Defective control of latent Epstein-Barr virus infection in systemic lupus erythematosus

EBV infection is more common in patients with systemic lupus erythematosus (SLE) than in control subjects, suggesting that this virus plays an etiologic role in disease and/or that patients with lupus have impaired EBV-specific immune responses. In the current report we assessed immune responsiveness to EBV in patients with SLE and healthy controls, determining virus-specific T cell responses and EBV viral loads using whole blood recall assays, HLA-A2 tetramers, and real-time quantitative PCR. Patients with SLE had an approximately 40-fold increase in EBV viral loads compared with controls, a finding not explained by disease activity or immunosuppressive medications. The frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma was higher in patients with SLE than in controls. By contrast, the frequency of EBV-specific CD69+ CD8+ T cells producing IFN-gamma in patients with SLE appeared lower than that in healthy controls, although this difference was not statistically significant. These findings suggest a role for CD4+ T cells in controlling, and a possible defect in CD8+ T cells in regulating, increased viral loads in lupus. These ideas were supported by correlations between viral loads and EBV-specific T cell responses in lupus patients. EBV viral loads were inversely correlated with the frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma and were positively correlated with the frequencies of CD69+ CD8+ T cells producing IFN-gamma and with EBV-specific, HLA-A2 tetramer-positive CD8+ T cells. These results demonstrate that patients with SLE have defective control of latent EBV infection that probably stems from altered T cell responses against EBV.     

Exhausted cytotoxic control of Epstein-Barr virus in human lupus

Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8⁺ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8⁺ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8⁺ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8⁺ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8⁺ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.     

Reconstitution of EBV latent but not lytic antigen-specific CD4+ and CD8+ T cells after HIV treatment with highly active antiretroviral therapy

The incidence of (EBV-related) malignancies in HIV-infected subjects has declined since the introduction of highly active antiretroviral therapy (HAART). To investigate the effect of HAART on EBV infection, we performed a longitudinal analysis of the T cell response to both a latent and a lytic Ag and EBV viral load in 10 subjects from early in HIV infection up to 5 years after HAART. All individuals responded to HAART by a decline in HIV viral load, a restoration of total CD4+ T cell numbers, and a decline in T cell immune activation. Despite this, EBV load remained unaltered, even after 5 years of therapy, although a decline in both CD4+ and CD8+ T cells specific for the lytic EBV protein BZLF1 suggested a decreased EBV reactivation rate. In contrast, latent EBV Ag EBNA1-specific CD4+ and CD8+ T cell responses were restored after 5 years of treatment to levels comparable to healthy individuals. In two individuals who were treated by HAART late during HIV progression, a lymphoma developed shortly after initiation of HAART, despite restoration of EBV-specific CD4+ and CD8+ T cells. In conclusion, long-term HAART does not alter the EBV DNA load, but does lead to a restoration of EBNA1-specific T cell responses, which might allow better control of EBV-infected cells when applied early enough during HIV infection.     

HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.     

CD4+ and CD8+ T-Cell Responses to Latent Antigen EBNA-1 and Lytic Antigen BZLF-1 during Persistent Lymphocryptovirus Infection of Rhesus Macaques

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4⁺ and/or CD8⁺ T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4⁺ T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8⁺ T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.     

CD4+ T cell-induced differentiation of EBV-transformed lymphoblastoid cells is associated with diminished recognition by EBV-specific CD8+ cytotoxic T cells

EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4(+) T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4(+) T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8(+) CTL. In contrast, CD4(+) T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8(+) CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8(+) CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8(+) T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4(+) T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8(+) CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4(+) T cell contact was prevented, indicated a major role for CD4(+) T cell cytokines in the differentiation of LCL.     

Phenotypic and functional heterogeneity of EBV epitope-specific CD8+ T cells

High frequencies of EBV-specific CD8(+) T cells have been detected during acute EBV infection, yet persistent infection inevitably results. To address this issue, we characterized the phenotype and function of epitope-specific CD8(+) T cell populations from presentation with acute through latent infection. Considerable phenotypic and functional heterogeneity within, as well as between, two different epitope-specific populations was observed over time following acute infection. B7 EBV-encoded nuclear Ag (EBNA)-3A-specific CD8(+) T cells expressed only CD45RO from acute through latent EBV infection. A2 BMLF-1-specific CD8(+) T cells expressed CD45RO during acute infection and either CD45RA or CD45RO during latent EBV infection. This difference in CD45 isoform expression between the two epitope-specific populations did not translate into differences in perforin content, the ability to produce IFN-gamma, or the ability to proliferate in response to Ag in vitro. In individuals with latent EBV infection, the frequencies of A2 BMLF-1- or B7 EBNA-3A-specific CD8(+) T cells that expressed CD45RA, CD45RO, CD62 ligand, CCR7, and perforin were stable over time. However, the expression of CD62 ligand and CCR7 was significantly higher among EBNA-3A-specific CD8(+) T cells than among BMLF-1-specific CD8(+) T cells. Further work is necessary to understand how phenotypic and functional differences between EBV epitope-specific CD8(+) T cells are related to the biology of the virus and to the equilibrium between the virus and the host during persistent infection.     

