Established Lymphoblastoid Cell Lines Research Articles

PROMOTER CHOICE: THE IDEAL PROMOTER TO DRIVE CHIMERIC ANTIGEN RECEPTOR (CAR) EXPRESSION IN NK CELLS

Aims Natural Killer (NK) cells have gained much recent attention as a promising alternative platform for CAR engineering owing to their unique biological attributes, their specialized cytotoxicity against tumors, their safety profile, and their potential use as an off-the-shelf cellular therapy. However, NK transgene expression level is a major technical obstacle to the development of NK cell immunotherapy. Promoters, present in vectors, are responsible for maintaining the expression of CAR in host cells. Furthermore, promoters may differ in their ability to produce lentiviral particles and in their transduction efficiency. The present study aimed to evaluate whether different internal promoters result in a different percentage of transduced cells and transgene expression level in NK cells and thus to be able to choose an ideal promoter to drive the expression of CAR.CD19 increasing the antitumor potency of NK cells. Methods Lentiviral particles with EF1a and SFFV promoters driving GFP gene expression or CAR.CD19 were produced, and the virus titer was measured by flow cytometry. Lentiviral particles were used for transduction of NK-92, NK from peripheral blood and NK from cord blood. CAR positive cells were detected by flow cytometry using anti F(ab)2 antibody. CAR-NK cell cytotoxic potential and specificity were assessed using established human Raji and NALM-6 lymphoblastoid cell lines (CD19+) as targets, which express varying antigen levels and MDA-MB-453 (CD19-) cells were used as negative control. Results and discussion First, we explored the potential EF1a and SFFV promoters to drive GFP gene expression. SFFV promoter produced high lentiviral titers and achieved the best transduction efficacy in NK cells. The major difference between the promoters was the ability of SFFV to maintain longer and more robust GFP expression in NK cells. Therefore, we chose this promoter to drive CAR.CD19 expression and interleukin (IL)-15 co-expression in NK cells. There was a transduction efficiency of 13% (+/- 13.7) for NK-92 cells, assessed 48h after transduction. NK cells from Peripheral Blood (NK-PB) and Cord Blood (NK-CB) were also transduced with an efficiency of 27% and 49%, respectively. After 3 weeks in culture, the percentage of CAR-NK cells progressively decreased in primary cells. Cell growth of transduced cells from both sources was not statistically different; despite wild type NK-CB cells had a higher fold-change expansion than wild type NK-PB. CAR-NK-92 cells modified with SFFV were enriched with magnetic beads to 98% of NK cells positive for CAR expression and showed efficient and stable transgene expression during ex vivo expansion. The enriched CAR.CD19-NK-92 cells showed a higher killing potential against Raji CD19+ cells, compared with non-transduced NK-92 cells. Further, we evaluated the CAR-NK primary cells in vitro cytotoxic potential. CAR-NKs both from PB and CB had a higher killing potential against Raji and NALM-6 CD19+ cell lines than non-transduced NK cells. Conclusions Taken together, these results indicate that the developed vector is functional and able to transduce NK cells from different sources, enhancing their anti-tumoral activity against tumoral CD19+ cells. Our results highlight that the promoter choice is essential to generate CAR-NK cells suitable for therapeutic applications. Funding Supported by FAPESP 2019/25309-0, 2013/08135-2, 2008/578773; CNPq 573754-2008-0; Capes (88887.140966/2017-00 and 88881.199630/2018-01).

Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program.

CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments.

Expression of the chemokine receptors CCR1 and CCR2B is up-regulated in peripheral blood B cells upon EBV infection and in established lymphoblastoid cell lines

In immunocompetent individuals, EBV establishes in B cells an asymptomatic lifelong latent infection controlled by the immune system. Chemokine receptors regulate immune system function. CCR1 and CCR2 share protein sequence similarity and exert responses to multiple chemokines. The role of these receptors in B cells is largely unknown. We show that the mRNA and functional protein expression of CCR1 and CCR2 is induced in ex vivo B cells upon EBV infection and in established lymphoblastoid cell lines (LCLs). The CCR1 and CCR2B ORF transcripts were determined in LCLs. In contrast, in both the EBV-negative and EBV-positive Burkitt lymphoma cell lines, neither the CCR1, CCR2A, and CCR2B ORF transcripts nor their corresponding proteins were detected. Our data suggest that CCR1/CCR2B could be involved in clearing EBV-infected latency III B cells in immunocompetent individuals via directing the migration of these cells and attracting the chemokines-expressing immune cells.

