EBV-positive tumours with limited antigen expression focuses attention on EBNA1 and the LMPs as target antigens. Of these, the richest source of CD8 epitopes, LMP2, is expressed in EBV-positive HL and NPC; furthermore EBV-positive T/NK lymphomas, which express very low levels of fulllength LMP2 transcripts by standard RT-PCR assays, are now known to express an alternative transcript encoding a protein that still contains all known target epitopes [52]. CD4+ T cells are also potential effectors against such tumours with the description of cytotoxic clones against CD4 epitopes in LMP1 [53,54], LMP2 [53] and EBNA1 [55], the latter with proven activity against T/NK lymphoma cells in vitro. Various methods have been designed Current Opinion in Immunology 2011, 23:258–264
to produce effector populations enriched in appropriate specificities, for example by ex vivo selection with EBVspecific pentamers [56] or by in vitro stimulation either with epitope peptides [57,58], with the autologous LCL over-expressing the particular antigen of choice [59], or with an adenovirus encoding EBNA1/LMP1/LMP2 epitope strings [60] or a scrambled EBNA1, LMP1, LMP2 protein sequence [61]. Such chimaeric constructs could also be used to boost relevant antigen-specific responses in the patient by vaccination. Indeed a Modified Vaccinia Ankara (MVA) recombinant expressing the CD4 epitoperich C-terminal fragment of EBNA1 and the full length sequence of LMP2 [62] has just completed Phase I trials in NPC patients with clear evidence of dose-dependent CD4 and CD8 response induction [E.P. Hui et al.; G.S. Taylor et al., unpublished]. An important further development involves genetically manipulating these EBV-specific preparations to render them therapeutically more effective in vivo. Firstly, one aims to maximise the chances of adoptively transferred cells expanding in their new host. One approach being tested in SCID mouse models is to express IL7 receptoralpha (CD127) [63] or IL15 [64] in the cells in order to exploit cytokine circuits that naturally mediate T cell expansion and/or survival. Such manipulations could be combined with prior lymphodepletion of the patient, a procedure designed to make immunological space for expansion that is now being tested in clinical trials in the NPC setting [65]. Secondly, the effector populations need to be able to traffic to the tumour site and resist the immunosuppressive environment that may well exist there. HL provides a useful example in this context, since the malignant Hodgkin–Reed Sternberg (HRS) cells appear to employ multiple immunosuppressive strategies. These range from expression of the PD-1 ligand, which has the capacity to inhibit activated PD1-positive effector cells [66], secretion of the CCL17 and CCL22 chemokines inducing local infiltration of TH2like and regulatory T cells [67], and production of other immunosuppressive cytokines such as IL-10, TGF-b [67], and the immunoregulatory glycan-binding protein, galectin-1 [68], to which EBNA1 and LMP-specific T cells appear selectively susceptible [69]. Several manipulations of adoptively transferred cells are being tested in response to these challenges. For example, one might turn tumour-homing signals to therapeutic advantage by expressing the relevant CCL17/CCL22 receptor CCR4 in the infused cells [70] or overcome local TGF-b-mediated suppression through introduction of a dominant negative TGF-bRII receptor [71]. Finally, there are many situations where T cells are being transferred into patients who are themselves on immunosuppressive therapy, typically involving calcineurin inhibitors such as tacrolimus (FK506) and cyclosporin A (CSA). This has prompted recent studies in which www.sciencedirect.com
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EBV-specific T cell preparations have been genetically manipulated to express a calcineurin mutant protein [72] or used as targets for siRNA knock-down of tacrolimus binding protein (FKBP12) [73], in both cases conferring functional resistance to these immunosuppressive drugs. Note that many of the above genetic modifications have been achieved by retroviral transfer and, as the work moves from the laboratory into the clinic, the issue of safety will become increasingly important. Interestingly, some of the early studies in PTLD therapy actually involved polyclonal EBV-CTL preparations retrovirally transduced with a neomycin transferase gene marker; to date, such cells have been detected in patients up to 105 months after infusion, without adverse effect and without evidence of any neo-marked clone becoming numerically dominant [39].
