EBV-induced Lymphoproliferation Research Articles

Successful hematopoietic stem cell transplantation in a 4-1BB deficient patient with EBV-induced lymphoproliferation

Complete remission from recurrent EBV-positive lymphoma is not mandatory before HSCT to achieve long-term cure in a patient suffering from a recently described immunodeficiency affecting the T-cell coactivation molecule 4-1BB.

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350+ 293T cells in vitro. The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV+ B95-8 cell line, showing selectivity against gp350+ cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34+ cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8+gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER+ B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8+gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV+ lymphoproliferation and lymphomagenesis.

Novel heterozygous PIK3CD mutation presenting with only laboratory markers of combined immunodeficiency

Introduction: Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta (PIK3CD) is one part of a heterodimer forming the enzyme phosphoinositide 3-kinase (PI3K), found primarily in leukocytes. PIK3CD generates phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and is involved in cell growth, survival, proliferation, motility, and morphology. An increasing number of patients have been described with heterozygous PIK3CD gain-of-function (GOF) mutations, leading to combined immunodeficiency with both B- and T-cell dysfunction. Patients suffer recurrent respiratory infections, often associated with bronchiectasis and ear and sinus damage, as well as severe recurrent or persistent infections by herpesviruses, including EBV-induced lymphoproliferation. Aim: To present the clinical phenotypic variability of a novel PI3KCD mutation within a family. Methods: Patient information was collected prospectively and retrospectively from medical records. Comprehensive immune work up, genetic, and signaling evaluation was performed. Results: We describe here 2 patients, daughter and mother, with heterozygous PIK3CD mutation identified by whole exome sequencing and Sanger confirmation. The child was screen-positive by newborn screening for severe combined immunodeficiency (SCID). Cellular assays revealed an increase in the baseline phosphorylation of T cells in the patient. Furthermore, both patients had hyper-activation of the catalytic domain, resulting in increased phosphorylation of AKT upon activation. Discussion: GOF mutations affecting the PIK3CD gene are associated with an increased risk for lymphoproliferation leading to Activated PIK3-delta syndrome (APDS). The clinical course of APDS is highly variable, ranging from combined immunodeficiency with recurrent infections, autoimmune complications, and requiring stem cell transplantation, through isolated antibody deficiency, to asymptomatic adults. Our patient is the first to be identified by newborn screening for SCID. Surprisingly, the clinical course has so far been unremarkable, as well, the mother appears to be completely asymptomatic. Nevertheless, the persistent lymphopenia indicates PIK3CD dysfunction. Because of the wide gap between laboratory findings and clinical manifestations, this kindred poses both a diagnostic as well treatment challenge. Statement of novelty: We report here a novel PIK3CD mutation diagnosed due to abnormal newborn screen for SCID.

Immunodeficiency and EBV-induced lymphoproliferation caused by 4-1BB deficiency

The tumor TNF receptor family member 4-1BB (CD137) is encoded by TNFRSF9 and expressed on activated Tcells. 4-1BB provides a costimulatory signal that enhances CD8+ T-cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. The ligand for 4-1BB is expressed on antigen-presenting cells and EBV-transformed B cells. We investigated the genetic basis of recurrent sinopulmonary infections, persistent EBV viremia, and EBV-induced lymphoproliferation in 2 unrelated patients. Whole-exome sequencing, immunoblotting, immunophenotyping, and invitro assays of lymphocyte and mitochondrial function were performed. The 2 patients shared a homozygous G109S missense mutation in 4-1BB that abolished protein expression and ligand binding. The patients' CD8+ T cells had reduced proliferation, impaired expression of IFN-γ and perforin, and diminished cytotoxicity against allogeneic and HLA-matched EBV-B cells. Mitochondrial biogenesis, membrane potential, and function were significantly reduced in the patients' activated T cells. An inhibitory antibody against 4-1BB recapitulated the patients' defective CD8+ T-cell activation and cytotoxicity against EBV-infected B cells invitro. This novel immunodeficiency demonstrates the critical role of 4-1BB costimulation in host immunity against EBV infection.

