EBV-associated Post-transplant Lymphoproliferative Disease Research Articles

Autopsy Case of Epstein-Barr Virus-associated T-cell Post-transplant Lymphoproliferative Disorder with Mono- and Polymorphic Lesions: Possibility of 'Polymorphic T-cell Post-transplant Lymphoproliferative Disorder'.

Background Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is predominantly of B cell origin. The concept of clonal evolution from poly- to monoclonal lymphoproliferation has been put forward, but T-cell PTLDs are rare with an unknown etiology. Case Presentation In a unique autopsy case of a 53-year-old man with EBV-associated T-cell PTLD, we observed polymorphic T-cell proliferation across several organs and monomorphic T-cell proliferation in the perforated ileum. Interestingly, both manifestations exhibited identical monoclonal peaks in the T-cell receptor rearrangement polymerase chain reaction (PCR) analyses. Conclusion These findings suggest the existence of clonal evolution in EBV-associated T-cell PTLD, leading to the proposal of the novel concept of polymorphic T-cell PTLD.

Persistent Hypogammaglobulinemia after Receiving Rituximab Post-HSCT Is Not Caused by an Intrinsic B Cell Defect.

In the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or autoimmune phenomena such as autoimmune hemolytic anemia (AIHA). Persistent hypogammaglobulinemia and immunoglobulin substitution dependence has been observed in several patients after RTX treatment despite the normalization of total B cell numbers. We aimed to study whether this is a B cell intrinsic phenomenon. We analyzed four patients with different primary diseases who were treated with myeloablative conditioning and matched unrelated donor HSCT who developed persistent hypogammaglobulinemia after receiving RTX treatment. They all received RTX early after HSCT to treat EBV infection or AIHA post-HSCT. All patients showed normalized total B cell numbers but absent to very low IgG positive memory B cells, and three lacked IgA positive memory B cells. All of the patients had full donor chimerism, and none had encountered graft-versus-host disease. Sorted peripheral blood naïve B cells from these patients, when stimulated with CD40L, IL21, IL10 and anti-IgM, demonstrated intact B cell differentiation including the formation of class-switched memory B cells and IgA and IgG production. Peripheral blood T cell numbers including CD4 follicular T-helper (Tfh) cells were all within the normal reference range. In conclusion, in these four HSCT patients, the persistent hypogammaglobulinemia observed after RTX cannot be attributed to an acquired intrinsic B cell problem nor to a reduction in Tfh cell numbers.

Low incidence and morbidity of Epstein-Barr virus reactivation following donor lymphocyte infusions.

Low incidence and morbidity of Epstein-Barr virus reactivation following donor lymphocyte infusions.

Impact of Pre-Emptive Rituximab Treatment for Epstein-Barr Virus Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation: A Longitudinal Study of 110 Patients

