EBV-specific Cytotoxic T Lymphocytes Research Articles

Epigenetic reprogramming sensitizes immunologically silent EBV+ lymphomas to virus-directed immunotherapy

AbstractDespite advances in T-cell immunotherapy against Epstein-Barr virus (EBV)-infected lymphomas that express the full EBV latency III program, a critical barrier has been that most EBV+ lymphomas express the latency I program, in which the single Epstein-Barr nuclear antigen (EBNA1) is produced. EBNA1 is poorly immunogenic, enabling tumors to evade immune responses. Using a high-throughput screen, we identified decitabine as a potent inducer of immunogenic EBV antigens, including LMP1, EBNA2, and EBNA3C. Induction occurs at low doses and persists after removal of decitabine. Decitabine treatment of latency I EBV+ Burkitt lymphoma (BL) sensitized cells to lysis by EBV-specific cytotoxic T cells (EBV-CTLs). In latency I BL xenografts, decitabine followed by EBV-CTLs results in T-cell homing to tumors and inhibition of tumor growth. Collectively, these results identify key epigenetic factors required for latency restriction and highlight a novel therapeutic approach to sensitize EBV+ lymphomas to immunotherapy.

The efficacy and safety of Epstein-Barr virus-specific antigen peptide-activated cytotoxic T-cells treatment for refractory or recurrent angioimmunoblastic T-cell lymphoma: A prospective clinical observational study.

The efficacy and safety of Epstein-Barr virus (EBV)-specific antigen peptide-activated cytotoxic T lymphocytes (CTLs) in the treatment of refractory or recurrent angioimmunoblastic T-cell lymphoma (AITL) was determined in this prospective one-arm clinical study. Seven males and two females were enrolled with a median age of 70 years. The tumor stages were all stage III and IV. All patients had group B symptoms and IPI scores of 3 to 5 points. All patients received chemotherapy before CTLs infusion which the median chemotherapy cycle was three. The diseases states before CTLs included five cases of disease progression (PD), two cases of recurrence (R), and two cases with residual lesions after chemotherapy. Eight patients received HLA-haploidentical EBV-specific CTLs, and one patient chose autologous CTLs. The number of transfused cells was 1.67 to 2.38 × 1010 for one course of CTLs therapy. One patient was treated with three courses of CTLs, three patients were treated with two courses of CTLs, and five patients were treated with one course of CTLs. During the infusion, eight patients had fever, one patient had rash, and no graft-vs-host diseases were observed. The EBV-DNA decreased by more than two orders of magnitude in six patients, and the response rate was 66.7%. Two patients of PD status achieved complete remission (CR), one patient of PD status achieved partial remission, two patients with residual lesions after chemotherapy achieved CR, and four patients had no response. The objective remission rate was 55.6%. After the median follow-up of 14.5 months, five patients died, and three patients were completely relieved while one patient was lost during follow-up. The 3-year overall survival was 44.4% and 3-year progression-free survival was 33.3%. EBV-specific antigen peptide-activated CTLs showed positive effect in certain patients with refractory and recurrent AITL with high clinical safety.

Management of Epstein-Barr virus-related post-transplant lymphoproliferative disorder after allogeneic hematopoietic stem cell transplantation.

Epstein–Barr virus-related post-transplant lymphoproliferative disorder (EBV-PTLD) is a rare but life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). T-cell immunodeficiency after transplantation and EBV primary infection/reactivation play major roles in the pathogenesis. Unspecific clinical manifestations make the diagnosis difficult and time consuming. Moreover, this fatal disease usually progresses rapidly, and leads to multiple organ dysfunction or death if not treated promptly. Early diagnosis of EBV-DNAemia or EBV-PTLD generally increases the chances of successful treatment by focusing on regular monitoring of EBV-DNA and detection of symptomatic patients as early as possible. Rituximab ± reduction of immunosuppression (RI) is currently the first-line choice in preemptive intervention and targeted treatment. Unless patients are suffering from severe graft versus host disease (GvHD), it is better to combine rituximab with RI. Once a probable diagnosis is made, the first-line treatment should be initiated rapidly, along with, or ahead of, biopsy, although histopathologic confirmation is requisite. In addition, EBV-specific cytotoxic T lymphocytes (EBV-CTLs) or donor lymphocyte infusion (DLI) has shown promise in cases of suboptimal response. Chemotherapy ± rituximab might lend more opportunities to refractory/relapsed patients, who might also benefit from ongoing clinical trials. Herein, we discuss our clinical experience in detail based on the current literature and our five cases.

