How does ebv immortalized b cells

Finally, EBV has evolved several tactics to prevent apoptosis of the infected cell in order to augment viral persistence. A functional bcl-2 homolog encoded by BHRF1 can inhibit apoptosis induced by a range of stimuli at least in part by binding to the pro-apoptotic protein Bim LMP1 signaling in human B lymphoma cell lines induces expression of the cellular protein c-FLIP that interferes with formation of the death inducing signaling complex DISC , required to initiate activation of caspase 8 following ligation of death receptors.

LMP1 modulation of cell death pathways may be especially relevant to the germinal center model for EBV discussed above. In this scenario LMP1, a functional homologue of CD40, can provide survival signals, in concert with another latent cycle protein, LMP2a, which mimics B cell receptor BCR signaling, to infected B cells that are otherwise destined to die in the absence of encounter with antigen. These are just a few salient examples of the myriad maneuvers through targeting of immune cell function, antigen presentation, and cell death pathways, by which EBV modulates the host immune response.

Understanding the manner in which EBV-encoded proteins cooperate to evade and subvert the immune response during lytic and latent infection is crucial to advancing new approaches to vaccine development and to understand the pathogenesis of EBV-associated malignancies.

LMP1 is required for transformation of human B cells 35 and is sufficient to transform rodent fibroblasts in vitro LMP1 is an integral membrane protein containing six transmembrane-spanning domains and a long C-terminal tail Fig.

The transmembrane domain acts to induce oligomerization of LMP1 complexes in the membrane to aggregate in lipid rafts. This clustering of LMP1 proteins brings individual C-terminal tails into proximity, creating suitable docking sites for cytoplasmic signaling adaptor proteins, thereby allowing LMP1 to signal in a constitutive fashion. We have examined in detail the signal transduction pathways elicited by LMP1 and the corresponding downstream functions in latently infected B cells.

It is likely that IL produced by EBV infected B cells also acts to negatively regulate the immune response, akin to regulatory IL producing B cells, that have been more recently described to play a role in peripheral tolerance in autoimmunity, cancer, and organ transplantation By understanding the signal transduction pathways elicited by LMP1 we have been able to pinpoint specific molecules within key signaling nodes as potential therapeutic targets.

EBV was the first virus shown to encode microRNA miRNA 46 , a family of small, approximately 22 nucleotide, non-coding RNA that post-transcriptionally regulate gene expression for control of cellular events. Subsequently it has been established that virally-encoded miRNA participate in viral-host cell interactions including immune evasion, prolonging survival of infected cells, regulation of viral genes and potentially in the pathogenesis of viral-associated disease Interestingly, overexpression of miR is characteristic of many B cell lymphomas 50 , 51 and in a mouse model constitutive expression of miR in B cells lead to uncontrolled proliferation of pre-B cells and subsequent malignancy We and others have found that EBV infection induces the expression of specific host B cell miRNAs, including miR and miR, which potentially play a role in viral oncogenesis These studies suggest that further analysis of viral diversity and the impact on cellular function may provide new insights in to the underlying viral mechanisms that drive B cell lymphomagenesis and may be exploited to develop biomarkers for disease.

In particular, a high proportion of LMP1 molecules isolated from cell lines derived from Chinese patients with NPC were shown to have a characteristic 30 base pair deletion, compared to the B Consequently, it was proposed that the 30 base pair deletion may be important in development of NPC, however, subsequent studies showed a similarly high frequency of this deletion in LMP1 isolated from healthy, seropositive Chinese subjects Several other classification schemes for EBV have been described and at least three systems are based on amino acid changes in the C-terminal region of LMP1.

Raub-Traub and colleagues 62 proposed a system based on sequences from aa—aa of LMP1 variants compared to the laboratory strain, B Another classification scheme focused on amino acid sequences in short segments surrounding the 33 bp repeat and identified 25 variant forms The various criteria used for each of these classification schemes and the limited number of samples that were analyzed make it challenging to draw comparisons across studies and to establish whether specific variants or sequences of the EBV genome are linked to disease.