Changes in Mitochondrial Morphology and Bioenergetics in Human Lymphoblastoid Cells With Four Novel OPA1 Mutations.

Mutations in the optic atrophy 1 gene (OPA1) have been reported in patients with autosomal dominant optic atrophy (ADOA). OPA1 plays important roles in mitochondrial dynamics and cell apoptosis. The link between OPA1 mutations and changes in bioenergetics is still not fully resolved. The aim of this study was to investigate the effects of OPA1 mutations on the mitochondrial tubular network and bioenergetics. We established lymphoblastoid cell lines from four ADOA families harboring different OPA1 mutations, unaffected relatives (internal control cell lines), and unrelated normal controls (normal control cell lines). OPA1 splice variants and mRNA were analyzed by reverse transcription-PCR and quantitative real-time PCR. Protein isoforms were examined by Western blotting. The mitochondrial network was visualized by confocal microscopy. Mitochondrial bioenergetics were assessed using a Seahorse XF24 flux analyzer. Mitochondrial membrane potential and oxidative damage were analyzed by flow cytometry. OPA1 mutant cell lines showed significant decreases in OPA1 mRNA and protein expression, mitochondrial membrane potential, and ATP synthesis. A marked deficiency of the long isoform of OPA1 was observed in cells with OPA1 mutations in the middle domain and GTPase effector domain. Confocal microscopy revealed increased mitochondrial fragmentation in OPA1 mutant cells. OPA1 mutant cells also displayed reduced oxygen consumption and underwent glycolysis to produce ATP. Moreover, OPA1 mutations caused the accumulation of oxidative damage. Our experiments demonstrated that OPA1 mutations induced mitochondrial fragmentation, uncoupled mitochondrial respiration, and elicited dysfunctional bioenergetics. However, there were no significant differences among the various OPA1 mutations.

A ChIP-seq-Defined Genome-Wide Map of MEF2C Binding Reveals Inflammatory Pathways Associated with Its Role in Bone Density Determination

Genome-wide association studies (GWAS) have demonstrated that genetic variation at the MADS box transcription enhancer factor 2, polypeptide C (MEF2C) locus is robustly associated with bone mineral density, primarily at the femoral neck. MEF2C is a transcription factor known to operate via the Wnt signaling pathway. Our hypothesis was that MEF2C regulates the expression of a set of molecular pathways critical to skeletal function. Drawing on our laboratory and bioinformatic experience with ChIP-seq, we analyzed ChIP-seq data for MEF2C available via the ENCODE project to gain insight in to its global genomic binding pattern. We aligned the ChIP-seq data generated for GM12878 (an established lymphoblastoid cell line) and, using the analysis package HOMER, a total of 17,611 binding sites corresponding to 8,118 known genes were observed. We then performed a pathway analysis of the gene list using Ingenuity. At 5 kb, the gene list yielded 'EIF2 Signaling' as the most significant annotation, with a P value of 5.01 × 10(-26). Moving further out, this category remained the top pathway at 50 and 100 kb, then dropped to just second place at 500 kb and beyond by 'Molecular Mechanisms of Cancer'. In addition, at 50 kb and beyond 'RANK Signaling in Osteoclasts' was a consistent feature and resonates with the main general finding from GWAS of bone density. We also observed that MEF2C binding sites were significantly enriched primarily near inflammation associated genes identified from GWAS; indeed, a similar enrichment for inflammation genes has been reported previously using a similar approach for the vitamin D receptor, an established key regulator of bone turnover. Our analyses point to known connective tissue and skeletal processes but also provide novel insights in to networks involved in skeletal regulation. The fact that a specific GWAS category is enriched points to a possible role of inflammation through which it impacts bone mineral density.

