The overall goal of this experimental procedure is to generate De Novo T Cell Receptors or TCRS, recognizing a specific antigen of interest. This method can help isolate optimal T Cell Receptors in a field of gene engineered immunotherapy. The main advantage of this technique is only a single alpha or beta hemi-chain needs to be isolated to generate new entrance specific T Cell Receptors.
After eluting the TCR, PCR product clone the gene by combining each linear fragment with a commercially available assembly master mixery agent and incubating the mixture at 50 degrees Celsius for one hour. At the end of the incubation, transform chemically confident E Coli with the assembled plasmin following the manufacturers protocol and seed the transformed bacteria onto agar plates for 18 hour incubation at 37 degrees Celsius. The next day, transfer a single bacterial colony into 50 milliliters of lysogeny broth medium for 16 hours in a shaking incubator at 37 degrees celsius and 200 revolutions per minute.
Then, using a commercially available Midiprep kit, according to the manufacturer's protocol, purify the plasmin and dilute the DNA to a concentration of around one microgram per microliter for the 293GPG cell transfection. One day after the transfection, replace the transfection medium from the 293GPG cell culture with fresh DMEM medium. Two days after changing the medium, collect the culture medium in a syringe, followed by it's passage through a 0.45 micron filter to collect the virus.
Next seed one times ten to the fifth PG-13 cells in a T-25 flask overnight. The next day aspirate the culture medium and add 1.5 milliliters of the 293GPG derived virus, 1.5 milliliters of DMEM medium, and eight micrograms per milliliter of Polygrain. Change the medium once a day for four days to establish a stable PG-13 packaging cell line for producing a retro virus, encoding a TCR hemi-chain of interest.
24 hours after the last infection, replace the culture medium with fresh DMEM medium. To harvest the virus from the transduced cell line, culture two times ten to the sixth PG-13 packaging cells in a T-75 flask with 10 milliliters of fresh DMEM medium. And collect the virus three days after seeding, as just demonstrated.
For T cell activation, culture two times ten to the seventh PBMCs per well in a six well plate in five milliliters of RPMI medium supplemented with Recombinant Human IL-2 and anti-CD3 monoclonal antibody. Three days post stimulation, harvest the activated T Cells for centrifugation and resuspend 0.5 to one times ten to the six cells in one milliliter of PG-13 virus and one milliliter of RPMI medium. Supplement it with Recombinant Human IL-2 per well, proceeding in a 24 well plate.
When all of the cells have been deposited, centrifuge the plate and transfer the plate into a cell cultured incubator. After 24 hours harvest the cells for centrifugation. Then, resuspend the T Cells pellet in one milliliter of PG-13 virus and one milliliter of RPMI by medium supplemented with IL-2 and centrifuge the plate as just demonstrated.
24 hours after the last infection, harvest the cells for centrifugation and resuspend the T Cells in RPMI medium supplemented with IL-2 alone. Return the cells to the incubator for two to three more days of culture. Then stain the cells with a HLA multimer for 30 minutes at four degrees Celsius.
Followed by anti-human CD3 and clone receptor monoclonal antibodies at four degrees Celsius for 15 minutes. Sort the multimer positive cells by flow cytometry using the appropriate controls. Seed five times ten to the fourth Jurkat cells per well in a 24 well plate in one milliliter of RPMI medium and one milliliter of 293GPG virus per Jurkat cell transfection with the centric TCR hemi-chain of interest.
Centrifuge the plate, and place the plate in the cell culture incubator. After 24 hours collect the cells for centrifugation and resuspend the hemi-chain transduced Jurkat 76 cell pellet in fresh RPMI medium for further culture. To fully reconstitute the T Cell Receptor, produce 293GPG virus encoding a TCR counter-chain, cloned from the sorted multimer positive cells.
Enter the centric hemi-chain expressing Jurkat 76 cells as just demonstrated. Three to five days post CD3 purification, stain the clonal tipic TCR expressing transfectants with the appropriate HLA multimer and cell surface staining antibodies of interest. And analyze the cells by flow cytometry to verify the antigen specificity.
Without prior knowledge of which hemi-chain is chain centric, The TCR alpha and TCR beta chains should be separately clone and transduced to peripheral blood T-cells. As for this HLA A-24 WT1 reactivate TAK1 T Cell Receptor. TAK1 Beta transsection yields a noticeably higher frequency of antigen specific T Cell.
While the transduction of a non centric hemi-chain does not yield to know well positive T Cells as observed for the TAK1 alpha chain. Conversely a single centric hemi-chain can be transduced if it is known to be dominant. Further, T Cells specific for CD1d increase in frequency upon transduction of the centric TCR V alpha 24 hemi-chain.
Together these data demonstrate that the introduction of a centric TCR alpha or beta hemi-chain into peripheral blood T cells can generate De Novo TCRs with the antigen specificity of interest. Moreover De Novo TCR alpha counter-chains, paired with TAK1 beta, stained with either a lower or higher intensity by the WT1 antigen complex than the parental TAK1 alpha beta pairing. Similarly, SAK-35 alpha paired with unique counter-chains, recognizes HLA A2 MART1 with a moderate to high avidity.
While attempting this procedure to obtain the maximal transection efficiency, it is important to maintain primary T Cells under the optimal culture conditions. Following this procedure, novel HRE class 2 restricted entrance specific T Cell Receptors can be cloned by exploiting the chain centricity. After watching this video you should have a good understanding of how to isolate and reconstitute T Cell Receptor genes by molecular cloning and retroviral transduction.
Don't forget that working with replication defective retrovirus can potentially be hazardous and that precaution such as wearing gloves and protective garments should always be taken while performing this procedure.
