The overall goal of this procedure is to generate CD 1D based artificial antigen presenting cells or A A PC for the expansion of NK T cells. This is accomplished by first adding proteins to functionalize the magnetic beads. In the second step, the initial NK T cell population from the peripheral blood is enriched.
Next, the enriched T cells are co cultured with the A A PC.In the final step, the phenotype and function of A A PC cell population are assessed. Ultimately, the purity of the expanded T-cell subsets can be determined by flow cytometric analysis. The main advantage of a APCs over current methods that utilize dendritic cells is that there can be high donor variability with dendritic cells.
A APCs provide a reproducible off the shelf and standardized method for the expansion of NK T cells. We first had the idea for this method when I was working with Dr.Schneck and oaky at Johns Hopkins. They have pioneered the field of using artificial antigen presenting complexes for the expansion of classical CD eight T-cell subsets.
Demonstrating the procedure will be Dr.James East, a postdoctoral fellow, and Dr.Wendy's son. A researcher associate from my laboratory Begin by transferring one milliliter of dyna bead M four 50 epoxy beads into a five milliliter clear bo silicate glass threaded vial. Then rinse the beads with three milliliters of sterile bore eight buffer in a separate 1.5 milliliter micro centrifuge tube.
Add 100 micrograms of human CD 1D IG dimer, and 20 micrograms of the desired co-stimulatory molecules to one milliliter of calcium and magnesium free PBS. Next, place the bead containing glass vial on a magnet and aspirate the borate buffer. Then add the protein mixture to the glass vial and replace the cap.
Mix the solution immediately by inverting the vial, then cover the cap with paraform and place it on a rotator overnight at four degrees Celsius the next day. Place the glass vial on the magnet again and remove the protein mixture carefully avoiding the beads. Wash the beads three times by adding three milliliters of bead wash buffer and incubating them on the rotator at four degrees Celsius for five minutes.
To confirm that the proteins are stably loaded onto the beads, stain an aliquot of the beads with antibodies and analyze them by flow cytometric analysis. Now transfer five times 10 to the seventh beads to a small 1.5 milliliter glass vial and rinse the beads with one milliliter of sterile PBS. Then 48 to 72 hours before use.
Load the washed beads with antigen in one milliliter of sterile PBS while the beads are being loaded with antigen. Collect the PBMC by first diluting the heparinized donor blood with an equal volume of PBS at room temperature. Then add 15 milliliters of room temperature PHI, call to 50 milliliter conical tubes, and slowly overlay 25 milliliters of the diluted blood mixture on top of the phi.
Call then after centrifuging the samples for 30 minutes at 700 times G and room temperature with the break off. Use a pasture pipette to carefully transfer the lymphocyte interface to a new 50 milliliter conical tube. Next, wash the lymphocytes in 50 milliliters of PBS, discarding the supernatant and combining the tubes from a single donor into a single tube.
After washing the PBMC again with 20 milliliters of PBS, count the cells and then resuspend them at a concentration of five times 10 to the seventh cells per milliliter in max buffer. To isolate the T-cell fraction, incubate 100 microliters of PAN T-cell enrichment solution from the EP human T-cell enrichment kit with two milliliters of PBMC for 10 minutes at room temperature. Then incubate the solution with 100 microliters of magnetic beads at room temperature for another 10 minutes.
Bring the final volume of the solvent to 2.5 milliliters and then place the tube in the magnet. After five minutes, quickly pour off the CD three positive fraction into a 15 milliliter conical tube. Now wash the cells in five milliliters cold max buffer, and count the viable cells.
Remove an aliquot for fact staining to select the CD 1 61 positive cells first. Resuspend the enriched T cells in 980 microliters of ice cold max buffer, and then incubate the cells in 10 micrograms of anti CD 1 61 monoclonal antibody for 10 minutes at four degrees Celsius. After washing the cells reconstitute the pellet in 800 microliters of max buffer and 200 microliters of anti-US IgG one microbeads.