Development of lymphoma from the donor of haploidentical stem cell transplantation: A case report.

Post-transplantation lymphoproliferative disease (PTLD) is a serious complication following hematopoietic stem cell transplantation (HSCT). The majority of the cases develop during the first year after the transplantation and are associated with reactivation of the Epstein-Barr virus (EBV); the EBV-induced lymphoproliferation usually includes donor-derived B cells. We herein describe the case of a 28-year-old female patient who developed EBV-negative PTLD, namely diffuse large B-cell lymphoma (DLBCL), 6 months after receiving a haploidentical HSCT from her father. Chimerism analysis performed with XY fluorescence in situ hybridization revealed a B-cell PTLD originating from the donor. Unfortunately, the donor also developed DLBCL 380 days after donating progenitor cells, although he was hematologically normal at the time of donation. The present case demonstrated that disease transmitted from the donor may be a possible cause of PTLD.

Management of post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disease (PTLD) is a major complication of hematopoietic stem cell and solid organ transplantation. The incidence of transplantation in childhood has been steadily rising, making PTLD the most common form of lymphoproliferation in childhood. The purpose of this review is to summarize the role of the Epstein-Barr virus (EBV) in the pathophysiology and discuss the management of PTLD. More than 90% of pediatric PTLD is EBV-positive. In immunocompetent hosts, the virus is controlled by cytotoxic T-cells, the cells targeted by immunosuppression to avoid graft-versus-host disease and/or organ rejection in transplant patients. The majority of pediatric transplant candidates are EBV-negative prior to transplant increasing the risk of EBV-induced lymphoproliferation upon seroconversion after transplant. Treatment options include reduction of immunosuppression, anti-CD20 monoclonal antibodies, and/or chemotherapy. Advanced understanding of the importance of cellular immunity in controlling lymphoproliferation has led to the development of cellular therapies targeting virus-specific antigens. PTLD is the most common form of lymphoproliferation in childhood due to the rising incidence of transplantation. EBV plays a pivotal role in the pathophysiology. Cellular therapies targeting viral antigens may replace chemotherapy in the treatment of PTLD in the near future.

Epstein-Barr virus oncoprotein super-enhancers control B cell growth.

Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.

Bendamustine, Ofatumumab and High-Dose Methylprednisolone (BOMP) in Relapsed/Refractory CLL: Results of a Planned Interim Analysis of the French CLL Intergroup ICLL01 Phase II Trial