With respect to the immune response, there is evidence that genetic diversity can correlate with differences in T cell immunity such that LMP1 variants derived from NPC tumors elicit augmented T regulatory cell function and diminished cytokine production 66 , The full impact of variation in EBV genotypes, with respect to the immune response and to disease, has not likely been appreciated yet, in part, because the analyses have focused on distinct parts of the viral genome.

The whole genome sequence of the B Current technologies including high throughput sequencing platforms are likely to provide a more comprehensive, integrated assessment of EBV genotypes and may lead to an improved understanding of the relationship between EBV diversity, health, and disease.

In conclusion, EBV infection initiates a complex, ongoing interplay between the virus, the host B cell and the immune response. EBV has successfully employed a variety of strategies to promote viral persistence in healthy individuals, however, dysregulation of these pathways, or perturbation of host immunity, may contribute to the development of EBV-associated malignancies.

Future studies to elucidate the mechanisms by which EBV coopts B cell function, the immune response to EBV, and the significance of viral diversity will be important in understanding the outcome of EBV disease.

National Center for Biotechnology Information , U. Immunol Res. Author manuscript; available in PMC Oct Olivia L. Krams , and Olivia M. Author information Copyright and License information Disclaimer. Corresponding author: Olivia M. Martinez, Ph. Copyright notice. The publisher's final edited version of this article is available at Immunol Res. See other articles in PMC that cite the published article. The Viral Life Cycle To fully understand the host-viral interactions following infection with EBV requires a closer look at the complex life cycle of this virus Fig 1.

Open in a separate window. Figure 1. Figure 2. References 1. Shannon-Lowe C, Rowe M. Epstein-Barr virus infection of polarized epithelial cells via the basolateral surface by memory B cell-mediated transfer infection. Virus particles were identified in cultured infected cells, and the results were published in the Lancet journal in by Epstein MA, Anchor B and Barr Y. In the following years serological markers were identified in cell lines and in mimicking some forms of EBV—related infections, was discovered that EBV can directly immortalize B cells after infection.

The human EBV preferentially infects B cells, but occasionally infects other cell types, especially epithelial cells. EBV is usually rapidly cleared by healthy human immune system. Human herpes viruses have a unique capacity to establish a life-long latent infection in the host, whereby the virus can persist within specific host cells, and protects itself from immune recognition by limiting viral gene expression. The establishment of EBV latency is the final phase of the four different EBV infection stages: the growth phase in which the virus activates resting B cells to became proliferating lymphoblasts, the default phases in which EBV provides survival signals to infected lymphoblasts to induce their differentiation into memory B cells and the maintenance of persistently infected memory cells and finally the latency phase that allows persistence of the virus in resting recirculating memory cells in a way that is non-pathogenic and not detectable by the immune system.

The human immune response is generally very successful at controlling infections and minimizing symptoms during primary and persistent infections; however, herpes viruses are responsible for several diseases including conditions associated with primary infections. These clinical problems are more common and severe in immune-compromised individuals such as transplant patients on immunosuppressive medication and human immunodeficiency virus HIV -infected individuals, because of an impaired adaptive immune system.

However, there is a dark side for this seemingly inoffensive virus. The EBV latency phase in combination to environmental and genetic cofactors could links EBV with neoplastic malignancy. We have explored whether cryo-preservation of 0. Ten millilitre ACD blood samples were provided by 10 healthy volunteers.

On completion of the programme the cryotubes were transferred to storage in the vapour phase of a liquid nitrogen refrigerator and maintained until required. On thawing, the contents were transferred by pipette into sterile centrifuge tubes and were diluted gradually with cold hepes buffered RPMI. Tubes were centrifuged at rpm for 10 min at room temperature and the process repeated. Any large particulate debris and clumps were removed by pipette.