Vaccination against EBV infection and EBVassociated disease The burden of disease currently linked to EBV infection, namely IM, the various EBV-associated malignancies and possibly also an autoimmune response, multiple sclerosis [74], is fuelling renewed interest in the possibility of a prophylactic vaccine to prevent EBV infection. In recent clinical trials a viral envelope glycoprotein (gp340)-based vaccine, designed primarily to induce neutralising antibody responses, did not protect young EBV-naı¨ve adults from primary infection but did appear to reduce the risk of that leading to IM [75,76]. The challenge now is to incorporate appropriate T cell target antigens into such a vaccine construct, thereby rendering the T cell response able to recognise and destroy the de novo B cell infections upon which establishment of the virus carrier state depends. One of the barriers to such a programme in the past has been the total reliance upon primates as animal models of gamma-1 herpesvirus infection. Very encouragingly, the ability to reconstitute immunodeficient mouse strains with a functional human immune system is now providing new models which not only allow EBV colonisation of the B cell system [77] but also support the induction of EBV-specific CD4+ and CD8+ T cell responses [78,79]. This clears an important obstacle on the road to developing an effective EBV vaccine.
Acknowledgements The authors would like to acknowledge grant funding from the Medical Research Council, Cancer Research UK, Leukaemia & Lymphoma Research and the Gregor MacKay Memorial Fund.
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Hislop AD, Palendira U, Leese AM, Arkwright PD, Rohrlich PS, Tangye SG, Gaspar HB, Lankester AC, Moretta A, Rickinson AB: Impaired Epstein-Barr virus-specific CD8+ T-cell function in X-linked lymphoproliferative disease is restricted to SLAM family-positive B-cell targets. Blood 2010, 116:3249-3257. Demonstrates selective impairment of B cell target recognition by EBVspecific CD8+ T cells from XLP patients. 10. Snow AL, Marsh RA, Krummey SM, Roehrs P, Young LR, Zhang K, van Hoff J, Dhar D, Nichols KE, Filipovich AH et al.: Restimulationinduced apoptosis of T cells is impaired in patients with Xlinked lymphoproliferative disease caused by SAP deficiency. J Clin Invest 2009, 119:2976-2989. 11. Ressing ME, Horst D, Griffin BD, Tellam J, Zuo J, Khanna R, Rowe M, Wiertz EJ: Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of multiple gene products. Semin Cancer Biol 2008, 18:397-408. A review of recent advances in the fast-moving field of EBV evasion of CD8+ immunity. 12. Hislop AD, Ressing ME, van Leeuwen D, Pudney VA, Horst D, Koppers-Lalic D, Croft NP, Neefjes JJ, Rickinson AB, Wiertz EJ: A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates. J Exp Med 2007, 204:1863-1873. 13. Horst D, van Leeuwen D, Croft NP, Garstka MA, Hislop AD, Kremmer E, Rickinson AB, Wiertz EJ, Ressing ME: Specific targeting of the EBV lytic phase protein BNLF2a to the transporter associated with antigen processing results in impairment of HLA class I-restricted antigen presentation. J Immunol 2009, 182:2313-2324. 14. Croft NP, Shannon-Lowe C, Bell AI, Horst D, Kremmer E, Ressing ME, Wiertz EJ, Middeldorp JM, Rowe M, Rickinson AB et al.: Stage-specific inhibition of MHC class I presentation by the Epstein-Barr virus BNLF2a protein during virus lytic cycle. PLoS Pathog 2009, 5:e1000490. 15. Zuo J, Currin A, Griffin BD, Shannon-Lowe C, Thomas WA, Ressing ME, Wiertz EJ, Rowe M: The Epstein-Barr virus Gprotein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation. PLoS Pathog 2009, 5:e1000255. Current Opinion in Immunology 2011, 23:258–264
