Figure 6. CTLs induced with lot 60506 IL-4 and stimulated with anti-CD3 antibodies produced soluble toxic products in the presence of triglyceride lipids.

These experiments had 2 steps. In step 1, supernatants were generated after stimulation with anti-CD3 or without stimulation and collected in the presence or absence of triglyceride lipids. In step 2, these supernatants were tested for release of either lipases or lipid byproducts that might be toxic when incubated with P815 cells. In each step, lipid was a variable, to test for the presence of lipases. CTLs induced with lot 60506 IL-4 were stimulated to undergo exocytosis using anti-CD3 coated beads for 48 hours in the presence or absence of triglycerides. Control supernatants were collected from CTLs under matched culture conditions that contained beads without anti-CD3. Following the initial 48 hour stimulated exocytosis, the CTL cell-free supernatants were collected and added to eGFP-P815 cells. The P815 cells were in media with or without fresh triglycerides in the hope of detecting released lipases and were incubated for 48 hours with the cell-free supernatants. The controls for this experiment that determined 100% viability were the supernatants from the un-stimulated CTLs (beads minus anti-CD3). These supernatants were without effect on P815 growth and survival (not illustrated). The cell-free supernatants generated in the presence of triglycerides (bars 3 & 4 numbered 1–4 from the left) were toxic compared to the cell-free supernatants produced without triglycerides during exocytosis. The lipid was essential as in its absence in during primary exocytosis (bars 1 & 2) there was little cytotoxicity elicited by anti-CD3 stimulation. Furthermore, these experiments indicate that anti-CD3 stimulation was essential to produce soluble toxins in the presence of lipids.