▪Background: Epstein-Barr Virus (EBV) reactivation and EBV-associated post-transplant lymphoproliferative disease (PTLD) are well recognized complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with a high mortality. Pre-emptive anti-CD20 therapy with Rituximab has proved successful to reduce PTLD incidence in the setting of EBV reactivation. However, since CD20 expression is not confined to the malignant cells, normal B cells are also targeted by Rituximab, which can be of concern in patients who are already immunosuppressed, especially with regard to long-term infectious complications. This study aimed to analyze immune recovery, and to define the incidence of infectious complications and outcomes following Rituximab pre-emptive therapy.Patients and Methods: One hundred and ten consecutive patients from a single center, who underwent allo-HSCT between 2009 and 2016 and treated with rituximab for EBV reactivation, were included. Based on ECIL recommendations, EBV-DNAemia was routinely monitored using real-time quantitative PCR to quantify EBV genomes. Patients with two consecutive positive results received weekly Rituximab until EBV-DNAemia clearance. Clinical data [infections, overall survival (OS), event-free survival (EFS), non-relapse mortality (NRM), relapse incidence (RI), acute graft-versus host-disease (GVHD) and chronic GVHD] were assessed together with sequential evaluation of the following immunological parameters (T cells, B cells, Ig levels and neutrophil counts). Moreover, in a subset of 38 patients, immune B-cell subset quantification were performed using multi-color flow cytometry at months (M) 1, 3, 6 and 12 after Rituximab administration.Results: In this cohort, median age was 55 (range, 16-71) years. Diagnoses were myeloid malignancies (71%), lymphoid malignancies (25%) or severe aplastic anemia (4%). Thirty-two percent of patients received a graft from a matched sibling donor, 44% from an unrelated donor, and 24% from a haplo-identical donor. Ninety-three patients received a reduced intensity conditioning regimen based on fludarabine, busulfan +/- thiotepa. Ninety-two percent of the patients received ATG as part of the conditioning regimen. In the haplo subgroup, patients received standard post-transplant cyclophosphamide. All patients received a combination of cyclosporine A and mycophenolate mofetil for GVHD prophylaxis, except for patients with a sibling donor who received cyclosporine A only.Median times from allo-HSCT to EBV reactivation and from allo-HSCT to rituximab administration were 52 and 60 days, respectively. Median number of Rituximab administrations was 3 (range, 2-8). Five patients developed PTLD despite pre-emptive Rituximab (including one case of PTLD-related death). The cumulative incidence (CI) of overall infections at 2 years was 36% (95%CI: 27-46%). Twenty-one percent developed at least one bacterial infection, 19% a viral infection, and 6% a fungal infection. At 2 years, EFS was 63% (95% CI: 53-73), OS was 69% (95% CI: 60-78), NRM was 18.6% (95% CI: 11-27) and RI 18% (95% CI: 11-27). CI of grade 2-4 acute GVHD was 6.4% (95% CI: 11-27). CI of chronic GVHD 34.5% (95% CI: 3-12) and CI of extensive cGVHD was 9%. After taking into account GVHD, donor lymphocyte infusion, Ig IV substitution and relapse, at M1, M3, M6, M12, the median lymphocytes counts were 0.62/µL, 0.7/µL, 0.88/µL and 1.62/µL, and Ig levels 6.3g/L, 6g/L, 6.1g/L and 6.6g/L, respectively. Twenty-three percent of patients developed a neutropenia that was considered to be related to Rituximab administration. A slow B cell reconstitution was observed with median counts of 0.12/µL at M1, 0.55/µL at M3, 0.70/µL at M6 and 0.90/µL at M12, with over time a decreased proportion of naïve B cells (CD24 low, CD27-, IgD+, CD38 low) contrasting with an increased proportion in transitional B cells (CD24+, CD27-, IgD+, CD38+).Conclusion: Pre-emptive Rituximab treatment for EBV reactivation after HSCT is associated with high infection rate and delayed immunological reconstitution, with a quarter of the patients developing Rituximab-related neutropenia. However, these unfavorable effects did not negatively impact general outcomes, and the relatively very low CI of extensive cGVHD may suggest a favorable impact of anti-CD20 therapy on this late complication. DisclosuresMohty:Sanofi: Honoraria, Speakers Bureau.

EBV-Associated Post-Transplant Lymphoproliferative Disease (PTLD) in Allogeneic Transplantation

Post-Transplant Lymphoproliferative Disease (PTLD) following both Solid Organ Transplantation (SOT) and Hematopoietic Stem Cell Transplantation (HSCT) is a rare life-threatening complication. The majority of PLTDs are associated to Epstein Bar Virus (EBV) [1] reactivation, usually in the early phase [2] after transplant, when the patient is severely immunocompromised and is unable to control virus replication [3]. Despite the mortality of EBV-associated PTLD has been reduced over the years, the different histological patterns of its presentation, ranging from indolent to high grade B cell lymphoma, still play a role in the outcome. Herein, we report the case of a 60-years-old man diagnosed with acute myeloid leukemia who underwent allogeneic transplantation and developed a fatal Hemophagocytic Histiocytosis (HLH) secondary to an aggressive EBV-associated PTLD, not responding to a rituximab-based treatment.

The Role of Antiviral Prophylaxis for the Prevention of Epstein-Barr Virus-Associated Posttransplant Lymphoproliferative Disease in Solid Organ Transplant Recipients: A Systematic Review.

