Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

The overall goal of this experiment is to generate a non cellular HLA IG based killer artificial antigen presenting cell, or K-A-A-P-C for the depletion of autoreactive or unwanted T-cell populations. This is achieved by first covalently linking HLA IG molecules and Antifa monoclonal antibodies onto cell sized iron dextran particles for the generation of the functional K-A-A-P-C In the second step, the K-A-A-P-C are characterized by flow cytometry to ensure their proper functionality. Next, the K-A-A-P-C are loaded with different peptides of interest to target the unwanted antigen specific T-cell population.

Ultimately, flow cytric analysis can be performed to measure the antigen specific anti-fat mediated killing of the T cells by the cognate loaded.K-A-A-P-C. We first had the idea for this method when we encountered problems working on the development of a cell based killer antigen presenting cell while we, at the same time we're working on the development of a beat based platform. Technology for immune cell modulation To generate K-A-A-P-C first, add 250 microliters of epoxy beads from a stock and 250 microliters of freshly prepared borate buffer into a sterile screw top glass vial.

Place the vial on a magnet to let the beads adhere after two minutes, aspirate the buffer and remove the vial from the magnet. Wash the beads once with another milliliter of bore eight buffer, and to then resuspend them in 550 microliters of HLA A two IG and anti-human CD 95 MAB. Shake the vial gently to mix the beads with the antibodies and then incubate the beads on a rotator end over end at four degrees Celsius for 24 hours.

The next day, spin down the beads for two minutes at 300 Gs and room temperature and place the vial back onto the magnet. After two minutes, aspirate the loading buffer. Remove the vial from the magnet and wash the beads in one milliliter of wash buffer with careful shaking.

After the third wash, rotate the vial in one milliliter of fresh bead wash buffer to block any residual protein binding sites. The next day, spin down the beads again and place the vial back onto the magnet. After two minutes, aspirate the wash buffer.

Remove the vial from the magnet and add one milliliter of PBS to the KAPC. To check the quality of the beads, transfer 1.5 times 10 to the fifth of the K-A-A-P-C into a fax tube and wash them with one milliliter of fax wash buffer Resus. Suspend the bead pellet in 100 microliters of the wash buffer and add one microliter of anti mouse IgG one PE to detect the HLA A two IG and one microliter of anti mouse IG MFI TC to detect the anti-human CD 95 MAB.

Then after a 15 minute incubation at four degrees Celsius, wash the beads in one milliliter of fax wash buffer Resus. Suspend them in 250 microliters of wash buffer and read the samples on a flow cytometer for peptide loading. Transfer two times 10 to the seventh of the K-A-A-P-C into a new sterile glass vial.

Then after placing the beads onto the magnet for two minutes, aspirate the PBS and resuspend the beads in 500 microliters of fresh PBS containing five micrograms of peptide. Label the vial with the date and the name of the peptide loaded, K-A-A-P-C. Then store the loaded K-A-A-P-C at four degrees Celsius for at least three days to allow sufficient peptide loading onto the HLA A two IG dimer to perform A-K-A-A-P-C in vitro killing assay.

Wash the peptide loaded K-A-A-P-C as demonstrated at least six times to remove all free peptide. Then transfer K-A-A-P-C and two times 10 to the fifth antigen specific T cells to one well per sample of a 96 well round bottom plate in 200 microliters of co-culture medium at a one to one ratio after 48 hours of culture in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. Transfer the samples to the appropriate corresponding fax tubes and wash them in one milliliter of a nexin five binding buffer.

Next, resuspend the pellets in 100 microliters of a nexin five binding buffer, and then incubate the samples in one microliter of a nexin five FITC and five microliters of seven a a d at room temperature. After 10 to 15 minutes, wash the samples with one milliliter of a nexin five binding buffer and resuspend the pellets in 250 microliters of a nin five binding buffer. Then immediately read the samples on a flow cytometer.

K-A-A-P-C display HLA A two IG, and Antifa MAB at the same time, which in comparison to cell-based approaches, is not altered by coding or transduction efficacies and can be easily modulated during production. K-A-A-P-C are capable of depleting antigen specific T cells in vitro when loaded with cognate peptide, while non cognate loaded. K-A-A-P-C do not deplete T-cell, thus K-A-A-P-C induced T-cell apoptosis in an antigen specific manner.

Subsequently, when K-A-A-P-C are used for the selective depletion of antigen-specific T cells from a mixture of different T-cell specificities only, minimal cytotoxicity leaks into neighboring T cells. Thus, K-A-A-P-C represent an exquisite tool for in vitro antigen, antigen-specific depletion of human activated antigen-specific T cells with a potential clinical applicability for the treatment of autoimmune diseases and allograft rejections After its development. This technique paved the way for researchers in the field of cellular immunology to explore new approaches for the treatment of T-cell mediad autoimmune diseases and transplant rejections.