CD4 and CD8 T cell responses to tumour-associated Epstein–Barr virus antigens in nasopharyngeal carcinoma patients

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-γ release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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

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

Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses

Human CD4(+) T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8(+) memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4(+) epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4(+) T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4(+) T-helper 1 cells (EBNA1, EBNA3C > LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8(+)-T-cell responses (EBNA3C > EBNA1 > LMP2 > LMP1). Furthermore, the range of CD4(+) memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8(+)-T-cell frequencies.     

鼻咽癌病人和正常人群中EB病毒特异性T细胞对靶抗原的识别和应答

为探讨痘苗病毒表达的Eoskein-Barr病毒（EBV）核抗原1、4（EBNA1、4）和潜伏膜蛋白1、2（LMP1、2）,在不同人群的特异性T细胞杀伤9CTL）中的作用,采集EBV阴性正常人、未经治疗的鼻咽癌（NPC）病人的EBV－IgA/VCA阳性者各10人的周围血淋巴单核细胞（PBMC）,用EBV转化B淋巴细胞,建立类淋巴母细胞（LCL）,用LCL刺激自休的T淋巴细胞作为效应细胞,以LCL感染重组痘苗病毒表达的EBNA1、4和LMP1、2为靶细胞,以^51Cr释放法检测EBV特异性CTL所识别的靶抗原。结果表明,EBV－LMP1、2可能既是EBV特异性T细胞的刺激抗原,又是其识别的靶抗原。将采集的30例试验者的各5 单克隆T细胞株分别检测HLA-Ⅰ型（A、B、C）,按照不同型别寻找相对应的EBNA1、4和LMP1、2的不同合成肽,应用酶免疫吸附斑点法（Elispot）检测EBV特异性CD8^ 的CTL应答。结果显示：10例正常人中9人有特异的LMP2应答,4人有特异的EBNA4应答;10例未治疗的NPC病人中3人有特异的LMP2,2人有特异的EBNA1,3个有特异的EBNA4应答;10例未治疗的NPC病人中3人有特异的EBNA1,3人有特异的EBNA4应答,在10例EBV－IgA/VCAbj ntg k ,6人有特异的LMP2,5人有特异的EBNA4应答。所有的试验者均未发现LMP1的特异性应答。     

Type 2 cytokines predominate in the human CD4(+) T-lymphocyte response to Epstein-Barr virus nuclear antigen 1

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persistently infects 85% of the adult population worldwide. In this report, we examine the proliferative response and cytokine secretion profile of CD4(+) T lymphocytes from a panel of unrelated EBV-positive donors against two EBV latent antigens, EBNA1 and EBNA3C. Substantial proliferative responses by CD4(+) lymphocytes were demonstrated to both antigens in multiple, randomly selected donors. Surprisingly, we observed a striking and consistent difference in cytokine response to EBNA1 and EBNA3C. EBNA1-specific CD4(+) T lymphocytes from multiple unrelated donors preferentially produced type 2-like cytokines in response to antigenic stimulation, while the response to EBNA3C was a characteristic type 1 response. The implications of these findings for EBV persistence and the development of EBV-associated malignancies are discussed.     

Epstein-Barr nuclear antigen 1-specific CD4(+) Th1 cells kill Burkitt's lymphoma cells

The gamma-herpesvirus, EBV, is reliably found in a latent state in endemic Burkitt's lymphoma. A single EBV gene product, Epstein-Barr nuclear Ag 1 (EBNA1), is expressed at the protein level. Several mechanisms prevent immune recognition of these tumor cells, including a block in EBNA1 presentation to CD8(+) killer T cells. Therefore, no EBV-specific immune response has yet been found to target Burkitt's lymphoma. We now find that EBNA1-specific, Th1 CD4(+) cytotoxic T cells recognize Burkitt's lymphoma lines. CD4(+) T cell epitopes of EBNA1 are predominantly found in the C-terminal, episome-binding domain of EBNA1, and approximately 0.5% of peripheral blood CD4(+) T cells are specific for EBNA1. Therefore, adaptive immunity can be directed against Burkitt's lymphoma, and perhaps this role for CD4(+) Th1 cells extends to other tumors that escape MHC class I presentation.     