Latent Membrane Protein 1 Contributes to Deficient Cellular Immunity in NKTCL Patients

Introduction Extranodal NK/T cell lymphoma (NKTCL) is an aggressive malignance that is correlated closely with persistent Epstein-Barr virus (EBV) infection and belongs to a latency II EBV disease. Chronic persistent viral infection is known to result in T cell exhaustion, which will dramatically impede anti-tumor immunity. Delineating mechanisms of immunosuppression contributes to the development of novel immuno-therapeutically strategies. Herein, we described the immune status and possible role of latent membrane protein 1(LMP1) in NKTCL. Methods Peripheral blood mononuclear cells (PBMCs) from newly diagnosed NKTCL patients and age-matched healthy donors (HDs) were isolated and stained with surface marker or intracellular marker after permeabilization. The frequency of EBV-specific cytotoxic T cells (CTLs) was analyzed by staining with HLA-A2 tetramers assembled with synthetic peptides from LMP1. T cell proliferation was detected by dilution of stained CFSE, and apoptosis of T cells was performed via annexinV and 7-AAD dual-staining. Cytotoxicity assay of natural killer cells (NK) was measured by detecting apoptosis of CFSE-labeled K562 cells after co-culturing with PBMCs. All flow cytometry was performed by BD FACS CantoII, and analyzed with FlowJo software. Results PBMCs from 19 NKTCL patients and 18 HDs were analyzed, which showed that the patients had higher ratio of CD4+/CD8+ cells and lower frequency of CD3-CD56+NK cells, higher percentage of immunosuppressive CD4+CD25hiCD127low T regulatory cells (Tregs) and HLA-DR-CD11b+CD33+ myeloid derived suppressor cells (MDSCs). Notably, the ratio of CD8+/Tregs, a parameter representing immune effector cells status, was obviously decreased in NKTCL patients (Figure-a). Given the potential association of T cell exhaustion and NKTCL, we detected expression of T cell exhaustion markers on T cells from NKTCL patients, and found that both CD4+ and CD8+ T cells expressed much higher level of inhibitory molecules PD1, CTLA4, TIGIT and TIM3 in NKTCL patients than that of HDs (Figure-b). To further explore cellular immunity in NKTCL, T cells were divided into four subsets: CD45RA-CCR7+ central memory T cells (Tcm), CD45RA+CCR7+ terminally differentiated effector T cells (Temra), CD45RA+CCR7- naïve T cells (Tn), CD45RA-CCR7- effector memory T cells (Tem) (Figure-c). It showed that CD4+ T cells had lower Tcm, Temra and Tn but much higher proportion of Tem compared with HDs (Figure-d). Moreover, we found that expression of PD1 and CTLA4 were upregulated on all lymphocyte subsets in NKTCL patients than HDs (Figure-e). We collected additional 13 NKTCL patients and 10 HDs with positive HLA-A2 phenotype to measure percentages of EBV-specific CTLs, which showed that frequency of EBV-specific CTLs was remarkably decreased in NKTCL patients (Figure-f). In addition, EBV-specific CTLs from NKTCL patients were more likely to express higher levels of exhaustion markers but produced much less IFN-γ (Figure g-h). To explore the potent mechanism of immunosuppression in NKTCL, LMP1 attracted our attention, which had been proven capable of activating multiple signaling pathways to exert its oncogenesis function. To elucidate effect of LMP1 on immune cells, we constructed two LMP1-derived peptides and found that LMP1-derived peptides suppressed the proliferation of CD4+ and CD8+ T cells and inhibited IFN-γ production of CD8+ T cells and NK cells (Figure i-j). Meanwhile, LMP1 promoted apoptosis of both CD4+ and CD8+ T cells (Figure-k). Subsequently regulation of LMP1 on exhaustion markers of T cells were detected, which showed that PD1, CTLA4, TIGIT and TIM3 were significantly upregulated on both CD4+ and CD8+ T cells after treatment with LMP1 derived peptides (Figure-m). Furthermore, LMP1 impaired NK cytotoxicity ability, evidenced by decrease apoptosis of target cells (Figure-n). Besides its effect on immune effector cells, we also found that LMP1-expressing NKTCL cell lines obviously induced both Tregs and MDSCs after co-culture. Interestingly, the extent of induction by LMP1 of Tregs and MDSCs were in line with the expression level of LMP1 on NKTCL cells (Figure o-p). Conclusion Our study showed that LMP1 contributes to deficient cellular immunity in NKTCL patients, providing us with more insight into immunosuppressive in this entity of disease, which would lead to identification of novel treatment strategies. Figure Disclosures No relevant conflicts of interest to declare.

