Lymphoblastoid Cell Lines Cells Research Articles

Characterizing asparagine synthetase deficiency variants in lymphoblastoid cell lines.

Asparagine synthetase (ASNS) catalyzes the synthesis of asparagine (Asn) from aspartate and glutamine. Biallelic mutations in the ASNS gene result in ASNS Deficiency (ASNSD). Children with ASNSD exhibit congenital microcephaly, epileptic-like seizures, and continued brain atrophy, often leading to premature mortality. This report describes a 4-year-old male with global developmental delay and seizures with two novel mutations in the ASNS gene, c.614A > C (maternal) and c.1192dupT (paternal) encoding p.H205P and p.Y398Lfs*4 variants, respectively. We employed the novel use of immortalized lymphoblastoid cell lines (LCL) to show that the proliferation of the heterozygotic parental LCL was not severely affected by culture in Asn-free medium, but growth of the child's cells was suppressed by about 50%. Asn production by the LCL from both the father and the child was significantly decreased relative to the mother's cells. mRNA and protein analysis of the paternal LCL cells for the Y398Lfs*4 variant revealed reductions in both. Attempts to ectopically express the truncated Y398Lfs*4 variant in either HEK293T or ASNS-null cells resulted in little or no detectable protein. Expression and purification of the H205P variant from HEK293T cells revealed enzymatic activity similar to wild-type ASNS. Stable expression of WT ASNS rescued the growth of ASNS-null JRS cells in Asn-free medium and the H205P variant was only slightly less effective. However, the Y398Lfs*4 variant appeared to be unstable in JRS cells. These results indicate that co-expression of the H205P and Y398Lfs*4 variants leads to a significant reduction in Asn synthesis and cellular growth.

Dendrosomal nanocurcumin prevents EBV-associated cell transformation by targeting the lytic cycle genes of the Epstein-Barr virus in the generation of lymphoblastoid cell line.

Targeting the lytic cycle of the Epstein-Barr virus (EBV) has been considered a new treatment strategy for malignancies caused by this virus. This study aimed to investigate the effect of Dendrosomal NanoCurcumin (DNC) to prevent cell transformation and inhibit the expression of viral lytic gene expression in the generation of lymphoblastoid cell line (LCL). Cell viability of LCLs and PBMCs was performed by MTT assay, and flow cytometry (Annexin/PI) was used for evaluation of apoptosis. CD markers on the surface of generated LCL (CD19) cells were examined for cell validation. The effect of DNC on transformation was evaluated by examining cell morphology and determining the expression level of lytic genes BZLF1, Zta, BHRF1, and BRLF1 of EBV using Real-time PCR. Student's t-test was used for statistical analysis. The MTT assay showed that DNC can inhibit the proliferation of LCL in a dose-dependent manner. The 50% cytotoxic concentration (CC50) of DNC and curcumin for LCL was determined 38.8 µg/ml and 75 µg/ml, respectively after 72 hr. Also, Real-time PCR data analysis showed that DNC in 30 µg/ml concentration significantly inhibited cell transformation in the LCL and significantly reduced viral lytic genes such as BZLF1, Zta, BHRF1, and BRLF1expression compared to control. Overall, these findings show that DNC reduces the expression of the viral lytic cycle genes and also the induction of cell apoptosis and finally prevents the generation of LCL.

Nuclear αvβ3 integrin expression, post translational modifications and regulation in hematological malignancies.

αvβ3 integrin, a plasma membrane protein, is amply expressed on an array of tumors. We identified nuclear αvβ3 pool in ovarian cancer cells and were interested to explore this phenomenon in two rare and aggressive types of leukemia, T-cell acute lymphoblastic leukemia (T-ALL) and Mast cell leukemia (MCL) using Jurkat and HMC-1 cell lines, respectively. Moreover, we collected primary cells from patients with chronic lymphocytic leukemia (CLL, n=11), the most common chronic adult leukemia and used human lymphoblastoid cell lines (LCL) generated from normal B cells. Nuclear αvβ3 integrin was assessed by Western blots, confocal microscopy, and the ImageStream technology which combines flow-cytometry with microscopy. We further examined post translational modifications (phosphorylation/glycosylation), nuclear trafficking regulation using inhibitors for MAPK (U0126) and PI3K (LY294002), as well as nuclear interactions by performing Co-immunoprecipitation (Co-IP). αvβ3 integrin was identified in all cell models within the nucleus and is N-glycosylated. In primary CLL cells the β3 integrin monomer is tyrosine Y759 phosphorylated, suggesting an active receptor conformation. MAPK and PI3K inhibition in Jurkat and CLL cells led to αvβ3 enhancement in the nucleus and a reduction in the membrane. The nuclear αvβ3 integrin interacts with ERK, Histone H3 and Lamin B1 in Jurkat, Histone H3 in CLL cells, but not in control LCL cells. To conclude, this observational study provides the identification of nuclear αvβ3 in hematological malignancies and lays the basis for novel cancer-relevant actions, which may be independent from the membrane functions.