The role of antiviral prophylaxis for the prevention of posttransplant lymphoproliferative disease (PTLD) remains controversial for solid organ transplantation (SOT) recipients who are seronegative for Epstein-Barr virus (EBV) but who received organs from seropositive donors. We performed a systematic review and meta-analysis to address this issue. Two independent assessors extracted data from studies after determining patient eligibility and completing quality assessments. Overall, 31 studies were identified and included in the quantitative synthesis. Nine studies were included in the direct comparisons (total 2366 participants), and 22 were included in the indirect analysis. There was no significant difference in the rate of EBV-associated PTLD in SOT recipients among those who received prophylaxis (acyclovir, valacyclovir, ganciclovir, valganciclovir) compared with those who did not receive prophylaxis (nine studies; risk ratio 0.95, 95% confidence interval 0.58-1.54). No significant differences were noted across all types of organ transplants, age groups, or antiviral use as prophylaxis or preemptive therapy. There was no significant heterogeneity in the effect of antiviral prophylaxis on the incidence of PTLD. In conclusion, the use of antiviral prophylaxis in high-risk EBV-naive patients has no effect on the incidence of PTLD in SOT recipients.

Rituximab-based treatments followed by adoptive cellular immunotherapy for biopsy-proven EBV-associated post-transplant lymphoproliferative disease in recipients of allogeneic hematopoietic stem cell transplantation

ABSTRACTTo improve prognosis of post-transplant lymphoproliferative disease (PTLD), a sequential therapeutic strategy that rituximab-based treatments followed by donor lymphocyte infusion (DLI) or autologous EBV-specific cytotoxic T lymphocytes (EBV-CTL) for biopsy-proven EBV-associated PTLD in recipients of allogeneic hematopoietic stem cell transplantation was designed. 84 patients with EBV-PTLD were enrolled in this prospective study. After two cycles of the rituximab-based treatments, 68 of 84 patients (81% [95% CI 71–88]) responded and 52 (62% [51–72]) had CRs. This increased to 73 of 77 patients (95% [87–98]) with completion of sequential cell infusions, and 70 of 77 (91% [82–96]) achieved CRs after DLI or autologous EBV-CTL infusion. 22 patients experienced acute GVHD (aGVHD) (grade I in 5 and grade II in 13, grade III in 4) and 13 chronic GVHD (limited cGVHD in 7 and extensive cGVHD in 6) in 62 patients undergoing a median of three doses of DLI. The incidences of GVHD were similar between DLI and EBV-CTL group (aGVHD 35% vs. 33%, p = 0.876; cGVHD 21% vs. 13%; p = 0.503). EBV-CTL activity after the rituximab-based treatments did not change, while increased after cell infusions and reached its maximum in the 3rd or 6th month after EBV-CTL or DLI treatment, respectively. The 5-y cumulative incidence of PTLD relapse was 4.5% ± 3.3%. The 5-y overall survival (OS) and progression-free survival (PFS) after PTLD were 70.7% ± 5.2% and 68.9% ± 5.3%, respectively. Rituximab-based treatments combined with adoptive cellular immunotherapy might elevate CR rates and reduce relapse of PTLD after allo-HSCT.

Greatly reduced risk of EBV reactivation in rituximab-experienced recipients of alemtuzumab-conditioned allogeneic HSCT.

EBV-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed the incidence and risk factors for EBV reactivation in 186 adult patients undergoing consecutive allo-HSCT with alemtuzumab T-cell depletion at a single centre. The cumulative incidence of EBV reactivation was 48% (confidence interval (CI) 41–55%) by 1 year, with an incidence of high-level EBV reactivation of 18% (CI 13–24%); 8 patients were concurrently diagnosed with PTLD. Amongst patients with high-level reactivation 31/38 (82%) developed this within only 2 weeks of first EBV qPCR positivity. In univariate analysis age⩾50 years was associated with significantly increased risk of EBV reactivation (hazard ratio (HR) 1.54, CI 1.02–2.31; P=0.039). Furthermore, a diagnosis of non-Hodgkin lymphoma (NHL) was associated with greatly reduced risk of reactivation (HR 0.10, CI 0.03–0.33; P=0.0001) and this was confirmed in multivariate testing. Importantly, rituximab therapy within 6 months prior to allo-HSCT was also highly predictive for lack of EBV reactivation (HR 0.18, CI 0.07–0.48; P=0.001) although confounding with NHL was apparent. Our data emphasise the risk of PTLD associated with alemtuzumab. Furthermore, we report the clinically important observation that rituximab, administered in the peri-transplant period, may provide effective prophylaxis for PTLD.

Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event

AbstractAllogeneic stem cell transplantation (allo-HSCT) provides a unique opportunity to track Epstein-Barr virus (EBV) infection in the context of the reconstituting B-cell system. Although many allo-HSCT recipients maintain low or undetectable levels of EBV DNA posttransplant, a significant proportion exhibit elevated and rapidly increasing EBV loads which, if left untreated, may lead to potentially fatal EBV-associated posttransplant lymphoproliferative disease. Intriguingly, this high-level EBV reactivation typically arises in the first 3 months posttransplant, at a time when the peripheral blood contains low numbers of CD27+ memory cells which are the site of EBV persistence in healthy immunocompetent donors. To investigate this apparent paradox, we prospectively monitored EBV levels and B-cell reconstitution in a cohort of allo-HSCT patients for up to 12 months posttransplant. In patients with low or undetectable levels of EBV, the circulating B-cell pool consisted predominantly of transitional and naive cells, with a marked deficiency of CD27+ memory cells which lasted >12 months. However, among patients with high EBV loads, there was a significant increase in both the proportion and number of CD27+ memory B cells. Analysis of sorted CD27+ memory B cells from these patients revealed that this population was preferentially infected with EBV, expressed EBV latent transcripts associated with B-cell growth transformation, had a plasmablastic phenotype, and frequently expressed the proliferation marker Ki-67. These findings suggest that high-level EBV reactivation following allo-HSCT may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.

Epstein-Barr virus LMP2A suppresses MHC class II expression by regulating the B-cell transcription factors E47 and PU.1

AbstractOncogenic Epstein-Barr virus (EBV) uses various approaches to escape host immune responses and persist in B cells. Such persistent infections may provide the opportunity for this virus to initiate tumor formation. Using EBV-immortalized lymphoblastoid cell lines (LCLs) as a model, we found that the expression of major histocompatibility complex (MHC) class II and CD74 in B cells is repressed after EBV infection. Class II transactivator (CIITA) is the master regulator of MHC class II–related genes. As expected, CIITA was downregulated in LCLs. We showed that downregulation of CIITA is caused by EBV latent membrane protein 2A (LMP2A) and driven by the CIITA-PIII promoter. Furthermore, we demonstrated that LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression. Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was critical for the repression of E47 and PU.1 promoter activity via Syk, Src, and the phosphatidylinositol 3-kinase/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach showed that the expression levels of E47, PU.1, CIITA, MHC class II, and CD74 are reversed. These data indicated that the LMP2A may reduce MHC class II expression through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that MHC class II may be detected in tonsils and EBV-negative Hodgkin disease but not in EBV-associated posttransplant lymphoproliferative disease and Hodgkin disease.

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

Rituximab-Based Treatments Followed By Adoptive Cellular Therapies For EBV-Associated Post-Transplant Lymphoproliferative Disease In Recipients Of Allogeneic Hematopoietic Stem Cell Transplantation