Introduction:For relapsed or refractory (R/R) CLL patients (pts), combination of bendamustine and rituximab appears safe and effective (Fischer 2011). Ofatumumab monotherapy gives 58% ORR in heavily pre-treated (median 4 prior lines) R/R CLL pts (Wierda JCO 2010). High doses (HD) steroids are also active in poor prognosis pts with bulky nodal involvement or p53 impairment (Castro 2008, Xu 2010). We report a planned interim analysis of the ICLL01-BOMP phase II trial evaluating the association of Bendamustine, Ofatumumab and high-dose MethylPrednisolone for fit R/R CLL pts after 1-3 previous lines (NCT01612988).Patients and Methods:Primary endpoint was CR rate after 6 cycles (cy) of the BOMP regimen [i.e. bendamustine (70 mg/m2 d1, d2), ofatumumab (1000 mg d1;15 on cy#1-2 and d1 on cy#3-6) and HD methylprednisolone (1 g/m2 d1-3)]. The c#1 was preceded by an ofatumumab (300 mg) prephase. Response evaluation (IWCLL 2008) was done 3 months (m) after the last cy along with blood and bone marrow 10-color flow MRD analysis.Results:Among the 55 pts of this analysis, median age was 64 years (44-76). CIRS-G comorbidity score was 2-6 in 61% and pts had received 2-3 lines in 37% of the cases. Prior FCR-like regimens (50 (91%) patients) had been followed by relapse within 2y in 22/55 and 5/55 pts were fludarabine-refractory (FR). IGVH was unmutated (UM) in (47/52) 90.4%. Karyotypes were complex in 18/46 (39%) cases. Distribution according to FISH hierarchical model was: del(17p) in 15 (27%), del(11q) in 14 (26%), trisomy 12 in 4 (7%), del(13q) in 17 (31%) and normal in 5 (9%). Mutations on the TP53, SF3B1 and NOTCH1 genes occurred in 17 (31%), 14 (26%) and 5 (9%) pts, respectively.According to published risk stratification (Zenz, 2012), 34/55 pts (62%) belonged to the “highest-risk” group with either TP53 disruption (deletion and/or mutation) (n=19) and/or early relapse within 2 years post-FCR (n=22). The remaining patients belonged to either the “high-risk” group (UM-IGVH and/or Highb2mic and/or del11q) accounting for 17 pts (31%) or to the “low-risk” group (or non evaluable) accounting for 4 (7%) pts.Overall, 292 BOMP cy (mean 5.3 cy/pts) were delivered. Safety analysis recorded 158 grade 3-4 adverse events (G3-4/AE) with according to cy: neutropenia: 20.8%, thrombocytopenia: 11.3%, anemia: 2.4%, infection: 5,8%, hyperglycemia: 7,5%, liver enzyme elevation: 1,4%, cutaneous reaction: 1,4%, ofatumumab infusion related reaction: 0,3% and other AE: 3,4%. Overall 43 out of 55 pts (78.2%) had at least one G3-4/AE. Twenty-eight severe adverse events were reported in 20 pts. Treatment interruption before planned 6 cy occurred for pts’ decision (n=3), excessive toxicity (n=5) or early progressive disease (PD) (n = 4).Response in the ITT population was 76.4% ORR with 20% CR (n=11), 56.4% PR (n=31 including 5 nPR and 1 CRi), 9.1% stable disease (n=5), 10.9% PD (n=6) and 3.6% (n=3) non evaluable. Blood MRD obtained in 45 pts was negative (<10-4) in 13 (28.8%) cases. Following evaluation, 5 responding pts (9%) had RIC allogeneic (RIC-Allo) transplantation with a persistent remission.With median follow-up of 16.2 (5.1-23.6) months (m) we observed 9 deaths, related to PD (n=5), EBV-induced lymphoproliferation (n=1), PML encephalitis (n=1), sepsis/pancytopenia (n=1) or unknown origin (n=1). We recorded 22 relapses (including 4 Richter Syndromes) resulting in treatment in 17 cases, with a BTK inhibitor in 8 cases.The median OS has not been reached (estimation 84% at 18 m) (Fig 1B). The median PFS was 18.4 m (95%CI, 14.6-22.2) and the median time to next treatment 17.6 m (95%CI, 12.9-22.4). With 5 cases censored at time of RIC-Allo, the PFS (censored analysis) was 17.5 m (95%CI, 13.2-21.8). (Fig 1A) After univariate analysis, ORR was lower in the “highest-risk” (64,6%, p=0.01), del(17p) (40%, p=0.003), TP53 mutation (47.1%, p=0.01) and complex karyotype (61.2%, p=0.024) groups. PFS was shorter in the “highest-risk” (14 m, p=0.046), FR (4.96 m, p<0.001), del(17p) (9.5 m, p=0.017) and TP53 mutation (9.5 m, p=0.007) groups.Conclusion:Relapse treatment of CLL is a challenge especially after prior FCR-like treatments, accounting for >90% of this trial population. These results in terms of response and survival appear noteworthy considering that >60% are “highest-risk” pts. This study provides important information for forthcoming comparison with next emerging CLL therapies. [Display omitted] Disclosuresde Guibert:Roche: Honoraria. Feugier:Roche: Honoraria. Schuh:Roche, Gilead, GSK, NAPP, Celgene: Honoraria. Leblond:Roche: Honoraria, Speakers Bureau. Tournilhac:mundipharma: Honoraria, Other, Research Funding; GSK: Honoraria, Other, Research Funding; Roche: Honoraria, Other, Research Funding.