Epstein-Barr virus-related post-transplant lymphoproliferative disease (EBV-PTLD) in the setting of allogeneic stem cell transplantation: a comprehensive review from pathogenesis to forthcoming treatment modalities.

Epstein-Barr virus (EBV) is a ubiquitous herpes virus that infects the majority of the population worldwide. The virus can establish a lifelong latent infection in host B-lymphocytes. In the setting of immunocompromise as is the case post transplantation, the virus can reactivate and cause one of the deadliest complications post hematopoietic stem cell transplantation (HSCT), post-lymphoproliferative disease (PTLD), the incidence of which has been increasing. Multiple risk factors have been associated with the onset of PTLD such as age, reduced intensity conditioning, EBV serology mismatch and cytomegalovirus (CMV) reactivation. The rarity of clinical trials involving PTLD and the lack of approved treatment modalities renders the management of PTLD challenging. While the first-line treatment involves weekly administration of rituximab, there is no consensus when treating rituximab-refractory PTLD. There is a handful of clinical trials that investigate the role of EBV-specific cytotoxic T-lymphocytes (CTLs) and novel agents, such as bortezomib, lenalidomide, everolimus, panobinostat, and brentuximab. This article aims to explore the entity of EBV-PTLD in HSCT recipients, expanding on clinical presentation, risk factors, modes of monitoring and treatment, and so highlighting the gaps in knowledge that are needed in order to build a treatment paradigm suitable for all patients at risk.

31O - T cell therapy with EBV-specific cytotoxic T-lymphocytes for patients with nasopharyngeal carcinoma

31O - T cell therapy with EBV-specific cytotoxic T-lymphocytes for patients with nasopharyngeal carcinoma

Risk Factors and Predictive Scoring System for Post-Transplant Lymphoproliferative Disorder after Hematopoietic Stem Cell Transplantation

Risk Factors and Predictive Scoring System for Post-Transplant Lymphoproliferative Disorder after Hematopoietic Stem Cell Transplantation

Clinical Analysis of Treating EBV Infection Accompanied with GVHD after Allon-HSCT by EBV-Specific Cytotoxic T Lymphocytes

To investigate the efficiency and safety of treating Epstein-Barr virus (EBV) infection of acute graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by EBV specific cytotoxic T lymphocytes (EBV-CTL). The Clinical characteristics, therapeutic efficacy and safety of 12 patients with EBV infection treated by EBV-CTL infusion after allo-HSCT in Department of Hemahlogy of Aero Space Center Hospital between Jan 2015 and May 2017 were analyzed retrospectioely. Our of 12 cases received EBV-CTL infusion after transplantation, 9 did not received Rituximab therapy due to the active infection, 4 cases including 3 received Ritaximab progressed into posttransplantation lymphoroliferetive disease (PTLD). The median time of EBV infection was 47 (22-71) days, median time of antivirus therapy before tramplantation was 10 (8-33) days, median time of first CTL infusion was 59(34-86) days after transplatation. The 43 cases-time CTL infusion was performed smoothly, no related harmful evnts occoured, no progression of GVHD was observed. After the first course of infusion, complete remission (CR), Partial remssion (PR) and no remssion (NR) were obtained in 9, 1 and 2 patients respectively, the relapse was observed in 4 patients who then received the socond course of infusion and all reached CR, the patient in PR did not reathed CR finally and died of GVGD at 5 months after transpplantation . Only 1 out of 2 cases of NR obtained CR, another 1 still was in NR, and died of transplantation related infection at 5 months after transplantation. 4 cases of PTLD were all cared. Preliminary results of this study suggest that EBV-CTL infusion is safe for the EBV infection combined with acute GVHD after all-HSCT. However, a further larger scale clinical studies are needed to prove the efficiency.