Inactivation of CDK/pRb Pathway Normalizes Survival Pattern of Lymphoblasts Expressing the FTLD-Progranulin Mutation c.709-1G>A

BackgroundMutations in the progranulin (PGRN) gene, leading to haploinsufficiency, cause familial frontotemporal lobar degeneration (FTLD-TDP), although the pathogenic mechanism of PGRN deficit is largely unknown. Allelic loss of PGRN was previously shown to increase the activity of cyclin-dependent kinase (CDK) CDK6/pRb pathway in lymphoblasts expressing the c.709-1G>A PGRN mutation. Since members of the CDK family appear to play a role in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults, we investigated the role of CDK6/pRb in cell survival/death mechanisms following serum deprivation.Methodology/Principal FindingsWe performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carriers of c.709-1G>A PGRN mutation, asymptomatic and FTLD-TDP diagnosed individuals. Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A bearing lymphoblasts. Apparently, this feature allows PGRN-deficient cells to escape from serum withdrawal-induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the vulnerability of lymphoblasts from FTLD-TDP patients to trophic factor withdrawal.Conclusion/SignificanceThe use of PGRN-deficient lymphoblasts from FTLD-TDP patients may be a useful model to investigate cell biochemical aspects of this disease. It is suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDP.

Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia

A recent challenge for investigators studying the progressive neurological disease ataxia-telangiectasia (A-T) is to identify mutations whose effects might be alleviated by mutation-targeted therapies. We studied ATM mutations in eight families of Japanese A-T patients (JPAT) and were able to identify all 16 mutations. The probands were compound heterozygotes in seven families, and one (JPAT2) was homozygous for a frameshift mutation. All mutations--four frameshift, two nonsense, four large genomic deletions, and six affecting splicing--were novel except for c.748C>T found in family JPAT6 and c.2639-384A>G found in family JPAT11/12. Using an established lymphoblastoid cell line (LCL) of patient JPAT11, ATM protein was restored to levels approaching wild type by exposure to an antisense morpholino oligonucleotide designed to correct a pseudoexon splicing mutation. In addition, in an LCL from patient JPAT8/9, a heterozygous carrier of a nonsense mutation, ATM levels could also be partially restored by exposure to readthrough compounds (RTCs): an aminoglycoside, G418, and a novel small molecule identified in our laboratory, RTC13. Taken together, our results suggest that screening and functional characterization of the various sorts of mutations affecting the ATM gene can lead to better identification of A-T patients who are most likely to benefit from rapidly developing mutation-targeted therapeutic technologies.

DNA ligand designed to antagonize EBNA1 represses Epstein–Barr virus‐induced immortalization

Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized cells in vitro and is associated with various malignancies in vivo. EBNA1, which is expressed in the majority of EBV-infected cells, recognizes specific DNA sequences at the cis-acting latent origin of plasmid replication (oriP) element of the EBV genome. EBNA1 plays a critical role in the viral episome maintenance and transactivates viral transforming genes in latently infected cells. Therefore, DNA-targeting agents that can disrupt the EBNA1-oriP interaction will offer novel functional inhibitors of EBNA1. Pyrrole-imidazole polyamides, sequence-specific DNA ligands, can be designed to interfere with the binding of various transcriptional factors. Here, we synthesized pyrrole-imidazole polyamides targeting EBNA1-bound DNA sequences and developed an inhibitor for the EBNA1-oriP interaction. A pyrrole-imidazole polyamide, designated as DSE-3, bound adjacent to the EBNA1 recognition sequences located in the dyad symmetry element of oriP, and selectively inhibited EBNA1-oriP binding both in vitro and in vivo. DSE-3 also inhibited the proliferation of established lymphoblastoid cell lines by eradicating EBV episomes from the cells. In addition, DSE-3 repressed the expression of viral transforming genes after infecting human peripheral blood mononuclear cells with EBV and, as a consequence, inhibited EBV-mediated B-cell immortalization. These results suggest that EBNA1 functions will be an attractive pharmacological target for EBV-associated diseases.