Hsa-miR-5581-3p and Hsa-miR-542-3p Target the F8 Gene in Hemophilia A without F8 Mutations.

ObjectiveThis study aims at uncovering the effects of microRNAs (miRNAs) on the F8 gene and FVIII protein in hemophilia A (HA).MethodsF8-targeting miRNAs were predicted by TargetScan, miRDB, and starBase. MiRNAs, predicted by at least two of the three databases, were selected for further study, and their expressions in the blood of HA patients without F8 mutations and healthy controls were detected. A dual-luciferase reporter assay was performed to verify the binding between hsa-miR-5581-3p/hsa-miR-542-3p and F8. In addition, the regulation of F8 by hsa-miR-5581-3p/hsa-miR-542-3p was investigated in human umbilical vein endothelial cells (HUVECs) and lymphoblastoid cell line (LCL) that displayed endogenous expression of FVIII. qRT-PCR was used to detect the expressions of miRNAs and F8 gene, and Western blotting was conducted to measure the expression of FVIII protein.ResultsA total of 42 F8-targeting miRNAs were predicted by at least two of the three databases. Among these miRNAs, hsa-miR-5581-3p and hsa-miR-542-3p were highly expressed in the blood of HA patients and have not been reported in previous studies of HA. Both hsa-miR-5581-3p and hsa-miR-542-3p could bind the 3′UTR of F8 mRNA. Upregulation of hsa-miR-5581-3p or hsa-miR-542-3p suppressed the expressions of F8 mRNA and FVIII protein in HUVECs and LCL cells.ConclusionHsa-miR-5581-3p and hsa-miR-542-3p target the F8 gene and suppress the expression of FVIII protein, which may contribute to the development of HA without F8 mutations.

Conditional expression of a dominant-negative form of Epstein-Barr virus (EBV) nuclear antigen EBNALP inhibits EBV-positive lymphoblastoid cell growth.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that transforms primary B lymphocytes, yielding lymphoblastoid cell lines (LCLs). EBV-encoded nuclear antigen 2 (EBNA2) and EBV-encoded nuclear antigen leader protein (EBNALP) are the first viral products expressed after EBV infection of primary B lymphocytes and are essential for EBV-induced B-lymphocyte growth transformation. EBNA2 functions as a transcriptional activator of viral and cellular genes, with EBNALP as a coactivator for EBNA2-mediated transcriptional activation. We previously reported that mutant EBNALP with a C-terminal 10-amino-acid truncation loses the ability to coactivate, and has a dominant-negative effect on wild-type-EBNALP-mediated coactivation. However, the functional relevance of EBNALP in maintenance of LCL cell growth has not been investigated. To address this, we have constructed LCL-derived cell clones in which this dominant-negative form of EBNALP (DNLP) is conditionally expressed by the Cre-loxP system. We used these cells to evaluate the effect of DNLP expression on EBV-induced cell proliferation. After drug treatment, the DNLP-expressing LCL clones showed reduced cell proliferation and viability. These results indicate that EBNALP is critical for maintaining LCL growth and EBV-induced cell proliferation.