PNEUMONITIS SECONDARY TO EBV-SPECIFIC CYTOTOXIC T-LYMPHOCYTES

SESSION TITLE: Diffuse Lung Disease 1 SESSION TYPE: Fellow Case Reports PRESENTED ON: 10/07/2018 03:30 pm - 04:30 pm INTRODUCTION: Novel immune based therapies are now being used for a wide range of malignancies including post-transplantation lymphoproliferative disorders (PTLD), however, the short and long term risk of these therapies remains unclear. Here, we present a case of a patient who developed hypoxemia one day after receiving an infusion of EBV-specific cytotoxic T-lymphocytes. CASE PRESENTATION: A 66-year-old male presented to the hospital with 4 days of progressive dyspnea, cough, and hypoxemia. He had a history of end stage renal disease and EBV-positive cadaveric renal transplantation which was complicated by EBV viremia and PTLD. Two months prior, the patient began therapy with HLA-matched EBV-specific T-lymphocytes after failing several immunosuppressive and chemotherapeutic regimens. His most recent infusion was 1 day prior to symptom onset. On admission, oxygen saturation was 91% on room air and 96% on 2 liters of oxygen by nasal cannula. Physical exam at that time was notable for faint crackles throughout all lung fields. Chest radiograph was negative for infiltrates or effusions although computed tomography imaging of the chest revealed diffuse bilateral ground glass opacities and patchy airspace disease with an upper lobe predominance (see Figure 1). Bronchoscopy with transbronchial biopsy revealed interstitial inflammation and fibrosis, hyperplasia of type II pneumocytes, and scattered foci of organizing pneumonia consistent with active pneumonitis. Bronchoalveolar lavage demonstrated a lymphocyte predominant cell distribution with 56% lymphocytes, 41% macrophages, and 3% neutrophils. BAL and biopsy were negative for viral, fungal, and bacterial elements. With supportive care alone, the patient improved over the course of 5 days and was discharged to a short term rehabilitation facility. DISCUSSION: PTLD is a serious complication for patients undergoing organ transplantation. EBV-specific cytotoxic T-cells have shown promising results in the treatment of some of these patients, however, emerging evidence suggests that pulmonary complications are a consequence of this therapy. Indeed, in a series of 39 patients there was 1 reported death due to pneumonitis. Moreover, outcomes in another study of 114 patients found that 1 patient developed severe pulmonary infiltrates within one week of receiving EBV-specific T-cells. This patient did not suffer long-term sequelae from this complication. Our case suggests that pneumonitis from EBV-specific T-lymphocytes is characterized by a histopathological pattern of organizing pneumonia and a lymphocyte predominant cellular distribution. CONCLUSIONS: EBV-specific cytotoxic T-cell therapy causes acute pneumonitis in some patients. Additional studies will be important to understand the true incidence of pneumonitis after EBV-specific therapies, and for determining the factors that predispose or prevent the development of this therapeutic complication. Reference #1: Heslop, H. E., Slobod, K. S., Pule, M. A., et al. Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients. Blood. 2010; 115(5):925-935. Reference #2: Rooney, C. M., Smith, C. A., Ng, C. Y., et al. Infusion of Cytotoxic T Cells for the Prevention and Treatment of Epstein-Barr Virus–Induced Lymphoma in Allogeneic Transplant Recipients. Blood. 1998; 92(5):1549-1555. Reference #3: Dierickx, D., and Habermann, T.M. Post-Transplantation Lymphoproliferative Disorders in Adults. N Engl J Med. 2018; 378:549-562. DISCLOSURES: No relevant relationships by Robert Hilton, source=Web Response No relevant relationships by Carl Nieweld, source=Web Response No relevant relationships by ross summer, source=Web Response

Abstract CT049: Long term outcome of EBV-associated lymphoma patients treated with post-remission therapy using EBV LMP1 and LMP2a specific CTLs