Resveratrol, a Natural Antioxidant From Grapes and Red Wines, Prevents EBV-Associated Lymphoproliferation and Transformation through Inducing Apoptosis

AbstractAbstract 2543Epstein-Barr virus (EBV) infection and reactivation is an increasing complication in immune deficient patients, particularly after allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). Whereas tapering of immunosuppression, donor lymphocyte infusion, and rituximab may be effective for this complication after HSCT, severe adverse events such as fatal GVHD and infections could develop thereafter. Resveratrol, a natural antioxidant compound found in grapes and red wines, possesses various biological activities including antitumor, anti-inflammatory, and antimicrobial efficacies “(Nature Reviews Drug Discovery 5, 493–506, 2006). We report here anti-EBV effects of resveratrol in eliminating EBV infected cells and preventing EBV-associated B-cell transformation. Resveratrol, at nontoxic concentrations to normal cells, effectively prevented the transformation of primary B cells derived from a panel of 16 healthy donors as well as cord blood mononuclear cells derived from 10 different donors. In addition, resveratrol induced dose-dependent apoptosis, assessed by FACS-based detection of Annexin V, in 7 different established EBV-transformed lymphoblastoid cell lines (LCLs) and in the Burkitt lymphoma EBV+ Raji and Daudi cell lines. These effects were consistently associated with the activation of caspase-8 and a 60 % down-regulation of the anti-apoptotic protein BCL-2. The addition of the caspase-8 inhibitor c-FLIPL to the cell culture vigorously inhibited the resveratrol induced apoptosis. Moreover, the resveratrol treatment decreased by 80% the EBV nuclear antigen 1 (EBNA1) expression, a critical protein required for the persistency of EBV infection. These results suggest that resveratrol may have potential roles in preventing EBV-associated lymphoproliferative diseases in immune compromised patients. Since resveratrol is a nontoxic, inexpensive and clinically available agent, it may be useful in preventing EBV- infection and reactivation after HSCT and SOT. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr virus (EBV)-associated miRNAs are important for the maintenance of EBV transformed B cells

The human oncogenic gamma-herpesvirus, Epstein-Barr virus (EBV), infects approximately 95% of the adult human population and causes B-cell lymphomas in immune-compromised individuals, such as AIDS patients. EBV transforms primary human B cells into indefinitely proliferating lymphoblastoid cell lines (LCLs) in vitro, which represents an important model system for the transformation of B cells by EBV. EBV encodes 25 viral miRNA, but their role in virus replication and pathogenesis is unclear. Three of these miRNAs, miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3, are expressed during the latency III stage of EBV, the EBV gene expression pattern observed in AIDS-associated B cell lymphomas and LCLs. We have performed functional studies examining the roles of the three BHRF1 miRNAs during the transformation of primary B cells by EBV in vitro as well as in the maintenance of the transformed state in established LCL cultures. Our preliminary data suggest that at least one EBV miRNA is necessary for LCL outgrowth. EBV is also known to up-regulate a number of cellular microRNAs, including miR-155, and our preliminary data also suggest a role of miR-155 in the maintenance of LCLs. We are currently examining the mechanisms by which EBV and cellular microRNAs contribute to the growth and maintenance of LCLs. Together, our data point to a role for EBV-encoded and EBV-induced miRNAs in EBV-induced cell transformation.

Toll-Like Receptor Agonists Synergistically Increase Proliferation and Activation of B Cells by Epstein-Barr Virus

Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cells in vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection.

