EBV-transformed B-cell Research Articles

Pan-Aurora Kinase Inhibitor Danusertib Induces Apoptosis of Epstein–Barr Virus-transformed B-Cells Through Caspase and Endoplasmic Reticulum Stress Signaling

Evidence for the relevance of Epstein-Barr virus (EBV) in various types of cancer has expanded; however, the definitive mechanism of EBV-induced oncogenesis remains ambiguous. The purpose of this study was to identify the relevance of aurora kinases in EBV-induced carcinogenesis, and the cellular responses to danusertib, a pan-aurora kinase inhibitor. The underlying signaling mechanism in EBV-transformed B-cells was also investigated. Western blotting was performed on EBV-transformed B-cells and EBV-positive lymphoma cells to identify aurora kinase expression. Cellular responses of EBV-transformed B-cells to danusertib were investigated using AlamaBlue assay and apoptosis analysis. To evaluate the underlying signaling mechanisms of danusertib-induced apoptosis, cleavage of caspase cascade molecules, endoplasmic reticulum (ER) stress-associated molecule activation, and intracellular Ca2+ levels were evaluated using western blotting, flow cytometry, and inhibition assays. Expression of both aurora kinase A and B was gradually increased in EBV-infected B-cells and two EBV-positive B lymphoma cell lines. Danusertib significantly suppressed EBV-transformed B-cell proliferation in a dose-dependent manner. Danusertib induced apoptosis and cell cycle arrest through disruption of mitochondrial membrane potential in EBV-transformed B-cells in a dose-dependent and time-dependent manner. Moreover, danusertib induced cleavage of caspases, ER stress-associated molecule activation, and intracellular Ca2+ release from ER to cytoplasm in EBV-transformed B-cells, while BAPTA-AM, a calcium chelator, inhibited danusertib-induced apoptosis. Danusertib treatment led to apoptosis of EBV-transformed B-cells through ER stress-associated proteins and mitochondrial caspase activation. These results suggest that aurora kinases may be valuable targets for potential therapeutic agents against EBV-associated carcinoma.

Gonadotropin-releasing hormone receptor pathway affects the function of human EBV-transformed B lymphocytes in an age-independent way.

Immune system function changes during aging, but the molecular mechanisms of this phenomenon are not fully understood. The present study identified pathways that are associated with age-associated changes in human B lymphocytes. Initial in silico analysis of 1355 genes involved in aging revealed the strongest association (p=4.36E-21) with the gonadotropin-releasing hormone receptor (GnRHR) pathway. Extended analysis of 2736 aging-related genes using updated databases confirmed such association (p=2.41E-16). Genes involved in both aging and the GnRHR pathway were significantly involved in lymphocyte B and T activation and aging-related phenotypes, including hyperinsulinemia and diabetes, arthritis, cerebrovascular disease, and cancers. We, therefore, examined non-tumorigenic Epstein-Barr virus (EBV)-transformed B-lymphocyte cell lines that originated from 12 young subjects (20-31years old) and 10 centenarians (100-102years old). Gonadotropin-releasing hormone I (GnRH-I) and GnRHR levels did not depend on the age of the cell donors. Inhibition of the GnRHR pathway age-independently decreased cell proliferation (p<0.001) and increased apoptosis (p<0.001). However, the decrease in immunoglobulin G synthesis (p<0.01) was twice as high in centenarian cells than in young cells. In conclusion, the GnRHR pathway regulated essential properties of B lymphocytes. However, upon EBV transformation, memory class-switched B cells became the dominant cell subpopulation. Therefore, the observed effects of GnRHR inhibition were attributable to this subpopulation.

