Determining virological, serological and immunological parameters of EBV infection in the development of PTLD

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.     

Adoptive cellular immunotherapy for EBV lymphoproliferative diseases

Summary: Reactivation of EBV (Epstein-Barr virus) after bone marrow transplantation can result in EBV-associated lymphoproliferative disease (EBV-LPD), We have administered donor-derived EBV-specific cytotoxic T lymphocytes (CTL) to patients who are at high risk of this complication after receiving a T-cell-depleted allograft from a matched unrelated or mismatched related donor. The cells were marked with (he neo GENE U fore infusion so that we could evaluate their persistence and efficacy, CTL infusion produced a virus-specific immune response to EBV that persisted for up to 2 years. None of the 36 patients who received prophylactic CTLs have developed EBV-LPD compared with a cumulative risk of 14% in patients who did not receive this treatment. Strong evidence of clinically valuable immune activity conies from 6 of these 36 patients whose pre-CTL levels of EBV DNA were elevated to a degree strongly predictive of the onset of lymphoma. In each of these cases, the levels returned to baseline after CTL infusion. 2 patients who were treated for clinically evident EBV-LPD attained prolonged remission after CTL infusion and in situ hybridization and semiquantitative PCR showed that the gene-marked CTL had selectively accumulated at disease sites, The prophylactic CTL treatment lacked acute adverse effects, whereas 1 patient who received CTLs for bulky established disease developed initial tumor swelling and respiratory obstruction. We conclude that EBV-specific CTLs are a safe and effective prophylaxis for EBV lymphoma and can also eradicate established disease. This approach is now being extended lo other viruses that produce post-trans-plant morbidity and to other EBV-associated malignancies.     

Epstein-Barr virus, B cell lymphoproliferative disease, and SCID mice: modeling T cell immunotherapy in vivo

Epstein‐Barr virus (EBV)‐associated post‐transplant lymphoproliferative disease (PTLD) arises in up to 10% of organ transplant recipients and is fatal in ～50% of cases. PTLD can be modeled in SCID mice using EBV+ve human B lymphoblastoid cell lines (BLCLs), and the current study investigated intraperitoneal (ip) inoculation of such animals in experiments which assessed the effect of EBV‐specific cytotoxic T lymphocytes (CTLs) and cytokines on PTLD growth. Ip transfer of one dose of autologous CTLs, or CD8‐enriched T cells, into ip BLCL‐inoculated animals significantly delayed tumor development (P = 0.001) and prevented tumor formation in a significant proportion (40%) of mice (P = 0.001). A combination of interleukin (IL)2, 7, and 15 conditioning of CTLs prior to ip injection significantly delayed ip BLCL‐derived tumor formation in vivo when compared to CTLs expanded in vitro using only IL2 (P = 0.04) and prevented tumor outgrowth in a significant proportion (60%) of mice (P = 0.02). Daily ip IL2 dosing of ip CTL‐inoculated mice significantly delayed tumor development in vivo (P = 0.004) and prevented tumor outgrowth in a significant proportion (78%) of mice (P = 0.02) when compared to animals dosed with vehicle only. In SCID mice, autologous CTLs, and CD8‐enriched T cells, have significant capacity to hinder development of PTLD‐like tumors. Whilst studies are needed to delineate the role of cytokine conditioning and CD4‐enriched T cells, the results suggest that IL2 plays a key role in supporting CTL funtion in vivo. J. Med. Virol. 83:1585–1596, 2011. © 2011 Wiley‐Liss, Inc.     