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16. Zuo J, Quinn LL, Tamblyn J, Thomas WA, Feederle R, Delecluse HJ, Hislop AD, Rowe M: The Epstein-Barr virusencoded BILF1 protein modulates immune recognition of endogenously processed antigen by targeting MHC class I molecules trafficking on both the exocytic and endocytic pathways. J Virol 2010. 17. Zuo J, Thomas W, van Leeuwen D, Middeldorp JM, Wiertz EJ, Ressing ME, Rowe M: The DNase of gammaherpesviruses impairs recognition by virus-specific CD8+ T cells through an additional host shutoff function. J Virol 2008, 82:2385-2393. 18. Nachmani D, Stern-Ginossar N, Sarid R, Mandelboim O: Diverse herpesvirus microRNAs target the stress-induced immune ligand MICB to escape recognition by natural killer cells. Cell Host Microbe 2009, 5:376-385. 19. Li D, Qian L, Chen C, Shi M, Yu M, Hu M, Song L, Shen B, Guo N: Down-regulation of MHC class II expression through inhibition of CIITA transcription by lytic transactivator Zta during Epstein-Barr virus reactivation. J Immunol 2009, 182:1799-1809. 20. Smith C, Wakisaka N, Crough T, Peet J, Yoshizaki T, Beagley L, Khanna R: Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation. Blood 2009, 113:6148-6152. 21. Lee DY, Sugden B: The latent membrane protein 1 oncogene modifies B-cell physiology by regulating autophagy. Oncogene 2008, 27:2833-2842. 22. Brooks JM, Lee SP, Leese AM, Thomas WA, Rowe M, Rickinson AB: Cyclical expression of EBV latent membrane protein 1 in EBV-transformed B cells underpins heterogeneity of epitope presentation and CD8+ T cell recognition. J Immunol 2009, 182:1919-1928. 23. Apcher S, Daskalogianni C, Manoury B, Fahraeus R: Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation. PLoS Pathog 2010, 6:e1001151. 24. Apcher S, Komarova A, Daskalogianni C, Yin Y, Malbert-Colas L, Fahraeus R: mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1. J Virol 2009, 83:1289-1298. 25. Tellam J, Smith C, Rist M, Webb N, Cooper L, Vuocolo T, Connolly G, Tscharke DC, Devoy MP, Khanna R: Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition. Proc Natl Acad Sci U S A 2008, 105:9319-9324. Evidence that sub-optimal codon usage in the GAr coding sequence of EBNA1, by slowing protein translation, reduces CD8 epitope display in cis. 26. Mackay LK, Long HM, Brooks JM, Taylor GS, Leung CS, Chen A, Wang F, Rickinson AB: T cell detection of a B-cell tropic virus infection: newly-synthesised versus mature viral proteins as antigen sources for CD4 and CD8 epitope display. PLoS Pathog 2009, 5:e1000699. 27. Leung CS, Haigh TA, Mackay LK, Rickinson AB, Taylor GS: Nuclear location of an endogenously expressed antigen. EBNA1, restricts access to macroautophagy and the range of CD4 epitope display. Proc Natl Acad Sci U S A 2010, 107:2165-2170. 28. Heller KN, Arrey F, Steinherz P, Portlock C, Chadburn A, Kelly K, Munz C: Patients with Epstein Barr virus-positive lymphomas have decreased CD4(+) T-cell responses to the viral nuclear antigen 1. Int J Cancer 2008, 123:2824-2831. 29. Moormann AM, Heller KN, Chelimo K, Embury P, Ploutz-Snyder R, Otieno JA, Oduor M, Munz C, Rochford R: Children with endemic Burkitt lymphoma are deficient in EBNA1-specific IFN-gamma T cell responses. Int J Cancer 2009, 124:1721-1726. 30. Fogg MH, Wirth LJ, Posner M, Wang F: Decreased EBNA-1specific CD8+ T cells in patients with Epstein-Barr virusassociated nasopharyngeal carcinoma. Proc Natl Acad Sci U S A 2009, 106:3318-3323. Current Opinion in Immunology 2011, 23:258–264