CROCIN PROMOTES APOPTOSIS IN HUMAN EBV-TRANSFORMED B-LYMPHOCYTE VIA INTRINSIC PATHWAY

BackgroundAs a major carotenoid in saffron, crocin demonstrates potent anti-cancer impacts. However, its anti-lymphoma effects remain vague, especially in the human EBV-associated B-cell lymphoproliferative disorders. This study examined crocin’s apoptogenic potential and its underlying mechanism in CO 88BV59-1 cell line vs. normal human peripheral blood B cells.MethodsCO 88BV59-1 cells were treated with crocin alone or in combination with vincristine for up to 72 h. The cell viability was examined using a resazurin assay. Flow cytometry using annexin V and propidium iodide labeling was performed to detect apoptotic cells. Also, the expression levels of genes and proteins involved in apoptosis (CASP3, CASP8, CASP9, P53, Bax, and Bcl-2) were respectively determined via real-time PCR and Western blot analysis.ResultsCrocin concentration-dependently reduced cell viability in CO 88BV59-1 cells with no significant toxicity toward normal B cells. Similar to vincristine, crocin significantly increased apoptosis in these cells during 72 h of incubation. Furthermore, the combination of crocin (80 μM) and vincristine (1 μM) enhanced apoptosis in CO 88BV59-1 cells. Therefore, this synergistic effect was detected in human EBV-transformed B-lymphocyte. CASP3, CASP9, P53, and Bax/Bcl-2 ratio expressions were significantly raised in CO 88BV59-1 cells, whereas CASP8 was unaltered. It was proposed that crocin promoted apoptosis in CO 88BV59-1 cells in a time- and concentration-dependent manner via the induction of the intrinsic pathway.ConclusionThe results suggest that crocin may serve as a good alternative/coadjuvant to vincristine in EBV-associated B-cell lymphoproliferative disorders.

Targeted Delivery of BZLF1 to DEC205 Drives EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world’s population and is linked to development of cancer. In immune-competent individuals, EBV infection is mitigated by a highly efficient virus-specific memory T-cell response. Risk of EBV-driven cancers increases with immune suppression (IS). EBV-seronegative recipients of solid organ transplants are at high risk of developing post-transplant lymphoproliferative disease (PTLD) due to iatrogenic IS. While reducing the level of IS may improve EBV-specific immunity and regression of PTLD, patients are at high risk for allograft rejection and need for immune-chemotherapy. Strategies to prevent PTLD in this vulnerable patient population represents an unmet need. We have previously shown that BZLF1-specific cytotoxic T-cell (CTL) expansion following reduced IS correlated with immune-mediated PTLD regression and improved patient survival. We have developed a vaccine to bolster EBV-specific immunity to the BZLF1 protein and show that co-culture of dendritic cells (DCs) loaded with a αDEC205-BZLF1 fusion protein with peripheral blood mononuclear cells (PMBCs) leads to expansion and increased cytotoxic activity of central-effector memory CTLs against EBV-transformed B-cells. Human–murine chimeric Hu-PBL-SCID mice were vaccinated with DCs loaded with αDEC205-BZLF1 or control to assess prevention of fatal human EBV lymphoproliferative disease. Despite a profoundly immunosuppressive environment, vaccination with αDEC205-BZLF1 stimulated clonal expansion of antigen-specific T-cells that produced abundant IFNγ and significantly prolonged survival. These results support preclinical and clinical development of vaccine approaches using BZLF1 as an immunogen to harness adaptive cellular responses and prevent PTLD in vulnerable patient populations.

Expression quantitative trait loci of genes predicting outcome are associated with survival of multiple myeloma patients.

Gene expression profiling can be used for predicting survival in multiple myeloma (MM) and identifying patients who will benefit from particular types of therapy. Some germline single nucleotide polymorphisms (SNPs) act as expression quantitative trait loci (eQTLs) showing strong associations with gene expression levels. We performed an association study to test whether eQTLs of genes reported to be associated with prognosis of MM patients are directly associated with measures of adverse outcome. Using the genotype-tissue expression portal, we identified a total of 16 candidate genes with at least one eQTL SNP associated with their expression with P < 10-7 either in EBV-transformed B-lymphocytes or whole blood. We genotyped the resulting 22 SNPs in 1327 MM cases from the International Multiple Myeloma rESEarch (IMMEnSE) consortium and examined their association with overall survival (OS) and progression-free survival (PFS), adjusting for age, sex, country of origin and disease stage. Three polymorphisms in two genes (TBRG4-rs1992292, TBRG4-rs2287535 and ENTPD1-rs2153913) showed associations with OS at P < .05, with the former two also associated with PFS. The associations of two polymorphisms in TBRG4 with OS were replicated in 1277 MM cases from the International Lymphoma Epidemiology (InterLymph) Consortium. A meta-analysis of the data from IMMEnSE and InterLymph (2579 cases) showed that TBRG4-rs1992292 is associated with OS (hazard ratio=1.14, 95% confidence interval 1.04-1.26, P=.007). In conclusion, we found biologically a plausible association between a SNP in TBRG4 and OS of MM patients.

Three Copies of Four Interferon Receptor Genes Underlie a Mild Type I Interferonopathy in Down Syndrome.

