Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The goal of this procedure is to isolate clinical grade antigen specific T cells for adoptive immunotherapy using a cytokine capture system. This is accomplished to by first incubating mononuclear cells obtained from A CMV positive donor with a panel of overlapping peptides derived from CMV PP 65 to activate the T cells. Next, the activated antigen specific CD 45 positive IFN gamma expressing T cells are isolated by cytokine capture.

The number and purity of the CMV specific T cells are then assessed by flow cytometry. Ultimately, a high percentage of CD four and CD eight positive IFN gamma expressing CMV specific T cells can be harvested using this cytokine capture system. The main advantages of this technique over existing methods are that it is completely automated and that a facility operating in compliance with good manufacturing practice is not required to isolate the captured clinical grade T cells.

This method can help answer key questions in the adoptive cell therapy field, such as how the costs associated with cell production be reduced while simultaneously increasing the accessibility of these cells. For a large number of patients, Though, this method can provide insight into the isolation of CMB antigen specific T cells. It may also be applied to the response of T cells to other pathogens by intubating the cells with pep antigens derived from the specific infectious agents of interest.

This automated system also simplifies the cytokine capture process and enables the captured T cell products to be produced on demand without the direct supervision of operators. Before beginning the procedure, use a lure spike interconnector to transfer freshly reconstituted C-M-V-P-P 65 peptide cocktail into a 50 milliliter volume freezing bag when all of the peptide has been transferred. Clamp the bag with locking forceps and open the cell enriching tubing set under sterile conditions, remove the vented vial adapter and the sterile filter from the tubing set and connect the peptide cocktail bag by screwing the lure spike interconnector onto the lure connection.

Then suspend 1 billion total nuclear cells in a total volume of 50 milliliters of freshly prepared P-B-S-E-D-T-A buffer and human serum albumin, and inject the cellular product into a 150 milliliter transfer bag. Now switch on the cell enrichment system and select the cytokine capture system IFN Gamma Enrichment Program. A user interface showing screens with instructions and pictures for the procedure will appear.

Enter the operator and tubing, set parcel number parameters. Then using the instructions displayed on the interactive monitor screen, install the prodigy tubing set to the automated cell enrichment device. After recording the catalog and lot numbers of the reagents, follow the step-by-step instructions displayed on the screen to connect the medium and buffers to the device.

Then after a final check of the tubing set, open the peptide cocktail and medium bags and initiate the automatic priming of the tubing. When the priming is complete, use a sterile tubing welder to add human serum albumin to the sodium chloride buffer in the reservoir bag and to transfer the starting cellular product into the application bag. Next, use adapters to connect the cytokine capture system reagents to their respective tubes and enter the preferred time for the end of the cell enrichment process.

Review and verify the accuracy of all the data and parameters entered. Then remove the quality control bag seal and weigh the bag and store it at four degrees Celsius. Now start the enrichment process.

The target cells will be collected in elution buffer from the reservoir bag. At the end of the enrichment, collect two aliquots from each of the non-target cell negative and target cell positive fractions. Then seal and weigh the non-target cell and target cell bags and store them at four degrees Celsius.

Finally, remove the tubing set from the cell enrichment instrument and transfer the log file to A USB drive for later use. To determine the number enriched cells from each of the samples, add CD 45 violet blue to one of the aliquots from the quality control non-target cell and target cell for 10 minutes in the dark at four degrees Celsius. Next, add 1.5 milliliters of freshly prepared red blood cell lysis solution to the quality control and negative fractions and 450 microliters to the positive fraction for 15 minutes.

At room temperature, immediately before the analysis, add propidium iodide to the samples at a final concentration of one microgram per milliliter and load the samples onto an automatic cell counter. To determine the absolute cell number of each sample, use the quality control sample to set the time gate as demonstrated. Gate the single cells in a forward scatter height by forward scatter area plot, followed by a monocyte and lymphocyte gate on the CD 45 positive cells then exclude the debris in the forward by side scatter plot to allow identification of the viable cells.

The number of viable cells within the original quality control sample can then be calculated to evaluate the quality of the enrichment wash the second aliquots from the original positive and negative fractions with pre chilled P-B-S-E-D-T-A buffer supplemented with a B serum. Re suspend the pellets in 100 microliters of the appropriate antibody fluorochrome staining mixture in the dark at four degrees Celsius. After 10 minutes, incubate the samples in one milliliter of freshly prepared red blood cell lysis solution for 15 minutes at room temperature.

Then spin down the cells again, resus suspending the pellets in fresh P-B-S-E-D-T-A supplemented with AB serum immediately before the analysis add propidium iodide as just demonstrated. Then used a following gating strategy to calculate the number of viable CD three positive T cells and the positive fraction after the cytokine capture system enrichment, as well as the frequencies of the CD four and CD eight, single positive and CD four IFN gamma and CD eight IFN gamma double positive T-cell populations in this representative experiment. An absolute count of interferon gamma positive T cells was assessed before and after the enrichment process.

The total number of interferon gamma positive T cells before the enrichment was 1, 140, 000 as derived from the 1 billion starting total nuclear cells. Whereas after the enrichment 309, 000 interferon gamma positive T cells were obtained, there were point 16%IFN gamma positive CD four positive T cells identified prior to the processing, which increased to the 47.5%after the enrichment. While the purity of the CD eight positive IFN gamma positive T cells was enriched from 0.47%to 90.3%The sample recovery and the captured positive fraction was 32.9%for the single positive CD four T cells and 31.8%for the single positive CD eight T cells.

Based on the measurement of the IFN gamma positive T cells in the starting population taken together, these data indicate that both CD four positive and CD eight positive CMV PP 65 specific T cells can be harvested automatically and in a suitable manner for their human application. Following this procedure, other assays like endotoxin and mycoplasma tests can be performed to answer additional questions about the sterility of the final product After its development. This technique paved the way for researchers in the field of adoptive cell therapy to explore the therapeutic use of multi antigen specific T cells for controlling various opportunistic infections.

After watching this video, you should have a good understanding of how clinical grade T-cells can be isolated using an automated system. Don't forget that working with biological samples can be extremely hazardous and that precautions such as using the appropriate personal protective equipment should always be taken while performing this procedure.