Generation of EBV-specific CTLs suitable for adoptive immunotherapy of EBV-associated lymphoproliferative disease following allogeneic transplantation

Lymphoproliferative disease induced by EBV (EBV-LPD) is a complication of allogeneic transplantation caused by T-cell immunodeficiency. EBV-LPD responds poorly to conventional treatment modalities. Recently, adoptive transfer of EBV-specific, HLA-class I-restricted CTL lines was shown to be effective both as prophylaxis and as treatment of EBV-LPD. The CTL lines are produced by in vitro selection of EBV-specific memory T cells contained in MNC of EBV-seropositive individuals by repeated stimulation with irradiated EBV-transformed cells. The non-EBV-reactive and potentially alloreactive cells remain unactivated. Twenty-one CTL lines were initiated. All were successfully generated and shown to be HLA-restricted and EBV-specific as well as CD8/CD4 and CD45RO positive T cells. The majority of the CTL lines were generated from BMT donors, but two CTL lines were obtained from solid organ transplant recipients receiving immunosuppression. Evidence of probable in vivo efficacy is presented. Development of efficient adoptive T-cell strategies for EBV-positive malignancies could serve as a model for treatment of other viral or malignant disorders.     

Dendritic cells expand Epstein Barr virus specific CD8+ T cell responses more efficiently than EBV transformed B cells

Adoptive transfer of Epstein Barr virus (EBV) specific cytotoxic T lymphocytes (CTLs) has been successfully applied in the treatment of EBV associated post-transplant lymphoproliferative disease (PTLD). In most studies EBV transformed B cells (LCLs) have been used for the induction of EBV specific T cell lines. Application of this approach to other EBV associated tumors is difficult, because LCLs focus T cell expansion toward immunodominant EBV antigens that are not expressed in EBV associated Hodgkin's lymphoma and nasopharyngeal carcinoma. Therefore, we compared dendritic cells (DCs) with LCLs for CD8+ T cell stimulation against dominant and subdominant EBV antigens. DCs expanded tenfold more EBNA3A and LMP2 specific CD8+ T cells than LCL and also stimulated EBV specific CTL from PTLD patients. Both, DCs and LCLs stimulations led to the expansion of high affinity T cells, capable to target EBV transformed B cells. While LCLs and DCs expressed MHC class I and II products at similar levels, DCs showed a higher expression of costimulatory and adhesion molecules. This resulted in more efficient T cell conjugate formation with DCs than with LCLs. We propose the use of DCs for stimulation of EBV specific T cells in active or passive immunotherapy of EBV associated malignancies.     

Using Epstein-Barr Viral Load Assays To Diagnose,Monitor, and Prevent Posttransplant Lymphoproliferative Disorder

Summary: Epstein-Barr virus (EBV) DNA measurement is being incorporated into routine medical practice to help diagnose, monitor, and predict posttransplant lymphoproliferative disorder (PTLD) in immunocompromised graft recipients. PTLD is an aggressive neoplasm that almost always harbors EBV DNA within the neoplastic lymphocytes, and it is often fatal if not recognized and treated promptly. Validated protocols, commercial reagents, and automated instruments facilitate implementation of EBV load assays by real-time PCR. When applied to either whole blood or plasma, EBV DNA levels reflect clinical status with respect to EBV-related neoplasia. While many healthy transplant recipients have low viral loads, high EBV loads are strongly associated with current or impending PTLD. Complementary laboratory assays as well as histopathologic examination of lesional tissue help in interpreting modest elevations in viral load. Circulating EBV levels in serial samples reflect changes in tumor burden and represent an effective, noninvasive tool for monitoring the efficacy of therapy. In high-risk patients, serial testing permits early clinical intervention to prevent progression toward frank PTLD. Restoring T cell immunity against EBV is a major strategy for overcoming PTLD, and novel EBV-directed therapies are being explored to thwart virus-driven neoplasia.     

Post-transplant lymphoproliferative disorders

Post-transplant lymphoproliferative disorders (PTLD) are the second most frequent neoplasia in transplant patients after skin carcinomas. They occur following both solid organ transplants (SOT) and haematopoietic stem cell transplants (HSCT). Most PTLD in solid organ recipients are of host origin, whereas in HSCT recipients they are most often of donor origin. The EBV status of the recipient and the donor, the type of transplant, the type of immunosuppressive therapy used, and the time after transplant are all important parameters that have been associated with the incidence and the type of PTLD. Although most PTLD are B-cell lesions up to 15% may be T-cell or NK-cell type. Most PTLD are associated with EBV, but EBV-negative PTLD are also clearly recognized. In the 2008 WHO classification of lymphoid neoplasms (ref) PTLD are subclassified according to their histological and immunophenotypic characteristics into early lesions, polymorphic type, monomorphic type, and classical Hodgkin lymphoma-type. Overall PTLD mortality rates are around 50%, but new therapies that include early treatment with rituximab and novel anti-EBV therapies promise better outcomes.     

Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.     

Epstein‐Barr virus related post‐transplant lymphoproliferative disorder prevention strategies in allogeneic hematopoietic stem cell transplantation

Epstein‐Barr virus associated post‐transplant lymphoproliferative disorders (EBV PTLD) are recognized as a significant cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). The number of patients at risk of developing EBV PTLD is increasing, partly as a result of highly immunosuppressive regimens, including the use of anti‐thymocyte globulin (ATG). Importantly, there is heterogeneity in PTLD management strategies between alloHSCT centers worldwide. This review summarizes the different EBV PTLD prevention strategies being utilized including the alloHSCT and T‐cell depletion regimes and the risk they confer; monitoring programs, including the timing and analytes used for EBV virus detection, as well as pre‐emptive thresholds and therapy with rituximab. In the absence of an institution‐specific policy, it is suggested that the optimal pre‐emptive strategy in HSCT recipients with T‐cell depleting treatments, acute graft vs host disease (GVHD) and a mismatched donor for PTLD prevention is (a) monitoring of EBV DNA post‐transplant weekly using plasma or WB as analyte and (b) pre‐emptively reducing immune suppression (if possible) at an EBV DNA threshold of >1000 copies/mL (plasma or WB), and treating with rituximab at a threshold of >1000 copies/mL (plasma) or >5000 copies/mL (WB). There is emerging evidence for prophylactic rituximab as a feasible and safe strategy for PTLD, particularly if pre‐emptive monitoring is problematic. Future management strategies such as prophylactic EBV specific CTLs have shown promising results and as this procedure becomes less expensive and more accessible, it may become the strategy of choice for EBV PTLD prevention.     

Intact antigen presentation for Epstein-Barr virus (EBV)-specific CTL by a lymphoblastoid cell line established from a patient with severe chronic active EBV infection

Severe chronic active Epstein-Barr virus (EBV) infection is a lymphoproliferative disease characterized by extremely high antibody titers to EBV, fever, lymphadenopathy, hepatosplenomegaly, and pancytopenia, without any prior immunological abnormality. A spontaneous lymphoblastoid cell line was established from a 4-year-old boy with severe chronic active EBV infection. Immunofluorescence and Western blotting analyses showed that the cell line was of B cell origin and expressed Epstein-Barr nuclear antigens 1, 2 3a, 3b and 3c, and latent membrane protein 1, which are reported to be targets for EBV-specific cytotoxic T lymphocytes (CTL). The cytotoxicity of peripheral blood mononuclear cells derived from the patient and his HLA-identical sister was assayed against the cell line. The cell line was recognized and killed by anti-EBV CTL derived from the HLA-identical sister, but the patient's peripheral blood mononuclear cells had no cytotoxicity. We conclude that antigen presentation in the EBV-infected cells from the patient is intact and sufficient for generation of an EBV-specific CTL response. These observations suggest that severe chronic active EBV infection may not be caused by impaired EBV-antigen presentation of the infected cells but by impaired cellular immune responses to the virus. Our results also suggest the therapeutic possibility that this disease may be treated by adoptive transfer of EBV-specific CTL or bone marrow transplantation from an HLA-matched donor whose immune response to EBV is intact.     

Detection of Epstein-Barr virus DNA in sera from transplant recipients with lymphoproliferative disorders

Early diagnosis of Epstein-Barr Virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is important because many patients respond to reduction in immunosuppression, especially if PTLD is detected at an early stage. Previous studies have found elevated EBV DNA levels in blood from patients with PTLD, but these assays required isolation of cellular blood fractions and quantitation. We evaluated the presence of cell-free EBV DNA in serum from solid-organ transplant recipients as a marker for PTLD. Five of 6 transplant recipients with histopathologically documented PTLD had EBV DNA detected in serum at the time of diagnosis (sensitivity = 83%), compared with 0 of 16 matched transplant recipients without PTLD (specificity = 100%) (P < 0.001 [Fisher's exact test]). Furthermore, EBV DNA was detected in serum 8 and 52 months prior to the diagnosis of PTLD in two of three patients for whom stored sera were analyzed. Detection of EBV DNA in serum appears to be a useful marker for the early detection of PTLD in solid-organ transplant recipients. Further studies to define the role of such assays in evaluating solid-organ transplant patients at risk for PTLD are warranted.     