Down syndrome (DS) is characterized by the occurrence of three copies of human chromosome 21 (HSA21). HSA21 contains a cluster of four interferon receptor (IFN-R) genes: IFNAR1, IFNAR2, IFNGR2, and IL10RB. DS patients often develop mucocutaneous infections and autoimmune diseases, mimicking patients with heterozygous gain-of-function (GOF) STAT1 mutations, which enhance cellular responses to three types of interferon (IFN). A gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with DS. We report high levels of IFN-αR1, IFN-αR2, and IFN-γR2 expression on the surface of monocytes and EBV-transformed-B (EBV-B) cells from studying 45 DS patients. Total and phosphorylated STAT1 (STAT1 and pSTAT1) levels were constitutively high in unstimulated and IFN-α- and IFN-γ-stimulated monocytes from DS patients but lower than those in patients with GOF STAT1 mutations. Following stimulation with IFN-α or -γ, but not with IL-6 or IL-21, pSTAT1 and IFN-γ activation factor (GAF) DNA-binding activities were significantly higher in the EBV-B cells of DS patients than in controls. These responses resemble the dysregulated responses observed in patients with STAT1 GOF mutations. Concentrations of plasma type I IFNs were high in 12% of the DS patients tested (1.8% in the healthy controls). Levels of type I IFNs, IFN-Rs, and STAT1 were similar in DS patients with and without recurrent skin infections. We performed a genome-wide transcriptomic analysis based on principal component analysis and interferon modules on circulating monocytes. We found that DS monocytes had levels of both IFN-α- and IFN-γ-inducible ISGs intermediate to those of monocytes from healthy controls and from patients with GOF STAT1 mutations. Unlike patients with GOF STAT1 mutations, patients with DS had normal circulating Th17 counts and a high proportion of terminally differentiated CD8+ T cells with low levels of STAT1 expression. We conclude a mild interferonopathy in Down syndrome leads to an incomplete penetrance at both cellular and clinical level, which is not correlate with recurrent skin bacterial or fungal infections. The constitutive upregulation of type I and type II IFN-R, at least in monocytes of DS patients, may contribute to the autoimmune diseases observed in these individuals.

A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disorder (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy (ALLELE)

Background In immunocompromised individuals where T-cell function is impaired, EBV-transformed B-cells can proliferate resulting in lymphoproliferative disorders or lymphoma (EBV+LPD). EBV+LPD following solid organ transplant (SOT) is known as EBV+ post-transplant lymphoproliferative disorder (EBV+PTLD). Treatments for EBV+PTLD following SOT include reduction in immunosuppression and off-label rituximab ± multi-agent chemotherapy (CT). Limitations of current EBV+PTLD treatments include lack of response, relapse, potential for graft rejection, short- and long-term toxicity and high mortality, highlighting a clear unmet medical. Tabelecleucel (tab-cel) is an off-the-shelf allogeneic EBV-specific T cell immunotherapy generated from healthy donors that is selected for each patient (pt) from a library of T cells using the most appropriate human leukocyte antigen (HLA) restriction to address the patient's disease. Tab-cel has been evaluated in several clinical studies; One published interim analysis showed that in ALLELE-eligible patients with EBV+PTLD following SOT, the objective response rate (ORR) was 83% (5/6 patients) (Prockop et al, ASH 2017). Here we describe the design of an ongoing study of tab-cel for SOT subjects with EBV+PTLD after failure of rituximab ± CT. Study Design and Methods ALLELE is a multicenter, open-label, single arm, phase 3 study evaluating the clinical benefit of tab-cel in pts with EBV+PTLD following SOT (n=33) after failure of (1) rituximab monotherapy or (2) rituximab plus CT (NCT03394365). Key inclusion criteria are a diagnosis of locally assessed, biopsy-proven EBV+PTLD and failure of rituximab or rituximab plus concurrent/sequential CT. Exclusion criteria include untreated or in-progress treatment of central nervous system PTLD and suspected or confirmed grade >2 graft-vs-host disease. Tab-cel is partially matched to each patient from a library that is HLA-characterized using 1 EBV HLA restriction allele and at least 1 other matched HLA allele. Tab-cel is administered in cycles lasting 5 weeks (35 days). During each cycle, subjects receive intravenous (IV) tab-cel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by observation through day 35. Response per clinical and radiographic assessment are evaluated at the end of each cycle by the investigator and independent review using modified Lugano Classification criteria. Up to 2 different HLA restrictions are allowed per protocol. Treatment is continued until maximal response, unacceptable toxicity, initiation of non-protocol therapy, or failure of the maximum allowable HLA restrictions. After treatment is completed or discontinued, subjects are assessed every 3 months, up to 24 months and every 6 months thereafter up to 5 years for survival status. The primary endpoint of the study is overall response rate (ORR) following the administration of tab-cel.