Abstract Background: Conventional therapeutic approaches, such as chemotherapy and radiotherapy, have been limited in successfully achieving durable response in Epstein Barr virus (EBV)-associated malignancies causing frequent relapse, low overall survival rate and poor prognosis. EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach to treat EBV associated lymphoma by enhancing EBV-specific immunity. Methods: To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated ten EBV+ ENKTCL patients and two EBV+ PTLD patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation were eligible to receive eight doses of 2 x107 LMP1/2a CTLs/m2. Results: Following infusion, there were no immediate or delayed toxicities. The seven-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71·4% to 100%) for ENKTCL patients respectively with a median follow-up of 94 months. Similarly, PTLD patients showed long-term remission with a follow-up of up to 70 months. Overall, circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Conclusion: Adoptive transfer of LMP1/2a CTLs in patients is a safe and effective post-remission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of EBV-associated lymphoma. Citation Format: Nayoun Kim, Hyun-Jung Sohn, Keon-Il Im, Young-Woo Jeon, Young-Sun Nam, Yunejin Song, Jun Seok Lee, Tai Gyu Kim, Seok-Goo Cho. Long term outcome of EBV-associated lymphoma patients treated with post-remission therapy using EBV LMP1 and LMP2a specific CTLs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT049.

Abstract 4694: The contribution of Epstein-Barr virus to the impaired T-cell responses in nasopharyngeal carcinoma

Abstract Undifferentiated nasopharyngeal carcinoma (NPC) is a geographically skewed disease in which more than 80% cases are from southern China and Southeast Asia. NPC is consistently associated with Epstein-Barr virus (EBV) infection and characterized by a prominent lymphocyte infiltration. The consistent expression of EBV proteins in NPC cells has led to several clinical trials of adoptive T-cell therapy or vaccination to boost the immune response to EBV antigens. However, these treatments yield relatively modest clinical benefit, which may be due to varying degrees of immunosuppression in the tumor microenvironment. The lack of model systems to analyze the ability of therapeutically relevant T cells to recognize and kill NPC cells represents a fundamental limitation in our attempts to harness the immune system for clinical benefit. We used three NPC cell lines as targets in T-cell assays, together with a panel of established HLA-matched EBV-specific CD8 and CD4 T-cell clones. T-cell activity and killing were measured by IFN-γ ELISA and outgrowth assays, respectively. We demonstrate, for the first time, that NPC cells can be recognized and killed by EBV-specific T-cell clones in vitro, which suggests that tumor-derived factors inhibit such responses in vivo. These observations are consistent with the previous reported functional inactivation of EBV-specific cytotoxic T lymphocytes (CTL) in NPC patients. We hypothesized that EBV infection of NPC cells induces the secretion of immunosuppressive factors. The effect of conditioned media derived from an EBV-infected premalignant nasopharyngeal cell line, NP460htert, on T-cell activity was examined by treating several clones of EBV-specific CTLs in vitro. The conditioned media inhibited the secretion of IFN-γ from peptide-stimulated CTLs, indicating that secreted factors that could suppress CTL functions are induced following EBV infection in NP460htert cells. RNA sequencing analyses on NP460hTert and three NPC cell lines following EBV infection revealed a significant enrichment of genes whose proteins are located at extracellular region/matrix, including a number of immunomodulators. Our results suggest that EBV infection might induce the production of soluble molecules, which in turn suppress the antitumor T-cell responses. In conclusion, we have developed a system with which to study NPC-specific immune responses and shown that soluble factors induced by EBV infection can suppress T-cell activity. These model systems will be critical in the future to examine the effects of checkpoint inhibitors, secreted factors from cancer-associated fibroblasts or NPC cells upon T-cell activity, which will ultimately enhance the success of EBV-targeted immunotherapy strategies. Citation Format: Hui Min Lee, Tracey Haigh, George SW Tsao, Paul G. Murray, Graham Taylor, Ian C. Paterson, Lee Fah Yap. The contribution of Epstein-Barr virus to the impaired T-cell responses in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4694.

