This protocol enables the purification of INK T-cells, which are very rare, and allows a 30-fold expansion in their number. The purification can be performed in one day and produce a large number of ready-to-use INK T-cells for in vivo and in vitro studies in two weeks. Demonstrating the procedure will be Gloria Delfanti, a post-doc in the lab.
After dissecting the mouse spleen, smash it through a 70 nanometer cell strainer to obtain a single cell suspension in 10 milliliters of PBS with 2%FBS. Centrifuge at 300 times G for five minutes. Remove the supernatant and resuspend the cell pellet with one milliliter of sterile ammonium chloride potassium lysine buffer.
Incubate the cells for three minutes at room temperature, then block with five milliliters of PBS with 2%FBS. Centrifuge at 300 times G for five minutes. After removing the supernatant, resuspend the cell pellet in three milliliters of PBS with 2%FBS and remove fat residues by pipetting.
For the enrichment steps, keep the cells cold and use solutions pre-cooled at four degrees Celsius and then kept on ice. Centrifuge at 300 times G for five minutes. Resuspend all the cells in the appropriate amount of PBS with 2%FPS and Fc blocker and incubate for 15 minutes at room temperature.
Wash with one to two milliliters of MACS separation buffer per 10 million cells and centrifuge at 300 times G for 10 minutes. After removing the supernatant, stain the cells with CD19-FITC and H2-IAb-FITC and incubate for 15 minutes in the dark at four to eight degrees Celsius. Wash cells by adding one to two milliliters of MACS buffer per 10 million cells and centrifuge at 300 times G for 10 minutes.
Remove supernatant and resuspend the cell pellet in 90 microliters of MACS buffer per 10 million cells. Add 10 microliters of anti-FITC microbeads per 10 million cells. Mix well and incubate for 15 minutes in the dark at four to eight degrees Celsius.
Wash the cells by adding one to two milliliters of MACS buffer per 10 million cells and centrifuge at 300 times G for 10 minutes. Remove the supernatant and resuspend up to 125 million cells in 500 microliters of MACS buffer. Place an LD column in the magnetic field of the MACS separator.
To avoid clogging, apply a pre-separation filter on the LD column and rinse it with two milliliters of MACS buffer. Apply the cell suspension onto the filter and collect the unlabeled cells that pass through the column. Wash the empty column reservoir three times with one milliliter of MACS buffer.
Collect the T-cell enriched effluent and count the cells, keeping 50 microliters for FACS analysis. After centrifuging at 300 times G for five minutes, remove the supernatant and stain the cells with CD1d tetramer-PE. Mix well and incubate for 30 minutes in the dark on ice.
Wash the cells by adding one to two milliliters of MACS buffer per 10 million cells. After centrifugation at 300 times G for 10 minutes, remove the supernatant and resuspend the cell pellet in 80 microliters of MACS buffer per 10 million cells. Add 20 microliters of anti-PE microbeads per 10 million cells.
Mix well and incubate for 15 minutes in the dark at four to eight degrees Celsius. Wash cells by adding one to two milliliters of MACS buffer per 10 million cells and centrifuge at 300 times G for 10 minutes. Remove the supernatant and resuspend up to 100 million cells in 500 microliters of MACS buffer.
According to the cell count, place the LS or MS column in the magnetic field of the MACS separator and rinse the column with MACS buffer. Apply the cell suspension onto the column, collect unlabeled cells that pass through and wash the column three times as described previously. This is the negative fraction.
Remove the column from the magnetic field and place it on a new collection tube. Pipette MACS buffer onto the column and push the provided plunger into the column to flush out the positive fraction enriched in INK T-cells. To further increase INK T-cell recovery, centrifuge the negative fraction at 300 times G for 10 minutes and repeat previous steps with a new LS or MS column.
Pull the positive fractions and determine cell count. Keep 50 microliters of both positive and negative fractions for FACS analysis to check the purity. To activate INK T cells at a one-to-one ratio, transfer the appropriate volume of anti-CD3 and CD28 magnetic beads to a tube and at an equal volume of PBS, then vortex for five seconds.
Place the tube on a magnet for one minute and discard the supernatant. Remove the tube from the magnet and resuspend the washed magnetic beads in the proper volume of RPMI. Centrifuge purified INK T-cells at 300 times G for five minutes and mix them with mouse T-activator anti-CD3 or CD28 magnetic beads.
Plate one milliliter of the cell suspension, anti-CD23 and CD28 magnetic beads in a 48-well plate with 20 units per milliliter IL-2, then incubate at 37 degrees Celsius. After five days, add 10 nanograms per milliliter IL-7. Split the cells in half when they reach 80 to 90%confluence, always adding 20 units per milliliter IL-2 and 10 nanograms per milliliter IL-7.
Under these conditions, INK T-cells can be expanded for up to 15 days. Using this protocol, INK T-cells are enriched from the spleen of transgenic mice through an immunomagnetic separation process. After enrichment, the cells can be expanded with anti-CD3 and CD28 beads, resulting in a 30-fold expansion on average by day 14 of the culture.
The strong activation with anti-CD3 and anti-CD beads induced the downregulation of the IMK T-cell TCR expression on the cell surface and a double negative population appeared. The majority of expanded INK T-cells are CD4 negative. Characterization of the lineage-specific transcription factors PLZF and ROR gamma T made it possible to identify the NKT1, NKT2, and NKT17 phenotypes on enriched INK T-cells at day zero and 14.
The enriched INK T-cells show a T80-like effector phenotype, which is conserved after 14 days of expansion as confirmed by the secretion of both interferon gamma and IL-4 after PMA and ionomycin stimulation. During the purification steps, remember to work fast with pre-cooled solutions and get rid of any fat residues that may be seen while pipetting. Expanded INK T-cells can be exploited for in vitro assays and in vivo transfers into mice, even after functional modifications via gene transfer or editing.
In vitro expansion of INK T-cells overcomes the limitation given by the paucity, making them exploitable in preclinical studies in the fields of tumor immune surveillance, infectious diseases, and auto-immunity.
