Epstein-Barr virus (EBV)-immortalized B cells, also known as lymphoblastoid cell lines (LCLs), are widely used in research for various applications, particularly in studies of human genetics, immunology, and infectious diseases. EBV immortalization allows B cells to proliferate indefinitely while maintaining many of their functional properties, such as antibody production and the ability to present antigens.
Mechanism of EBV-Mediated Immortalization
Infection with EBV
Epstein-Barr virus, a member of the herpesvirus family, infects B lymphocytes by binding to the CD21 receptor on their surface.
Once inside the cell, EBV establishes a latent infection. During latency, the virus expresses several proteins, including Epstein-Barr Nuclear Antigen 2 (EBNA-2) and Latent Membrane Protein 1 (LMP-1).
Cell Cycle Regulation
EBNA-2: Activates the transcription of viral and cellular genes that are essential for B cell proliferation.
LMP-1: Mimics the signaling of CD40, a crucial molecule in B cell activation. This signaling prevents apoptosis and promotes continuous cell division.
Immortalization
The expression of EBV genes drives the B cell into a state of continuous proliferation, allowing it to divide indefinitely while retaining many functional characteristics of primary B cells.
EBV Immortalization Protocol
Materials
Peripheral blood mononuclear cells (PBMCs) or purified B cells
Epstein-Barr Virus (EBV)-containing supernatant or EBV-producing cell line (e.g., B95-8)
RPMI-1640 medium (or other appropriate growth medium for B cells)
Fetal bovine serum (FBS)
Penicillin-Streptomycin
Phytohemagglutinin (PHA, optional)
Cyclosporine A (optional, to suppress T cell activation)
37°C incubator with 5% CO₂
Step-by-Step Protocol
Isolation of PBMCs or B Cells
Collect blood from the donor and isolate peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation (e.g., using Ficoll-Paque).
Alternatively, purify B cells from the PBMCs using magnetic beads (CD19+ or CD20+ beads) if you require higher purity.
Infection with EBV
Seed the PBMCs or purified B cells in a 24-well or 6-well plate at a concentration of 0.5-1 million cells per well in RPMI-1640 medium supplemented with 10-15% FBS and penicillin-streptomycin.
Add the EBV-containing supernatant (typically from an EBV-producing cell line such as B95-8) to the wells. Use a 1:1 or 1:2 ratio of EBV supernatant to growth medium.
Optional: Add phytohemagglutinin (PHA) to a final concentration of 1-2 µg/mL to stimulate initial B cell activation.
Optional: Add cyclosporine A at a concentration of 0.5-1 µg/mL to inhibit T cell proliferation, which can interfere with B cell immortalization.
Incubation
Incubate the cells at 37°C in a 5% CO₂ incubator. After 24-48 hours, gently replace the medium with fresh RPMI-1640 containing 10-15% FBS, without removing the cells.
Continue changing the medium every 3-4 days, ensuring not to discard any of the adherent or semi-adherent B cells during the medium changes.
Growth and Expansion
After 1-2 weeks, clusters of proliferating B cells (lymphoblastoid cell lines) should start to form. These clusters will grow as LCLs, exhibiting rapid proliferation.
After 2-3 weeks, the cell culture should be expanded into larger flasks or plates as the LCLs continue to grow.
Monitor the culture for contamination or overgrowth of non-B cell populations, although the addition of cyclosporine A can help suppress contaminating T cells.
Cryopreservation
Once the immortalized B cells reach sufficient numbers, they can be cryopreserved in freezing medium (e.g., 10% DMSO in FBS) for long-term storage and future use.
Characterization of EBV-Immortalized B Cells
Phenotypic Confirmation
Confirm the immortalization by testing for the expression of B cell markers (e.g., CD19, CD20) via flow cytometry or immunofluorescence.
The cells should also express EBV latent proteins such as EBNA-2 and LMP-1, which can be detected by Western blotting or PCR.
Proliferation Assays
EBV-immortalized B cells should show continuous proliferation in culture. Proliferation assays, such as cell counting or MTT assays, can be used to measure their growth rate.
Functional Studies
EBV-immortalized B cells retain many functional properties of primary B cells, such as antibody production and antigen presentation. These cells can be used in immune response studies, including cytokine secretion, antigen processing, and antibody secretion assays.
Applications of EBV-Immortalized B Cells
Human Genetic Studies
EBV-immortalized B cells are commonly used in large-scale genetic studies because they provide a renewable source of DNA. Immortalized B cells are particularly valuable for genome-wide association studies (GWAS) and for studying inherited disorders.
Immunology and Infectious Disease Research
LCLs are useful for investigating the biology of B cells, including studies on antibody production, cytokine secretion, and antigen presentation.
Researchers use EBV-immortalized B cells to study how the virus interacts with the immune system and contributes to diseases such as autoimmune disorders and B cell lymphomas.
Vaccine Development
EBV-immortalized B cells are often used to study the development of vaccines targeting viral infections, as they provide insight into how the immune system responds to viral antigens.
Monoclonal Antibody Production
These immortalized cells can be used to generate B cell clones that produce specific monoclonal antibodies for therapeutic or diagnostic purposes.
Functional Assays
LCLs can be used in functional assays to study B cell signaling, antigen presentation, and their interaction with T cells or other immune cells. They are valuable in studies related to transplantation immunology or autoimmunity.
Cancer Research
Because EBV immortalization mimics certain steps in B cell transformation leading to lymphoma, these cells serve as a model for studying B cell malignancies, such as Burkitt’s lymphoma and Hodgkin’s lymphoma.
Advantages and Limitations of EBV-Immortalized B Cells
Advantages
Unlimited Proliferation: LCLs provide a continuous, renewable source of B cells.
Maintenance of B Cell Functions: These cells retain their ability to produce antibodies and present antigens, making them useful for functional immunology studies.
Long-Term Genetic Stability: EBV-immortalized B cells are a stable source of genetic material for research, providing consistent results over time.
Limitations
Viral Gene Expression: The presence of EBV genes (e.g., EBNA-2, LMP-1) may interfere with some cellular processes, potentially limiting the use of these cells in certain types of experiments.
Risk of Transformation: Although rare, EBV-immortalized B cells may undergo additional mutations over time, leading to transformation or cancer-like growth.
Variable Yield: The efficiency of EBV-mediated immortalization can vary, and some B cell populations may not immortalize as efficiently as others.
EBV-immortalized B cells are a powerful tool in immunology, genetics, and infectious disease research, providing researchers with a stable and renewable source of functional human B cells for long-term studies.