A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy

Background: Epstein-Barr virus (EBV), a member of the herpesvirus family, normally infects individuals in early adolescence and results in either asymptomatic infection or infectious mononucleosis. Following a lytic primary infection, EBV latently infects B-cells with control of latent infection primarily relying on T-cell function. In immunocompromised individuals where T-cell function is impaired, EBV-transformed B-cells can proliferate resulting in lymphoproliferative disorders or lymphoma (EBV+LPD). EBV+LPD that occurs following solid organ transplant (SOT) is also called EBV+ post-transplant lymphoproliferative disorder (EBV+PTLD). Current treatments for EBV+PTLD following SOT include reduction in immunosuppression (RIS), use of off-label rituximab alone or in combination with multi-agent chemotherapy (CT). Challenges with current treatments include lack of response, relapse, potential for graft rejection, short- and long-term toxicity and high mortality rates. These challenges highlight a clear unmet need for patients who fail or relapse after initial treatment with rituximab. Tabelecleucel is an off-the-shelf, allogeneic EBV-specific T-cell immunotherapy generated from healthy donors. Tabelecleucel is selected for each patient from a library of T cells using the most appropriate HLA restriction to address the patient's disease and has been evaluated in clinical studies (NCT00002663, NCT01498484, NCT02822495). One published interim analysis showed that in ALLELE-eligible patients with EBV+PTLD following SOT, the objective response rate (ORR) was 83% (5/6 patients) (Prockop et al, ASH 2017). Here we describe a multicenter, open label, phase-3 study of tabelecleucel for solid organ transplant subjects with EBV+PTLD after failure of rituximab or rituximab and chemotherapy. Study Design and Methods: This multicenter, open label, single arm, phase 3 study is designed to determine the clinical benefit of tabelecleucel in subjects with EBV+PTLD following SOT after failure of (1) rituximab monotherapy or (2) rituximab plus chemotherapy (NCT03394365). Key inclusion criteria include a diagnosis of locally-assessed, biopsy-proven EBV+PTLD and treatment failure of rituximab or rituximab plus concurrent or sequential chemotherapy. Exclusion criteria include untreated central nervous system (CNS) PTLD or CNS PTLD where treatment is not yet complete or subjects with suspected or confirmed grade &gt; 2 graft-vs-host disease. Tabelecleucel is partially matched to each patient from a library of HLA-characterized tabelecleucel using 1 EBV HLA restriction allele and at least 1 other matched HLA allele. Tabelecleucel is administered in cycles lasting 5 weeks (35 days). During each cycle, subjects receive intravenous (IV) tabelecleucel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by observation through day 35. At the end of each cycle, each subject's response will undergo clinical and radiographic assessment by the investigator based on Lugano response criteria (Cheson et al, 2014). Subjects' responses to tabelecleucel are evaluated by investigators to determine treatment duration and evaluate HLA restriction based on the tabelecleucel-treatment algorithm. Treatment with tabelecluecel is continued until maximal response, unacceptable toxicity, initiation of non-protocol therapy, or failure in response to tabelecluecel with up to 2 different HLA restrictions. After treatment is completed or discontinued, subjects are assessed for disease response every 3 months, up to 24 months from cycle 1 day 1, and every 6 months thereafter up to 5 years from cycle 1 day 1 for survival status. The primary endpoint of the study is overall response rate (ORR) following the administration of tabelecleucel. Disclosures Prockop: Atara Biotherapeutics: Other: Support for industry sponsored trails ; Mesoblast: Other: Support for industry sponsored trails . Hiremath:Atara Biotherapeutics: Employment, Equity Ownership. Ye:Atara Biotherapeutics: Employment, Equity Ownership. Gamelin:Atara Biotherapeutics: Employment, Equity Ownership. Navarro:Atara Biotherapeutics: Employment, Equity Ownership, Patents &amp; Royalties; Pfizer: Equity Ownership; Bluebird Bio: Equity Ownership; GE: Equity Ownership. Mahadeo:Recipient of unrestricted medical education grant from Jazz: Research Funding; PI for ATARA EBV CTL Trials: Other: Other .

Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival.

In T-cell lines dually sensitized with antigens from EBV and CMV or from EBV and WT1 the T-cells specific for CMV or WT1 are often restricted by hla alleles that differ from those restricting T-cells specific for EBV

Background & Aim Virus-specific T-cells generated from transplant or 3rd party donors can induce durable remissions of post-transplant severe infections or EBV lymphomas. T-cells simultaneously sensitized with antigens from multiple viruses have also shown promise. We hypothesized that in any individual donor, immunogenic peptides from different viruses might elicit T-cell responses restricted by different HLA alleles. In HLA non-identical patients, the T-cells, specific for one virus or tumor antigen will be effective only if they are restricted by an HLA allele shared by the patient. Methods, Results & Conclusion We analyzed HLA restrictions (HLA-R) of 42 CTL lines generated by dual sensitization with either autologous EBV-transformed B-cells (BLCL) loaded with a pool of overlapping 15-mer peptides spanning the sequence of CMVpp65 (BLCL/CMV CTL) (n=20) or autologous BLCL loaded with a pool of 15-mers spanning the oncofetal protein WT-1 (BLCL/WT1 CTL) (n=22). EBV CTL lines were generated by sensitization with BLCL alone. The HLA-R of the CMVpp65-specific T-cells(CMV-T), WT1 specific T cells (WT1-T) and EBV specific T cells (EBV-T) in each T cell product were assessed by their cytotoxic activity against a panel of Cr51 labeled peptide-loaded dendritic cells and BLCL sharing a single HLA allele with the T cell donor. In 13/20 BLCL/CMV CTL(65%) and 17/22 BLCL/WT1 CTL(77%) the CMV-T or WT1-T were restricted by single HLA alleles. In 10/20(50%) BLCL/CMV CTL, CMV-T and EBV-T in the same line were restricted by the same HLA allele. However, in the other 10(50%) the CMV-T were restricted by an HLA allele different from that of the EBV-T. Similarly, in 13/22(59%) BLCL/WT1 CTL, WT1-T and EBV-T in the same line were restricted by different HLA alleles. In 2/4 BLCL/CMV CTL and 2/5 BLCL/WT1 CTL, in which the HLA-R of CMV-T or WT1-T differed from that of EBV-T in the same culture, the HLA allele differentially presenting CMV or WT1 antigen but not an EBV antigen was a prominent restricting allele of EBV-T in the EBV CTL of the same origin. This suggests that while immunodominant EBV and CMV epitopes presented by certain prevalent HLA alleles such as HLA B0702 or HLA A0201 often induce responses to both viruses, in certain dual-sensitized cultures, immunogenic epitopes from different antigens may compete for presentation by a specific HLA allele or differ in their potential to induce a dominant T-cell response.

T-cell mitochondrial dysfunction and lymphopenia in DOCK2-deficient patients

This study delineates the mitochondrial defects in DOCK2-deficient T cells contributing to the T cell lymphopenia characteristic of this primary immunodeficiency.

Dual-Sensitized T-Cells Responding to EBV Blcl and Either CMVpp65 or WT-1 Peptide Pools Have Distinct or Shared HLA Restrictions That May Depend on the Presenting HLA Alleles