Incubate this solution at four degrees Celsius for another 10 minutes. While the cells are incubating with the microbeads, add three milliliters of max buffer to equilibrate an LS column. After washing this cell fraction, slowly pipette the cells in three milliliters of max buffer into the column.
Now rinse the column three times with three milliliters of fresh max buffer. Then add three more milliliters of fresh max buffer to the column and transfer the column to a 15 milliliter conical tube. Plunge out the purified CD 1 61 positive CD three positive cells, and then count the NKT cell enriched fraction.
Begin this step by co culturing 10 to the sixth of the enriched CD 1 61 positive CD three positive T cells and 10 to the sixth, A A PC in 16 milliliters of complete medium plate. 160 microliters per well of the co-culture to a 96 well to tissue culture treated polystyrene U bottom plate with a low evaporation lid on day 12 to 14, harvest and count the cells. Analyze the populations by flow cytometric analysis, then repl and co-culture.
The expanded NKT cells as just demonstrated freezing the extra NKT cells following the second round of expansion. To test the functionality of the A A PC cells first co-culture, five times 10 to the fourth NKT cells per well with five times 10 to the fifth, A A PC in a 200 microliter final volume of complete medium and a 96 well you bottom plate after 24 to 48 hours, harvest the cell culture supernatant for analysis by Eliza. This first figure demonstrates that the CD 1D IG proteins are stably immobilized onto the surface of the magnetic beads as shown here.
CD 1D IG and anti CD 28 antibodies are both expressed on the surface of the magnetic beads as demonstrated in this graph. The V alpha 14 positive mouse NK T cell hybridoma DN 30 2D three was co cultured with either medium soluble alpha galera antigen unloaded A A PC or alpha gal loaded A A PC.The culture supernatants were then harvested, and IL two production was measured by Eliza providing a simple method for testing each batch of antigen presenting cells. In this experiment, human CD 1 61 positive CD three positive peripheral blood T cells were enriched by magnetic bead separation.
The cells were then stimulated once a week for 14 days with alpha galera loaded A A PC and analyzed for V alpha 24 and V beta 11 expression. Importantly, it was found that even with a relatively low initial NKT cell population of 0.03%the cells were able to be expanded to about a 67%V alpha 24 positive V beta 11 positive population. Here, C one R CD 1D cells were incubated with NKT cells at the indicated ratios in the presence or absence of Alpha Gal SER antigen in 96, well, U bottom plates for 20 to 24 hours.
NKT cell mediated cell lysis of the B cell lymphoma line was then assessed by a chromium release assay. A A PC expanded NKT cells were able to ly this lymphoma cell line. In the presence or absence of alpha galera, NKT cells have been expanded from the PBMC of both healthy volunteers and cancer patients, and the alpha galera loaded.
A A PC have been able to expand the NKT cell populations from both groups. When using a starting population of 2 million CD 1 61 positive CD three positive T cells greater than 10 to the seventh cells can be obtained after two rounds of expansion expanded. NK NKT cells can be used for functional studies as demonstrated here.
The ex vivo expanded NKT cells remain responsive to Alpha GAL stimulation and our potent producers of IL 17 A TNF alpha and interferon gamma. It should be noted that if the initial T-cell enrichment population is low and the second CD 1 61 enrichment step is unable to be performed, the A A PC mediated expansion may not yield the expected results. However, if the percentage of circulating NKT cells is higher than 0.1%a significant expansion of invariant NKT cells should still be able to be obtained.
Collectively, these data demonstrate that CD 1D based A A PC can be used to effectively expand and stimulate primary human NK T cells Following this procedure. Other methods such as QPCR can be performed in order to answer additional questions such as what are the kinetics of NK T cell activation After its development. This technique pa the way for researchers in the field of hepatology to explore the role of NKT cells in fatty liver disease.
