Interferon--gamma control of EBV-transformed B cells: a role for CD8+ T cells that poorly kill EBV-infected cells

Control of Epstein-Barr virus (EBV) infection requires CD8+ T cells. Surprisingly, many EBV-specific CD8+ T cells kill autologous EBV-transformed B lymphoblasts poorly. We investigated the effector functions used by poorly cytotoxic EBV-specific CD8+ D7 cloned T cells and by EBV-stimulated peripheral blood lymphocytes. D7 T cells did not inhibit B lymphoblast growth in long-term coculture, but prevented the outgrowth of newly infected autologous B cells. Optimally stimulated D7 T cells and EBV-stimulated peripheral blood lymphocytes produced interferon (IFN)-y at levels that inhibited EBV-transformed B cell outgrowth. Inhibitory factor activity was neutralized by anti-IFN-gamma monoclonal antibodies (mAb), but not by antibodies to several other cytokines. These data suggest an in vivo role for IFN-y secreting EBV-specific CD8+ T cells.     

Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-γ-producing CD8⁺ cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4⁺ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients. Received: 4 October 2000     

Severe chronic EBV infection associated with specific EBV immunodeficiency and an EBNA+ T-cell lymphoma containing linear, EBV DNA

A patient with severe chronic Epstein-Barr virus (EBV) infection (CEBVI) of 6 years duration developed an EBV+ T-cell lymphoma. To determine whether the development of the T-cell tumor was linked to EBV, we studied this patient's EBV-specific immune response and her T-cell tumor tissue for evidence of EBV infection. Peripheral blood lymphocytes from this patient were systematically studied for immune function and response to EBV. Tumor tissue was examined for EBV genome and for evidence of EBV replication. This patient failed to develop anti-EBV nuclear antigen (EBNA) antibodies and had decreased mitogen responsiveness. Her T-cells showed a broad suppression of both autologous and allogeneic B-cells, which was coincident with clinical hypoimmunoglobulinemia. A selective cytotoxic T-cell defect toward autologous EBV-infected B lymphoblasts, which could not be corrected by the addition of lymphokine-mediated T-cell help, was also documented. A lymph node biopsy taken 5 years after her clinical presentation revealed lymph node architecture completely effaced by a diffuse CD3+, CD4+, Ia+, CR2+ T-cell lymphoma containing EBNA and linear, replicating EBV DNA. Select CEBVI patients with humoral and combined cellular aberrations in the immune response to EBV may be predisposed to the development of EBV+ T-cell tumors.     

Factors influencing the human cytotoxic T cell response to autologous lymphoblastoid cell lines in vitro

Epstein-Barr virus (EBV)- and fetal calf serum (FCS)-specific cytotoxic human T cells can be generated in vitro, and have been shown to be HLA-antigen restricted. In the present work, peripheral blood mononuclear cells were stimulated with the gamma-irradiated autologous lymphoblastoid cell line (LCL) grown previously in FCS, human serum, or without serum. The induction and generation of cytotoxic T cells was carried out exclusively in culture medium containing autologous serum. With EBV-seronegative responders, FCS-grown stimulator LCL generated a FCS-specific cytotoxic T cell response. AB serum-grown LCL generated only a weak response, except at a high stimulatory dose, where the response tended to be essentially nonspecific. EBV-seropositive responders, in contrast, gave a typical secondary EBV-specific response regardless of the serum in which the LCL had been grown previously; no FCS response was detected. Dose-response and cold target inhibition studies confirmed these results. EBV immunity obviously plays a major role in the T cell response to the autologous LCL, which can no longer be viewed simply as a form of autologous mixed leucocyte reaction.     

