Immortalizing T cells is a complex process due to their highly specialized and differentiated nature. Immortalization is used to generate long-term T cell lines that retain their antigen-specific properties, allowing for continuous studies on T cell biology, immunotherapy, and drug development. Several methods have been developed for T cell immortalization, including the use of viral oncogenes, telomerase overexpression, and hybridoma technology (for mouse T cells). Below are the most common methods and protocols used to immortalize T cells.
Methods of T Cell Immortalization
hTERT (Human Telomerase Reverse Transcriptase) Overexpression
Mechanism
Similar to immortalizing other primary cells, overexpression of hTERT in T cells reactivates telomerase, preventing telomere shortening and allowing T cells to bypass senescence.
Advantages
Preserves most of the T cell’s normal functions and is less likely to induce genetic instability compared to viral oncogenes.
Disadvantages
T cells are difficult to transduce, and hTERT expression may not fully immortalize some T cell subsets.
Applications
Widely used in T cell research for understanding long-term immune responses, aging, and immunotherapy.
SV40 Large T Antigen
Mechanism
SV40 Large T antigen inactivates tumor suppressor proteins p53 and Rb, pushing T cells into continuous proliferation.
Advantages
Efficient and allows for immortalization of T cells from various species, including humans and mice.
Disadvantages
Cells immortalized with SV40 Large T antigen can accumulate genetic mutations over time, which may alter their functional properties.
Applications
Used for long-term studies on T cell activation, signaling, and differentiation.
Human Papillomavirus (HPV) E6/E7 Oncoproteins
Mechanism
The E6 and E7 oncoproteins of HPV inactivate p53 and Rb, enabling T cells to evade senescence and continue proliferating.
Advantages
Effective in immortalizing primary T cells, especially when combined with hTERT.
Disadvantages
T cell functions may be altered over time due to the expression of viral oncogenes, and there is a risk of transformation.
Applications
Immortalized T cells can be used to study T cell receptor (TCR) signaling, immune responses, and cancer immunology.
EBV-Immortalization of T Cells
Mechanism
Epstein-Barr Virus (EBV) is typically used to immortalize B cells, but it can also be used for T cells, especially T regulatory cells (Tregs). EBV infection leads to continuous proliferation of lymphocytes by expressing LMP1 and EBNA-2.
Advantages
Effective for long-term cultures of specific T cell subsets.
Disadvantages
Limited use in certain T cell populations and requires specific conditions to maintain functionality.
Retroviral or Lentiviral Expression of Oncogenes (e.g., c-myc, Bcl-xL)
Mechanism
T cells can be immortalized by retroviral or lentiviral expression of oncogenes like c-myc or Bcl-xL, which promote cell survival and proliferation. c-myc drives cell cycle progression, while Bcl-xL inhibits apoptosis.
Advantages
Effective at immortalizing T cells while maintaining some level of functionality.
Disadvantages
Oncogene expression can alter T cell function over time and may lead to transformation.
Applications
Used for generating long-lived T cell lines for research into T cell receptor signaling, antigen specificity, and immune modulation.
Hybridoma Technology (for Mouse T Cells)
Mechanism
For mouse T cells, a technique similar to B cell hybridoma generation can be used. T cells are fused with an immortalized T cell line (e.g., BW5147 cells) to create a hybrid that retains the antigen specificity of the T cell while being able to proliferate indefinitely.
Advantages
The resulting hybridomas maintain T cell specificity and can be used to study antigen-specific responses.
Disadvantages
The technique is limited to mouse T cells and may not fully retain all functional characteristics of the original T cells.
Applications
Used for generating mouse T cell clones with specific antigen recognition.
Protocol for T Cell Immortalization Using hTERT and SV40 Large T Antigen
This protocol focuses on a combination of hTERT overexpression and SV40 Large T antigen, which is commonly used to immortalize human T cells while maintaining their functional properties.