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.     

Establishment of anti-Epstein–Barr virus (EBV) cellular immunity by adoptive transfer of virus-specific cytotoxic T lymphocytes from an HLA-matched sibling to a patient with severe chronic active EBV infection

We describe an experience of a specific immune transfer treatment in a patient with chronic active EBV infection. The patient had low anti-EBV T cell-mediated cytotoxic activity in his peripheral blood mononuclear cells (PBMC), which may have been the primary cause of the disease. An EBV-specific cytotoxic T lymphocyte (CTL) line was established from PBMC obtained from the patient’s sister whose human leucocyte antigens (HLA) are identical to patient's. The patient received three courses of intravenously administered CTL at 3-week intervals. The number of the cells was increased with each course of treatment. After infusion of the T cell line, anti-EBV CTL activity was detected in the patient's PBMC. CTL activity increased markedly after the second course of immune transfer therapy. The amount of EBV DNA in the patient's plasma showed transient but repeated decreases. Serum levels of tumour necrosis factor-alpha (TNF-α), which had elevated before treatment, began to decrease after initiation of treatment. No adverse effects were directly associated with CTL infusions. Despite having previously received a pneumococcal vaccine and prophylactic antibodies, the patient died of infection caused by Streptococcus pneumoniae bacteraemia 27 days after the third infusion. Although the long-term efficacy and safety of this therapy remains to be established, our findings suggest that adoptive transfer of CTL specific for EBV obtained from an HLA-matched donor might be a promising treatment for patients with chronic active EBV infection.     

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.     

Expression of leukocyte immunoglobulin-like receptors and natural killer receptors on virus-specific CD8+ T cells during the evolution of Epstein-Barr virus-specific immune responses in vivo

Antigen-primed cytotoxic T lymphocytes (CTL) may express leukocyte immunoglobulin-like receptors (LILRs) and natural killer receptors (NKRs). Published work suggests that expression of some of these receptors confers survival advantage, leading to the idea that cells expressing such receptors may accumulate as an antigen-specific response evolves. Here we tested this hypothesis by analyzing expression of CD85j (also known as LILRB1 or ILT2), KIRs, CD94, and CD161 by Epstein- Barr virus (EBV)-specific CTL during the primary and persistent phases of EBV infection in humans. During primary infection, few EBV-specific CTL expressed these receptors and this proportion was equally low in early persistent infection. Thus, expression of these molecules does not influence capacity to survive downregulation of the primary response. However, in donors persistently infected with EBV for many years, a significantly higher proportion of EBV-specific CTL expressed CD85j and NKRs, suggesting that cells expressing these receptors can accumulate with time. Using FACS analysis, we confirmed, at a single cell level, that expression of CD85j, defined by staining with the antibody VMP55, was associated with reduced capacity of EBV-specific CD8+ T cells to respond to antigen. Thus, in the later stages of persistent infection, protective immunity to EBV may be reduced due to the preferential accumulation of hyporesponsive EBV-specific CD8+ T cells.     

Lytic and latent EBV gene expression in transplant recipients with and without post-transplant lymphoproliferative disorder

Background

Epstein–Barr virus (EBV) is associated with post-transplant lymphoproliferative disorder (PTLD), which has significant morbidity and mortality in transplant recipients. To devise prophylactic measures, we need predictors of PTLD and a better understanding of the physiopathogenesis of the disease.

Objectives

To identify a molecular pattern of EBV gene products in blood that is specific to PTLD and can be used for the diagnosis of this disease.