Adoptive transfer of EBV specific CD8+ T cell clones can transiently control EBV infection in humanized mice.

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.

165. Towards Gene Therapy for EBV-Associated Post-Transplant Lymphoproliferative Disease: Genetically Modified EBV-Specific Cytotoxic T Lymphocytes Induce Regression of Autologous EBV-Induced Lymphoproliferation Despite Immunosuppression

165. Towards Gene Therapy for EBV-Associated Post-Transplant Lymphoproliferative Disease: Genetically Modified EBV-Specific Cytotoxic T Lymphocytes Induce Regression of Autologous EBV-Induced Lymphoproliferation Despite Immunosuppression

Whole-exome sequencing identifies Coronin-1A deficiency in 3 siblings with immunodeficiency and EBV-associated B-cell lymphoproliferation

Primary immunodeficiencies are a rare group of inborn diseases characterized by a broad clinical and genetic heterogeneity. Substantial advances in the identification of the underlying molecular mechanisms can be achieved through the study of patients with increased susceptibility to specific infections and immune dysregulation. We evaluated 3 siblings from a consanguineous family presenting with EBV-associated B-cell lymphoproliferation at an early age (12, 7½, and 14 months, respectively) and profound naive T-cell lymphopenia. On the basis of the hypothesis of a rare inborn immunodeficiency of autosomal recessive inheritance, we sought to characterize the underlying genetic defect. We performed genome-wide homozygosity mapping, followed by whole-exome sequencing. We identified a homozygous inherited missense mutation in the gene encoding Coronin-1A (CORO1A) in the 3 siblings. This mutation, p. V134M, results in the substitution of an evolutionarily conserved amino acid within the β-propeller domain, which abrogates almost completely the protein expression in the patients' cells. In addition to a significant diminution of naive T-cell numbers, we found impaired development of a diverse T-cell repertoire, near-to-absent invariant natural killer T cells, and severely diminished mucosal-associated invariant T cell numbers. Our findings define a new clinical entity of a primary immunodeficiency with increased susceptibility to EBV-induced lymphoproliferation in patients associated with hypomorphic Coronin-1A mutation.

EBV lytic infection enhances transformation of B‐lymphocytes infected with EBV in the presence of T‐lymphocytes

Epstein-Barr virus (EBV) establishes lifelong latency in B-lymphocytes following infection. Although in immune-competent individuals EBV remains in a quiescent state, in immunodeficient individuals, such as those with AIDS or transplant recipients, B-lymphocytes infected with EBV proliferate to give rise to lymphoproliferative diseases. Similarly, in vitro, EBV transforms human B-lymphocytes into indefinitely growing lymphoblastoid cell lines (LCLs) in the absence of cytotoxic T-lymphocytes. Although LCLs harbor the entire EBV genome as an episome, in most cells the virus remains in a latent state expressing a fraction of EBV genes, and lytic infection occurs spontaneously but only in a small percentage of cells. Here, we report that lytic infection contributes to EBV-induced lymphoproliferation by a paracrine mechanism. An EBV immediate-early protein, BZLF1, induces IL-13, thus facilitating the proliferation of EBV-transformed B-lymphocytes in the presence of T-lymphocytes. These data suggest that lytic gene products could contribute to virus-induced oncogenesis by a paracrine mechanism.