Treatment options for post-transplant lymphoproliferative disorder and other Epstein-Barr virus-associated malignancies

Epstein-Barr virus (EBV) is associated with a range of malignancies that largely arise from a defect in EBV-specific cytotoxic T lymphocyte (CTL) immunity and function. Much work has focused on the reconstitution of CTL immunity to EBV in transplant patients, in whom immunosuppression modalities render them susceptible to post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of autologous CTLs is effective at both preventing and curing PTLD in solid organ transplant recipients and can produce a long-term memory response and protection against recurring disease. In this review, the benefits and restrictions of administering EBV-specific CTLs for the treatment of PTLD are discussed and compared with emerging therapies including the generation of allogeneic human leukocyte antigen-matched CTL banks and the anti-CD20 monoclonal antibody therapy, MabThera. Furthermore, studies involving other EBV-associated disorders have described the potential benefit of adoptive transfer of EBV-specific CTLs for Hodgkin's disease, nasopharyngeal carcinoma, chronic active EBV infection, and Burkitt's lymphoma. The challenges of tailor-making therapies for individual diseases and EBV antigen expression latencies are highlighted, in addition to considering vaccination strategies for optimal treatment.     

Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones.

We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells (PBMCs) were cultured with EBV, and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+, CD4+, and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-superinfected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+, CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-superinfected and non-superinfected LCLs. Proliferative and cytotoxic responses to allogenic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections.     

Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly

Thymus transplantation in subjects with complete DiGeorge anomaly using postnatal allogeneic HLA-nonmatched cultured thymus tissue provides immunoreconstitution. Tolerance of the newly developed T cells toward the donor thymus has not previously been studied. Mixed lymphocyte cultures were used to test 12 thymus transplant recipients for long-term tolerance toward their thymus allografts. Two subjects tested for responses toward thymus donor peripheral blood mononuclear cells showed significantly less reactivity toward the donors compared to responses against third-party allogeneic cells. Peripheral blood mononuclear cells from 10 other subjects were less responsive toward cryopreserved donor thymic cells than toward allogeneic cells (P=0.00007). Adult control peripheral blood mononuclear cells proliferated strongly in response to the donor thymic cells. Both the subjects and controls showed similar proliferative responses against allogeneic cells and phytohemagglutinin. This study provides in vitro evidence for long-term tolerance of complete DiGeorge anomaly thymus transplantation recipients toward their HLA-nonmatched thymus grafts.     

T cell repertoire and Epstein-Barr virus-specific T cell response in chronic active Epstein-Barr virus infection: a case study

In a patient with chronic active Epstein-Barr virus infection associated with vasculitis and fulminant CD4+ T cell lymphoproliferative disorder, we probed the peripheral blood mononuclear cells (PBMC) for the presence of an EBV-specific T cell repertoire and tested the possible relationship between the lymphocytic infiltrate and the EBV-specific T cell response. Our results give credence to the presence of an apparently normal EBV-specific memory T cell response after in vitro reactivation of the patient's PBMC with autologous infected B lymphoblastoid cell lines. In keeping with the characterization of the vasculitis, certain T cell subsets were detected after expansion of skin lesion-infiltrating lymphocytes and were found to be infected with EBV. These particular T cell expansions were neither the effectors nor the targets of the in vitro reactivated EBV-specific T cells, thus excluding a simple relationship between EBV, the skin lesions, and the T cell expansions frequently observed in these patients.     

Bone marrow cells inhibit the generation of autologous EBV-specific CTL

Recently, we reported that human bone marrow cells (BMC) inhibited the proliferative (recall) response of lymphocytes to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) protein antigens [12]. To clarify further the effect of BMC on the immune response to viral antigens, we obtained PBL from EBV IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) 1 year after renal transplantation and generated EBV-specific CTL in vitro in the presence or absence of autologous BMC. The addition of freshly aspirated autologous iliac crest BMC from either R or LRD caused a significant inhibitory effect on the generation of EBV-specific CTL from CTL precursors, in contrast to the addition of autologous PBL used as controls (62.29 +/- 10.85% inhibition using BMC from the kidney transplant recipients; 74.47 +/- 15.21% inhibition using BMC from the living-related donors). This inhibitory effect was only exerted during the CTL generation phase; but not in the effector CTL killing phase. The expression of CD94, a component of the killer inhibitory receptor (KIR) on CD3(+) cells was elevated in the cultures with BMC, in contrast to the cultures without BMC. The BMC inhibitory effect was partially abrogated by pre-incubation of the CTL effectors with anti-CD94 monoclonal antibody, in contrast with its isotype control. In addition, supernatants obtained from the CTL generating cultures with BMC contained high levels of prostaglandin E(2) (PGE(2)), and EBV-specific CTL activity was inhibited by the addition of exogenous PGE(2) in the absence of BMC. The induction of CD40L cell surface expression by anti-CD3 was also decreased on the effector T cell population when BMC were added. There was a concomitant reduction in protein kinase C (PKC) activity. These studies demonstrate that BMC exert an inhibitory effect on T cell-mediated immunity to viral antigens in humans by regulating autologous effector T cell generation and early T cell activation signaling pathways.     