A range of experiments were performed to assess cell viability of lymphocytes and the relative proportions of T and B cells in the thawed samples of whole blood. The former was assessed using a colorimetric assay and incubation with the tetrazolium salt WST-1 Roche Applied Science. These were compared to a standard curve prepared using fresh lymphocytes, separated using density centrifugation.

This is a bench-top computer-controlled magnetic cell sorter designed for sterile cell separation from whole blood. The following protocol was finally used. After incubation 5 ml of cold running buffer were added and gently mixed. The tubes were then centrifuged at rpm for 10 min and the supernatant carefully removed leaving 2 mm of liquid above the pellet.

A further 1 ml of running buffer was added and gently mixed. A pre filter was placed onto a fresh 15 ml centrifuge tube and wetted with 1 ml of running buffer.

The samples were then allowed to pass through the wet pre-filters. New separation columns were installed and purged to manufacturer's instructions.

Prior to processing further samples, the uptake and positive 2 ports were again swabbed with ethanol and a rinse programme performed with 50 ml tubes on all ports. A number of experiments were performed to assess the viability and purity of the B cells produced by this procedure. The marmoset lymphoblastoid cell line B Cultures were transferred to sterile 50 ml centrifuge tubes and spun at rpm for 10 min.

Supernatants were removed and put through 0. All samples were routinely checked microscopically for signs of microbiological contamination and cell line transformation. As cultures transformed, they were gradually increased in volume before transfer to culture flasks. A pilot study was performed to determine whether ACD whole unfractionated blood samples collected to the UK Biobank protocol and cryo-preserved in liquid nitrogen can be effectively used as a source for subsequent EBV immortalization of B cell lines.

A further objective was to assess whether this could also be achieved for blood samples that had been maintained for 24 h at room temperature prior to cryo-preservation, thought to reflect the situation with UK Biobank more accurately. Initial blood samples from 40 different volunteers were collected and delivered in four batches of 10 samples. These samples were divided and cryo-preserved at two different time points; on the day of delivery and the following day.

Cryo-preservation was performed as described earlier. EBV transformation was performed on these samples using the method previously described. After thawing and washing whole blood sample, considerable haemolysis and clumping of cells was observed. Clumps appeared to consist largely of agglutinated platelets and dead neutrophils. An estimation of lymphocyte viability compared with a calibration curve of fresh viable cells revealed an average of viable cells per millilitre of thawed blood.

An average time of 5 weeks was taken for cell lines to become established in culture plates. The UK Biobank pilot samples that had been maintained at room temperature prior to cryo-preservation were thawed and assessed for their potential to EBV transform.

The majority Two samples were identified as having bacterial contamination after 3 days and one sample developed a fungal contaminant. The methodology developed and assessed clearly demonstrates that it is possible to cryo-preserve relatively small volumes of whole blood and subsequently generate EBV immortalized lymphoblastoid cell lines. Furthermore, we have been able to establish that such methodology would be appropriate for UK Biobank and they will be able to cryo-preserve whole blood samples confident with the knowledge that cell lines can be subsequently generated.

These findings have since been replicated elsewhere, where similar levels of transformation success have been achieved with small volumes of cryo-preserved whole blood Dr David Lewis, ECACC—personal communication.

The use of antibody conjugated magnetizable micro particles to produce a pure B lymphocyte population from cryo-preserved whole blood for EBV cell line transformation appears to be a procedure with great potential and worthy of further development. The B cell population produced is viable and free of T cells thus obviating the need for other steps to overcome cytotoxicity.

They appear to have no detrimental effects on long-term culture of cells. It is also apparent that long-term sterility of cultures using this method of separation is not a significant concern. The device used for separation has specially been designed for isolation of cells for long-term culture 18 and has also been used for the separation of cells used for in vivo therapeutic procedures. If used correctly in a biological safety cabinet there does not appear an issue regarding contamination of cultures.

It is likely that this methodology will be further developed and refined to produce a more automated and economic method for large-scale EBV cell line transformation from small volumes of cryo-preserved whole blood.