Quantitative evaluation of the role of Epstein–Barr virus immediate-early protein BZLF1 in B-cell transformation

The Epstein-Barr virus (EBV) immediate-early transactivator BZLF1 plays a key role in switching EBV infection from the latent to the lytic form by stimulating the expression cascade of lytic genes; it also regulates the expression of several cellular genes. Recently, we reported that BZLF1 is expressed in primary human B cells early after EBV infection. To investigate whether this BZLF1 expression early after infection plays a role in the EBV-induced growth transformation of primary B cells, we generated BZLF1-knockout EBV and quantitatively evaluated its transforming ability compared with that of wild-type EBV. We found that the 50% transforming dose of BZLF1-knockout EBV was quite similar to that of wild-type EBV. Established lymphoblastoid cell lines (LCLs) harbouring BZLF1-knockout EBV were indistinguishable from LCLs harbouring wild-type EBV in their pattern of latent gene expression and in their growth in vitro. Furthermore, the copy numbers of EBV episomes were very similar in the LCLs harbouring BZLF1-knockout EBV and in those harbouring wild-type EBV. These data indicate that disrupting BZLF1 expression in the context of the EBV genome, and the resultant inability to enter lytic replication, have little impact on the growth of LCLs and the steady-state copy number of EBV episomes in established LCLs.

MDM2-dependent inhibition of P53 is required for Epstein-Barr virus B cell growth transformation and infected cell survival

Epstein-Barr virus (EBV) growth transformation of primary B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs) depends on the concerted activities of a subset of viral proteins expressed during latency. EBV drives quiescent B cells into S phase and consequently a host response is activated that includes expression of p53 and its target genes. Since LCLs retain wild-type p53, it was of interest to determine what contribution the p53 pathway may have in controlling established LCL growth and EBV-mediated transformation of primary B cells. We found that liberation of p53 through chemical antagonism of one of its major ubiquitin ligases, MDM2, led to apoptosis of established LCLs and suppressed EBV-mediated transformation of primary B cells. The activation of latent p53 induced target genes associated with apoptosis and was antagonized by constitutive NFκB activity in LCLs. Furthermore, the NFκB-dependent antagonism of p53 was not at the p53-dependent transcriptional level, but rather involved increasing the level of steady-state MDM2 protein. The consequence of these effects through NFκB is to increase the MDM2/p53 ratio, thereby sensitizing cells to MDM2 antagonism. This mechanism, likely through increased MDM2 translation, may provide a novel means by which NFκB activating oncogenes suppress wild-type p53 activity and overcome the oncogenic stress checkpoint. Furthermore, the acquisition of Nutlin-sensitivity in EBV-infected cells provides a novel system for studying the pathways that dictate LCL survival and regulate EBV transformation. Finally, MDM2 antagonists may be considered alone or in combination with NFκB inhibition for therapeutic intervention in EBV-associated malignancies expressing wild-type p53.

Capacity of robot handling for Epstein-Barr virus transformation.

Epstein-Barr virus (EBV) transformation has been described as a routine method to establish human B lymphoblastoid cell lines. Each established lymphoblastoid cell line represents one unique genetic information carrier and can produce unlimited quantities of DNA materials available for downstream applications and research. Undoubtedly, it is of great value to human clinical and experimental genetic studies. However, the current process of EBV transformation requires much manpower in the routine renewal of medium, which is time-consuming. This situation can become a serious problem especially when establishing a human B lymphoblastoid cell bank. A modified and cost-effective protocol for EBV transformation should be considered. In the present study, process in EBV transformation was modified to fit the requirements of robot handling. 1 mL of whole blood was demonstrated to be sufficient to perform EBV transformation. Additionally, EBV transformation can performed in 96-deep-well plates that are directly and widely used with automatic work platforms. Based on these facts, a process of EBV transformation can be modified to fit the requirements of robot handling.

