The overall goal of this protocol is to isolate primary murine helper T cells, and transduce them with retroviral expression constructs for a gene of interest. Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here we describe a genetic approach using retroviral transduction, which is economic, time efficient, and highly informative in identifying regulatory pathways.
We have used this technique to analyze the function of microRNAs, which are important regulators of gene expression. Using retroviral transduction, we have been able to test the function of individual microRNAs and helper T Cell differentiation. My collaborator, Oliver Garden, will be demonstrating primary T cell isolation, and I will be demonstrating retroviral transduction.
24 hours prior to transfection, split a confluent plate of cells at a ratio of 1:10 in a 10 centimeter plate, so that the cells are about 50%confluent the next day. Approximately one hour prior to transfection, remove the medium from the cells, and carefully add 9 millileters of fresh HEK293 T medium, to prevent the cells detaching from the plate. In a six-milliliter round bottom, or a 15-milliliter conical tube, add five micrograms of retroviral DNA, five micrograms of helper virus DNA, and water to a volume of 420 microliters.
Then, add 80 microliters of 2.5 molar calcium chloride to bring the final volume to 500 microliters. After that, slowly add 500 microliters of 2x HBS drop by drop to the DNA mixture, while gently vortexing, so that the solution is continuously mixed, and the calcium phosphate precipitates in even sized crystals. Subsequently, transfer the calcium phosphate DNA mixture to a tissue culture hood.
Slowly add the mixture to the cells, while constantly and gently swirling the plate, before placing the cells back in the incubator. To collect the virus the next day, remove the medium and feed the cells with 10 milliliters of fresh HEK293 T medium, being careful not to dislodge the cells from the plate. In the evening, feed the cells with 3.5 milliliters of fresh HEK293 T medium, and incubate the cells at four degrees Celsius for 12 hours.
After approximately 12 hours, collect the medium and feed the cells with 3.5 milliliters of fresh HEK293 T medium again. Repeat the medium collection two more times, at roughly 12 hour intervals, which results in about 10 milliliters of virus stock being collected. In this procedure, add spleen and lymph nodes to the cell strainer, and macerate using the end of a five milliliter syringe plunger.
After that, rinse them with one to two milliliters of R10 medium. Then transfer the cells into a 15-milliliter conical tube and bring the volume up to 14 milliliters with R10. Centrifuge the cells at 600 times G for five minutes at four degrees Celsius.
Afterward, discard the supernatant by pouring it off with one clean movement, to prevent disturbing the cell palate. Next, add two milliliters of cold red cell lysis buffer to the cells. Mix, then place the cells on ice for 3.5 minutes with gentle mixing throughout.
Then, add 12 milliliters of R10 medium and immediately centrifuge at 600 times G for five minutes at four degrees Celsius. Perform the washing steps two more times with R10 medium to remove any residual red cell lysis buffer from the cells, and avoid lymphocyte death. In this step, aliquot eight to ten, times ten to the 7th cells per conical tube, and centrifuge them at 600 times G for five minutes at four degrees celsius.
Pour off the supernatant with one clean movement, to prevent disturbing the cell palate. Then, re-suspend the cells and 100 microliters of heat inactivated FBS. Next, add 100 microliters of antibody mix to the cells, and incubate for 30 minutes at four degrees celsius, with gentle mixing on a roller.
After that, re-suspend the beads in the vial, by vortexing them for more than 30 seconds. Then transfer the beads to a 15-milliliter conical tube, and add 2 milliliters of R10 medium per milliliter of beads. Place the tube in the magnet for one minute.
Then discard the supernatant, and resuspend the beads in four milliliters of R10 medium. Wash the cells at the end of the antibody incubation, by adding 10 milliliters of R10 medium per tube, and centrifuge at 600 times G for five minutes at four degrees celsius. Pour off the supernatant with one clean movement to prevent disturbing the cell palate, and then re-suspend the cells in 1 milliliter of R10 medium.