Materials
Primary T cells (isolated from peripheral blood mononuclear cells (PBMCs) or lymph nodes)
Lentiviral vector containing hTERT or SV40 Large T antigen
Lentiviral packaging plasmids (if generating your own virus)
Transfection reagent (e.g., Lipofectamine 2000)
Polybrene (for enhancing transduction efficiency)
Selection antibiotic (e.g., puromycin)
RPMI-1640 medium (or other suitable T cell medium)
T cell growth factors (e.g., IL-2, IL-7, IL-15)
Incubator set to 37°C with 5% CO₂
Step-by-Step Protocol
Day 1: Virus Production (if generating lentiviral particles)
- HEK 293T Cell Transfection:
Seed HEK 293T cells at 70-80% confluency in a 10 cm dish.
Transfect the HEK 293T cells with the hTERT or SV40 Large T antigen plasmid along with packaging plasmids using a transfection reagent (e.g., Lipofectamine 2000 or PEI).
Incubate for 48-72 hours.
- Collect Lentiviral Supernatant:
Harvest the virus-containing supernatant and filter it through a 0.45 µm filter to remove cell debris.
Concentrate the virus using ultracentrifugation or a commercial viral concentration kit (optional).
Day 3: Transduction of Primary T Cells
- Prepare Primary T Cells:
Isolate T cells from PBMCs using density gradient centrifugation or magnetic bead separation (e.g., CD3+ T cell isolation kit).
Seed the T cells in a 6-well plate in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and IL-2 (50-100 IU/mL).
- Lentiviral Transduction:
Add polybrene (5-10 µg/mL) to the medium to enhance transduction efficiency.
Add the concentrated lentiviral supernatant containing hTERT or SV40 Large T antigen to the T cells.
Incubate for 24 hours at 37°C.
- Medium Replacement:
After 24 hours, replace the lentiviral supernatant with fresh medium containing IL-2 (50-100 IU/mL) and continue to culture the cells.
Day 6: Selection of Transduced Cells (Optional)
- Antibiotic Selection:
If the lentiviral vector contains an antibiotic resistance gene (e.g., puromycin), begin selection by adding the appropriate antibiotic to the culture medium (e.g., puromycin at 1-2 µg/mL).
Select for several days until non-transduced cells are eliminated.
- Expand Surviving Cells:
Expand the surviving immortalized T cells in medium supplemented with IL-2, IL-7, or IL-15 to promote T cell growth.
Validation of Immortalization
- Check for hTERT or SV40 Large T Antigen Expression:
Validate the expression of hTERT or SV40 Large T antigen using Western blot, qPCR, or immunofluorescence.
Check for telomerase activity using the TRAP assay (Telomeric Repeat Amplification Protocol) if using hTERT.
- Proliferation Assay:
Monitor the growth rate of the immortalized T cells to confirm extended proliferative capacity compared to non-immortalized controls.
- Functional Assays:
Perform functional assays such as TCR stimulation, cytokine secretion (e.g., IFN-γ, IL-2), and cytotoxicity assays to confirm that the T cells retain their immune functions.
- Senescence Assay:
Perform β-galactosidase staining to ensure that the immortalized T cells are not undergoing senescence.
Applications of Immortalized T Cells
- Immunotherapy:
Immortalized T cells are used to study the role of T cells in cancer immunotherapy, including CAR-T cell therapies.
- T Cell Receptor (TCR) Research:
Immortalized T cells can be used to explore TCR signaling, antigen specificity, and T cell activation in response to pathogens or tumors.
- Autoimmune Disease Studies:
Immortalized T cell lines are used to study the role of autoreactive T cells in diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis.
- Infectious Disease Research:
These cells are employed to investigate T cell responses to viral, bacterial, and parasitic infections.
Conclusion
Immortalizing T cells is a powerful tool for generating stable and long-lasting T cell lines. Methods such as hTERT overexpression, SV40 Large T antigen, or oncogene-driven immortalization allow researchers to conduct long-term studies on T cell function, signaling, and immunotherapy. However, it is important to validate that the immortalized cells retain their functional properties and do not undergo unwanted transformation.