Adoptive Immunotherapy with virus-specific T-cells generated from transplant or third party donors can induce durable remissions of severe infections or EBV lymphomas post-transplant. T-cells sensitized with antigens from multiple viruses have also shown promise. However, in any individual donor, immunogenic peptides from different viruses might be expected to elicit T-cell responses restricted by different HLA alleles. In HLA non-identical patients, the efficacy of T-cells reactive against any one virus would be eliminated if the T-cells specific for that virus are restricted by an HLA allele not shared by the patient.To examine this hypothesis, we evaluated the HLA restrictions of T-cells generated from 42 healthy donors after dual sensitization with either autologous EBV-transformed B-cells (EBVBLCL) loaded with a pool of overlapping 15-mer peptides spanning the sequence of CMVpp65 (n=20) or autologous EBVBLCL loaded with a pool of 15-mers spanning the oncofetal protein WT-1 (n=22). The HLA restrictions of the CMVpp65-specific and WT1 specific T cells were assessed by their cytotoxic activity against a panel of Cr51 labeled dendritic cells sharing a single HLA allele with the T cells donor. The EBV restrictions of the dual sensitized EBV CTLs were identified by their cytotoxic activity against EBV BLCLs sharing the same single HLA alleles derived from the same donors. In 13/20 CMVpp65/EBV sensitized T cells (65%) and 17/22 WT1/EBV sensitized T cells (77%) the CMV or WT1 specific T cell lines were restricted by single HLA alleles. In 10 of the 20 (50%) lines sensitized with EBV BLCL and CMVpp65, CMVpp65 specific T cells were restricted by an HLA allele that was also one of the restricting alleles for EBV CTLs in the same line. However, in the other 10(50%) the CMVpp65 T cells were restricted by an HLA allele different from that of the EBV CTLs. In the 22 lines co-sensitized with EBV and WT1, WT1 specific T cells were restricted by an allele different from those of the EBV CTLs in 13 (59%) lines.Comparison of EBVCTLs from dual sensitized T cell lines with EBVCTLs contemporaneously generated from the same donors but sensitized with EBV BLCL alone revealed that in 2/4 CMVpp65/EBV lines and 2/5 WT1/EBV lines in which the HLA restriction of CMVpp65 or WT1 specific T cells differed from that of EBV T cells in the same culture, the HLA allele differentially presenting the CMV or WT1 antigen but not an EBV antigen in the dual sensitized cultures was a prominent restricting allele of T cells sensitized with an autologous EBV BLCL alone.In our bank of 135 CMVpp65-specific T-cells sensitized with autologous APCs loaded with the same pool of overlapping CMVpp65 peptides, T-cells specific for epitopes presented by HLA B0702 were dominant in 33/34 donors inheriting this allele. Furthermore, for T-cell lines generated from 50 donors inheriting HLA A0201, HLA A0201 restricted T-cells specific for the NLV peptide of CMVpp65 were dominant for all lines except those 13 that co-inherited HLA B0702. DisclosuresDoubrovina:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding. Hasan:GlaxoSmithKline: Employment. Prockop:Atara Biotherapeutics: Research Funding; Mesoblast: Research Funding. O'Reilly:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding.

A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection.

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.

Abstract 649: Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes

Abstract Background: High-throughput sequencing efforts in molecular tumor diagnostics detect increased numbers of novel genetic variants, including the ones that are predicted to affect splicing. Current approach for predicting whether a specific variant may result in aberrant RNA transcripts often consists of in-silico prediction using bioinformatics prediction tools, which is sometimes followed by RT-PCR analysis of the RNA extracted from blood, or functional splicing reporter assays. Although high-quality patient RNA analysis is usually preferred, such RNA is not always available, or the analysis is hampered due to degradation of aberrant transcripts through nonsense-mediated decay. To overcome this issue, we investigated the possibility of analyzing RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues, with the underlying hypothesis that formalin fixation may inhibit RNA degradation, enabling the detection of aberrant RNA products. Methods: RNA analysis was performed for 13 canonical splice site variants predicted or known to affect splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Of 11 variants, total nucleic acid was obtained from FFPE tumor tissues. For two variants, no FFPE material was available, but EBV-transformed B-cells carrying a splice variant were cultured. Primers were designed to amplify the exon-exon boundary predicted to be affected. Housekeeping gene expression was assessed to determine presence of RNA. Results: Total nucleic acid was successfully isolated for 10 variants from eight FFPE tumor tissues and two EBV-transformed B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known splice variant, present in the B-cell line, the known aberrant splice product could only be detected after formalin fixation of the cells, suggesting that formalin fixation possibly inhibited RNA degradation. Conclusion: Aberrant splicing was successfully identified in all known splice variants and three of four variants predicted to affect splicing, of which the splice effect was previously unknown. Using a RNA sequencing approach, somatic splice variants could be easily and rapidly analyzed, enabling retrospective analysis of variants predicted to result in splicing when only FFPE material is available in clinical settings. Citation Format: Anne M. Jansen, Heleen M. van der Klift, Jaap D. van Eendenburg, Carli M. Tops, Juul T. Wijnen, Frederik J. Hes, Ajay Goel, Hans Morreau, Tom van Wezel. Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes [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 649.

EBV transformation induces overexpression of hMSH2/3/6 on B lymphocytes and enhances γδT-cell-mediated cytotoxicity via TCR and NKG2D.