Study design

We evaluated the ratio between latent and replicating EBV nucleic acids in individuals with PTLD by comparison with transplant recipients without PTLD and immunocompetent hosts with EBV DNA-emia. Subjects were prospectively identified between July 2009 and October 2010 at the University of Colorado Hospital. EBV DNA, LMP-2A Latency III and BZLF1 Lytic genes mRNA were quantified using real-time PCR.

Results

We found that PTLD subjects (N = 7) had significantly higher EBV DNA-emia compared with non-transplant immunocompetent subjects (N = 69; p < 0.0001), and transplant recipients without PTLD (N = 105; p < 0.0001). The ratios between LMP-2A and BZLF1 mRNA in transplant recipients were significantly lower than in non-transplant subjects (p = 0.04). However, PTLD and non-PTLD transplant recipients displayed similar ratios.

Conclusions

These results suggest that EBV replication makes a larger contribution to the circulating EBV DNA in transplant recipients compared with immunocompetent hosts. Transplant recipients seem to lose control over EBV replication, which may contribute to the development of PTLD.     

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.     

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     

B lymphocytes and Epstein-Barr virus: the lesson of post-transplant lymphoproliferative disorders

Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpes virus that establishes a life-long asymptomatic infection in immunocompetent hosts by colonizing memory B lymphocytes and hijacking cellular signaling pathways that regulate antigen-dependent B-cell activation and differentiation. In patients with solid organ or hematopoietic stem cell transplantation, the defect in EBV-specific immune responses may allow the outgrowth of EBV-carrying B lymphocytes that may give rise to a spectrum of different clinico-pathologic entities encompassed by the term post-transplantation lymphoproliferative disorders (PTLD). EBV-driven immortalization of B-cells is mediated by the cooperative activity of viral proteins that derange critical cellular pathways controlling growth and/or survival of B lymphocytes. Full transformation of infected B-cells is achieved by the contribution of poorly defined additional co-factors, including microenvironmental stimuli, genetic and epigenetic alterations. The quantification of circulating EBV DNA and EBV-specific T cells are valuable tools in the clinical monitoring of EBV-associated PTLD. The recent advances in elucidation of the mechanisms underlying EBV-induced growth transformation will be instrumental in guiding the design of novel approaches for the treatment of these often life-threatening lymphoproliferative disorders.     

Epstein-Barr virus infection in paediatric liver transplant recipients: detection of the virus in post-transplant tonsillectomy specimens.

AIMS: Post-transplant lymphoproliferative disease (PTLD) is an important and serious complication in transplant patients. Recent studies have suggested that quantitative assessment of Epstein-Barr virus (EBV) infection in transplant patients might help to identify those at risk of developing PTLD. Therefore, tonsils from paediatric liver transplant recipients were studied for evidence of EBV infection. METHODS: Tonsils were studied by in situ hybridisation for the detection of the small EBV encoded nuclear RNAs (EBERs). The phenotype of EBV infected cells was determined by double labelling in situ hybridisation and immunohistochemistry. The expression of viral latent and lytic antigens was determined by immunohistochemistry. Tonsils from patients without known immune defects were studied as controls. RESULTS: Tonsils from transplant patients showed pronounced follicular hyperplasia and minor paracortical hyperplasia. In situ hybridisation revealed variable numbers of EBV infected B cells in the tonsils from transplant patients (range, 2-1000/0.5 cm(2); mean, 434/0.5 cm(2); median, 105/0.5 cm(2)). Lower numbers were detected in the control tonsils (range, 1-200/0.5 cm(2); mean, 47/0.5 cm(2); median, 9/0.5 cm(2)). The latent membrane protein 1 (LMP1) of EBV was not detected and there were only rare cells in two cases showing expression of the EBV encoded nuclear antigen 2 (EBNA2). There was no evidence of lytic infection. None of the patients developed PTLD within a follow up period of up to five years. CONCLUSIONS: These data indicate that tonsillar enlargement in paediatric liver transplant patients does not necessarily imply a diagnosis of PTLD. Furthermore, the presence of increased numbers of EBV infected cells in tonsils from liver transplant recipients by itself does not indicate an increased risk of developing PTLD.