The overall goal of this procedure is to use artificial antigen presenting cells or a A PC for ex vivo expansion of human antigen specific CTL for potential use in adoptive immunotherapy. This is accomplished by first conjugating HLA A two IG and anti-human CD 28 monoclonal antibody to magnetic dyna beads to make A A PC.The second step of the procedure is to perform quality control and peptide loading of the A A PC.The third step of the procedure is to isolate human peripheral blood CD eight positive T cells. The final step of the procedure is to incubate CD eight positive T cells with a A PC for a week.
The CTL are then harvested and characterized before they're either used or are re stimulated for another week to achieve the desired level of their specificity and expansion. Ultimately, teum or staining results can be obtained that show that CTL that are expanded in the presence of A A PC have an increased antigen specificity. Today I'm going to show you an HI IG based A A PC system and how to use this system to efficiently expand CMV specific human CTL exvivo compared with other existing methods.
Our A A PC technology offers several important advantages. It's off the shelf available, it's of unlimited quantity, and let's say A A PC can be used for all HRAA two positive donors. Okay, let's start Transfer one milliliter of M four 50 epoxy beads from a stock approximately 400 million beads into a sterile screwup glass file.
Wash the beads once by adding one milliliter of boid buffer and then placing the glass vial against a dyin all magnet NBC one while the beads are adhered to the side of the vial. Remove the snat by aspiration Resus. Suspend the beads in a mixture of boid buffer HLA A two IG dimer and anti-human CD 28 monoclonal antibody.
Then to facilitate protein to bead conjugation, rotate the glass vial on a rotator at four degrees Celsius for 24 hours. Next, place the tube in an MPC one magnet and remove all of the borate buffer. After the buffer has been removed, wash the beads twice with one milliliter of bead wash buffer.
Now incubate the beads in one milliliter of bead wash buffer and rotate the vial at four degrees Celsius for another 24 hours to block the residual protein binding sites on the beads. Then remove the buffer from the glass vial and replace it with one milliliter of fresh bead wash buffer. Transfer 100 microliters of fax washing buffer to fax tubes, and then add five times 10 to the fifth beads.
Stain the beads with one microliter of anti muse IgG, one PE and one microliter of anti muse IgG two a fite after staining for 20 minutes at four degrees Celsius. At three milliliters of fax washing buffer to each of the staining samples. Then pellet the A A PC by spending at 300 Gs for five minutes at four degrees Celsius.
Reus suspend the A A PC in fresh fax buffer and read the staining result immediately by flow cytometer. Wash the beads twice with sterile PBS as before. Next, load the beads with 10 microliters of CMV peptide.
Count the beads by hemo cytometer and then label them with the date and concentration of the beads. As beads per milliliter. Incubate the A A PC with the peptide for at least three days at four degrees Celsius centrifuge approximately 100 milliliters of fresh peripheral blood from an HLA A two positive donor in 10 sodium heparin vacutainer tubes at 300 Gs for 10 minutes.
At room temperature, carefully remove the top plasma layer by aspiration. Replace the collected plasma with sterile PBS and transfer the blood into a sterile T 75 culture flask. Mix the blood and PBS well by pipetting once all the blood has been collected at 15 milliliters of fial pack plus to four 50 milliliter conical tubes.
Slowly overlay 30 to 35 milliliters of the blood cells on top of the fial in each tube. Keeping the interface distinct between the fial and the blood cells centrifuge the blood and fial gradient at 500 GS for 20 minutes at room temperature with the break off and the acceleration as low as possible to maintain a clear interface between the layers after the spin aspirate off the top plasma layer and then use a serological pipette to carefully aspirate the PBMC layer. Transfer the PBMC to a fresh 50 milliliter conical tube when all the PBMC have been harvested at 30 milliliters of PBS to the cells and centrifuge them.
Next, discard the supernat and wash the pellet. One more time with 30 milliliters of PBS to remove the residual fi call then enrich for the CD eight positive T-cell population using a Milton Human CD eight positive T-cell isolation kit. According to the manufacturer's protocol, count the CD eight positive T-cells and confirm the purity by fax analysis.
Suspend 1 million CTL in eight milliliters of T-cell growth factor two x culture medium plus eight milliliters of complete RPMI medium and add one times 10 of the six A A PC mix. Well then use a multi-channel pipette to plate 160 microliters of cells per well onto a 96 well U bottom tissue culture plate. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for seven days.
Feed the cells on day four with 80 microliters per well of T-cell growth. Factor two x culture medium. Harvest the cells on day seven and then place the cells against the magnet to remove the old A A PC when all the beads adhere to the wall of the vial.
Harvest and transfer the cells into another 50 milliliter conical tube and count the cell number. Determine the antigen specificity of the A A PC by tetramer staining according to the manufacturer's protocol. Replay the harvested cells with new A A PC under the same conditions to generate increased cell numbers into antigen specificity.
Here is a representative tetramer staining result of CMV specific CTL generated by A A PC after one week of culture. Shown here is a representative intracellular cytokine staining result of CMV specific CTL generated by A-A-P-C-C-M-V specificity was 61%by tetramer staining. This method can be used to answer key questions in the T-cell field, such as how can we identify the optimal cultural conditions and the requirements for T-cell functional modulation.
Lowly method provided insights into treating viral diseases. It can also be applied into other fields such as cancer.
