The aim of this procedure is to express functional antigen specific T-cell receptors on mouse T cells. First, transfect a retroviral producing cell line with plasmid containing the TCR gene of interest to package TCR expressing retrovirus. Next, isolate and purify T cells from mouse spleens infect these purified T cells with the retrovirus produced in the first step.
Finally, expand the transduced T cells to express and obtain appreciable levels of receptor. Ultimately, results can be obtained that show the expression of functional antigen specific TCRs on mouse T cells through flow cytometry. Hi, my name is Michelle Crow Guard.
I'm at the NYU Cancer Institute at NYU Medical School. Today we're gonna show you how to transduce primary T-cell, the people who will be showing you how to do this will be shiong, a postdoc in the lab, Kaleena Melek, a graduate student in the lab, and Arian Perez Garcia, a postdoc in the lab. The main advantage of this technique over existing method like transgenic mice is that it is much less time consuming.
This method can help answer key immunological questions such as which TCR can give better response against antigens. This technique can be applied for adoptive T-cell transfer therapy, which has been shown to be promising for the treatment of cancer patients and consists in the in vitro modification of human T-cells To ensure equal expression of the T-cell receptor alpha and beta chains. Subclone the gene of interest into the same retroviral vector under control of the same promoter.
Prepare high quality plasma DNA using the Kyogen Maxi prep kit and make a stock of one microgram per microliter. Remove media from the plate with platinum E retroviral packaging cells and wash the plate once with one XPBS. Dislodge the cells by adding trypsin EDTA and incubate at 37 degrees Celsius for a few minutes.
Neutralize the dislodged cells with DMEM media and transfer to a falcon tube. Next centrifuge the cells at 1000 times G for five minutes. Aspirate the supinate and resuspend the cell pellet in DMEM media.
Determine the cell number and dilute the cells at 0.6 times 10 to the six per milliliter plate. 10 milliliters of cell suspension in a 10 millimeter polylysine coated tissue culture plate and grow overnight in a cell incubator at 37 degrees Celsius in 5%carbon dioxide. The next morning, examine the cells under a light microscope to verify approximately 80%co fluency.
Gently remove the media from the plate. Wash the cells once with one XPBS and add 10 milliliters of prewarm fresh DMEM media without penicillin or streptomycin. For the lip mean transfection complex, prepare two mixes and opti ME M1 of DNA and the other of lipectomy reagent equilibriate separately for five minutes at room temperature.
Then mix together gently and incubate for 20 minutes. At room temperature, slowly drip the mixture into the platinum E cells. Gently rock the plate back and forth to distribute the transfection mixture evenly.
Then incubate cells at 37 degrees Celsius and 5%carbon dioxide in a cell incubator. After six to eight hours, replace medium with 10 milliliters of fresh DMEM medium for viral production. Harvest a mouse spleen and transfer to RPMI medium.
Place the spleen in a cell strainer. Mash the spleen with a syringe plunger into a five centimeter tissue culture plate. Rinse cells off the cell strainer with sterile PBS in order to harvest the cells centrifuge at 1000 times G for five minutes.
Discard the sup natant. Continue by Resus suspending the cell pellet in a CK lysing buffer. Adding two milliliters per spleen incubate for two minutes.
At room temperature. Add RPMI medium up to 20 milliliters and centrifuge at 1000 times G for five minutes. Finally, resuspend the cell pellet in sterile PBS.
Determine the cell number and centrifuge at 1000 times G for five minutes at four degrees Celsius. In order to activate the mouse T cells before viral transduction coat plates with activating antibodies, namely anti CD three epsilon and anti CD 28. Prepare an antibody mixture of one microgram per milliliter of anti CD three epsilon and two micrograms per milliliter of anti CD 28 in sterile PBS dispense 250 microliters of the antibody mixture into each well of the 24 well tissue culture plate and incubate for two hours at 37 degrees Celsius.
Magnetically label the spleen cells according to the product manual. Place an LS column in the magnetic field. Apply labeled cell suspension onto the column.
Collect flow through of enriched mouse CD eight plus T cells. Wash the column three times with three milliliters of buffer and combined with the previous blow through. Stain the cells with anti CD three epsilon and anti CD eight alpha antibodies and analyzed flow cytometry.
Next, centrifuge the cells at 1000 times G for five minutes and resuspend the cell pelle to 1 million cells per milliliter in RPMI medium with recombinant human IL two. Remove the antibody solution from the activation plate. Rinse each well with sterile PBS.
Add one milliliter of the cell suspension to each well and place the plate at 37 degrees Celsius. 5%carbon dioxide in a cell incubator. After two days of virus production, the medium in the platinum ESE cell plate should become yellow.
Transfer the viral supinate to a 15 milliliter conical tube to remove cell debris. Centrifuge the virus S supernatant at 1000 times G for five minutes. Carefully transfer the virus supinate to a new tube.
Leave some liquid at the bottom of the tube and do not disturb the cells debris. Collect activated T cells from the plate into a 50 millimeter conical tube. Determine the cell number.
Save some cells in a separate tube to be used as a negative control centrifuge at 1000 times G for five minutes and discard the supine natant. Then resuspend the cell pellet in virus supine. Natant at 10 to the six cells per milliliter with 20 nanograms per milliliter of recombinant human, interleukin two and 10 micrograms per milliliter of protein sulfate.
Add one milliliter of cell suspension to each well of the 24 well plate for the control tube.Resus. Suspend the cells in RPMI medium at the same cell density with the same amount of recombinant human, interleukin two and protein sulfate. Add the cells to the same plate.
Wrap the plate with plastic film centrifuge for 90 minutes at 2000 times G at 32 degrees Celsius with no break. After the centrifugation, remove the plastic film. Add one milliliter of fresh RPMI medium with 20 nanograms per milliliter of recombinant human interleukin two to each, well put the plate back into the incubator.
It is important to examine the transduced T cells daily. Split the cells at one to three ratios when necessary. Do not let the cells overgrow or let the medium turn yellow at day six or day seven.
Stain the cells with antibody and MHC tetramer specific for the TCR to evaluate the expression of TCR on T-cell surface. Use the UNSD T cells as a control. These transduced T cells are now ready for further downstream applications.
Prior to viral transduction. It is advantageous to obtain high purity T-cell subsets using commercial magnetic beads to evaluate the expression level of TCRs on T cells. Antibody specific for TCR alpha or beta chains can be used.
Typically, 30%to 80%of the cells can express transduced TCR depending on the TCR genes and virus titers. MHC Tetramer specific for the TCR can also be used to further verify functional expression of TCRs. Once mastered, this technique can be done in one week if performed properly.
While attempting this procedure, it is important to remember to keep the cells in healthy condition Following this procedure. Other methods like cytotoxicity acid or cytokine release assays can be performed in order to answer additional questions like the specificity and the sensitivity of transduce. The TCRs.
After watching this video, you should be an expert in transducing your favorite TCR into primary T cells. So see you in the lab.