Poster 197: Molecular and Clinical Approach in a Japanese Family With CL/P and a Balanced Translocation t (9;17)(q32; q12)

Cleft lip and/or palate (CL/P) are common birth defects of multifactorial etiology by both genetic and environmental factors (1). Recently, we found a Japanese family with a balanced translocation t(9;17)(q32;q12). Of three generations with translocation, one subject has cleft lip and palate. It has been demonstrated that facial development is associated with genes on chromosome 9q and 17q (2), leading to a possibility that genes disrupted by the translocation may play an important role in clefting. The purposes of this study are following;1)Identification of genes underlying the breakpoints of the translocation.2)Determine the association of the disrupted genes with the occurrence of CL/P. Here, we report a molecular genetic analysis and clinical examination of this family. A male infant was born prematurely by cesarean section at 24th gestational age after an uneventful pregnancy. His birth weight and height were 706 g and 31.8 cm, respectively. He had ocular hypertelorism, a bilateral cleft lip and palate, and a mild flat nose. His father and paternal grandmother had the same translocation of 9q32 and 17q12 as seen in the patient, while no family history of malformations.1)We determined the breakpoints of the translocation by fluorescence in situ hybridization (FISH) analysis using BAC clones and subsequent long range PCR analysis using established lymphoblastoid cell lines.2)In order to study the role of these candidate genes in forming a cleft, we examined clinical data and findings. The PCR products covering the breakpoint were analyzed on ABI 3700 automated sequencer, after the reaction with BigDye. The Applied Biosystems sequence software (version 2.1.2) was used for lane tracking. Chromatograms were transferred to a UNIX workstation, base called with PHRED 4.0 (http://droog.mbt.washington.edu/poly_doc40.html) and results viewed with CONSED program (version 4.0) (3). The UCSC (http://genome.ucsc.edu/cgi-bin/hgGateway) database was used to detect potential genes around the breakpoint. Then, the gene function is examined by biochemical test of the patient. 1) Sequence analysis demonstrated that two genes, SLC31A1 (Solute Carrier Family 31 Member 1) on chromosome 9 and CCL2 (Chemokine Ligand 2) on chromosome 17, were rearranged with the breaks occurring within their introns.2) Although, SLC31A1 is associated with a transportation element essential for life, copper, so far, we did not find any clinical evidence for the Copper metabolism disorder in the patient. Although, the present data support that genes may play an important role in facial development, it is necessary to extend an additional investigation of Copper metabolism.

High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines.

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.

Somatic mutations and activation-induced cytidine deaminase (AID) expression in established rheumatoid factor-producing lymphoblastoid cell line

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs), allowing the production of specific antibody-secreting cell lines. We and others have previously found that in contrast to peripheral blood B cells, EBV-transformed lymphoblastoid cell lines express the activation-induced cytidine deaminase (AID) gene. The opposite is true for the germinal center-specific BCL6 gene: it is expressed in adult peripheral blood B cells and is no longer expressed in LCLs. The present work extends our findings and shows that whereas AID expression is rapidly induced following EBV infection, BCL6 expression is gradually down-regulated and is fully extinguished in already established LCLs. The question of whether AID activation induces the process of somatic hypermutation (SHM) was investigated in adult-derived LCLs. It was found that the V H gene from the rheumatoid factor-producing RF LCL (derived from a rheumatoid arthritis patient), accumulated somatic point mutations in culture. Overall, nine unique mutations have accumulated in the rearranged V H gene since the generation of the RF cell line. Four additional intraclonal mutations were found among 10 cellular clones of the RF cells. One out of the four was in CDR1 and could be correlated with loss of antigen-binding activity in three out of the 10 clones. Altogether, these 13 mutations were preferentially targeted to the DGYW motifs and showed preference for CG nucleotides, indicating that they were AID-mediated. By contrast, mutations were not detected among 3700–4000 nucleotides each of the Vλ, Cμ and GAPDH genes derived from the same RF cell cultures and the cellular clones. Our results thus show that AID may generate point mutations in the rearranged Ig V H during in vitro cell culture of adult-LCLs and that these mutations may be responsible, at least in part, for the known instability and occasional loss of antigen-binding activity of antibody-secreting LCLs.

