Nonstimulatory peptide MHC complexes do not activate T cells but can enhance T cell responses to agonist peptide MHC. This protocol describes an experimental system to investigate co-agonism during human CD8 T cell activation. We express MHC molecules as single-chained complexes with covalently-linked heavy chain, beta-2-microglobulin, and the peptide.
This allows introducing mutations into MHC molecules presenting predetermined peptides. This protocol uses human CTN clone, a specific for an epitope of hepatitis B virus. Understanding the mechanism of co-agonism during T cell responses against hepatitis can contribute to development of immunotropics against this viral infection.
The presented methods can be used in research and other fundamental aspects of T cell activation in human T cell systems with known peptide MHC specificity. This protocol uses single-chain human MHC class I molecules, consisting of signal sequence, peptide of interest, linker, human beta-2-microglobulin linker, and MHC heavy chain. E1A3 peptide from hepatitis B is used as agonist peptide.
Gag from HIV is used as nonstimulatory peptide. The single-chain human MHC molecules are expressed in a hamster cell line containing tetracycline repressor. Agonist single-chain MHC is expressed using tetracycline-inducible plasmid, resulting in very low expression levels in the absence of tetracycline, and high expression levels in the presence of tetracycline.
CHO cells expressing low levels of agonist peptide MHC are transfected with constitutively expressed nonstimulatory peptide MHC. Use of this experimental system allows comparing T cell responses to agonist peptide MHC in the presence or absence of nonstimulatory peptide MHC. To begin this procedure, cultivate CHO cells in a T-75 flask, as outlined in the text protocol.
On the day before the T cell activation experiment, wash the cells in the flask with PBS. Add a solution containing 05%trypsin and 2%EDTA in PBS, and incubate for five to ten minutes at room temperature. Using a light microscope, observe the cells to make sure they have detached.
Then add complete F12 to the flask to stop the trypsinization. Transfer the cell suspension to a 15 milliliter tube. Centrifuge at 400G for five minutes, and remove the supernatant by either pipetting or decanting.
Re-suspend the cell pellet in five milliliters of complete F12. Use a hemocytometer to count the cells, and adjust the cell concentration to 200, 000 cells per milliliter in CF12. Add 50 to 60 nanograms per milliliter of tetracycline to the agonist-only samples to induce high amounts of agonist pMHC class I expression as a positive control.
After this, mix the CHO cells either by vortexing or pipetting. Add 100 microliters of the cell suspension to each well of a U-bottom 96-well plate. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.
The day before the experiment, centrifuge CTLS cells at 400 times G and at four degrees Celsius for five minutes. Use a hemocytometer to count the cells, and re-suspend them at a density of one million cells per milliliter in complete human T cell media without cytokines or PHA. Incubate the CTLs overnight at 37 degrees Celsius with 5%carbon dioxide.
The next day, centrifuge the T cells at 400 times G and at four degrees Celsius for five minutes. Discard the supernatant, and re-suspend the cells in 2 milliliters of complete serum-free media. Use a hemocytometer to count the T cells, and adjust their density to one million live cells per milliliter in complete human T cell media.
Add anti-human CD107a antibody at a one to 100 dilution, and Brefeldin A at a one to 1000 dilution. Next, retrieve the 96-well plate containing the CHO cells. Using a glass Pasteur pipette, aspirate the media from the CHO cells.
Add 200 microliters of the T cell suspension to each well. Co-culture the T cells with CHO cells at 37 degrees Celsius with 5%carbon dioxide for three to four hours. At the end of the co-culture, centrifuge the 96-well plate at 400 times G and four degrees Celsius for five minutes.
Remove the supernatant by aspirating or flicking. Then add 2.5 microliters of CD3 antibodies and 2.5 microliters of CD8 antibodies in 50 microliters of 5%BSA in PBS to each well, for cell surface antigen staining, and mix by pipetting. Incubate the samples in the dark, and on ice, for 30 minutes.
After this, add 150 microliters of wash buffer to each well to wash the samples. Centrifuge the samples at 400 times G and at four degrees Celsius for five minutes. Remove the supernatant by aspirating or flicking.
Set out a fixation/permeabilization kit, and add 100 microliters of fixation/permeabilization solution to each well, and pipette to mix. Incubate on ice for 20 minutes. Then, centrifuge the plate at 400 times G at four degrees Celsius for five minutes.
Discard the supernatant and add 200 microliters of 1X perm/wash buffer to each well. Repeat this process from centrifugation through adding the perm/wash buffer once. After this, re-suspend the cells in 50 microliters of perm/wash buffer containing anti-interferon-gamma at a concentration of 3 micrograms per milliliter.
Incubate on ice in the dark for 30 minutes. Next, add 150 microliters of 1X perm/wash buffer. Centrifuge at 400G, four degrees Celsius, for five minutes.
Remove the supernatant and add 200 microliters of 1X perm/wash buffer. Centrifuge the plate again at 400 times G, four degrees Celsius, for five minutes, and remove the supernatant by aspirating or flicking. Re-suspend the sample in 200 microliters of wash buffer and proceed with cytometric analysis.
One day before the experiment, mix the CHO cells either by vortexing or pipetting. Next add one milliliter of the cell suspension to each well of a 12-well plate. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.
On the day of the experiment, use a cell scraper to detach the CHO cells from the bottom of each well. Transfer the one milliliter of media containing the cells from each well to FACS tubes. Centrifuge at 400 times G and at four degrees Celsius for five minutes.
Re-suspend in 100 microliters of anti-HLA antibody diluted in wash buffer. Incubate on ice for 30 minutes. Then add one milliliter of wash buffer to each CHO sample.
Centrifuge at 400 times G and at four degrees Celsius for five minutes, and discard the supernatant. Re-suspend each sample in 3 milliliters of wash buffer, and proceed to flow cytometry analysis. In this study, a robust tool is presented for the investigation of molecular interaction during human CD8 positive T cell activation.
An engineered xenogenic system is generated that presents low levels of agonist pMHC in the presence or absence of co-agonist pMHC. These engineered APCs are then used to stimulate an E183-specific human CD8 positive T cell clone. Untransfected CHO cells, and CHO cells that express the nonstimulatory single-chain Gag-MHC complex do not induce activation.
Expression of high levels of the agonist single-chain E183-MHC complex, which is the positive control, is seen to induce very efficient interferon-gamma production, and degranulation, demonstrating that the single-chain E183 construct can be recognized by a specific TCR to activate T cell effector functions. Expression of low levels of single-chain E183-MHC complex induce lower levels of interferon-gamma production and degranulation, which was enhanced by presence of a nonstimulatory Gag-MHC complex. Note that very high percentages of CD107a+T cells are observed even in response to low levels of antigenic pMHC, which is consistent with previous studies.
However, presence of nonstimulatory Gag-MHC complex increases CD107a MFI. During this procedure, it is critical to control agonist peptide MHC expression to ensure equal agonist presentation with or without nonstimulatory peptide. It is critical to quantify agonist peptide MHC expression using TCR-like antibodies in each experiment.
An alternative approach is to use supported lipid bilayers, or beads, presenting fixed amount of agonist peptide MHC in the presence of nonstimulatory peptide MHC. Those experimental system should allow testing of multiple nonstimulatory peptide sequences for co-agonism. The single-chain MHC technology can be used to investigate molecular interactions in CD4 T cell activation, as well as to investigate non-classical MHC molecules.
This protocol uses human blood and human cells. Follow all the necessary precautions when working with human blood to reduce the risk of transmission of bloodborne diseases.