Inflammatory pseudotumor of the spleen associated with a clonal Epstein-Barr virus genome. Case report and review of the literature

We report a case of an inflammatory pseudotumor (IPT) of the spleen occurring in an 81-year-old woman with a history of a monoclonal gammopathy of undetermined significance. Eighteen-month follow-up after splenectomy demonstrated no tumor recurrence or progression of underlying plasma cell disease. Histologic examination of the tumor demonstrated a polymorphic population of inflammatory and epithelioid and spindle cells. Immunophenotyping showed large numbers of T cells, B cells, and polyclonal plasma cells. The epithelioid and spindle cells were positive for vimentin and CD68 but lacked expression of follicular dendritic cell markers and actin. Epstein-Barr virus (EBV) genome was identified in the epithelioid and spindle cell population by in situ hybridization using probes specific for EBV-encoded RNAs (EBER1 and EBER2). Southern blot analysis of digested DNA extracted from the tumor using an EBV-specific probe (XhoI) demonstrated the presence of a single high-intensity band, indicative of EBV monoclonality. While there have been 2 previous reports of hepatic IPTs containing a monoclonal population of EBV-infected tumor cells, this is the first report of such an association occurring in the spleen. The presence of clonal EBV DNA suggests some splenic IPTs may be true neoplasms.     

Thymus transplantation

Thymus transplantation is a promising investigational therapy for infants born with no thymus. Because of the athymia, these infants lack T cell development and have a severe primary immunodeficiency. Although thymic hypoplasia or aplasia is characteristic of DiGeorge anomaly, in “complete” DiGeorge anomaly, there is no detectable thymus as determined by the absence of naive (CD45RA⁺, CD62L⁺) T cells. Transplantation of postnatal allogeneic cultured thymus tissue was performed in sixty subjects with complete DiGeorge anomaly who were under the age of 2 years. Recipient survival was over 70%. Naive T cells developed 3–5 months after transplantation. The graft recipients were able to discontinue antibiotic prophylaxis, and immunoglobulin replacement. Immunosuppression was used in a subset of subjects but was discontinued when naive T cells developed. The adverse events have been acceptable with thyroid disease being the most common. Research continues on mechanisms underlying immune reconstitution after thymus transplantation.     

Epstein-Barr virus and rheumatoid arthritis

The cause of rheumatoid arthritis (RA) is still unknown. Both genetic and environmental factors may help its development. For 25 years, the Epstein-Barr Virus (EBV) has been suspected to contribute to RA pathogenesis. RA patients have higher levels of anti-EBV antibodies than healthy controls. EBV-specific suppressor T cell function is defective in RA. HLA-DRB1*0404, an RA predisposing allele, is associated with low frequencies of T cells specific for EBV gp110, a replicative phase glycoprotein critical for the control of EBV infection. Patients with RA have higher EBV load in peripheral blood lymphocytes (median 8.84 copies per 500 ng DNA) than healthy controls (median 0.6 copies/500 ng DNA). EBV, a widespread virus, highly recognized by antibodies but never eliminated, is an ideal candidate to trigger chronic immune complex disease. Anti-EBV antibody responses should be considered as one of the chronic autoantibody responses that are most relevant to the development of RA.     

T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNgamma production

EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.     

Diagnosis of 22q11.2 Deletion Syndrome and Artemis Deficiency in Two Children with T-B-NK+ Immunodeficiency

Two infants are described who presented with 22q11.2 deletion and a T^-B^-NK⁺ immune phenotype. For both infants, the initial diagnosis was athymia secondary to complete DiGeorge anomaly. The first infant underwent thymus transplantation but 6 months after transplantation had circulating thymus donor T cells; the patient did not develop recipient naïve T cells. Genetic analyses revealed that both patients had Artemis deficiency, a rare form of severe combined immunodeficiency (SCID). Both infants have subsequently undergone bone marrow transplantation. These cases illustrate the importance and paradox of differentiating SCID from complete DiGeorge anomaly because hematopoietic stem cell transplantation (HSCT) is the preferred treatment for SCID but is ineffective for complete DiGeorge anomaly. However, if the thymus is completely absent, donor stem cells from a HSCT would not be able to be educated.     