P043 The effects of IBD biologic drugs on the propagation of lymphomatous transformation of EBV-infected peripheral blood mononuclear cells

Immunomodulators have been implicated in increasing the risk of EBV-driven B-cell non-Hodgkin's lymphomas (NHL) in inflammatory bowel disease (IBD) patients. Whether tumour-necrosis alpha inhibitors (anti-TNFs) can also propagate this lympho-proliferative transformation remains unsettled, but their use was associated with increased NHL risk when used in combination with thiopurines. The mechanisms underlying this lymphoproliferation are poorly understood. a NHL in-vitro model was established by co-incubation of EBV-infected-human peripheral-blood mononuclear-cells (PBMC) with Cyclosporine. The resultant lymphoblastoid cell-line (LCL) were characterised by flow-cytometry staining of surface CD markers, telomerase activity assay and by next-generation sequencing (NGS) of V,D,J immunoglobulin heavy-chains after 4 weeks of culture. In parallel, EBV-titers were measured weekly by qPCR. PBMC-EBV cultures were in parallel performed in the presence or absence of major therapeutic agents for IBD (anti-TNFs, anti-integrin vedolizumab, 6mp, methotrexate) for the entire duration of the four-weeks experiment or only during the three-days of initial exposure to EBV after which drugs were washed out of the culture. Incubation of PBMC with EBV and cyclosporine caused the appearance of LCL cells, which were characterised as an expanded population of CD58hiCD23hiCD19+ B-cells. This population uniquely presented an oligo-mono-clonal profile on NGS, as well as high telomerase activity, which was absent in cells exposed to EBV alone. LCL formation was accompanied by increase in intracellular EBV-viral copies until week three and extracellular titer until week two of incubation. When thiopurines and biologics were tested, the frequency of transformed EBV-induced LCLs was significantly higher following co-incubation with anti-TNFs (Infliximab: median 20.88%, IQR 11.84-20.88, vs. median 11.12%, IQR 4.65–18.51 for EBV alone; p = 0.033, n = 20; adalimumab: median 21.31%, IQR 7.9–25.27, vs. median 9.83%, IQR 4.56–18.18 for EBV alone; p = 0.01, n = 19), and with washed-out 6MP (6MP-washout: median 31.39%, IQR 28.97–45.54, vs. EBV alone: median 17.23%, IQR 14.03–35.39; p = 0.018, n = 7), but not with the anti-integrin vedolizumab (vedolizumab: median 9.76%, IQR 3.5–19.43, vs. EBV alone: median 9.83%, IQR 4.56–18.62; p = 0.295, n = 19). These results suggest that anti-TNFs and immunomodulators - but not vedolizumab - propagate EBV-driven lymphoblastoid transformation in vitro. This model may prove useful for studying mechanisms underlying NHL risk in immunosuppressed IBD patients.

Combination of betulinic acid and chidamide synergistically inhibits Epstein-Barr virus replication through over-generation of reactive oxygen species.

Epstein-Barr virus (EBV) has widely infected more than 90% of human populations. Currently, there is no efficient way to remove the virus because the EBV carriers are usually in a latent stage that allows them to escape the immune system and common antiviral drugs. In the effort to develop an efficient strategy for the removal of the EBV virus, we have shown that betulinic acid (BA) slightly suppresses EBV replication through SOD2 suppression with subsequent reactive oxygen species (ROS) generation and DNA damage in EBV-transformed LCL (lymphoblastoid cell line) cells. Chidamide (CDM, CS055), a novel histone deacetylase inhibitor (HDACi), could significantly switch EBV from the latent stage to the lytic stage with increased gene expression of BZLF1 and BMRF1, but has a small effect on EBV replication due to the suppression effect of CDM-mediated ROS generation. Interestingly, a combination of BA and CDM synergistically inhibits EBV replication with ROS over-generation and subsequent DNA damage and apoptosis. Overexpression of SOD2 diminishes this effect, while SOD2 knockdown mimics this effect. An in vivo xenograft tumor development study with the tail vein injection of EBV-transformed LCL cells in nude mice proves that the combination of BA and CDM synergistically increases superoxide anion release in tumor tissues and suppresses EBV replication and tumor growth, and significantly prolongs mouse survival. We conclude that the combination of BA and CDM could be an efficient strategy for clinical EBV removal.

EBV-miR-BHRF1-2 targets PRDM1/Blimp1: potential role in EBV lymphomagenesis.