Background The introduction of rituximab has improved the complete remission(CR) rate of EBV-associated posttransplant lymphoproliferative disease(PTLD), thus the recurrence of PTLD becomes the main cause affecting long-term survival, which is closely related with the reestablishment of immune functions. Unfortunately, PTLD happens generally at the early stage of transplants and the reconstitution of immunocompetence needs for 3-5 years in the recipients of allo-HSCT. In this study, a sequential therapeutic strategy that based on rituximab followed by adoptive cellular therapies (G-CSF mobilized donor lymphocyte infusion (DLI) or EBV-specific cytotoxic T lymphocyte infusion (EBV-CTL)) was evaluated for decreasing relapse of PTLD. Methods Fifty-two patients with EBV-PTLD were enrolled in this prospective study. Once PTLD was diagnosed,immunosuppressants would be withdrawn in a stepwise fashion (ie, total dose reduced by 20%/week)if the condition of the patient was acceptable. The rituximab-based treatments (rituximab alone or combined with chemotherapy) were administrated based on PTLD histopathology and the blood cells counts. After CR or 2 cycles of rithuximab-based treatments, DLI or EBV-CTL therapy would be performed in this cohort. The rituximab-based treatments would be discontinued once patient obtained CR, and DLI would be performed once Monthly till GVHD occurred or for a total of 4 doses and EBV-CTL infusion would be performed every two weeks till GVHD occurred or for a total of 8 doses . Results After 2 cycles of the rituximab-based treatments, 37 patients obtained complete remission (CR), 8 obtained partial remission (PR), and 7 no remission (NR), including 4 died of PTLD progression. The CR rate of 2 cycles of rituximab-based treatments was 71.2%. After rituximab-based treatments combined with the adoptive cellular therapies, 9 obtained CR and 2 died of PTLD progression in 11 patients who did not achieve CR within 2cycles of rituximab-based treatments. The CR rate of 2 cycles of rituximab-based treatments combined with cellular therapies was 88.5%. Seven patients experience acute GVHD (aGVHD) (grade I in 1 and grade II in 6) and 8 chronic (cGVHD) (limited cGVHD in 5 and extensive cGVHD in 3) in 39 patients underwent a median 4 doses of DLI. One patient experienced grade II aGVHD and 2 limited cGVHD in 8 patients underwent a median of 7 doses of EBV-CTL. There were no differences in incidence of aGVHD(P=1.000) and cGVHD(P=1.000) between the patients received DLI and CTL. Within a median follow-up of 632 (range, 21 to 1651) days, one patient experienced PTLD relapse, and the 4 year cumulative incidence of PTLD relapse and primary malignancy relapse was 5.3%±5.1% and 6.2±4.3%, respectively. The 4 year cumulative overall survival (OS) after PTLD and disease(PTLD)-free survival were 66.2%±7.1% and 65.9±7.3%, respectively. Conclusions Rituximab-based treatments combined with the adoptive cellular therapies might elevate PTLD CR rate, and decrease the relapse in the recipients of allo-HSCT. Disclosures: Liu: It was supported by 863 Program (No. 2011AA020105), National Public Health Grand Research Foundation (Grant No. 201202017): Research Funding; It was supported by National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China(11A72121174): Research Funding.

Genetically Modified EBV-Specific Cytotoxic T Lymphocytes Induce Regression Of EBV Lymphoproliferation Despite Immunosuppression In Xenografted Mice: A Novel Strategy For EBV-Associated Post-Transplant Lymphoproliferative Disease