Longitudinal analysis of frequency and reactivity of Epstein-Barr virus-specific T lymphocytes and their association with intermittent viral reactivation

Persistent Epstein-Barr virus (EBV) infection is controlled tightly by virus-specific T cells. EBV infection is reactivated intermittently over time, even in apparently healthy carriers. Changes in frequency and reactivity of memory T cells, particularly of CD8(+) origin, have not been assessed in this context. It is hypothesized that viral reactivation is facilitated by diminished EBV-specific T-cell immunity. To this end, blood samples from 14 healthy donors were collected at irregular time intervals for a period of about 1 year. Samples were screened for both EBV plasma viremia and increases in viral load in PBMCs as parameters of EBV reactivation. PBMCs were subject to IFN-γ ELISPOT analysis using the autologous EBV-transformed lymphoblastoid cell line (EBV-LCL) or appropriate HLA class I-restricted EBV peptides as stimulators. Frequencies of epitope-specific CD8(+) T cells were monitored further using HLA tetramers and flow cytometry. Twelve of 14 donors exhibited signs of asymptomatic EBV reactivation. Viral reactivation was accompanied by either substantially decreased IFN-γ responses against autologous EBV-LCL (eight of 12 study participants) and/or increased responses against particular EBV peptides (six of 12 donors). In seven persons with HLA-A2 and/or -B8 alleles numbers of HLA tetramer-positive CD8(+) T cells also varied over time, but showed no correlation to episodes of detectable viral activity. In summary, IFN-γ reactivity of EBV-specific T cells is not constant. Viral reactivation is detected preferably at times of diminished EBV-LCL-specific cellular immunity. However, increased reactivity of single immunodominant CD8(+) EBV-specific T-cell clones may occur in response to virus replication.     

A global appraisal of immunodominant CD8 T cell responses to Epstein-Barr virus and cytomegalovirus by bulk screening

Knowledge of the immunodominant responses to Epstein-Barr Virus (EBV) and human cytomegalovirus (HCMV) should help to generate cytotoxic T cell lines to these herpesviruses. Here we report on the analysis of CD8 T cell responses to EBV and HCMV in the blood of kidney transplant recipients undergoing viral reactivation (n = 16) and in healthy virus carriers (n = 10). We used a transient COS transfection assay that permits semi-quantitative estimation of CD8+ T cell responses against a larger number of HLA/viral protein combinations within polyclonal T cell lines and thus allows a rapid identification of major epitopes. From the comparison of these responses to those that we previously described in the synovial fluid of patients suffering from various forms of chronic arthritis (n = 32), it appears that EBV-specific T cells are mainly directed against a restricted set of immunodominant epitopes, primarily generated during the early lytic cycle and that both IE1 and pp65 are targets of the anti-HCMV response. We suggest that this method could be generally applied to the rapid identification of immunodominant T cell epitopes in viral and tumor immunity, and could help selecting HLA-peptide complexes that could be used to detect and sort specific T cell populations.     

Effect of 12-O-tetradecanoyl-phorbol-13-acetate on the replication of Epstein-Barr virus. I. Characterization of viral DNA.

The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), induces replication of Epstein-Barr virus (EBV) DNA in a virus-producing human lymphoblastoid cell line, P3HR-1, but not in a nonproducer cell line, Raji. A 6-fold increase in EBV genome copies per P3HR-1 cell parallels the increase in percentage of cells synthesizing viral capsid antigen. In situ cytohybridization with EBV-specific cRNA shows that most of the TPA-treated population participates in the virus-productive cycle. In Raji cells there is abortive induction with an increase in cells showing early antigen from <0.01% to approximately 10%, but there is no increase in EBV genome copies per cell. The optimal TPA concentration for induction of viral DNA replication is 10 ng/ml. EBV DNA synthesized in Raji cells superinfected by virus prepared from TPA-induced P3HR-1 cells is increased approximately 15-fold above that of Raji cells superinfected with control virus. The buoyant density of EBV DNA isolated from virus from TPA-induced cells or of DNA from Raji cells superinfected with TPA-induced and control virus is identical; viral DNA from all sources had the same S value. The XhoI restriction endonuclease digestion patterns of TPA-induced viral DNA and control viral DNA were the same as the viral DNA recovered from Raji cells superinfected with TPA-induced and control virus. Some differences were noted in the molar ratios of some of the fragments.     