31. Lin X, Gudgeon NH, Hui EP, Jia H, Qun X, Taylor GS, Barnardo MC, Lin CK, Rickinson AB, Chan AT: CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients. Cancer Immunol Immunother 2008, 57:963-975. 32. Diepstra A, Niens M, Vellenga E, van Imhoff GW, Nolte IM, Schaapveld M, van der Steege G, van den Berg A, Kibbelaar RE, te Meerman GJ et al.: Association with HLA class I in EpsteinBarr-virus-positive and with HLA class III in Epstein-Barrvirus-negative Hodgkin’s lymphoma. Lancet 2005, 365:2216-2224. The first evidence (based on microsatellite marker analysis) that HLA-A allele polymorphism specifically influences EBV-positive HL risk. 33. Niens M, Jarrett RF, Hepkema B, Nolte IM, Diepstra A, Platteel M, Kouprie N, Delury CP, Gallagher A, Visser L et al.: HLA-A*02 is associated with a reduced risk and HLA-A*01 with an increased risk of developing EBV+ Hodgkin lymphoma. Blood 2007, 110:3310-3315. 34. Hjalgrim H, Rostgaard K, Johnson PC, Lake A, Shield L, Little AM, Ekstrom-Smedby K, Adami HO, Glimelius B, Hamilton-Dutoit S et al.: HLA-A alleles and infectious mononucleosis suggest a critical role for cytotoxic T-cell response in EBV-related Hodgkin lymphoma. Proc Natl Acad Sci U S A 2010, 107:64006405. 35. Brennan RM, Burrows SR: A mechanism for the HLA-A*01associated risk for EBV+ Hodgkin lymphoma and infectious mononucleosis. Blood 2008, 112:2589-2590. 36. McAulay KA, Higgins CD, Macsween KF, Lake A, Jarrett RF, Robertson FL, Williams H, Crawford DH: HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection. J Clin Invest 2007, 117:3042-3048. Evidence that IM risk is linked to the same microsatellite markers in the HLA locus as described in ref 31 for EBV-positive HL risk. 37. Tse KP, Su WH, Chang KP, Tsang NM, Yu CJ, Tang P, See LC, Hsueh C, Yang ML, Hao SP et al.: Genome-wide association study reveals multiple nasopharyngeal carcinoma-associated loci within the HLA region at chromosome 6p21.3. Am J Hum Genet 2009, 85:194-203. 38. Bei JX, Li Y, Jia WH, Feng BJ, Zhou G, Chen LZ, Feng QS, Low HQ, Zhang H, He F et al.: A genome-wide association study of nasopharyngeal carcinoma identifies three new susceptibility loci. Nat Genet 2010, 42:599-603. 39. Heslop HE, Slobod KS, Pule MA, Hale GA, Rousseau A, Smith CA, Bollard CM, Liu H, Wu MF, Rochester RJ et al.: Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients. Blood 2010, 115:925-935. An important summary of 15 years’ clinical experience treating PTLD in stem cell transplant patients with EBV-CTL preparations. 40. Haque T, McAulay KA, Kelly D, Crawford DH: Allogeneic T-cell therapy for Epstein-Barr virus-positive posttransplant lymphoproliferative disease: long-term follow-up. Transplantation 2010, 90:93-94. 41. Amir AL, D’Orsogna LJ, Roelen DL, van Loenen MM, Hagedoorn RS, de Boer R, van der Hoorn MA, Kester MG, Doxiadis II, Falkenburg JH et al.: Allo-HLA reactivity of virusspecific memory T cells is common. Blood 2010, 115:3146-3157. 42. Melenhorst JJ, Leen AM, Bollard CM, Quigley MF, Price DA, Rooney CM, Brenner MK, Barrett AJ, Heslop HE: Allogeneic virus-specific T cells with HLA alloreactivity do not produce GVHD in human subjects. Blood 2010, 116:4700-4702. 43. Barker JN, Doubrovina E, Sauter C, Jaroscak JJ, Perales MA, Doubrovin M, Prockop SE, Koehne G, O’Reilly RJ: Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBVspecific cytotoxic T lymphocytes. Blood 2010, 116:5045-5049. 44. Stanevsky A, Goldstein G, Nagler A: Umbilical cord blood transplantation: pros, cons and beyond. Blood Rev 2009, 23:199-204. www.sciencedirect.com
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45. Hanley PJ, Cruz CR, Savoldo B, Leen AM, Stanojevic M, Khalil M, Decker W, Molldrem JJ, Liu H, Gee AP et al.: Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes. Blood 2009, 114:1958-1967.
60. Duraiswamy J, Bharadwaj M, Tellam J, Connolly G, Cooper L, Moss D, Thomson S, Yotnda P, Khanna R: Induction of therapeutic T-cell responses to subdominant tumorassociated viral oncogene after immunization with replication-incompetent polyepitope adenovirus vaccine. Cancer Res 2004, 64:1483-1489.
46. Louis CU, Straathof K, Bollard CM, Ennamuri S, Gerken C, Lopez TT, Huls MH, Sheehan A, Wu MF, Liu H et al.: Adoptive transfer of EBV-specific T cells results in sustained clinical responses in patients with locoregional nasopharyngeal carcinoma. J Immunother 2010, 33:983-990.
