Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

Tumor cells activate effector immune cells to release cytokines and cytolytic granules containing perforin and granzymes. Perforin forms pores in the tumor cell membrane, facilitating granzyme entry into the cell and initiating apoptosis, causing cell death.

To analyze the cytolytic function of human peripheral blood mononuclear cells, PBMCs, following tobacco product preparation, TPP, exposure, take a multi-well plate with increasing TPP concentrations. A medium-containing well acts as the control.

Add PBMC suspension. TPPs' primary immunosuppressive agent, nicotine, binds to effector PBMCs' nicotinic acetylcholine receptors, downregulating the pro-inflammatory cytokine production.

Centrifuge. Remove the TPP-containing supernatant. Resuspend the PBMCs in media. Add fluorescently labeled K562 cells, human leukemic cells having an increased susceptibility to PBMC effector cell cytotoxicity, to the wells. 

TPP-treated effector cells with suppressed immune responses exhibit reduced perforin expression and cytokine release, decreasing the PBMC's cytolytic ability.

Centrifuge. Resuspend the cells in a buffer followed by a cell-impermeable fluorescent dye. The dye enters the cells with compromised membranes and stains the DNA.

Using a flow cytometer, determine the proportion of dead and live K562 cells in the wells. A lower percentage of dead K562 cells in wells with higher TPP concentrations than the control indicates suppression of K562 cell killing by TPP-treated PBMCs.

After preparing a plate of tobacco product-treated PBMCs, as just demonstrated, wash a culture of K562 cells harvested at an 80% confluency in 10 milliliters of DPBS. Resuspend the pellet in 5 milliliters of DPBS, and count the cells. Then, add 1 milliliter of freshly-prepared CFSE working solution to 1 to 2 times 10 to the seventh K562 cells in 1 milliliter of complete medium, and disperse the cell stain by vortexing.

Incubate the cells at room temperature. After exactly 2 minutes, add 200 microliters of FBS, and vortex the cells again, incubating them for another two minutes.

After the second incubation, wash the cells in 10 milliliters of complete medium. Then, resuspend the pellet in another 10 milliliters of complete medium and count the cells. Now, centrifuge the tobacco-treated PBMCs, and decant the supernatant. Then, replace the cover, resuspend the cells by vortexing, and wash them in 200 microliters of complete medium per well.

To initiate the killing assay, add the CFSE-labeled K562 cells to the PBMCs at a 1 to 15 ratio, and incubate the co-culture at 37 degree Celsius and 5% CO2.

After five hours, spin down the cells and discard the supernatant. Resuspend the pellets by vortexing, and then wash the cells with 200 microliters of running buffer per well. Then, add 95 microliters of running buffer followed by 5 microliters of 7-AAD, for a total volume of 100 microliters per well.

Finally, incubate the cells in the dark at room temperature for 15 minutes, then, add 100 microliters of running buffer to each well, and transfer the entire volume to the appropriate corresponding FACS tubes to determine the percentage of 7-AAD and CFSE-positive cells by flow cytometric analysis.