BackgroundEBV associated lymphoproliferative disease (PTLD) remains a major cause of morbidity and mortality after stem cell (SCT) or solid organ (SOT) transplant. Adoptive transfer of ex vivo-derived EBV-specific cytotoxic T lymphocytes (EBV-CTL) to reconstitute immunity to the oncogenic virus EBV has been highly effective for both the prophylaxis and treatment of PTLD after SCT, where immunosuppression could be withdrawn before transfer of EBV-CTLs. In contrast, the application of this approach for the treatment of PTLD in SOT patients, although feasible, has been challenging with restricted expansion, persistence and efficacy of the adoptive transferred T cells. This difference is likely to reflect the need for the on-going immunosuppression to prevent graft-rejection post SOT which inhibits the virus-specific T cell responses. Our group has previously genetically engineered EBV-CTL to enable them to function in vitro in the presence of the calcineurin inhibitor Tacrolimus (FK506) through retroviral transfer of a calcineurin mutant (CNA12). AimTo examine the ability of genetically engineered EBV-CTLs resistant to FK506 to control EBV+ B cell lymphoma progression in vivoin a xenogeneic mouse model in the presence of FK506. MethodsNOD/SCID/IL2rgnull (NSG) mice were inoculated subcutaneously with 5×106 EBV-transformed lymphoblastoid B cell lines (LCL) labelled with F-Luc to develop human EBV+ lymphoma. To evaluate in vivo antitumor activity, 5×106 CTLs retrovirally transduced with CNA12 or eGFP control CTLs were injected intravenously after 7 days in the presence or absence of FK506 (10mg/kg/day). Tumour growth was analysed using the Xenogen-IVIS system. ResultsAdoptive transfer of autologous CNA12 transduced CTLs induced EBV+ lymphoma regression in the presence of FK506, as assessed both by IVIS and tumour size, whereas FK506 treated mice receiving eGFP control CTLs had tumour progression (P<0.05). This resulted in significantly improved survival of mice treated with CNA12-CTL in the presence of FK506 than eGFP-CTLs treated animals (P<0.0001). CNA12 transduced EBV CTLs persisted longer and expanded more (P<0.0001) than eGFP-CTLs in peripheral blood of mice treated with FK506 demonstrating a selective growth advantage and enrichment of CTLs resistant to immunosuppression. Immunohistochemical staining showed that adoptively transferred CTLs home to the tumour and an increase of tumour infiltrating T cells in CNA12-CTL compared with eGFP-CTLs treated mice in the presence of FK506 was observed. ConclusionsOur results demonstrate that CNA12 modified EBV-CTL can induce regression of EBV-associated tumours in vivo in the face of on-going immunosuppression with FK506. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy. Disclosures:No relevant conflicts of interest to declare.

EBV-associated post-transplant lymphoproliferative disorder following in vivo T-cell-depleted allogeneic transplantation: clinical features, viral load correlates and prognostic factors in the rituximab era

EBV-associated post-transplant lymphoproliferative disease (PTLD) following Alemtuzumab-based allo-SCT is a relatively uncommon and challenging clinical problem but has not received detailed study in a large cohort. Quantitative-PCR (qPCR) monitoring for EBV reactivation post allo-SCT is now commonplace but its diagnostic and predictive value remains unclear. Sixty-nine patients with PTLD following Alemtuzumab-based allo-SCT were studied. Marked clinicopathological heterogeneity was evident; lymphadenopathy was frequently absent, whereas advanced extranodal disease was common. The median viral load at clinical presentation was 49 300 copies/mL (50-65 200 000 copies/mL) and, notably, 23% and 45% of cases, respectively, had 10 000 and 40 000 copies/mL. The overall response rate to rituximab as first-line therapy was 70%. For rituximab failures, chemotherapy was ineffectual but DLIs were successful. A four-parameter prognostic index predicted response to therapy (OR 0.30 (0.12-0.74); P=0.009] and PTLD mortality (hazard ratio (HR) 1.81 (1.12-2.93) P=0.02) on multivariate analysis. This is the largest detailed series of EBV-associated PTLD after allo-SCT. At clinical presentation, EBV-qPCR values are frequently below customary thresholds for pre-emptive therapy, challenging current paradigms for monitoring and intervention. A four-point score identifies a proportion of patients at risk of rituximab-refractory disease for whom alternative therapy is needed.

De novo malignant disease after liver transplantation? Risk and surveillance strategies

De novo malignant disease after liver transplantation? Risk and surveillance strategies

Targeting EBV‘s Achilles‘ Heel with Antigen-Specific T Cells

Evaluation of: Icheva V, Kayser S, Wolff D et al. Adoptive transfer of Epstein-Barr virus (EBV) nuclear antigen 1-specific T cells as treatment for EBV reactivation and lymphoproliferative disorders after allogeneic stem-cell transplantation. J. Clin. Oncol. 31(1), 39-48 (2013). Adoptive transfer of donor-derived EBV-specific T cells is an effective strategy for the prevention and treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD). However, the time-consuming process of EBV-specific T-cell generation using standard protocols has limited their broader use. Ex vivo IFN-γ capture assay is an attractive alternative for the rapid isolation of EBV-specific T cells. In the present study, Icheva et al. employ this method to rapidly isolate clinical-grade T cells that are specific for EBNA1. Adoptive transfer of EBNA1-specific T cells was safe and resulted in clinical benefit in 7 out of 10 patients with EBV-viremia and/or PTLD. Thus, T-cell therapy targeting a single EBV antigen, EBNA1, which is critical for EBV episome maintenance, may be sufficient for EBV-PTLD therapy in hematopoietic stem cells transplantation recipients.