Plasma Peptide YY and Ghrelin Levels in Infants and Children with Prader-Willi Syndrome

An insatiable appetite is a cardinal feature of Prader-Willi syndrome (PWS) with stomach rupturing as a reported consequence. Peptide YY, secreted by the intestine and released post-prandially, inhibits appetite, while ghrelin, secreted by the stomach during mealtime hunger, stimulates appetite. Both peptide YY and ghrelin act at the brain level, particularly the hypothalamus. Recently, plasma ghrelin levels were reported to be elevated in children and adults with PWS but peptide YY levels have not been studied in this syndrome or ghrelin in infants with PWS. To further address the abnormal eating behavior in PWS, we obtained fasting plasma peptide YY and ghrelin levels in 12 infants and children with PWS ranging in age from 2.5 months to 13.3 years and compared them with values from normal populations reported in the literature. Plasma ghrelin levels in our patients with PWS were similar to those of other children with PWS and were significantly higher than those reported in obese children without PWS. Our infants with PWS had similar plasma ghrelin levels compared with our children with PWS but peptide YY levels in our children and infants with PWS were lower than reported in similarly aged individuals without PWS. In addition, we performed preliminary gene expression analysis of ghrelin and peptide YY and their receptors in patients with PWS using established lymphoblastoid cell lines but gene expression did not correlate with plasma ghrelin or peptide YY levels.

In vitro genotoxicity testing of the furylethylene derivative UC-245 in human cells

The possible genotoxic potential of the 2-furylethylene derivative UC-245 has been evaluated in vitro using human cells as a test system. This compound was synthesized at the Centro de Bioactivos Químicos, Universidad Central de Las Villas (Cuba) and it appears to be effective against leishmaniosis. The induced genetic damage was determined by scoring the frequency of micronuclei (MN) and the frequency of sister chromatid exchanges (SCE) in primary lymphocyte cultures set up from two different donors. The DNA breakage level was also evaluated by the Comet assay, using an established human lymphoblastoid cell line (TK6). For the MN and SCE studies, to detect eventual metabolic modification in the genotoxicity of this compound, the cultures were treated with S9 microsomal fraction. The results obtained indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of presence/absence of the metabolic fraction, which would indicate a lack of clastogenic and/or aneugenic potential. Nevertheless, a clear and significant increase in the SCE frequency was observed in the treatments without S9. This would support the 2-furylethylene derivative UC-245 inducing DNA primary damage. In addition, the results obtained in the Comet assay also show that UC-245 induces a significant increase in the level of DNA breakage, which would confirm its genotoxicity.

Cytolytic CD4(+)-T-cell clones reactive to EBNA1 inhibit Epstein-Barr virus-induced B-cell proliferation.

In the absence of immune surveillance, Epstein-Barr virus (EBV)-infected B cells generate neoplasms in vivo and transformed cell lines in vitro. In an in vitro system which modeled the first steps of in vivo immune control over posttransplant lymphoproliferative disease and lymphomas, our investigators previously demonstrated that memory CD4(+) T cells reactive to EBV were necessary and sufficient to prevent proliferation of B cells newly infected by EBV (S. Nikiforow et al., J. Virol. 75:3740-3752, 2001). Here, we show that three CD4(+)-T-cell clones reactive to the latent EBV antigen EBNA1 also prevent the proliferation of newly infected B cells from major histocompatibility complex (MHC) class II-matched donors, a crucial first step in the transformation process. EBNA1-reactive T-cell clones recognized B cells as early as 4 days after EBV infection through an HLA-DR-restricted interaction. They secreted Th1-type and Th2-type cytokines and lysed EBV-transformed established lymphoblastoid cell lines via a Fas/Fas ligand-dependent mechanism. Once specifically activated, they also caused bystander regression and bystander killing of non-MHC-matched EBV-infected B cells. Since EBNA1 is recognized by CD4(+) T cells from nearly all EBV-seropositive individuals and evades detection by CD8(+) T cells, EBNA1-reactive CD4(+) T cells may control de novo expansion of B cells following EBV infection in vivo. Thus, EBNA1-reactive CD4(+)-T-cell clones may find use as adoptive immunotherapy against EBV-related lymphoproliferative disease and many other EBV-associated tumors.