EBV-Induced Lymphoproliferation

Abstract SCI-9The similar genome structures, methods of entry, replication, and egress of herpesviruses suggest a common evolutionary origin dating back about 200 million years. EBV, a gammaherpesvirus, has co-evolved with its human host alone for over 80 million years and has developed a relationship that is seldom detrimental to its host and may even be beneficial. For example, EBV-specific T cells with heterologous specificity may protect against other pathogens. Unlike RNA viruses, the sequence of EBV, including its strong immunogenic epitopes, is remarkably conserved even across geographically separated populations. This underscores the comfortable relationship that EBV has developed with the host immune response, which tightly controls but never eliminates the virus. Rarely, this relationship breaks down resulting in diverse diseases and malignancies with underlying causes that are poorly understood. Is mononucleosis restricted to adolescents because of a too active or an inadequate immune response? Why is post-transplant lymphoma (PTLD) not more common in T cell depleted stem cell recipients? How can EBV-positive malignancies develop in patients who have circulating T cells specific for the viral tumor antigens expressed in those malignancies and who control their general EBV infection? Patients with nasopharyngeal carcinoma (NPC) or EBV-positive lymphoma seldom display an elevated virus load in peripheral blood lymphocytes. To prevent EBV-associated diseases we must understand their root causes, which, in turn, require an understanding of the virus' complex life cycle that comprises involve four types of latency in B cells and replication in both B cells and mucosal epithelium. Each phase of the life cycle is represented both in normal infected cells and in one or more diseases. For example, virus replication is observed in tonsillar epithelium and in the oral hairy leukoplakias of HIV patients. Virus replication is also indirectly associated with NPC; its development is preceded by elevated antibody titers to the viral lytic cycle antigens despite the fact that NPC cells express only latency proteins. There are four patterns of viral latency and three are observed in tumor cells. Type 1 latency (expression of only two of over 90 viral proteins) is observed in normal circulating B cells, Burkitt's lymphoma and gastric carcinoma. Type 2 latency is shared by the T, B, and NK lymphomas that occur in immunocompetent individuals. Type 3 latency, the most immunogenic form of latency in which nine latency-associated proteins are expressed, is seen in tonsils, in lymphomas of the immunosuppressed and elderly, and in leiomyosarcoma. Therapies may specifically target the function or immunogenicity of the viral proteins, while prophylaxis, usually by vaccination, aims to prevent them. We will discuss the types of latency observed in EBV-associated lymphoproliferations in the context of immunotherapies that might be used to control them. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr Virus—Induced Lymphoproliferative Disorder After Rituximab Combined with CHOP Therapy

We present the case of a 35-year-old woman with follicular lymphoma who developed an Epstein-Barr virus (EBV)-induced B-cell lymphoproliferation with secondary viral-induced hemophagocytosis 13 months after treatment with rituximab and CHOP (cyclophosphamide/doxorubicin/vincristine/prednisone) chemotherapy. The EBV-induced lymphoproliferation was successfully treated with single-agent rituximab. To our knowledge, this is the first such case reported in the literature. Herein, we briefly review the spectrum of immunodeficiency-associated lymphoproliferative disorders (LPDs), discuss the immune deficit induced by rituximab, and speculate on its possible association with EBV reactivation in this patient.

Donor T lymphocyte infusion following ex vivo depletion of donor anti-host reactivity by a specific anti–interleukin-2 receptor P55 chain immunotoxin