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

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

Increased frequency of EBV-specific effector memory CD8+ T cells correlates with higher viral load in rheumatoid arthritis

EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8(+) T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8(+) T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4(+) T cell responses to EBV and T cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific effector T cell and Ab responses are driven by an elevated EBV load in RA.     

Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus

Alteration of T cell suppression function has been recognized in patients with systemic lupus erythematosus (SLE). Recently, CD8(+) T suppressor lymphocytes (CD8(+) Ts) have been generated in vitro by incubating purified CD8(+) T cells with IL-2 and GM-CSF. Using this method, we generated CD8(+) Ts from patients affected by SLE. No major differences were found in the CD8(+) Ts phenotype between SLE patients and healthy subjects. CD8(+) Ts from SLE patients with active disease did not inhibit the anti-CD3 mAb-induced proliferation of autologous PBMC, whereas CD8(+) Ts from SLE patients in remission exerted an inhibitory activity comparable to normal subjects. The inhibitory effect of CD8(+) Ts cells was neither mediated by cytotoxic activity nor by apoptosis induction. Two cytokines, IFN-gamma and IL-6, were found to be responsible for the function of CD8(+) TS: In fact, counteraction of CD8(+) Ts suppression activity was obtained by blocking IFN-gamma with a specific Ab or by inhibiting CD8(+) Ts-mediated IL-6 secretion by an antisense oligonucleotide. Interestingly, CD8(+) Ts from SLE patients showed a peculiar cytokine pattern characterized by an impaired secretion of IL-6 and an increased secretion of IL-12. Thus, it appears that an altered balance between inhibitory (IL-6) and stimulatory (IL-12) cytokines might be responsible for the functional impairment of CD8(+) Ts in SLE patients.     

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.     

CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.     

Killing kinetics of simian immunodeficiency virus-specific CD8+ T cells: implications for HIV vaccine strategies

Both the magnitude and function of vaccine-induced HIV-specific CD8+ CTLs are likely to be important in the outcome of infection. We hypothesized that rapid cytolysis by CTLs may facilitate control of viral challenge. Release kinetics of the cytolytic effector molecules granzyme B and perforin, as well as the expression of the degranulation marker CD107a and IFN-gamma were simultaneously studied in SIV Gag(164-172) KP9-specific CD8+ T cells from Mane-A*10+ pigtail macaques. Macaques were vaccinated with either prime-boost poxvirus vector vaccines or live-attenuated SIV vaccines. Prime-boost vaccination induced Gag-specific CTLs capable of only slow (after 3 h) production of IFN-gamma and with limited (<5%) degranulation and granzyme B release. Vaccination with live-attenuated SIV resulted in a rapid cytolytic profile of SIV-specific CTLs with rapid (<0.5 h) and robust (>50% of tetramer-positive CD8+ T cells) degranulation and granzyme B release. The cytolytic phenotype following live-attenuated SIV vaccinations were similar to that associated with the partial resolution of viremia following SIV(mac251) challenge of prime-boost-vaccinated macaques, albeit with less IFN-gamma expression. High proportions of KP9-specific T cells expressed the costimulatory molecule CD28 when they exhibited a rapid cytolytic phenotype. The delayed cytolytic phenotype exhibited by standard vector-based vaccine-induced CTLs may limit the ability of T cell-based HIV vaccines to rapidly control acute infection following a pathogenic lentiviral exposure.     

Functional CD4(+) and CD8(+) T-cell responses induced by autologous mitomycin C treated Epstein-Barr virus transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4(+) and CD8(+) T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4(+) and (granzyme B positive) CD8(+) T-cells rosetting with McLCL. Granzymes A and B, IFN-gamma, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4(+) and CD8(+) T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.     

Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.     

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.     

Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus latent membrane protein 2B: a new modality for vaccination

Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8(+) T lymphocytes from HLA-A2.1(+), EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-gamma spot formation (T-cell frequency, <0.003%). IFN-gamma ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8(+) effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies.     

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8⁺ and CD4⁺ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8⁺ and CD4⁺ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4⁺ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4⁺ T cells was significantly correlated with that of CD8⁺ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4⁺ T cell responses showed more significant changes than CD8⁺ T cell responses. CD8⁺ and CD4⁺ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4⁺ T cells secreted significantly higher cytokine levels than did CD8⁺ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.     

Programmed Death-1 expression on Epstein Barr virus specific CD8+ T cells varies by stage of infection, epitope specificity, and T-cell receptor usage

Background

Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence.

Methodology/Principal Findings

Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage.

Conclusions/Significance

These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage.     

Functional reversion of antigen-specific CD8+ T cells from patients with Hodgkin lymphoma following in vitro stimulation with recombinant polyepitope

Recent studies on Hodgkin's lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8+ T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8+ T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8+ T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8+ T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8+ T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-gamma production and cytolytic function. More importantly, these activated CD8+ T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62L(high) and CD27(high), and CCR7(low), consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma.