Epstein-Barr Virus-transformed Cell Lines Research Articles

Human B cells and dendritic cells are susceptible and permissive to enterovirus D68 infection.

Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.

Modulation of peroxisomal compartment by Epstein-Barr virus.

Viruses utilize clever strategies of interacting with various cellular factors, to remodel an organelle function, for the establishment of successful infection. In recent decades, numerous studies revealed the exploitation of the peroxisomal compartment by viruses. Epstein-Barr virus (EBV) is a ubiquitous virus linked with various cancers and neurological disorders. Till now, there is no report regarding the impacts of EBV infection on peroxisomal compartment. Therefore, we investigate the modulation of peroxisomal proteins in EBV transformed cell lines and during acute EBV infection. EBV positive Burkitt lymphoma cells of different origins as EBV transformed cells along with EBV negative Burkitt lymphoma cells as a control were used in this study. For acute EBV infection experiments, we infected peripheral blood mononuclear cells with EBV for three days. Thereafter, analyzed the gene expression patterns of peroxisomal proteins using qPCR. In addition, quantification of lipid content was performed by using fluorescence microscopy and biochemical assay. Our results revealed that, the peroxisomal proteins were distinctly regulated in EBV transformed cells and during acute EBV infection. Interestingly, PEX19 was significantly upregulated in EBV infected cells. Further, in correlation with the altered expression of peroxisomes proteins involved in lipid metabolism, the EBV transformed cells showed lower lipid abundance. Conversely, the lipid levels were increased during acute EBV infection. Our study highlights the importance of investigating the manipulation of the peroxisomal compartment by putting forward various differentially expressed proteins upon EBV infection. This study provides a base for further investigation to delve deeper into EBV and peroxisomal interactions. The future research in this direction could provide involvement of novel signaling pathways to understand molecular changes during EBV mediated pathologies.

American Heart Association Cardiovascular Genome-Phenome Study: foundational basis and program.

The American Heart Association has established the Cardiovascular Genome-Phenome Study (CV-GPS) as a means by which to achieve the goal of using modern genomics and phenotyping optimally to combat cardiovascular disease (CVD). CVD remains the leading cause of death in the United States and has become a major cause of morbidity and mortality worldwide. The course of CVD begins at an early age and evolves throughout life. Typically, risk factor precursors (eg, prehypertension, borderline abnormal lipids) are first observed in adolescence or young adulthood. By middle age, they develop into full-fledged cardiometabolic risk factors and subclinical disease, which usually become clinically apparent in older age. Importantly, risk factors for CVD reflect the complex interplay between genetic and environmental factors and demonstrate the complexity of the multiple determinants of disease. Editorial see p 17 Many of the genetic and environmental determinants of CVD have been identified over the past 50 years, and successful preventive and therapeutic strategies have been developed as a result. However, many potential genetic or acquired disease drivers remain unaccounted for, as illustrated by the observation that the major CVD risk factors combined account for only a fraction of the population-attributable risk.1 In addition, variations in CVD risk exist among individuals with similar risk profiles. Part of these differences may reflect differences in profiles of known risk factors that have not yet been fully refined but also individual differences in underlying genetic modifiers of such risk factors. Modern genomics, with its ability to provide ideally unbiased analysis of the entire genome, offers an approach to the ascertainment of all genetic determinants of CVD. Furthermore, the increasingly broad range of “omics” methods, including RNA-Seq, modern proteomics, metabolomics, and metabonomics, provide deeper and more refined molecular detail by which to define an individual’s genome and its relationship to (patho)phenotype. …

SNP array-based copy number and genotype analyses for preimplantation genetic diagnosis of human unbalanced translocations

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalanced translocations in cleavage-stage embryos. A total of 36 cleavage-stage embryos that were diagnosed as unbalanced by initial PGD FISH analysis were dissociated (n=146) and amplified by multiple displacement amplification (MDA). SNP CNs and genotypes were determined using SNP array. Epstein-Barr Virus-transformed cell lines with known CR were used for optimising the genomic smoothing (GS) length setting to increase signal to noise ratio. SNP CN analysis showed 23 embryos (64%) that were unbalanced in all blastomeres for the chromosomes involved in the translocation, 5 embryos (14%) that were normal or balanced in all blastomeres and 8 embryos (22%) that were mosaic. SNP genotyping, based on analysis of informative SNP loci with opposing homozygous parental genotypes, confirmed partial monosomies associated with inheritance of unbalanced translocation in surplus embryos. We have developed a universal MDA-SNP array technique for chromosome CN analysis in single blastomeres. SNP genotyping could confirm partial monosomies. This combination of techniques showed improved diagnostic specificity compared with FISH and may provide more reliable PGD analysis associated with higher embryo transfer rate.

Methylome-wide comparison of human genomic DNA extracted from whole blood and from EBV-transformed lymphocyte cell lines

DNA from Epstein-Barr virus-transformed lymphocyte cell lines (LCLs) has proven useful for studies of genetic sequence polymorphisms. Whether LCL DNA is suitable for methylation studies is less clear. We conduct a genome-wide methylation investigation using an array set with 45 million probes to investigate the methylome of LCL DNA and technical duplicates of WB DNA from the same 10 individuals. We focus specifically on methylation sites that show variation between individuals and, therefore, are potentially useful as biomarkers. The sample correlations for the methylation variable probes ranged from 0.69 to 0.78 for the WB duplicates and from 0.27 to 0.72 for WB vs LCL. To compare the pattern of the methylation signals, we grouped adjacent probes based on their inter-correlations. These analyses showed ∼29 000 and ∼14 000 blocks in WB and LCL, respectively. Merely 31% of the methylated regions detected in WB were detectable in LCLs. Furthermore, we observed significant differences in mean difference between WB and LCL as compared with duplicates of WB (P-value =2.2 × 10(-16)). Our study shows that there are substantial differences in the DNA methylation patterns between LCL and WB. Thus, LCL DNA should not be used as a proxy for WB DNA in methylome-wide studies.

The WST survival assay: an easy and reliable method to screen radiation-sensitive individuals

An easy, fast and reliable method was developed to screen hundreds of Epstein-Barr virus-transformed cell lines (lymphoblastoid cell lines, LCLs) for radiation sensitivity that were generated from lymphocytes isolated from young lung cancer patients. The WST-1 test explores the metabolic activity of the mitochondria as an indicator for the vital status of cells. Cell proliferation as well as indirect cell death can be quantified by this method on a large scale in microtiter plates. Cell survival was measured at 24- and 48-h post-irradiation with 10 Gy ((137)Cs source) by the WST-1 assay and Trypan blue staining. To set up the experimental screening conditions and to establish a positive and a negative control, an ATM-mutated cell line from a radiation-sensitive ATM patient and an ATM proficient cell line from a healthy brother were compared. An optimal differentiation between the two cell lines was demonstrated for 10 Gy and 24- and 48-h cell growth after irradiation. Upon screening 120 LCLs of young lung cancer patients under these conditions, 5 of them were found to be radiation sensitive to a high degree of statistical significance. The results have been confirmed by a second laboratory by means of Trypan blue testing. The WST-1 test represents an efficient and reliable method by means of screening for radiation-sensitive cell lines.

Comparison of Immunologic Assays for Detecting Immune Responses in HIV Immunotherapeutic Studies: AIDS Clinical Trials Group Trial A5181

This study was designed to evaluate which of several T-cell-specific, immune response assays are the most relevant in measuring the key characteristics of an effective immune response to HIV-1. Using 5 HIV-1 antigens as stimulants, we assessed lymphocyte proliferation, supernatant gamma interferon (IFN-gamma) cytokine production (CP), single-cell IFN-gamma production by enzyme-linked immunospot (ELISPOT) assay, with and without Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs), and intracellular cytokine production (ICC) for IFN-gamma and interleukin 2 (IL-2) by flow cytometry. We used these to compare specimens from HIV-1-infected subjects who were virally suppressed with a stable antiretroviral therapy (ART) regimen (group A) with specimens from subjects not on ART but with HIV-1 viremia of <3,000 copies/ml (group B). The lymphocyte proliferation assay (LPA) did not significantly differentiate between the two groups. Using fresh peripheral blood mononuclear cells (PBMCs), the CP and ELISPOT assays for IFN-gamma detected the greatest differences between the two groups, specific for three of the five HIV-1 antigens, whereas significant differences were seen only in response to one antigen when cryopreserved cells were used. The strongest correlations were seen between the CP and ELISPOT assays. The ELISPOT B-LCL assay showed a cell concentration-dependent increase in IFN-gamma production compared to that shown by the standard ELISPOT assay but did not differentiate between the groups. In the ICC assay, greater numbers of IFN-gamma-producing T cells were seen in group B, and little or no detectable IL-2 production was seen in both groups. These studies highlight complexities of immunologic monitoring of T-cell responses in multisite clinical trials in HIV infection and outline considerations for optimizing these efforts.

Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium

Background and Purpose To yield large amounts of DNA for many genotype analyses and to provide a renewable source of DNA, the Type 1 Diabetes Genetics Consortium (T1DGC) harvested DNA and peripheral blood mononuclear cells (PBMCs) from individuals with type 1 diabetes and their family members in several regions of the world.Methods DNA repositories were established in Asia-Pacific, Europe, North America, and the United Kingdom. To address region-specific needs, different methods and sample processing techniques were used among the laboratories to extract and to quantify DNA and to establish Epstein-Barr virus transformed cell lines.Results More than 98% of the samples of PBMCs were successfully transformed. Approximately 20–25 µg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 µg per cell pack. In addition, the extracted DNA from whole blood or transformed cells was successfully utilized in each regional human leukocyte antigen genotyping laboratory and by several additional laboratories performing consortium-wide genotyping projects.Limitations Although the isolation of PBMCs was consistent among sites, the measurement of DNA was difficult to harmonize.Conclusions DNA repositories can be established in different regions of the world and produce similar amounts of high-quality DNA for a variety of high-throughput genotyping techniques. Furthermore, even with the distances and time necessary for transportation, highly efficient transformation of PBMCs is possible. For future studies/trials involving several laboratories in different locations, the T1DGC experience includes examples of protocols that may be applicable. In summary, T1DGC has developed protocols that would be of interest to any scientific organization attempting to overcome the logistical problems associated with studies/trials spanning multiple research facilities, located in different regions of the world.

Report on the 14th international society of blood transfusion platelet immunology workshop

The aims of the 14th ISBT Platelet Immunology Workshop were to evaluate in-house methods for detection of antibodies to human platelet antigens, to compare the sensitivity and specificity of antibody detection using a panel of monoclonal antibodies and to evaluate genotyping methods and establish procedures for drug-dependent antibody detection. Forty-two laboratories from 23 countries participated. Samples and reagents provided for the five different exercises. The ability of participating laboratories to correctly identify the HPA antibody specificity in the nine samples ranged from 20% to 97%. The greatest difficulty was observed with samples that contained antibodies against HPA-3b and GPIV. The significant differences in optical density values by monoclonal antibody of immobilization of platelet antigens (MAIPA) assay were observed when testing the same platelet-specific antibodies. HPA genotyping of DNA with novel mutations did not significantly affect the results. The overall average discrepancy rate was 2·15% for genotyping of 10 DNA samples from well-characterized Epstein–Barr virus transformed cell lines. For detection of drug-dependent antibodies, excellent results for specificity and sensitivity were obtained by the laboratories using the MAIPA and flow cytometry. Most laboratories were able to identify the majority of HPA antibodies; however, significant disparities were observed in proficiency testing. MAIPA assay sensitivity is influenced by the monoclonal antibody clone used. DNA with new mutations and EBV cell lines are valuable samples to ensure accurate genotyping. A sensitive and specific drug-dependent antibody assay performed well in the hands of participants.

Burkitt lymphoma-associated molecules impair HLA class II activation of CD4+ T cells (100.20)

Abstract Burkitt Lymphoma (BL) is a very fast growing form of non-Hodgkin's lymphoma. The common characteristic of all BL is the translocation and constitutive activation of the c-myc oncogene. Our present study suggests that BL and Epstein-Barr Virus-transformed B-lymphoblastoid cell lines (B-LCL) constitutively express HLA class II proteins and bind peptide ligands. However, unlike B-LCL, BL cells were not capable of activating CD4+ T cells. While BL and B-LCL possessed similar levels of HLA class II, invariant chain (Ii) and HLA-DM, BL cells expressed lower levels of co-stimulatory molecules (CD80/CD86). Biochemical and functional assays showed that addition of external co-stimulatory signals to BL cells failed to functionally deliver HLA class II antigens to CD4+ T cells. Although both BL and B-LCL were capable of binding peptides at neutral and acidic pH, BL cells were only able to activate T cells at low pH (5.5), suggesting that BL-associated inhibitory molecules were eluted at the acidic pH. BL conditioned medium, as well as acid eluent from BL cells and primary B-cell tumors, inhibited functional class II presentation by both B-LCL and mature dendritic cells, while those eluent from B-LCL and primary B-cells did not. No characteristic inhibitory cytokines were identified in the acid eluent from BL or primary B-cell tumors. Taken together, these results suggest that BL-associated molecules may inhibit HLA class II activation of CD4+ T cells.

Consensus Characterization of 16 FMR1 Reference Materials: A Consortium Study

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Activity of the LMP1 gene promoter in Epstein–Barr virus-transformed cell lines is modulated by sequence variations in the promoter-proximal CRE site

The Epstein-Barr virus (EBV)-encoded tumour-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We have previously identified a cyclic AMP-responsive element (CRE) in the B95-8 LMP1 promoter that is essential for transcription activation. Sequencing of LMP1 promoter in the P3HR1-derived EREB2.5 cell line revealed 25 single base pair substitutions in comparison to the B95-8 virus, one of them localized in the CRE element. Sequence variations in this element have been identified in several EBV isolates of both African and Asian origins. The effect of the P3HR1 CRE site variation on binding of factors to the LMP1 promoter sequence (LRS) and promoter activation was investigated with electrophoretic mobility-shift assays and reporter gene transfection assays. ATF1 and CREB1 transcription factors bound with reduced efficiency to the P3HR1 variant and below the detection level to the other tested variants. Accordingly, reporter plasmids carrying the P3HR1 CRE sequence in a B95-8 LRS context displayed 50 % lower activity in all tested cell lines. The impaired ability to activate transcription caused by the C to A substitution in CRE was not apparent when the mutated site was placed in a P3HR1 LRS context and the reporter transfected into Jijoye cells, most likely as a consequence of the other base pair substitutions in P3HR1 LRS. Overall, our results suggest that the mutations in the LRS CRE site have been conserved to adjust LMP1 expression to levels that favour cell survival in certain cellular and environmental contexts.

Quantitative analysis of human leucocyte antigen expression during culture of Epstein-Barr virustransformed cell lines using the Dako QIFIKIT

Pre-existing donor-specific human leucocyte antigen (HLA) antibodies in renal allograft recipients result in hyperacute and accelerated graft failure. These antibodies can be detected in flow cytometric assay systems using HLA-characterised Epstein-Barr virus (EBV)-transformed B-lymphocyte cell lines. Confident assay performance is predicated by the expression of HLAs on the EBV-transformed B-cell line surface. Surface HLA expression of three EBV-transformed B-cell lines that had previously been used as part of a potential organ recipient serum screening panel at St James’ University Hospital, Leeds, are assessed for changes in the level of HLA expression over the nominal culture duration of eight days using the QIFIKIT (Dako, Denmark), a quantitative flow cytometry kit for assessing cell surface antigens. A comparison of the mean fluorescence intensity (MFI) of the known antigen levels of the beads via a calibration graph permits determination of the antibody binding capacity of the cell lines. Results showed that HLA expression is not consistent throughout the cell culture, with optimal expression occurring during day 2 of culture. Inconsistent HLA expression demonstrated during the cell culture means that no assumption of the level of HLA expression can be made, and that cell lines used as part of a screening panel should have their HLA expression in cell culture determined.

Novel Pathway for Induction of Latent Virus from Resting CD4+T Cells in the Simian Immunodeficiency Virus/Macaque Model of Human Immunodeficiency Virus Type 1 Latency

Although combination therapy allows the suppression of human immunodeficiency virus type 1 (HIV-1) viremia to undetectable levels, eradication has not been achieved because the virus persists in cellular reservoirs, particularly the latent reservoir in resting CD4(+) T lymphocytes. We previously established a simian immunodeficiency virus (SIV)/macaque model to study latency. We describe here a novel mechanism for the induction of SIV from latently infected resting CD4(+) T cells. Several human cell lines including CEMx174 and Epstein-Barr virus-transformed human B-lymphoblastoid cell lines mediated contact-dependent activation of resting macaque T cells and induction of latent SIV. Antibody-blocking assays showed that interactions between the costimulatory molecule CD2 and its ligand CD58 were involved, whereas soluble factors and interactions between T-cell receptors and major histocompatibility complex class II were not. Combinations of specific antibodies to CD2 also induced T-cell activation and virus induction in human resting CD4(+) T cells carrying latent HIV-1. This is the first demonstration that costimulatory signals can induce latent virus without the coengagement of the T-cell receptor, and this study might provide insights into potential pathways to target latent HIV-1.

