The overall goal of this procedure is to identify Qa-1 epitopes in proteins. This method can help answer key questions in the field of immune regulation and immunotherapy. The main advantages of this technique are that mapped peptides are known to stimulate Qa-1 restricted CD8 T cells, and the procedure is easy to perform.
The implications of this technique extend towards therapeutic designs for immune-mediated diseases, such as multiple sclerosis. Though this method can provide insight into Qa-1 mediated immune regulation in animals, it can also be applied to HLA-E mediated immune regulation in humans. To begin the procedure, design a library of 15-mer peptides, in which adjacent peptides overlap in 11 amino acids.
Obtain and reconstitute the peptides under sterile conditions. For each peptide, combine five milligrams of the peptide with 100 microliters of dimethyl sulfoxide. Combine 10 microliters of each peptide stock in a clean 1-milliliter cryo tube to prepare the OLP pool stock.
Then, load 20 microliters of each peptide stock into a V-bottomed 96-well plate, and dilute each stock with 80 microliters of sterile water. Store both the remaining 50 milligrams per milliliter peptide stocks and the plate of 10 milligrams per milliliter peptides stocks at 20 degrees Celsius. Next, add 10 microliters of the OLP pool stock solution to a 15 milliliter tube containing two milliliters of irradiated dendritic cells in serum-free medium, thus diluting the DMSO two hundred-fold.
Store the remaining OLP pool stock at 20 degrees Celsius. Incubate the DCs with the OLP pool at room temperature for three hours. Gently shake the cells every 15 minutes by hand.
During the incubation, use a kit to purify CD8+T cells from a harvested mouse spleen or lymph nodes. Prepare five milliliters of a solution with 10 million T cells per milliliter of serum-free medium with 50 units per milliliter of interleukin 2, and 100 units per milliliter of interleukin 7. The purified CD8 T cells should be free of beads, or untouched.
Place 0.5 milliliters of the T-cell solution in each of 10 wells of a 48-well plate. Once the incubation of the OLP pool-pulsed DC solution is complete, add 10 milliliters of serum-free medium to the DCs. Centrifuge the cells at 300 times G for 10 minutes at room temperature.
Then, reconstitute the cells in five milliliters of serum-free medium. Add 0.5 milliliters of the cell suspension to each of the 10 wells. Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide atmosphere for four days.
Then, remove 400 microliters of the culture medium from each well. Add 500 microliters of fresh serum-free medium containing 100 units per milliliter each of IL-2 and IL-7 to each well. Incubate the cells for another two to three days before re-stimulating the OLP pool-primed cells.
Prior to re-stimulation, obtain and irradiate mouse peritoneal macrophages. Adjust the macrophage concentration to five million cells per milliliter of serum-free medium. Combine 10 microliters of the OLP pool stock with two milliliters of the irradiated peritoneal macrophages for a final DMSO concentration of 0.5%by volume.
Supplement this solution with 100 units per milliliter of murine macrophage colony-stimulating factor. Fill a 48-well plate with 200 microliters of the OLP-pool macrophage solution in each well. Incubate the plate at 37 degrees Celsius for four hours.
Then, gently wash each well with 200 microliters of pre-warmed RPMI-0 medium. The macrophage solution will contain lot of beads. Before the OLP pool-primed CD8 T cells are added, the plate should be checked under a microscope to make sure that all the wells are free of beads.
Next, collect and pool the OLP pool-primed CD8+T cells that are ready for re-stimulation. Adjust the T-cell concentration to one million cells per milliliter in serum-free medium containing 25 units per milliliter of IL-2, and 50 units per milliliter of IL-7. Add one milliliter of the pooled T-cell solution to each well containing OLP pool-pulsed macrophages.
Culture the cells at 37 degrees Celsius in a 5%carbon dioxide atmosphere for four days. Then, remove about 400 microliters of the medium from each well. Add to each well 500 microliters of fresh serum-free medium containing 100 units per milliliter each of IL-2 and IL-7.
Resume incubation under the same conditions. After three to four days, examine the re-stimulated cells for an OLP pool-specific Qa-1 restricted response with an interferon-gamma enzyme linked immuno spot assay. Refresh the CD8+T-cell medium every three to four days while waiting for the results.
Perform an ELISPOT assay with the dilute peptide stocks to determine which peptides produce the OLP pool-specific Qa-1 restricted interferon-gamma secretion. Lastly, evaluate a truncated peptide series with ELISPOT to identify the optimal Qa-1 epitope. An interferon-gamma ELISPOT assay showed a significant increase in spot-forming cells when CD8+T cells stimulated by the pooled MOG overlapping peptide library were combined with C1R.
Qa-1b cells. This suggested that the T cells responded to the Qa-1 binding peptides, and that the MOG_pool contained one or more Qa-1 binding epitopes. Another assay was performed to screen the individual peptides in the pool.
Three peptides showed responses that were at least three times greater than the response in the presence of C1R. Qa-1b cells without peptides. Of these, the peptide with the greatest response was selected for further evaluation.
A library of truncated peptides based on the 15-mer peptide of interest was evaluated to identify the optimal Qa-1 epitope. The epitope would ideally be eight to 10 mer, with nine mer preferred. Further, the optimal epitope would show a similar or stronger interferon-gamma response than the 15-mer peptide.
The nine mer N-terminally truncated peptide best fit these criteria, making it the optimal Qa-1 epitope. After its development, this technique paved the way for researchers in the field of immune regulation to discover new Qa-1 epitopes for the study of immune regulation in other tissues, such as the pancreas. Don't forget, working with radiation can be extremely hazardous.
Researchers should strictly follow the institutional policy and be appropriately trained.