61. Lutzky VP, Corban M, Heslop L, Morrison LE, Crooks P, Hall DF, Coman WB, Thomson SA, Moss DJ: Novel approach to the formulation of an Epstein-Barr virus antigen-based nasopharyngeal carcinoma vaccine. J Virol 2010, 84:407-417.
47. McAulay KA, Haque T, Urquhart G, Bellamy C, Guiretti D, Crawford DH: Epitope specificity and clonality of EBV-specific CTLs used to treat posttransplant lymphoproliferative disease. J Immunol 2009, 182:3892-3901.
62. Taylor GS, Haigh TA, Gudgeon NH, Phelps RJ, Lee SP, Steven NM, Rickinson AB: 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. J Virol 2004, 78:768-778.
48. Haque T, Wilkie GM, Jones MM, Higgins CD, Urquhart G, Wingate P, Burns D, McAulay K, Turner M, Bellamy C et al.: Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood 2007, 110:1123-1131. Results of the first trial of adoptive T cell therapy for PTLD using 3rd party donor EBV-CTLs.
63. Vera JF, Hoyos V, Savoldo B, Quintarelli C, Giordano Attianese GM, Leen AM, Liu H, Foster AE, Heslop HE, Rooney CM et al.: Genetic manipulation of tumor-specific cytotoxic T lymphocytes to restore responsiveness to IL-7. Mol Ther 2009, 17:880-888.
49. Adhikary D, Behrends U, Boerschmann H, Pfunder A, Burdach S, Moosmann A, Witter K, Bornkamm GW, Mautner J: Immunodominance of lytic cycle antigens in Epstein-Barr virus-specific CD4+ T cell preparations for therapy. PLoS One 2007, 2:e583.
64. Hoyos V, Savoldo B, Quintarelli C, Mahendravada A, Zhang M, Vera J, Heslop HE, Rooney CM, Brenner MK, Dotti G: Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 2010, 24:1160-1170.
50. Taylor GS, Long HM, Haigh TA, Larsen M, Brooks J, Rickinson AB: 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 2006, 177:3746-3756.
65. Louis CU, Straathof K, Bollard CM, Gerken C, Huls MH, Gresik MV, Wu MF, Weiss HL, Gee AP, Brenner MK et al.: Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients. Blood 2009, 113:2442-2450. First paper indicating that prior lymphodepletion enhances in vivo expansion of EBV-CTLs adoptively transferred in a non-stem cell transplant patient setting.
51. Long HM, Zuo J, Leese AM, Gudgeon NH, Jia H, Taylor GS, Rickinson AB: CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus-transformed B cells. Blood 2009, 114:807-815. 52. Fox CP, Haigh TA, Taylor GS, Long HM, Lee SP, Shannon-Lowe C, O’Connor S, Bollard CM, Iqbal J, Chan WC et al.: A novel latent membrane 2 transcript expressed in Epstein-Barr viruspositive NK- and T-cell lymphoproliferative disease encodes a target for cellular immunotherapy. Blood 2010, 116:3695-3704. 53. Haigh TA, Lin X, Jia H, Hui EP, Chan AT, Rickinson AB, Taylor GS: EBV latent membrane proteins (LMPs) 1 and 2 as immunotherapeutic targets: LMP-specific CD4+ cytotoxic T cell recognition of EBV-transformed B cell lines. J Immunol 2008, 180:1643-1654. 54. Kobayashi H, Nagato T, Takahara M, Sato K, Kimura S, Aoki N, Azumi M, Tateno M, Harabuchi Y, Celis E: Induction of EBVlatent membrane protein 1-specific MHC class II-restricted Tcell responses against natural killer lymphoma cells. Cancer Res 2008, 68:901-908. 55. Demachi-Okamura A, Ito Y, Akatsuka Y, Tsujimura K, Morishima Y, Takahashi T, Kuzushima K: Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells. Cancer Sci 2008, 99:1633-1642. 56. Uhlin M, Okas M, Gertow J, Uzunel M, Brismar TB, Mattsson J: A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation. Cancer Immunol Immunother 2010, 59:473-477. 57. Jones K, Nourse JP, Morrison L, Nguyen-Van D, Moss DJ, Burrows SR, Gandhi MK: Expansion of EBNA1-specific effector T cells in posttransplantation lymphoproliferative disorders. Blood 2010, 116:2245-2252.