Improving T-Cell Therapy for Epstein-Barr Virus Lymphoproliferative Disorders

More than 90% of the population is infected with Epstein-Barr virus (EBV), which is an enveloped herpesvirus. EBV infection in immunocompetent individuals causes a mild self-limiting illness, but the virus persists lifelong as a latent infection in B cells and spreads in the community by productive replication in B cells and oral epithelial cells. EBV latency varies in the number and type of EBV proteins expressed by B cells. In type 3 latency, all nine latency-associated EBV proteins are expressed. Such B cells express cell adhesion and costimulatory molecules rendering them immunogenic and susceptible to immune-mediated killing by EBV-specific cytotoxic T lymphocytes (CTLs). After hematopoietic stem-cell transplantation (HSCT) without functional CTLs, type 3 latency EBVinfected B cells proliferate unchecked. EBV-infected B cells cause a lymphoproliferative disorder that can progress to lymphoma with increased levels of EBV DNA detected by polymerase chain reaction (PCR). EBV-associated post-transplant lymphoproliferative disease (PTLD) occurs in less than 1% to 25% of HSCT recipients depending on the type of graft, the degree to which the graft is T-cell depleted, and the post-HSCT immunosuppression. Rituximab is an effective monotherapy even in established PTLD after HSCT, with response rates of 55% to 100%. However, this B-cell depleting antibody increases risk of infection, and PTLD can recur if EBV-specific T-cell immunity is not restored. Because of its immunogenicity and occurrence in the context of immunodeficiency, PTLD is highly amenable to immunotherapy with EBVspecific CTLs. In the article that accompanies this editorial, Icheva et al used donor-derived Epstein-Barr nuclear antigen 1 (EBNA1) –specific T cells to treat post-HSCT PTLD. CTLs were generated with antigenpresenting cells that were pulsed either with whole protein or synthetic overlapping peptides to elicit EBNA1-specific T cells, which were then selected by interferon alfa capture (Fig 1). The selected T cells were either transfused directly without in vitro expansion or cryopreserved for future use. These rapidly generated EBNA1 CTLs were effective in seven of 10 cases of EBV-associated PTLD. Immunotherapeutic strategies to restore EBV-specific T-cell function have been used for more than 15 years to improve the outcome of PTLD. Initially, unselected donor lymphocytes from the EBV seropositive stem-cell donors (assumed to have a high frequency of EBV-specific precursors) were used to restore the immune response to EBV. This simple strategy is readily available at any transplant center and yields responses of approximately 70%. However, unselected donor lymphocyte infusions carry significant risk of severe graft versus host disease. Because of this, several groups generated and infused donor-derived EBV-specific T cells in patients after SCT. More than 100 patients have received such EBV-specific T cells, either as prophylaxis or as treatment for EBV PTLD with complete remission rates also in the order of 70%. Thus far, to generate EBV-specific CTLs, investigators have used EBV-transformed B cells (lymphoblastoid cell lines [LCLs]) that express type III latency like PTLD. It takes 10 to 12 weeks to generate LCLs and sufficient EBV-specific CTLs for infusion and release testing. This prolonged manufacturing time limits the ready use of this therapy for the urgent treatment of EBVassociated PTLD. The importance of the study by Icheva et al is that it demonstrates that rapidly generated EBV-specific CTLs can control PTLD similarly to the traditionally manufactured EBVspecific CTLs. Other investigators have successfully reported the rapid manufacture of EBV-CTLs by using HLA-A2 restricted tetramer selection, immunomagnetic selection of interferon-gamma–secreting T cells after stimulation of donor mononuclear cells with a spectrum of class I and II EBV peptides or by using latent membrane protein 2 (LMP2), EBNA1, and BamH1 Z leftward reading frame 1 (BZLF1, an immediate early transcription protein responsible for switching between latent and lytic EBV infection) plasmid transfected dendritic cells to expand EBV-specific T cells. The novelty of the approach by Icheva et al is that they chose (more for regulatory rather than scientific reasons) to target only the immunosubdominant EBV antigen EBNA1 rather than the immunodominant EBV proteins EBNA3, EBNA2, or LMP2 expressed in PTLD. Nevertheless, EBNA1 was shown to be an effective target and, moreover, promises to broaden the applicability of this immunotherapy to other EBV type 1 and 2 latency tumors (including Burkitt’s lymphoma and Hodgkin’s lymphoma, respectively) because all EBVassociated cancers express EBNA1. However, there are still problems to be addressed in additional studies. First, the targeting of a single antigen can increase the likelihood of tumor immune escape. For this reason, the authors intend to target additional EBV antigens such as LMP2 in future studies. In addition, because EBV reactivation and lymphoproliferation usually occurs from endogenous virus, seropositive recipients of transplants from EBV-naive, seronegative donors are at greatest risk for EBV LPD. Although EBV-specific CTLs have been JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 1 JANUARY 1 2013