Lymphocyte-mediated lysis of autologous and allogeneic B-cell lines in man

The lytic potential of human blood lymphocytes was assayed against autologous and allogeneic EBV-transformed B-cell lines (LCL). The effects--if present--were very weak. Short-term interferon (IFN) treatment of the lymphocytes induced cytotoxic potential which could be manifested against autologous and allogeneic LCL. The sensitivity of the targets increased after superinfection with the P3HR-1 strain of EBV. Thus the strongest lytic effects were obtained with IFN-treated lymphocytes acting on EBV superinfected targets. Individuals with and without previous EBV encounter reacted against autologous LCL, indicating that the lysis did not represent an EBV-specific cellular memory. No evidence for alloantigen recognition emerged from the tests.     

Multiple HLA class I-dependent cytotoxicities constitute the "non-HLA-restricted" response in infectious mononucleosis

Primary Epstein-Barr virus (EBV) infection, when manifest as infectious mononucleosis (IM), induces a broad-ranging and apparently non-HLA-restricted cytotoxic response whose nature has not been resolved. In the present experiments the ability to cryo-preserve IM mononuclear cell preparations, after depletion of CD16+ natural killer cells, has allowed detailed analysis of the response on appropriately constructed target cell panels. The results show that IM effector preparations are polyclonal with separate HLA class I antigen-dependent reactivities against the autologous EBV-transformed lymphoblastoid cell line (LCL) and particular HLA class I-mismatched LCLs. The autologous LCL-directed response shows the hallmarks of immunologically specific T cell cytotoxicity; only EBV+ B cell blasts are recognized and the interaction can be blocked by monoclonal antibodies to CD3 and CD8 on the effector cell surface and to HLA class I antigens on the target cell. Such findings demonstrate, for the first time, that the primary cytotoxic response to EBV infection includes a virus-specific HLA-restricted component like that found in the T cell memory of persistently infected individuals. Separate components of the response are preferentially active against some (but not all) HLA-mismatched LCLs, the pattern of reactivity being distinct for each individual IM patient and reproducible on repeated testing. Monoclonal antibody blocking experiments show that these HLA-mismatched interactions also involve CD3 and CD8 antigens on the effector cell and HLA class I antigens on the target cell. Where tested, such lysis affected both EBV+ and EBV- B cell blasts from the relevant HLA-mismatched donors. We postulate that a widespread primary infection of the B cell system by EBV leads to a generalized expansion not just of the virus-specific response but also of other T cell responses coincidentally active at the time. The unusual activity of IM effector preparations against HLA-mismatched LCLs arises from fortuitous cross-recognition of allogeneic cells by immunologically specific cytotoxic T cell clones coincidentally expanded in vivo alongside the EBV-specific response.     

Virological aspects of Epstein-Barr virus infections

Epstein-Barr virus (EBV) is usually maintained in an asymptomatic and latent form by the host immune system, and primarily by EBV-specific cytotoxic T cells (CTLs). However, EBV has been linked to several refractory diseases such as EBV-associated hemophagocytic syndrome(EBV-AHS) and chronic active EBV infection (CAEBV). In these ectopic diseases, EBV infects T/NK cells, causing severe immunodeficiency with a very high EBV load. In recent years, the laboratory procedure to assess these types of EBV infections has been improved. In particular, real-time polymerase chain reaction (PCR) has been used to quantify the EBV load, and the MHC: peptide tetramer assay has been used to quantitate EBV-specific CTLs; these tests have been employed for the management of the illnesses associated with EBV infection. Here, we have reviewed the recent progress in the clinical application of these assays. The pathogenesis of EBV-infected T/NK cells, and the host immune response to infection, including the roles carried out by innate immunity and inflammatory cytokines, are likely to be revealed in the future.