The importance of exogenous antigen in priming the human CD8+ T cell response: lessons from the EBV nuclear antigen EBNA1

Mouse models suggest that the processing of exogenous Ag by dendritic cells can be important for priming the CD8(+) CTL response. To study the situation in humans, we have exploited the CTL response to EBV infection. In this context EBV expresses eight latent proteins, of which EBV-encoded nuclear Ag (EBNA) 3A, 3B, and 3C appear to be immunodominant for CTL responses, whereas another nuclear Ag, EBNA1, which is completely protected from endogenous presentation via the MHC class I pathway, is thought to induce responses rarely, if ever. Here, using EBNA1 peptides and/or EBNA1 protein-loaded dendritic cells as in vitro stimuli, we have identified memory CTL responses to HLA-B*3501, -B7, and -B53-restricted EBNA1 epitopes that can be as strong as those seen in immunodominant epitopes from the "conventionally processed" EBNA3 Ags. Furthermore, we used HLA-peptide tetramers to show that the primary response to one such EBNA1 epitope constituted up to 5% of the CD8(+) T cells in infectious mononucleosis blood, the strongest latent Ag-specific response yet detected in this setting. We conclude that exogenous protein represents a significant source of Ag for priming the human CTL response.     

A role for intercellular antigen transfer in the recognition of EBV-transformed B cell lines by EBV nuclear antigen-specific CD4+ T cells

The CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directly recognize virus-transformed lymphoblastoid cell lines (LCLs) in vitro and inhibit their growth. In this study, we used a panel of EBNA1, 2, 3A, and 3C-specific CD4+ T cell clones to study the route whereby endogenously expressed EBNAs access the HLA class II-presentation pathway. Two sets of results spoke against a direct route of intracellular access. First, none of the clones recognized cognate Ag overexpressed in cells from vaccinia vectors but did recognize Ag fused to an endo/lysosomal targeting sequence. Second, focusing on clones with the strongest LCL recognition that were specific for EBNA2- and EBNA3C-derived epitopes LCL recognition was unaffected by inhibiting autophagy, a postulated route for intracellular Ag delivery into the HLA class II pathway in LCL cells. Subsequently, using these same epitope-specific clones, we found that Ag-negative cells with the appropriate HLA-restricting allele could be efficiently sensitized to CD4+ T cell recognition by cocultivation with Ag-positive donor lines or by exposure to donor line-conditioned culture medium. Sensitization was mediated by a high m.w. antigenic species and required active Ag processing by recipient cells. We infer that intercellular Ag transfer plays a major role in the presentation of EBNA-derived CD4 epitopes by latently infected target cells.     

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

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

Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy

Background aims

EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1.

Novel Approach to the Formulation of an Epstein-Barr Virus Antigen-Based Nasopharyngeal Carcinoma Vaccine

Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.Epstein-Barr virus (EBV) is a member of the herpesvirus family and is one of the most common human viruses. It occurs worldwide, and most people become infected with the virus sometime during their lives. EBV is associated with a range of neoplasms. These include various B- and T-cell non-Hodgkin''s lymphomas such as posttransplant lymphoproliferative disease (PTLD), Hodgkin''s lymphoma (HL), and several lymphoepithelioma-like carcinomas, of which nasopharyngeal carcinoma (NPC) is the archetype (1). The association of the virus with these malignancies and its oncogenic potential have been well established (19).Worldwide, NPC is characterized epidemiologically by foci of relatively high endemicity in certain geographic regions including southern China, Hong Kong, Taiwan, the Philippines, Singapore, Vietnam, Kenya, Tunisia, Sudan, and Uganda. The reason for the focal distribution of NPC is uncertain, although genetics and environmental factors have been suggested to be causes (14, 49).Currently, the mainstay for the treatment of NPC is radiation and chemotherapy. Indeed, this treatment is frequently successful when the extent of the tumor is small and confined. However, when disease is advanced at diagnosis and where metastatic spread has become apparent, more radical treatments may need to be adopted, including surgery. In either case, all these treatments are associated with severe short- and long-term side effects including secondary malignancies (16). Hence, there is a need for specific therapies that target the tumor itself rather than therapies that are associated with the destruction of normal tissue.Virus-associated malignancies offer a distinct advantage in this regard since therapy can be directed specifically toward viral proteins expressed in the tumor, thus avoiding collateral damage to normal tissue. This has been dramatically demonstrated in the case of PTLD, where the adoptive transfer of EBV-specific cytotoxic T lymphocytes (CTLs) activated in vitro by using autologous lymphoblastoid cell lines (LCLs) has resulted in a resolution of disease with a very low frequency of side effects (9, 18, 40). In this case, it is likely that the effector cells infused into these patients are directed mainly toward the dominant EBV nuclear antigen 3A (EBNA3A), EBNA3B, and EBNA3C. The concept of immunological intervention as a treatment option for NPC is greatly enhanced by a range of previously reported studies that indicated the presence of transport-associated proteins (TAP1 and TAP2) and major histocompatibility complex class I and class II in NPC (23, 37, 42, 48), all of which are required for efficient CTL recognition. In NPC, EBNA1, latent membrane protein 1 (LMP1), and LMP2 offer the best opportunity for specific targeting since these are the only EBV proteins expressed in this malignancy. This is particularly so in the case of immunotherapy since defined CD4⁺ and CD8⁺ T-cell determinants in each of these proteins have been defined (12, 15, 20, 31). However, the CTL response in the case of the LMPs is relatively weak (particularly LMP1), and the glycine-alanine repeat sequence within EBNA1 may affect immunological processing (29), although this may not be the absolute barrier that was first hypothesized (46). Recent studies have provided some encouragement that immunotherapeutic intervention may be a realistic treatment option for NPC (4, 5, 7, 8, 10, 30, 43, 45). For example, Straathof et al. (43) and Comoli et al. (10) adoptively transferred effector cells expanded in vitro by using LCLs to activate CTLs in patients with advanced NPC, resulting in some cases in the resolution of disease, although in other cases, efficacy was limited and transient (3). Those studies, however, have provided a promising hint that the immunotherapeutic control of NPC might be feasible.Indeed, recent studies have shown that multiple human leukocyte antigen (HLA) A2-restricted LMP1 CTL epitopes, when used as a polyepitope vaccine in a poxvirus vector, efficiently induced a strong CTL response, and this response could reverse the outgrowth of LMP1-expressing tumors in HLA-A2/Kb mice (13). The poxvirus-based LMP1-polyepitope vaccine tested in these studies contained only HLA A2-restricted epitopes, and targeting just one HLA allele will not be suitable for all ethnic groups. If a CTL-based therapy for NPC is to be universally applicable, the target epitopes must bind to a range of HLA alleles preferably present at a high frequency in patient populations and include determinants irrespective of whether they have previously been defined.It is likely that the essential difference between the very successful treatment of patients with PTLD and the partial success in the case of NPC is that in the former case, immunodominant targets are available, while in the latter case, only relatively weak responses are seen even for healthy individuals. The present communication has arisen in an attempt to maximize the possibility of activating a response toward the three proteins present in NPC rather than skewing the effector population toward the immunodominant EBNA3A, -B, and -C proteins. We have achieved this by generating a “scrambled-antigen vaccine” (referred to as SAVINE) incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2. This SAVINE has been incorporated into a replication-deficient adenovirus (Ad5/F35) as a 6.9-kb insert (Ad-SAVINE). An important feature of the Ad-SAVINE strategy is that it provides a platform for the activation of all possible immunological determinants (including helper cells and CTLs) within EBNA1, LMP1, and LMP2 and should be applicable to all populations for which NPC is endemic. This report describes the construction of this Ad-SAVINE construct and its utility in generating LMP1 and LMP2 responses from peripheral blood mononuclear cells (PBMCs) from healthy individuals and NPC patients.     

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

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

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy with high prevalence in Southern Chinese. In order to assess whether defects of EBV-specific immunity may contribute to the tumor, the phenotype and function of circulating T-cells and tumor infiltrating lymphocytes (TILs) were investigated in untreated NPC patients. Circulating naïve CD3⁺CD45RA⁺ and CD4⁺CD25⁻ cells were decreased, while activated CD4⁺CD25⁺ T-cells and CD3⁻CD16⁺ NK-cells were increased in patients compared to healthy donors. The frequency of T-cells recognizing seven HLA-A2 restricted epitopes in LMP1 and LMP2 was lower in the patients and remained low after stimulation with autologous EBV-carrying cells. TILs expanded in low doses of IL-2 exhibited an increase of CD3⁺CD4⁺, CD3⁺CD45RO⁺ and CD4⁺CD25⁺ cells and 2 to 5 fold higher frequency of LMP1 and LMP2 tetramer positive cells compared to peripheral blood. EBV-specific cytotoxicity could be reactivated from the blood of most patients, whereas the TILs lacked cytotoxic activity and failed to produce IFNγ upon specific stimulation. Thus, EBV-specific rejection responses appear to be functionally inactivated at the tumor site in NPC.     

Increased CD4+/CD8+ double-positive T cells in chronic Chagasic patients

Background

CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out.

Methodology/Principal Findings

Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor.

Conclusions

Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can migrate to the heart tissue.     

Human cytotoxic CD4+ T cells recognize HLA-DR1-restricted epitopes on vaccinia virus proteins A24R and D1R conserved among poxviruses

We previously demonstrated that vaccinia virus (VV)-specific CD4(+) cytolytic T cells can persist for >50 years after immunization against smallpox in the absence of re-exposure to VV. Nevertheless, there have been few studies focusing on CD4(+) T cell responses to smallpox vaccination. To ensure successful vaccination, a candidate vaccine should contain immunodominant CD4(+) T cell epitopes as well as CD8(+) T and B cell epitopes. In the present study, we established cytotoxic CD4(+) T cell lines from VV-immune donors, which recognize epitopes in VV proteins D1R and A24R in association with HLA-DR1 Ags. Comparisons of sequences between different members of the poxvirus family show that both epitopes are completely conserved among VV, variola viruses, and most mammalian poxviruses, including monkeypox, cowpox, and ectromelia. The CD4(+) T cell lines lysed VV-infected, Ag- and peptide-pulsed targets, and the lysis was inhibited by concanamycin A. We also detected these peptide-specific cytolytic and IFN-gamma-producing CD4(+) T cells in short-term bulk cultures of PBMC from each of the three VV-immune donors tested. These are the first VV-specific CD4(+) T cell epitopes identified in humans restricted by one of the most common MHC class II molecules, HLA-DR1, and this information may be useful in analyzing CD4(+) T cell responses to pre-existing or new generation VV vaccines against smallpox.     

Identification of naturally processed CD4 T cell epitopes from the prostate-specific antigen kallikrein 4 using peptide-based in vitro stimulation

Kallikrein (KLK)4 is a recently described member of the tissue kallikrein gene family that is specifically expressed in normal and prostate tumor tissues. The tissue-specific expression profile of this molecule suggests that it might be useful as a vaccine candidate against prostate cancer. To examine the presence of CD4 T cells specific for KLK4 in PBMC of normal individuals, a peptide-based in vitro stimulation protocol was developed that uses overlapping KLK4-derived peptides spanning the majority of the KLK4 protein. Using this methodology, three naturally processed CD4 epitopes derived from the KLK4 sequence are identified. These epitopes are restricted by HLA-DRB1*0404, HLA-DRB1*0701, and HLA-DPB1*0401 class II alleles. CD4 T cell clones specific for these epitopes are shown to efficiently and specifically recognize both recombinant KLK4 protein and lysates from prostate tumor cell lines virally infected to express KLK4. CD4 T cells specific for these KLK4 epitopes are shown to exist in PBMC from multiple male donors that express the relevant class II alleles, indicating that a CD4 T cell repertoire specific for KLK4 is present and potentially expandable in prostate cancer patients. The demonstration that KLK4-specific CD4 T cells exist in the peripheral circulation of normal male donors and the identification of naturally processed KLK4-derived CD4 T cell epitopes support the use of KLK4 in whole gene-, protein-, or peptide-based vaccine strategies against prostate cancer. Furthermore, the identification of naturally processed KLK4-derived epitopes provides valuable tools for monitoring preexisting and vaccine-induced responses to this molecule.     

Genomic sequencing and comparative analysis of Epstein-Barr virus genome isolated from primary nasopharyngeal carcinoma biopsy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt's lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8(+) T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.