Figure 5. Cytotoxic capability of NKT cell subsets.

Experiments were performed in triplicate per donor; standard deviation is depicted.

For A, B, C, and D, one representative donor out of three independent experiments is shown. (A) NKT cells were added at an Effector:Target (NKT cell: 3T3 cell) ratio of 1∶1 to adherent 3T3 cells in the presence of different α-GalCer concentrations.

After 18–20 hours, NKT cells were collected and stained with anti-CD25 (unlabelled followed by goat anti-mouse APC) to determine activation.

(B) As in A, NKT cells at an NKT:3T3 of 1∶1 were activated in the presence of varying concentrations of α-GalCer.

After 18–20 hours, cells were collected by trypsinization to remove any 3T3 cells that were still adherent and target 3T3 cells were stained with propidium iodide to determine their viability.

(C) α-GalCer was added at a concentration of 100 ng/ml to adherent 3T3 cells.

NKT cell subsets were then added at different NKT:3T3 ratios. Viability of 3T3 cells after an 18–20 hour incubation period was determined as in 5B.

(D) Polarized nCD4+ NKT cells were generated as in Figure 2.

The cytotoxic capacity of these NKT0, NKT1, and NKT2 lines was assayed as in Figure 5C.

One NKT:3T3 ratio for each donor is shown.