ALLOGENEIC stem cell transplantation is the treatment of choice for many hematologic malignancies or inherited disorders. In the partially incompatible HLA setting ex vivo T-cell depletion of the graft and posttransplantation immunosuppression prevent development of graft-versus-host disease (GVHD), but lead in turn to a delay in immune reconstitution and a concordant increase in the incidence of opportunistic infections and disease relapses. To deplete the T cells responsible for GVHD but retain T cells responsible for the anti-leukemic and antiinfectious activities, we have developed an in vitro procedure to allo-activate donor T cells responsible for GVHD and eliminate them with an immunotoxin that is reactive with the cell surface activation antigen CD25. We infused the remaining allodepleted T cells between day 15 and 47 into pediatric patients with acquired or congenital hematopoietic disorders who received hematopoietic stem cell transplants (HSCT) on day 0. We evaluated the rates of GVHD and opportunistic infections as well as the time to immune reconstitution. Sixteen infusions of allodepleted T cells, ranging from 1 to 8 10 cells/kg were administered to pediatric patients between March 1999 and April 2001, none of whom received prophylactic therapy for GVHD. Despite the risk of transfusing donor T cells activated against recipient alloantigens ex vivo, GVHD greater than grade II was not observed. Two patients (13%) developed grade 1 aGVHD limited to skin, and two others (13%) developed grade II aGVHD with one case involving both the skin and gastrointestinal tract. All cases resolved with treatment. Interestingly, all cases correlated with a residual alloproliferation of 1% (P .001) (data not shown). Three patients displayed an active cytomegalovirus (CMV) infection at the time of HSCT that required antiCMV treatment. In addition, one patient (#5) experienced pulmonary and cerebral EBV-induced lymphoproliferation and a varicella zoster virus infection. After the infusion of allodepleted T cells, two types of situations were observed. Patients who did not show major infectious complications or GVHD displayed a progressive increase in the CD4 T-cell compartment, particularly of naive CD45RA phenotype cells, which were detectable by week 14. In contrast, patients who were infected at time of T-cell infusion demonstrated a rapid expansion in the CD4 T-cell compartment with clinically significant numbers of cells, all expressing CD45RO by week 8. In an attempt to assess the ability of an allodepleted T-cell “add-back” to exert an anti-viral infection effect, immunity to EBV and CMV infections was tested at 1 month after infusion in two infected patients, included the one presenting with cerebral and pulmonary lesions. In these two cases, circulating T cells were highly responsive to CMV and EBV as shown by their capacity to secrete -interferon in the presence of an autologous EBV cell line (transfected or not with CMV pp65 antigen) and their capacity to kill CMV pp65-expressing target cells without in vitro restimulation. As shown by CT scan, pulmonary and central nervous system lesions in patient 5 caused by the EBV infection disappeared 11 weeks after the T-cell infusion. For the other patient, CMV antigen was negative 15 weeks post-HSCT (10 weeks postinfusion). Infused allodepleted T cells seem to not recirculate in noninfected patients, an observation that may depend upon the low numbers of infused cells. As illustrated by the three cases of relapse among the five leukemic patients included in the clinical trial, cellular therapy for viral infections is achieved with doses of lymphocytes within the range of the

Inhibition of EBV-induced lymphoproliferation by CD4(+) T cells specific for an MHC class II promiscuous epitope.

Posttransplant lymphoproliferative disorder (PTLD) and B cell lymphomas induced by EBV continue to be a major life-threatening complication in transplant patients. The establishment and enhancement of T cell immunity to EBV before transplantation and immunosuppressive therapy could help diminish these complications, but the lack of an effective vaccine has limited this prophylactic approach. We describe here the identification of a peptide epitope from the EBV EBNA2 Ag that is capable of inducing in vitro CD4(+) T cell responses that inhibit the EBV-mediated B lymphocyte proliferation associated with PTLD. Most significantly, T cell responses to the EBNA2 epitope were found to be restricted by numerous MHC class II alleles (DR1, DR7, DR16, DR52, DQ2, and DQ7), indicating that this peptide is highly promiscuous and would be recognized by a large proportion (>50%) of the general population. These results are relevant for the design of a simple, inexpensive and widely applicable peptide-based vaccine to prevent PTLD in solid organ transplant patients.

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)

ICAM-1, soluble-CD23, and interleukin-10 concentrations in serum in renal-transplant recipients with Epstein-Barr virus reactivation.