Subsequently, add 2 milliliters of the washed beads to the tube of antibody treated cells and incubate for 30 minutes at four degrees celsius, with gentle mixing on a roller. Then, re-suspend the cell bead mixture, by gently pi petting five times before placing the tube in the bead magnet for two minutes. Transfer the supernatant containing the negatively selected cells to a new tube, and repeat the negative selection one more time.
In this procedure, centrifuge CD4 positive T cells at 600 times G for five minutes at four degrees celsius and re-suspend in 150 to 200 microliters of R10 medium per ten to the eighth cells. Next, add two microliters of stock biotinulated CD25 monocoil antibodies to the cells and incubate for 30 minutes at four degrees celsius with gentle mixing on a roller. Afterward, wash the cells by adding 10 milliliters of cell separation column buffer, and centrifuge at 600 times G for five minutes at four degrees celsius.
Then, remove as much supernatant as possible, and re-suspend the cells to a final volume of approximately 90 microliters per 10 to the seventh cells. Subsequently, add 20 microliters of streptavidin beads per 10 to the seventh cells, and mix with gentle flicks. Then, incubate them for 15 minutes at four degrees celsius.
While incubating the cells, prepare the MS columns. At the end of the incubation, wash the cells by adding 10 milliliters of cell separation column buffer, and centrifuging at 600 times G for five minutes at four degrees celsius. Re-suspend the cells in 500 microliters of cell separation column buffer, per 10 to the eighth cells.
Afterward, apply 500 microliters of cell suspension to the column and collect the flow through. Pass it over the column one more time, and collect the flow through once again. Then, rinse the column three times with 500 microliters of cell separation column buffer, and add each flow through to the collected CD25 negative cells.
Perform the wash, streptavidin bead binding and cell separation column preparation as previously mentioned, but use the LS column instead. This time, discard the flow through and flush the cells from the column with the supplied plunger. While isolating the naive T cells, coat a 24 wild tissue culture plate with anti CD3 and anti CD28 antibodies diluted in DPBS, and incubate for two hours at 37 degrees celsius.
Immediately prior to culturing the cells, wash the plate with 250 microliters of DPBS per well, to remove unbound antibody. Then, remove DPBS and add 1 milliliter of naive CD4 positive T cells in R10 medium overnight. The following day, centrifuge the cells at 900 times G for 10 minutes at 30 degrees celsius.
Then collect the medium, and add 1 milliliter of virus culture supernatant, prepared from the virus production protocol. Subsequently, add 1 microliter of eight milligrams per milliliter polybrene, and 10 microliters of one molar hepes, to aid virus uptake before centrifuging the cells at 900 times G for 90 minutes at 30 degrees celsius. To differentiate the cells, carefully remove the virus culture supernatant and add 1 milliliter of the medium collected previously, as it contains IL2 and other T cell growth factors.
Then add the reagents indicated in Table 2 of the accompanying manuscript, and culture the cells for three to four days to differentiate cells into specific subsets. This figure shows the typical purity of pre and post selected populations at each stage of the naive helper T cell isolation protocol. The expression profiles of CD4, CD8A, MHC II, CD25, CD62L and CD44 illustrate the loss of MHC Class 2 cells, plus cytotoxic, regulatory, and memory T cells.
This results in a population that is greater than 95%percent CD4 positive T cells, with approximately 85%to 90%being naive, and the remaining 10%to 15%being effector cells. In this figure, GFP expression is showN in HEC293 T cells, that were either un-transfected or transfected, And analyzed after the collection of viral culture supernatants. GFP analysis was done on live cells from the gate on the FSE and SSE plot.
Typical transfection efficiencies range between 30%TO 90%Here, GFP expression is shown in retro viral transduced helper T cells after differentiation in Th0, Th1, Th2, Th9, Th17, and T reg polarization conditions for three days. Transduction efficiencies vary between 10%to 75%depending on the construct and the polarization conditions. Here are the representative cytokine profiles on helper T cell differentiation in different polarization conditions.
Once mastered, these techniques can be a powerful way to analyze gene function and helper T cell development and function. Just remember that informative results depend on the quality of the cells and reagents. Therefore it is important to optimize the transduction protocols and the isolation and differentiation of helper T cells.