Diagnostic Strategy for the Detection of Dystrophin Gene Mutations in Asian Patients and Carriers Using Immortalized Cell Lines

Duchenne muscular dystrophy and Becker muscular dystrophy are X-linked recessive diseases of muscle degeneration caused by mutations in the dystrophin gene. More than half of our local Asian patients have point mutations that cannot be detected by conventional multiplex polymerase chain reaction deletion screening. This study aimed to develop mutational screening and carrier detection for Duchenne and Becker muscular dystrophy using protein truncation analysis from Epstein-Barr virus-transformed lymphocyte cell lines. Messenger ribonucleic acid was extracted from fresh lymphocytes and Epstein-Barr virus-transformed lymphocyte cell lines of 14 patients. Reverse transcriptase polymerase chain reaction was performed in 11 overlapping segments, followed by in vitro protein translation and truncation analysis. DNA sequencing was carried out for the corresponding complementary DNA regions, which showed aberrant truncated protein products. Carrier studies using this method were also performed for two families. Half of the patients had frame-shifting deletions, and the remaining seven patients showed point mutations, of which four were novel. These mutations were detected in messenger ribonucleic acid extracted from both fresh lymphocytes and Epstein-Barr virus-transformed lymphocyte cell lines. Carrier status was confirmed in one family and was found to be negative in the other family studied. Protein truncation analysis is an efficient method of screening truncating point mutations from immortalized lymphocyte cell lines from patients. This approach not only serves to prove the pathogenicity of both deletion- and nondeletion-type mutations; it is also effective for carrier detection. The use of such cell lines obviates the need for repeated blood and muscle sampling in patients and offers a perpetual source of messenger ribonucleic acid that can be used long after the patient's demise.

Stable Isotope Tagging of Epitopes

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Long-Term Preservation of Antibody-Dependent Cellular Cytotoxicity (ADCC) of Natural Killer Cells Amplified In Vitro From the Peripheral Blood of Breast Cancer Patients After Chemotherapy

Twenty percent of breast cancer adenocarcinomas overexpress the oncogene c-erb-2 that is recognized by the humanized anti-Her2/neu monoclonal antibody Herceptin. Results from clinical studies suggest that antibody-dependent cellular cytotoxicity (ADCC) is involved in the clinical response of Herceptin-treated patients. The purpose of the current study was to evaluate the possibility of amplifying in vitro the CD3-/CD16+ natural killer (NK) cell subset that mediates ADCC from breast cancer patients after chemotherapy. Peripheral blood mononuclear cells from six breast cancer patients taken 2 months after chemotherapy completion were co-cultured with an autologous irradiated Epstein-Barr virus-transformed B-lymphoblastoid cell line (LCL) in the presence of interleukin-2 (IL-2) for 4-6 weeks. These LCL + IL2 activated cultures (ACs) were tested for ADCC potential, and their CD3/CD16 NK proportion was quantified. Among the ACs, the proportion of CD3-/CD16+ NK cells increased up to 64% over the first 2 weeks of culture and the ACs continued to expand for 1 month thereafter. Control and patient ACs displayed ADCC activity (tested in the presence of Rituximab against the autologous LCL to take into account any possible effect of inhibitory NK receptors) as well as against the MCF-7(Her2/neu) breast cancer cell line in the presence of Herceptin. This ADCC activity was maintained during the entire culture period. In conclusion, chemotherapy in breast cancer patients does not obviate the possibility of amplifying in vitro the NK cell subset that mediates ADCC. Consequently, adoptive transfer of lymphocytes mediating ADCC can be considered using this protocol to test its benefit in patients under Herceptin treatment.

Immunization against a low‐frequency human platelet alloantigen in fetal alloimmune thrombocytopenia is not a single event: characterization by the combined use of reference DNA and novel allele‐specific cell lines expressing recombinant antigens

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal immunization against a fetal platelet (PLT) alloantigen. In cases of FNAIT attributed to low-frequency PLT alloantigens, the laboratory diagnosis is often hampered by the lack of adequate PLTs. Three families with maternal immunization against fetal PLT antigens were analyzed. In Family 1, previous immunization of another female or woman has been observed. In Families 2 and 3, newborns presented with the typical clinical picture of FNAIT. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing with reference to DNA from Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Antibodies were characterized by glycoprotein (GP)-specific immunoassay with a panel of stable Chinese hamster ovary cell lines expressing low-frequency alloantigens. In three families, maternal immunization associated with the low-frequency alloantigens human PLT antigen (HPA)-8bw (Sra), HPA-11bw (Groa), and HPA-13bw (Sita) was identified. Maternal serum samples showed positive reactions in an antigen capture assay with cell lines carrying recombinant GP IIb/IIIa (HPA-8bw and -11bw) or GPIa/IIa (HPA-13bw), respectively. These results could be confirmed by genotyping analysis of fathers and newborns. This study demonstrates that cases of FNAIT attributed to low-frequency PLT alloantigens cannot be regarded as single events. The availability of reference DNA and cell lines expressing recombinant PLT alloantigens can facilitate their identification.

Spontaneous transgenesis of human B lymphocytes.

DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM(+)/IgD(+)/CD27(-)). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.

Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV.

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.