309 - Multi-parameter flow cytometry in manufacturing of therapeutic t cells: Assessing quality and potency in different gmp-compliant generation processes for ebv-specific T cells

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has proven to be an efficacious treatment for patients with EBV-related malignancies, particularly Post Transplant Lymphoproliferative Disease (PTLD). Clinical manufacture of allogeneic EBV-specific CTLs for therapy ideally requires a GMP-compliant, fast, scalable, cost-effective, simplified generation protocol. We are developing two generation protocols including the traditional EBV lymphoblastoid cell line (LCL) co-culture method, and the more recent EBV peptide-mediated isolation of interferon-gamma secreting virus-specific T cells. It was the aim of this study to compare both GMP-compliant systems and develop a flow-based assay which provides relevant quality criteria on immunophenotype and functional characterisation.

Association Between Epstein Barr Virus Infection and Lymphoproliferative Syndrome in a Renal Transplant Patient

Epstein-Barr virus (EBV), a virus of the human Herpes viridae class, has a particular importance in renal transplantation because of its complex interaction with the immune system. Indeed, it causes both infectious mononucleosis (IM), immunopathological responses of the host, and at the extreme a malignant tumor, when the responses are altered. The lymphoproliferative syndromes induced by the EBV infection are lymphoid proliferations resulting from the loss of an EBV-specific cytotoxic T-cell response. About 10% of B-type lymphoproliferative syndromes including diffuse large B-cell lymphoma are EBV-positive. We report the case of a 23-year-old man who developed a tumor in the nasal cavity 6 months after renal transplantation. His nephropathy was membranoproliferative glomerulonephritis. The induction therapy indicated was antithymoglobulin (ATG) and solumedrol. In maintenance therapy, mycofenolate mofetil (MMF) and tacrolimus were used. Nasal biopsy has proved CD20-positive B-cell lymphoma. No other site of the disease has been identified. The immunosuppressive agents were modulated with a switch of tacrolimus by an m-TOR inhibitor and a progressive reduction of MMF. After three cycles of R CHOP followed by radiotherapy, he achieved complete remission with a stable graft function. World J Nephrol Urol. 2018;7(2):56-59 doi: https://doi.org/10.14740/wjnu352w

Personalized adoptive immunotherapy for patients with EBV-associated tumors and complications: Evaluation of novel naturally processed and presented EBV-derived T-cell epitopes.

Morbidity and mortality of immunocompromised patients are increased by primary infection with or reactivation of Epstein-Barr virus (EBV), possibly triggering EBV+ post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) promises a non-toxic immunotherapy to effectively prevent or treat these complications.To improve immunotherapy and immunomonitoring this study aimed at identifying and evaluating naturally processed and presented HLA-A*03:01-restricted EBV-CTL epitopes as immunodominant targets. More than 15000 peptides were sequenced from EBV-immortalized B cells transduced with soluble HLA-A*03:01, sorted using different epitope prediction tools and eleven candidates were preselected. T2 and Flex-T peptide-binding and dissociation assays confirmed the stability of peptide-MHC complexes. Their immunogenicity and clinical relevance were evaluated by assessing the frequencies and functionality of EBV-CTLs in healthy donors (n > 10) and EBV+ PTLD-patients (n = 5) by multimer staining, Eli- and FluoroSpot assays. All eleven peptides elicited EBV-CTL responses in the donors. Their clinical applicability was determined by small-scale T-cell enrichment using Cytokine Secretion Assay and immunophenotyping. Mixtures of these peptides when added to the EBV Consensus pool revealed enhanced stimulation and enrichment efficacy. These EBV-specific epitopes broadening the repertoire of known targets will improve manufacturing of clinically applicable EBV-CTLs and monitoring of EBV-specific T-cell responses in patients.

Cytotoxic response against Epstein Barr virus coexists with diffuse large B-cell lymphoma tolerogenic microenvironment: clinical features and survival impact