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in aggressive lymphomas, including diffuse large B-cell lymphoma (DLBCL). It has been shown in DLBCL and Hodgkin lymphoma that PRDM1 is downregulated by cellular microRNAs. In this study, we identify the Epstein–Barr virus (EBV) microRNA (miRNA), EBV-miR-BHRF1-2, as a viral miRNA regulator of PRDM1. EBV-miR-BHRF1-2 repressed luciferase reporter activity by specific interaction with the seed region within the PRDM1 3' untranslated region. EBV-miR-BHRF1-2 inhibition upregulated PRDM1 protein expression in lymphoblastoid cell lines (LCL), supporting a role of miR-BHRF1-2 in PRDM1 downregulation in vivo. Discordance of PRDM1 messenger RNA and protein expressions is associated with high EBV-miR-BHRF1-2 levels in LCLs and primary post-transplant EBV-positive DLBCL. Enforced expression of PRDM1-induced apoptosis and cell cycle arrest in LCL cells. Inhibition of EBV-miR-BHRF1-2 negatively regulates cell cycle and decreases expression of SCARNA20, a small nucleolar RNA that is also downregulated by PRDM1 overexpression. The interaction between EBV-miR-BHRF1-2 and PRDM1 may be one of the mechanisms by which EBV-miR-BHRF1-2 promotes EBV lymphomagenesis. Our results support the potential of EBV-miR-BHRF1-2 as a therapeutic target in EBV-associated lymphoma.

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis.EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control.MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects.In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. DisclosuresNo relevant conflicts of interest to declare.

EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10−7) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10−5). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.

Thalidomide, Lenalidomide and Pomalidomide Disrupt Epstein-Barr Virus (EBV) Latency: Clinical Implications

IntroductionThalidomide (THAL), and the IMiDs® immunomodulatory agents lenalidomide (LEN), and pomalidomide (POM) are all approved for use in multiple myeloma (MM) either as single agents, or in combination with dexamethasone (DEX). Despite the enhanced efficacy of these novel agents, concern has arisen as to the increased incidence of secondary primary malignancies (SPM). For example, the IFM 2005-002 trial reported cases of lymphoblastic leukemia and Hodgkin's disease (HD) following LEN use (Attal, Lauwers-Cances et al. 2012) in MM patients on maintenance therapy. Also, a recent case report described a MM patient who developed HD who had been treated with salvage therapy containing THAL(Chim, Choi et al. 2013), and two publications reported EBV reactivation in MM patients treated with LEN (Kneppers, van der Holt et al. 2011; Kroger, Zabelina et al. 2013). As HD is causally linked to EBV, this raises the question as to whether the IMiDs reactivate latent EBV infection in normal memory B-cells, and thereby increase the risk of EBV-related malignancies. To this end, we have investigated the ability of the IMiD's to induce reactivation of latently infected B-cell lines. MethodsA panel of latently infected EBV-positive B-cell lines including Burkitt's lymphoma (BL) cells and lymphoblastoid cell lines (LCL) were treated with either LEN, THAL or POM, and the status of the EBV lytic cycle was evaluated using in vitro and in vivo models. ResultsTreatment of BL and LCL cell lines with physiological concentrations of IMiDs (1-5 μM) induced the immediate early gene BZLF1 and the early gene BMRF1. Interestingly, the ability to induce EBV reactivation was in their potency order (i.e. POM>LEN>THAL). The IMiD's also induced lytic cell death, as an LCL carrying a BZLF1-deleted EBV, which is incapable of undergoing a lytic cycle, showed no change in cell viability, compared to wild-type cells which had increased cell death. The addition of the nucleoside analogue ganciclovir (GCV) enhanced the cytotoxic effect of LEN and POM alone in BL cells lines. An in vivo xenograft model of BL demonstrated that the combination of LEN and GCV was highly efficacious at suppressing tumor cell growth, thus confirming the ability of LEN to stimulate the EBV-lytic life cycle. The ability to induce EBV reactivation was directly related to the stimulation of phosphatidylinositol-3 kinase (PI3K) signaling, which was completely blockaded by the PI3K-δ inhibitor, CAL101. The combination of LEN with either, DEX or rituximab, induced increased BMRF1 compared to the LEN alone. ConclusionsThe IMiD class of drugs has a potent ability to reactivate the lytic cycle in B-cells latently infected with EBV. We hypothesize that the IMiD's reactivate latently infected resting memory B cells through enhancing PI3K signaling. This reactivation may be further potentiated when the IMiDs are used in combination with rituximab or DEX, which may simultaneously enhance the EBV lytic cycle and suppress the host immune response. These findings suggest the possibility that immunocompromised patients who receive IMiDs should be monitored for evidence of EBV reactivation. Also, this may suggest a mechanism by which patients may develop EBV-associated SPM, an effect which is similar to the methotrexate induced EBV-positive lymphomas seen in rheumatoid arthritis patients (Feng, Cohen et al. 2004).ReferencesAttal, M., V. Lauwers-Cances, et al. (2012). “Lenalidomide maintenance after stem-cell transplantation for multiple myeloma.” The New England journal of medicine 366(19): 1782-1791.Chim, C. S., P. T. Choi, et al. (2013). “Hodgkin's lymphoma as a second cancer in multiple myeloma never exposed to lenalidomide.” Annals of hematology 92(6): 855-857.Feng, W. H., J. I. Cohen, et al. (2004). “Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas.” Journal of the National Cancer Institute 96(22): 1691-1702.Kneppers, E., B. van der Holt, et al. (2011). “Lenalidomide maintenance after nonmyeloablative allogeneic stem cell transplantation in multiple myeloma is not feasible: results of the HOVON 76 Trial.” Blood 118(9): 2413-2419.Kroger, N., T. Zabelina, et al. (2013). “Toxicity-reduced, myeloablative allograft followed by lenalidomide maintenance as salvage therapy for refractory/relapsed myeloma patients.” Bone marrow transplantation 48(3): 403-407. Disclosures:Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees.