The engagement of Epstein-Barr virus (EBV)-induced protein ligands in γδ T-cell-mediated anti-EBV immunity, especially in EBV-associated B-cell malignancies, has not been fully elucidated. Previously we reported the overexpression of human MutS homologue 2 (hMSH2), a stress-inducible protein ligand for human γδ T-cells, on EBV-transformed B lymphoblastic cell lines (B-LCLs). In this study, we first generated EBV-transformed B-LCLs from peripheral blood mononuclear cells of healthy volunteers with B95-8 cellular supernatant and cyclosporine A. Secondly, we demonstrated the significantly elevated cell surface protein expression and mRNA transcription of hMSH2 in EBV-transformed B-LCLs, 3D5 and EBV-positive B lymphoma cell line Daudi and Raji. Thirdly, hMSH2-mediated recognition of EBV-transformed B malignant cells by human γδ T-cells was confirmed by specific antibody blocking and siRNA interference. Both TCRγδ and NKG2D participated in hMSH2-mediated recognition of EBV-transformed B malignant cells. Furthermore, hMSH3 and hMSH6, the companion proteins of hMSH2, along with CD98, were found overexpressed on the surface of EBV-transformed malignant B-cells. We concluded that the induced overexpression of hMSH proteins might serve as early alerting biomarkers emerged in EBV-related B-cell malignances or as potential targets for establishing γδ T-cell-based therapeutic immunotherapies towards EBV infection.

Antibodies against the mono-methylated arginine-glycine repeat (MMA-RG) of the Epstein-Barr virus nuclear antigen 2 (EBNA2) identify potential cellular proteins targeted in viral transformation.

The Epstein-Barr virus is a human herpes virus with oncogenic potential. The virus-encoded nuclear antigen 2 (EBNA2) is a key mediator of viral tumorigenesis. EBNA2 features an arginine-glycine (RG) repeat at amino acids (aa)339-354 that is essential for the transformation of lymphocytes and contains symmetrically (SDMA) and asymmetrically (ADMA) di-methylated arginine residues. The SDMA-modified EBNA2 binds the survival motor neuron protein (SMN), thus mimicking SMD3, a cellular SDMA-containing protein that interacts with SMN. Accordingly, a monoclonal antibody (mAb) specific for the SDMA-modified RG repeat of EBNA2 also binds to SMD3. With the novel mAb 19D4 we now show that EBNA2 contains mono-methylated arginine (MMA) residues within the RG repeat. Using 19D4, we immune-precipitated and analysed by mass spectrometry cellular proteins in EBV-transformed B-cells that feature MMA motifs that are similar to the one in EBNA2. Among the cellular proteins identified, we confirmed by immunoprecipitation and/or Western blot analyses Aly/REF, Coilin, DDX5, FXR1, HNRNPK, LSM4, MRE11, NRIP, nucleolin, PRPF8, RBM26, SMD1 (SNRDP1) and THRAP3 proteins that are either known to contain MMA residues or feature RG repeat sequences that probably serve as methylation substrates. The identified proteins are involved in splicing, tumorigenesis, transcriptional activation, DNA stability and RNA processing or export. Furthermore, we found that several proteins involved in energy metabolism are associated with MMA-modified proteins. Interestingly, the viral EBNA1 protein that features methylated RG repeat motifs also reacted with the antibodies. Our results indicate that the region between aa 34-52 of EBNA1 contains ADMA or SDMA residues, while the region between aa 328-377 mainly contains MMA residues.

Application of Epstein-Barr Virus for Optimization of Immortalized B-lymphocyte Production as a Positive Control in Genetic Studies.

Background:Infection of B-cells with Epstein–Barr virus (EBV) leads to more and subsequent immortalization. This is considered as the method of choice for generating lymphoblastoid cell lines (LCLs). Producing LCLs, although very useful but is very time consuming and troublesome, drives the requirement for quicker and more reliable methods for EBV-driven B-cell transformation.Materials and Methods:After successfully production of LCLs, different parameters including temperature, serum concentration, type of culture medium, and CO2 concentration were evaluated on EBV-transformed B-cells. In this study, we were able to produce LCLs and optimize condition.Results:The best condition for generating LCLs was 37°C, 5% CO2, 20% fasting blood sugar, and RPMI 1640. The study results were to establish a reliable method for producing LCLs that can be used to produce immortalized B-cells from almost any sources.Conclusion:This can help with tumorgenecity studies, as well as producing control material for rare genetic disorders and so on. The aim of this study was to determine optimized condition for reliable and reproducible LCLs from different sources.