66. Yamamoto R, Nishikori M, Kitawaki T, Sakai T, Hishizawa M, Tashima M, Kondo T, Ohmori K, Kurata M, Hayashi T et al.: PD-1PD-1 ligand interaction contributes to immunosuppressive microenvironment of Hodgkin lymphoma. Blood 2008, 111:3220-3224. 67. Aldinucci D, Gloghini A, Pinto A, De Filippi R, Carbone A: The classical Hodgkin’s lymphoma microenvironment and its role in promoting tumour growth and immune escape. J Pathol 2010, 221:248-263. 68. Gandhi MK, Wilkie GM, Dua U, Mollee PN, Grimmett K, Williams T, Whitaker N, Gill D, Crawford DH: Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders. Am J Transplant 2007, 7:1293-1299. 69. Smith C, Beagley L, Khanna R: Acquisition of polyfunctionality by Epstein-Barr virus-specific CD8+ T cells correlates with increased resistance to galectin-1-mediated suppression. J Virol 2009, 83:6192-6198. 70. Di Stasi A, De Angelis B, Rooney CM, Zhang L, Mahendravada A, Foster AE, Heslop HE, Brenner MK, Dotti G, Savoldo B: T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood 2009, 113:6392-6402. 71. Foster AE, Dotti G, Lu A, Khalil M, Brenner MK, Heslop HE, Rooney CM, Bollard CM: Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor. J Immunother 2008, 31:500-505.
58. Moosmann A, Bigalke I, Tischer J, Schirrmann L, Kasten J, Tippmer S, Leeping M, Prevalsek D, Jaeger G, Ledderose G et al.: Effective and long-term control of EBV PTLD after transfer of peptide-selected T cells. Blood 2010, 115:2960-2970.
72. Brewin J, Mancao C, Straathof K, Karlsson H, Samarasinghe S, Amrolia PJ, Pule M: Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease. Blood 2009, 114:4792-4803.
59. Bollard CM, Gottschalk S, Leen AM, Weiss H, Straathof KC, Carrum G, Khalil M, Wu MF, Huls MH, Chang CC et al.: Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and Tlymphocyte transfer. Blood 2007, 110:2838-2845.
73. De Angelis B, Dotti G, Quintarelli C, Huye LE, Zhang L, Zhang M, Pane F, Heslop HE, Brenner MK, Rooney CM et al.: Generation of Epstein-Barr-virus-specific cytotoxic T lymphocytes resistant to the immunosuppressive drug tacrolimus (FK506). Blood 2009.
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74. Levin LI, Munger KL, O’Reilly EJ, Falk KI, Ascherio A: Primary infection with the Epstein-Barr virus and risk of multiple sclerosis. Ann Neurol 2010, 67:824-830. The most recent in a series of papers providing strong epidemiological evidence of a link between EBV infection and MS risk. 75. Moutschen M, Leonard P, Sokal EM, Smets F, Haumont M, Mazzu P, Bollen A, Denamur F, Peeters P, Dubin G et al.: Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults. Vaccine 2007, 25:4697-4705. 76. Sokal EM, Hoppenbrouwers K, Vandermeulen C, Moutschen M, Leonard P, Moreels A, Haumont M, Bollen A, Smets F, Denis M: Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults. J Infect Dis 2007, 196:1749-1753. 77. Cocco M, Bellan C, Tussiwand R, Corti D, Traggiai E, Lazzi S, Mannucci S, Bronz L, Palummo N, Ginanneschi C et al.:
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CD34+ cord blood cell-transplanted Rag2S/S gamma(c)S/S mice as a model for Epstein-Barr virus infection. Am J Pathol 2008, 173:1369-1378. 78. Strowig T, Gurer C, Ploss A, Liu YF, Arrey F, Sashihara J, Koo G, Rice CM, Young JW, Chadburn A et al.: Priming of protective T cell responses against virus-induced tumors in mice with human immune system components. J Exp Med 2009, 206:1423-1434. This paper along with Ref. [79] highlighting the potential to study human T cell responses to experimental EBV infection in humanised mouse models. 79. Shultz LD, Saito Y, Najima Y, Tanaka S, Ochi T, Tomizawa M, Doi T, Sone A, Suzuki N, Fujiwara H et al.: Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice. Proc Natl Acad Sci U S A 2010, 107:13022-13027. See annotation to Ref. [78].
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