Primary and reactivated Epstein-Barr virus (EBV) infections after organ transplantation are associated with the development of posttransplant lymphoproliferative malignancies. Since viral reactivation frequently stays asymptomatic, early diagnosis and treatment are challenges during posttransplant patient monitoring. Both soluble-CD23 (sCD23) and intercellular adhesion molecule 1 (ICAM-1) cell surface expression as well as interleukin-10 (IL-10) production are closely associated with viral gene expression. Therefore, immunoglobulin M (IgM), IgG, IgA, sCD23, ICAM-1, and IL-10 concentrations were measured in serum samples from patients during EBV reactivation (n = 14) and were compared with those in samples from patients without EBV reactivation (n = 10) following renal transplantation. In addition, serum sCD23, ICAM-1, and IL-10 concentrations were measured longitudinally in weekly to biweekly samples from 10 patients with EBV reactivation for at least 20 weeks following transplantation. A significant elevation of sCD23 was found during viral reactivation (P < 0.05), whereas ICAM-1 levels showed a nonsignificant increase. The finding of a highly significant elevation of the serum IL-10 concentration during EBV reactivation (P < 0.001) may support speculations about its role in EBV-induced lymphoproliferation and in the development of opportunistic infections and secondary malignancies. Maximum serum IL-10 levels at the time of EBV reactivation were found in 7 of 10 patients. Well-defined ICAM-1 and sCD23 concentration peaks were found in 9 of 10 and 8 of 10 patients, respectively. Although both markers are not specific for EBV reactivation and therefore may not be useful for primary diagnosis, sCD23 and ICAM-1 might be potent tools for the clinical monitoring of EBV activity and virus-induced lymphoproliferation.

Lymphoproliferative disease in scid mice reconstituted with human Sjögren's syndrome lymphocytes

Patients with Sjögren's syndrome (SS) have increased frequency of non-Hodgkin's B-cell lymphoma. These lymphomas frequently use a specific subclass of kappa light chain (encoded by variable region gene segment Hum KV325) and exhibit bcl-2 protooncogene translocation t(14;18). In order to determine whether expansion of this B-cell subset could be reproduced in an animal model, immunodeficient SCID (CB-17) mice were reconstituted with lymphocytes from 4 different SS patients at high risk of the development of lymphoma. Tumor-like nodules developed in all 11 SCID mice that received at least 5 x 10(5) lymphocytes from SS salivary glands or peripheral blood samples. However, the tumor-like nodules in the SCID mice differed from SS lymphomas in vivo in that they (1) exhibited multiple immunoglobulin gene rearrangements; (2) did not have expansion of B-cells expressing the Hum KV325 K-light chain; and (3) lacked detectable t(14;18) translocations. Characterization of the SCID tumor-like nodules revealed a high level of Epstein-Barr virus (EBV) DNA, EBV-associated antigens (EA-R, EBNA-2, AND LMP), and the EBV-encoded cytokine BCRF-1 that is structurally similar to IL-10. These results demonstrate that the lymphoproliferation occurring in the salivary glands of SS patients is not reproduced in the SCID/hu chimeric mouse. It is likely that specific factors in the human salivary gland are required for development of lymphoma in SS patients and that such factors are not present in the SCID/hu chimeric mouse. Furthermore, EBV-induced lymphoproliferation, as seen in the SCID/hu chimera, does not lead to expansion of the same lymphoid subsets that occurs in vivo.

Cyclosporin Abrogates Virus-Specific T-Cell Control of EBV-Induced B-Cell Lymphoproliferation

Renal and cardiac recipients undergoing aggressive immunosuppressive therapy with cyclosporin A have been reported to have unusually high incidences of Epstein-Barr virus (EBV) genome-positive lymphomas. T cells have been shown to be of critical importance in controlling the lymphoproliferative potential of the EB virus. EBV-specific T cell clones were generated in vitro by repeated antigenic restimulation in the presence of interleukin-2 (IL-2). These virus-specific cells were used to study cyclosporin A's effect on their activation, proliferation and cytotoxic function, and the possible abrogation of the cell's ability to control virus-induced lymphoproliferation. In this study, we show that cyclosporin A does not interfere with antigen recognition, since the T cell cytotoxic potential is unchanged. However, cyclosporin A induces a dose-dependent reduction in membrane IL-2 receptor expression which consequently limits the proliferation of the antigen-activated cell. This may translate in vivo to an insufficient expansion of the cytotoxic T cells which controls the outgrowth of the EBV-infected B cell.