Lentiviral Vectors Incorporating Ubiquitous Chromatin Opening Element Driving Canine CD18 Expression

Leukocyte adhesion deficiency type 1 (LAD1) in humans is caused due to mutations in the ITGB2 gene encoding the leukocyte CD18 subunit (b2 integrin). This results in defective leukocyte adhesion and migration leading to recurrent episodes of life-threatening bacterial infection. Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific large animal model of LAD1 in which new therapeutic approaches could be tested. Our previous studies have demonstrated variable efficiency of CD18 expression under the control of several promoters. These include cellular promoters such as those of human elongation factor 1a (hEF1a): long (1169bp) and short (248bp) fragments, human phosphoglycerate kinase (hPGK), human CD11b and human CD18 genes. In addition, murine stem cell virus (MSCV) promoter has also been demonstrated to lead to very high levels of CD18 expression in CLAD CD34+ cells thereby reversing the CLAD phenotype in dogs previously treated with both foamy and lentiviral vectors. But, due to potential genotoxicity associated with the use of viral promoters, we continued our efforts in search of novel cellular promoters. One such promoter is the ubiquitous chromatin opening element (UCOE) from the human heterogeneous ribonucleoprotein A2/B1 and chromobox homolog 3 (HNRPA2B1-CBX3) loci. UCOE has been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., Blood 2007).It has also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., Mol Ther. 2010). Since the full-length element is about 2.6 kb, we cloned and tested different fragment lengths of the UCOE promoter in a SIN lentiviral vector (pCL20) in CLAD CD34+ cells in vitro. Efficiency of expression of CD18 obtained with the six promoter fragments of UCOE (in bp), namely U3'631, U3'1262, U3'652, U5'1357, U5'723 and U5'655 were compared to those obtained with an MSCV promoter. Functional viral titers were first determined using a human LAD EBV-transformed B-cell line that lacks endogenous human CD18. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells after a 24h cytokine prestimulation in vitro, the percentage of CD18+ cells 5 days after transduction were as follows: U3'631 - 8.49%, U3'1262 - 15.9%, U3'652 - 21.3% (tested at MOI 100), U5'1357 - 2.05% (tested at MOI 30), U5'723 - 2.44% (tested at MOI 20), U5'655 - 3.01% (tested at MOI 50) and MSCV - 35.3% (tested at MOI 100). The CD18 expression levels driven by some of these promoter fragments were comparable to those driven by cellular promoters mentioned previously. The UCOE is promising in that it could overcome possible gene silencing effects when used in vivo, unlike promoters such as EF1a and PGK which were largely subjected to post-transcriptional gene silencing with sub-therapeutic levels of CD18 as previously tested in the dog model. Hence, functional correction of the CD18 defect could be achieved with candidate UCOE-incorporating SIN lentiviral vector(s) when used in the treatment of CLAD dogs. DisclosuresNo relevant conflicts of interest to declare.

UPR activation and CHOP mediated induction of GBA1 transcription in Gaucher disease

Chronic presence of mutant, misfolded proteins in the endoplasmic reticulum (ER) initiates ER stress and induces the Unfolded Protein Response (UPR).In Gaucher disease (GD), resulting from mutations in the GBA1 gene, encoding lysosomal acid β-glucocerebrosidase (GCase), a certain fraction of the mutant variants is retained in the ER and activates the UPR. We have previously shown UPR activation in GD derived fibroblasts, in fibroblasts that derived from carriers of GD mutations and in Drosophila models of carriers of GD mutations.In the present work we extended our studies to include a large collection of fibroblasts, EBV-transformed B-cells and white blood cells (WBCs) that derived from GD patients.The results showed UPR activation in all tested cells. They also indicated that transcription of the GBA1 gene is upregulated through activation of the UPR-induced CHOP transcription factor. Transcription of the MAN2B gene, encoding alpha-mannosidase and of the ACP gene, encoding acid phosphatase was also elevated presumably through CHOP activation.Our results highlight the existence of chronic stress in GD derived cells due to the presence of ER-retained mutant GCase, which leads to upregulation of GBA1 expression.

Lymphoblastoid Cell Lines as a Tool to Study Inter-Individual Differences in the Response to Glucose.

BackgroundWhite blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy.MethodsLymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species.ResultsCollectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001).ConclusionsExposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.