Epstein–Barr Virus (EBV) is present in neoplastic cells of 15% of Asian and Latin-American diffuse large B-cell lymphoma (DLBCL) patients. Even though a tolerogenic microenvironment was recently described in DLBCL, little is known concerning immunomodulatory features induced by EBV. As suggested in Hodgkin lymphoma, EBV-specific cytotoxic T-cells are increased but showing immune exhaustion features. Hence, host immunity suppression may play a critical role in tumor progression. This study aimed to investigate, whether an association between tumor microenvironment features and EBV presence is taking place, and its clinical correlate. The incidence of EBV+DLBCL NOS was 12.6% in this cohort. Cytokine and chemokine transcripts expression and immunophenotype analysis showed that EBV infection was associated with increased gene expression of immunosuppressive cytokine (IL-10) together with increased CD8+ T-cells and granzyme B+ cytotoxic effector cells. However, this specific response coexists with a tolerogenic milieu, by PD-1 expression, in EBV+ and EBV−DLBCL cases. High PD-1+ cell counts, EBV presence and low CCL22 expression were associated with worse survival, supporting our hypothesis that EBV-specific response is mounted locally and its inhibition by, for example PD-1+ cells, may negatively affect outcome. The better understanding of the interplay between lymphoma cells and microenvironment in a viral framework could thereby facilitate the discovery of new targets for innovative anti-lymphoma treatment strategies.

Exploring the effect of vitamin D3 supplementation on the anti-EBV antibody response in relapsing-remitting multiple sclerosis

Background: Epstein–Barr virus (EBV) infection and vitamin D insufficiency are potentially interacting risk factors for multiple sclerosis (MS). Objectives: To investigate the effect of high-dose vitamin D3 supplements on antibody levels against the EBV nuclear antigen-1 (EBNA-1) in patients with relapsing-remitting multiple sclerosis (RRMS) and to explore any underlying mechanism affecting anti-EBNA-1 antibody levels. Methods: This study utilized blood samples from a randomized controlled trial in RRMS patients receiving either vitamin D3 (14,000 IU/day; n = 30) or placebo (n = 23) over 48 weeks. Circulating levels of 25-hydroxyvitamin-D, and anti-EBNA-1, anti-EBV viral capsid antigen (VCA), and anti-cytomegalovirus (CMV) antibodies were measured. EBV load in leukocytes, EBV-specific cytotoxic T-cell responses, and anti-EBNA-1 antibody production in vitro were also explored. Results: The median antibody levels against EBNA-1, but not VCA and CMV, significantly reduced in the vitamin D3 group (526 (368–1683) to 455 (380–1148) U/mL) compared to the placebo group (432 (351–1280) to 429 (297–1290) U/mL; p = 0.023). EBV load and cytotoxic T-cell responses were unaffected. Anti-EBNA-1 antibody levels remained below detection limits in B-cell cultures. Conclusion: High-dose vitamin D3 supplementation selectively reduces anti-EBNA-1 antibody levels in RRMS patients. Our exploratory studies do not implicate a promoted immune response against EBV as the underlying mechanism.

Abstract B049: Immunotherapy of EBV associated gastric cancer using EBV specific T cells

Abstract Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is mediated by EBV-infected malignant cells in gastric intestinal tract. About 10% of gastric carcinoma are EBVaGC worldwide, and over 85,000 patients are developed EBVaGC annually. EBVaGC shows several different aspects with EBV-negative gastric cancer such as clinicopathology, CpG island methylation status and expression of EBV-related genes. Therefore, immunotherapy to treat EBVaGC would be a good approach because expression pattern of EBVaGC is evidently different with that of EBV-negative cells in stomach. Adoptive T cell transfer has been effective for treatment of EBV-positive nasopharyngeal carcinoma (NPC) and lymphomas, which express similar EBV antigens to those of EBVaGC cells. Thus, we wanted to use system of NPC clinical trials to EBVaGC. Targeted EBV antigens expressed both in NPC and EBVaGC were latent membrane proteins (LMPs)-1,2 and EBV-determined nuclear antigen (EBNA) 1. To generate EBV-specific cytotoxic T lymphocytes (EBV-CTLs), we firstly isolated peripheral blood mononuclear cells (PBMCs) from healthy human donor blood by using Ficoll and sorted dendritic cells (DCs) to deliver LMP2A and EBNA1 expressing-vectors by nucleofection into DCs. Nucleofected DCs were cocultured with PBMCs in order to cross-present EBV antigens toward CD4+, CD8+ T cells in DC maturation condition. Generated EBV-CTLs were incubated with IL-2, 7, 15-supplemented CTL media for inducing memory-like cells and expanded for 14 days to apply to adoptive transfer of EBVaGC model. Cytotoxicity of EBV-CTLs were specific for EBV-expressing gastric cancer cell line (SNU 719) in vitro, not EBV-negative gastric cancer cell line (MKN45). To confirm efficacy of EBV-CTLs in mouse model, we developed EBVaGC mouse model using SNU719 and injected 1 × 107 cells/mouse once a week for three times. EBV-CTLs had an effect on the reduction of tumor size of EBVaGC and effectively migrated into tumor site. Also, administered EBV-CTLs stably existed in blood and tumor tissue even over 3 days analyzed by FACS and immunohistochemistry. In conclusion, EBV-CTLs represented improved anti-tumor activity to treat EBVaGC and specific lysis to EBV-expressing cells. Also, any side effects or autoimmune diseases were not detected. Therefore, EBV-CTLs would be likely to play important roles in targeting many EBV-associated cancers and be applied to clinical trials. Note: This abstract was not presented at the conference. Citation Format: Jae Seung Moon, Jung Ho Kim, Sang Kyou Lee. Immunotherapy of EBV associated gastric cancer using EBV specific T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B049.