Constitutive Interferon-Inducible Protein 16-Inflammasome Activation during Epstein-Barr Virus Latency I, II, and III in B and Epithelial Cells

Epstein-Barr virus (EBV), etiologically linked with human B-cell malignancies and nasopharyngeal carcinoma (NPC), establishes three types of latency that facilitate its episomal genome persistence and evasion of host immune responses. The innate inflammasome responses recognize the pathogen-associated molecular patterns which lead into the association of a cytoplasmic sensor such as NLRP3 and AIM2 proteins or nuclear interferon-inducible protein 16 (IFI16) with adaptor ASC protein (apoptosis-associated speck-like protein with a caspase recruitment domain) and effector procaspase-1, resulting in active caspase-1 formation which cleaves the proforms of inflammatory interleukin-1β (IL-1β), IL-18, and IL-33 cytokines. Whether inflammasome responses recognize and respond to EBV genome in the nuclei was not known. We observed evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β, -IL-18, and -IL-33, in EBV latency I Raji cells, latency II NPC C666-1 cells, and latency III lymphoblastoid cell lines (LCL). Interaction between ASC with IFI16 but not with AIM2 or NLRP3 was detected in all three latencies and during EBV infection of primary human B cells. IFI16 and cleaved caspase-1, IL-1β, IL-18, and IL-33 were detected in the exosomes from Raji cells and LCL. Though EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) are common to all forms of EBV latency, caspase-1 cleavage was not detected in cells expressing EBNA1 alone, and blocking EBER transcription did not inhibit caspase-1 cleavage. In fluorescence in situ hybridization (FISH) analysis, IFI16 colocalized with the EBV genome in LCL and Raji cell nuclei. These studies demonstrated that constant sensing of latent EBV genome by IFI16 in all types of latency results in the constitutive induction of the inflammasome and IL-1β, IL-18, and IL-33 maturation.

Epstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis

The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.

The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.

Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.

Human herpesvirus 6 integrates within telomeric regions as evidenced by five different chromosomal Sites

Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal integration sites of human herpesvirus 6 (HHV-6) in phytohemagglutinin-stimulated leukocytes and B lymphocytes from Epstein-Barr virus transformed lymphoblastoid cell lines (LCLs). Five different chromosomal integration sites were found in nine individuals. Only one site was identified in each individual, each site was in the vicinity of the telomeric region and was on either the p or q arm of only one of the two chromosome homologues. The sites were 9q34.3, 10q26.3, 11p15.5, 17p13.3, and 19q 13.4, of which three have not been previously identified. For 9q34.3 the site of integration was further mapped using a locus-specific probe for 9q34.3 together with a pan-telomeric probe and both co-localized with the HHV-6 signal. Similarly an arm-specific telomeric probe for 19q co-localized with the HHV-6 signal. It was therefore concluded that the site of integration is actually within the telomere. The number of viral DNA copies/cell was calculated in blood, LCL cells and hair follicles and was one or more in every case for each of the nine individuals. This result was confirmed by FISH where 100% of cells gave an HHV-6 signal. These findings add to previous reports suggesting that integrated HHV-6 DNA is found in every cell in the body and transmitted vertically. Finally, including our data, worldwide seven different chromosomal sites of HHV-6 integration have now been identified. Large epidemiological studies of chromosomal integration are required to identify further telomeric sites, geographical or racial variation and possible clinical consequences.