Multiplex polymerase chain reaction for six herpesviruses after hematopoietic stem cell transplantation

Herpesviruses cause life-threatening diseases after hematopoietic stem cell transplantation (HSCT). It is necessary that viral diseases are identified early and safely diagnosed. The purpose of this study was to evaluate the efficacy of multiplex polymerase chain reaction (PCR) for qualitative detection of the six herpesviruses simultaneously: herpes simplex virus type 1 and type 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV) and human herpesvirus 6B. Multiplex PCR was applied to patients with various clinical manifestations including central nervous system, cutaneous and mucosal complications after allogeneic HSCT, and the data were retrospectively analyzed. Patients positive for cytomegalovirus in peripheral blood by multiplex PCR might need pre-emptive treatment, but a positive result for EBV had no specific correlation with EBV-associated post-transplant lymphoproliferative disease, and positive result for human herpesvirus 6B failed to show any clinical significance. The multiplex PCR was safe and helpful to diagnose viral diseases of local regions, for example, the central nervous system, skin and mucosa. It may be worthwhile to survey the six herpesviruses with multiplex PCR after HSCT.

Decreased IL-7 Signaling in T Cells From Patients With PTLD After Allogeneic HSCT

Interleukin (IL)-7, a nonredundant cytokine for B and T cells, plays a central role in cell survival and immune memory formation. Peripheral blood mononuclear cells (PBMCs) from 7 patients after hematopoietic stem cell transplantation (HSCT) diagnosed with posttransplantation lymphoproliferative disease (PTLD) and from 10 Epstein-Barr virus (EBV) polymerase chain reaction-positive HSCT patients (controls) were evaluated for IL-7- and IL-2 induced Stat5 phosphorylation in CD4+ and CD8+ T cells. PBMCs from PTLD+ and control patients exhibited detectable EBV specific CD8+ T cells defined by tetramer analysis. CD4+ and CD8+ T cells from patients with PTLD showed statistically significant reduction in responsiveness to IL-7 compared with PBMCs obtained from controls defined by Stat5 phosphorylation. CD20+ B cells from patients with PTLD and from some EBV+ polymerase chain reaction control individuals exhibited IL-7R expression. Dysregulated immune surveillance, reflected by deficient Stat5 phosphorylation, may facilitate PTLD development despite the presence of EBV-reactive CD8+ T cells. Reduced IL-7 responsiveness will aid to monitor patients after HSCT for increased risk to develop EBV-associated PTLD.

The role of virus-specific CD4+ T cells in the control of Epstein-Barr virus infection

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans and has been implicated in the pathogenesis of several human malignancies. Protective immunity against EBV is mediated by T cells, as indicated by an increased incidence of EBV-associated malignancies in immunocompromised patients, and by the successful treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) in transplant recipients by the infusion of polyclonal EBV-specific T cell lines. To implement this treatment modality as a conventional therapeutic option, and to extend this protocol to other EBV-associated diseases, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. To this aim, we studied the poorly defined EBV-specific CD4+ T cell response during acute and chronic infection.