The overall goal of this procedure is to evaluate the in vitro suppressive function of regulatory T cells, isolated from chronic virus infected mice. This method can help answer questions about the phenotype and in vitro suppressive function of activated regulatory T cells during a chronic virus infection. This technique can evaluate the direct effect of regulatory T cells on the individual with a positive response to T cells in vitro.
This is a great advantage of this technique. Begin by transferring five times ten to the fifth total splenic lymphocytes in 50 microliters of facs buffer into each well of a new U bottom 96 well plate, followed by 150 microliters of facs buffer. Centrifuge the plate and discard the supernatant.
Then agitate the cell pellets and wash the cells two more times in 200 microliters of facs buffer per well. After the second wash, resuspend the pellets in 50 microliters of antibody cocktail per well, for 20 minutes in the dark at four degrees Celsius. Then wash the cells two times in facs buffer, and decant the supernatant.
Next, fix the cells for 20 minutes in the dark at four degrees Celsius with 100 microliters of fixation buffer, followed by two washes with permeabilization wash buffer. After the second wash, resuspend the cells in 50 microliters of anti-FOXP3 antibody. To prepare the anti CD3 CD28 coated beads, transfer the appropriate volume of magnetic beads into a 15 milliliter tube, and add an equal volume of PPS.
Gently mix the solution by pipetting, and centrifuge the magnetic beads. At the end of the wash, discard the supernatant, and dilute the beads in 50 microliters of complete medium per well. Next, seed 50 microliters of one times ten to the fifth CD4 positive CD25 positive regulatory T cells per well in a 96 well U bottom plate, followed by 50 microliters of one times ten to the fifth CD8 positive responder T cells per well.
When all of the cells have been plated, add 50 microliters of the diluted anti CD3 CD28 coated beads to each well, and add up to 50 microliters of medium to bring the total volume of each well up to 200 microliters. Then cover the plate with foil and place it at 37 degrees Celsius in a carbon dioxide incubator for 72 hours. To analyze the CD8 positive T cell cytokine production, after three days of culture, transfer the supernatants into another 96 well plate and analyze the supernatants by ELISA, according to the manufacturer's instructions.
To analyze the proliferation of the CD8 positive T cells, wash the plate of cells with facs buffer three times. Then resuspend the pellets in 50 microliters of an appropriate antibody cocktail against proliferated CD8 positive T cells for 20 minutes in the dark at four degrees Celsius. At the end of the incubation, wash the cells two times in facs buffer and fix them for 20 minutes in the dark at four degrees Celsius with 100 microliters of fixation buffer.
Finally, wash the cells two more times in facs buffer and resuspend the pellets in 200 microliters of facs buffer. Then measure the percentage of phylate dilabelled CD8 positive T cell proliferation by flow cytometry. In splenic lymphocytes obtained from naive and infected mice, PD-1 is specifically up regulated in both conventional FOXP3 negative CD4 positive and FOXP3 negative CD8 positive cells of LCMV clone 13 infected mice.
The frequency of FOXP3 positive CD4 positive regulatory T cells is two times higher in LCMV clone 13 infected mice than in naive mice, with most of the FOXP3 positive CD4 positive cells also displaying an activated PD-1 high phenotype. CD8 positive responder T cells can be separated successfully without significant contamination by other immune cells. After regulatory T cell isolation, a dominant population of CD25 positive CD4 positive T cells with more than 80 percent purity can be obtained.
The stimulation of isolated regulatory and responder T cells by anti CD3 CD28 coated beads results in the inhibition of CD8 positive responder T cells in a dose dependent manner. Indeed, when CD8 positive responder T cells are co-cultured with chronic regulatory T cells at a one to one ratio, the CD8 positive responder T cell proliferation is significantly inhibited. Interferon gamma production by CD8 positive responder cells is also significantly inhibited when CD8 positive responder T cells are co-cultured with activated regulatory T cells from chronically infected mice, compared to co-culture with resting regulatory T cells from naive mice.
We have found this technique can be completed in 80 hours if it is performed properly. While you are attempting this procedure, it is important to remember to use freshly isolated regulatory T cells immediately after their isolation. After this development, this technique paved the way for researchers in the field of medicine to explore the function of regulatory T cells in a chronic inflammatory disease.
After watching this video, you should have a good understanding of how to set up an in vitro regulatory T cell suppression procedure, using a regulatory and responder T cell co-culture system.