Cell cycle arrest and lytic induction of EBV-transformed B lymphoblastoid cells by a histone deacetylase inhibitor, Trichostatin A

Latent infection of the Epstein-Barr virus (EBV) is strongly associated with the pathogenesis of several human tumor types. The restricted expression of the latent EBV antigens is critical for EBV-associated tumors to escape from immune surveillance. EBV lytic replication can be triggered by various treatments and the induced lytic genes cause strong cytotoxic T lymphocyte (CTL) responses. Histone acetylation or deacetylation is associated with chromatin remodeling and regulates gene expression. Histone deacetylase (HDAC) inhibitors affect cell cycle progression as well as gene expression in a wide variety of transformed cells. We examined whether an HDAC inhibitor, TSA, can affect cell cycle progression and induce EBV lytic replication in EBV-transformed lymphoblastoid cell lines (LCLs). TSA caused cell cycle arrest at low concentrations and induced apoptosis at higher (>300 nM) concentrations in the LCLs and EBV negative BJAB cells. To clarify the underlying mechanism of TSA-induced cell cycle arrest, expression of cell cycle regulatory factors was examined by RNase protection assay and Western blot analysis. Following TSA treatment, a reduced expression of cyclin D2 and an induction of p21 may have played an essential role for G1 arrest in LCLs, while p21 induction might have arrested BJAB cells in G1 phase. A Cdk inhibitor, p57, was increased by 300 nM TSA in both LCLs and BJAB cells, indicating its role in apoptosis. Moreover, immunofluorescene assay and Western blotting showed that TSA induced EBV lytic replication in LCL cells. These results suggest that TSA may exert an enhanced anti-tumor effect for EBV-associated tumors not only by inducing a cell cycle arrest and apoptosis, but also by triggering an EBV lytic cycle.

A Role for Intercellular Antigen Transfer in the Recognition of EBV-Transformed B Cell Lines by EBV Nuclear Antigen-Specific CD4+ T Cells

The CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directly recognize virus-transformed lymphoblastoid cell lines (LCLs) in vitro and inhibit their growth. In this study, we used a panel of EBNA1, 2, 3A, and 3C-specific CD4+ T cell clones to study the route whereby endogenously expressed EBNAs access the HLA class II-presentation pathway. Two sets of results spoke against a direct route of intracellular access. First, none of the clones recognized cognate Ag overexpressed in cells from vaccinia vectors but did recognize Ag fused to an endo/lysosomal targeting sequence. Second, focusing on clones with the strongest LCL recognition that were specific for EBNA2- and EBNA3C-derived epitopes LCL recognition was unaffected by inhibiting autophagy, a postulated route for intracellular Ag delivery into the HLA class II pathway in LCL cells. Subsequently, using these same epitope-specific clones, we found that Ag-negative cells with the appropriate HLA-restricting allele could be efficiently sensitized to CD4+ T cell recognition by cocultivation with Ag-positive donor lines or by exposure to donor line-conditioned culture medium. Sensitization was mediated by a high m.w. antigenic species and required active Ag processing by recipient cells. We infer that intercellular Ag transfer plays a major role in the presentation of EBNA-derived CD4 epitopes by latently infected target cells.

Inhibition of Hematopoietic Progenitor Cells by CD4+ T Cells Specific for an Endogenous Peroxisomal Protein, DRS-1: A Possible Mechanism for Bone Marrow Failure in Individuals Carrying HLA-DR15.