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.

Reconstitution of Epstein-Barr Virus-Specific T Lymphocytes at the Early Stage of Allogeneic Stem Cell Transplantation

Backgroud:Epstein-Barr virus (EBV) infection/reactivation is an common complication following allogeneic stem cell transplantation (allo-HSCT) which usually occure at the early stage after transplantation. EBV-specific cytotoxic T lymphocytes (EBV-CTLs) are considered to provide protection from EBV infection/reactivation and associated diseases in recipients of allo-HSCT. Herein, we evaluated the levels of EBV-CTLs early after transplantion and investigated the associaton between the reconstitution of EBV-CTLs and EBV infection/reactivation after allo-HSCT.Methods:Thirty patients undergoing allo-HSCT between November 2013 to September 2014 were enrolled in this study. Of these patients, 16 received HLA-matched donor and 14 received HLA-mismatched transplantation. Antithymocyteglobulin was given in 20 patients. EBV-CTLs were tested using Enzyme-linked immunospot assay (ELISPOT) presenting 9 peptides derived from 3 EBV proteins in +30, +90 and +180 days after transplantation.Results:EBV-CTLs, although low levels, were detected in 90% (27/30) patients in +30 days after HSCT. In most patients, EBV-CTLs levels in +90 days was significantly higher than +30 days and returned to normal levels within +180 days after HSCT. Thirteen patients developed EBV infection/reactivation within the median time of 48 (range, 23-110) after transplantation. EBV-CTLs levels had no significant difference among the patients with or without infection/reactivation in +30 days while the patients with EBV infection/reactivation had higher EBV-CTLs levels than those without in +90d after HSCT.Conclusion:Reconstitution of EBV-CTLs seems rapid in both HLA-matched and -mismatched donor transplantation. The association between EBV infection/reactivation and EBV-CTLs levels at monthly intervels after HSCT should be studied further. DisclosuresNo relevant conflicts of interest to declare.

Enhanced antitumor effect of combining chemotherapy with Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes in mice with EBV-related non-Hodgkin's lymphoma.

Epstein-Barr virus (EBV)-related non-Hodgkin's lymphoma (NHL) represents a major problem in hematological clinical studies due to its drug tolerance and refractoriness. EBV infection is a key factor driving the process of tumor growth. Immune therapy is an important biotherapeutic method of treating cancer, which is attracting increasing attention. We hypothesized that combining conventional chemotherapy with immune therapy in the treatment of EBV-related NHL may achieve better outcomes. First, we successfully cloned large numbers of EBV-specific T cells by immune stimulation ex vivo. Subsequently, the combined therapy was applied in a murine model of human EBV-related NHL. As expected, combined therapy inhibited tumor growth more effectively compared with monotherapy. In addition, we continuously tested the tumor-associated immune microenvironment and observed that the numbers of tumor-infiltrating cytotoxic T lymphocytes (CTLs) and macrophages were elevated following combined therapy. These effects suggest that EBV-specific CTLs may indirectly promote an innate immune reaction in lymphoma by activating tumor-infiltrating macrophage proliferation. Our findings may provide a guide for the prospective treatment of EBV-related NHL.