A large body of evidence has suggested that acquired aplastic anemia (AA) of patients carrying HLA-DR15 is a kind of organ-specific autoimmune disease where hematopoietic progenitor cells in bone marrow are attacked by CD4+ T cells recognizing endogenous antigens. We recently identified diazepam-binding inhibitor-related protein 1 (DRS-1) as a candidate autoantigen capable of provoking immune system attack against hematopoietic progenitor cells in AA (Blood, 2004). Although in other organ-specific autoimmune diseases such as insulin-dependent diabetes mellitus and primary biliary cirrhosis, cytoplasmic proteins including glutamic acid decarboxylase 65 and pyruvate dehydrogenase complex have been shown to serve as autoantigens and mediate organ damages by CD4+ T cells, it remains unclear whether a peroxisomal protein like DRS-1 can be processed in hematopoietic progenitor cells, presented by HLA-DR15, and eventually serve as a target antigen of specific CD4+ T cells, leading to killing of hematopoietic progenitor cells themselves. To clarify these issues, we established a CD4+ T-cell line specific to a DRS-1 peptide (amino acid residues 191–204) from an AA patient carrying HLA-DR15 who had exhibited a high titer of anti-DRS-1 antibody as well as a high frequency of T-cell precursors specific to DRS-1, and then examined the cytotoxicity of the DRS-1-specific T-cell line against (1) autologous lymphoblastoid cell line (LCL) cells transfected with full length DRS-1 cDNA using a lentiviral vector, (2) myeloid leukemia cell lines carrying HLA-DR15 (KH88 and SAS413) and a leukemia cell line not carrying HLA-DR15 (K562), and (3) CD34+ progenitor cells from normal individuals. When all leukemia cell lines and LCL cells were examined for DRS-1 expression using Western blotting with specific monoclonal antibodies, DRS-1 protein was detected in DRS-1-transfected LCL cells, KH88 and K562, but not in nontransfected LCL cells and SAS413. Overexpression of DRS-1 gene by the CD34+ cells from normal individuals was ascertained by real-time PCR. In the 51Cr release assay, DRS-1-specific T cells showed cytotoxicity against only DRS-1-transfected LCL cells and KH88 in a dose-dependent manner (Figure), indicating that the T cell line requires presence of both DRS-1 and HLA-DR15 on target cells to exert cytotoxicity. When the DRS-1-specific T cells were incubated with CD34+ cells isolated from normal individuals with or without HLA-DR15 at an 10:1 ratio for 4 hours and cultured in a methylcellulose medium supplemented with colony-stimulating factors, the numbers of CFU-GM and BFU-E colonies derived from an HLA-DR15+ individual were 60.0% and 52.9% of a control whereas those derived from an HLA-DR15− individual were 90.1% and 88.2%. These findings indicate that hematopoietic progenitor cells in individuals with HLA-DR15 can present DRS-1 through the DR molecule and a breakdown of immune tolerance to DRS-1 may lead to development of AA. [Display omitted]

Influence of Single-Nucleotide Polymorphisms in the Multidrug Resistance-1 Gene on the Cellular Export of Nelfinavir and Its Clinical Implication for Highly Active Antiretroviral Therapy

Protease inhibitors (PIs) such as nelfinavir (NFV) suppress HIV replication. PIs are substrates of P-glycoprotein (P-gp), the product of the multidrug-resistance-1 (MDR1) gene. Three single-nucleotide polymorphisms (SNPs) are present in exons of the MDR1 gene: MDR1 1236, MDR1 2677 and MDR1 3435. We speculated that these genetic polymorphisms affected PI concentration in the cell. To verify this hypothesis, we first genotyped these SNPs in 79 Japanese patients by the SNaPshot method and found incomplete linkage disequilibrium between the SNPs. Because the SNP at MDR1 3435 has been reported to be associated with P-gp expression, we evaluated the effect of that SNP on the export of NFV from HIV-positive patients' lymphoblastoid cell lines by measuring time-dependent decrease in the amount of intracellular NFV by high-performance liquid chromatography. We found the intracellular concentration of NFV in lymphoblastoid cell lines (LCLs) with the homozygous T/T genotype at MDR1 3435 were higher than that with C/C genotype with statistical significance. This suggests that the activity of P-gp in patients' LCL cells with the MDR1 3435 T/T genotype was lower. In a retrospective study we evaluated the effect of the SNPs on CD4 cell count recovery in response to antiretroviral treatment with PIs, and obtained statistically significant evidence that suggested marginal association of the SNP at MDR1 1236 but not at MDR1 2677 or MDR1 3435. As in vitro results were not consistent with the clinical evaluation, clinical importance of MDR1 genotyping for antiretroviral therapy remains to be investigated in a larger, case-controlled study.