Figure 6.
Figure 6. PPARγ ligands and PLA2 inhibitors cooperate to reverse M2-mediated CTL suppression. (A) Splenocytes from 5- to 7-week BW-Sp3 progressors were restimulated in vitro with irradiated BW-Sp3(B7-1), and a distinction was made between (1) CTL activity in a 5-day culture without inhibitors (Total). (2) CTL activity in 5-day cultures with inhibitors added at day 2 (Total + inhibitor). Inhibitors used were as follows: SOD (O2 –), catalase (H2O2), and neutralizing anti–PD-L2 mAb. (3) CTL activity of nonadherent splenocytes, removed from the adherent fraction after 2 days of culture and restimulated in a fresh plate for another 3 days (Nad). (4) Nad cultures supplemented with inhibitors (Nad + inhibitor). The cytotoxic activity in all cultures was tested using 111In-labeled BW-Sp3(B7-1) cells as targets at a 100:1 effector-target ratio. Results show the average 111In-release ± SD of triplicates. One exemplary experiment is shown, but all inhibitors have been tested in at least 3 independent experiments with comparable results. (B) After 3 days of restimulation, without inhibitors, plastic-adherent cells were recovered from the plate and incubated with 2 μM DHE for 1 hour or 2 μM DCFDA for 30 minutes at 37°C. After washing, cells were labeled with anti-CD11b/PE and analyzed. Histograms represent gated CD11b+ cells without dye (dotted line), or with either DHE or DCFDA (full line) from one experiment. The average percentage of DCFDA-positive (gate M1) ± SD from 3 independent experiments is given. (C) Similar experiment to that in panel A. Inhibitors used were as follows: baicalein (12/15 LOX), GW9662 (PPARγ). Agonists used were as follows: rosiglitazone (PPARγ), 15d-PGJ2 (PPARγ). One exemplary experiment of 4 is shown. Boxes represent average percentage of CTL recovery ± SD of 4 experiments. *P < .05, percentage of CTL recovery higher comparing Total + PPARγ ligands versus Total + baicalein; #P < .05, percentage of CTL recovery higher comparing Total + PPARγ ligands versus Total + GW9662. (D) Similar experiment as panel A. Inhibitor used was as follows: aristolochic acid (PLA2). Agonists used were as follows: rosiglitazone (PPARγ), 15d-PGJ2 (PPARγ). Again, one exemplary experiment of 4 is shown. Boxes represent average percentage of CTL recovery ± SD of 4 experiments.*P < .05, percentage CTL recovery higher comparing Total + aristolochic acid + PPARγ ligands versus Total + PPARγ ligands; #P < .05, percentage CTL recovery higher comparing Total + aristolochic acid + PPARγ ligands versus Total + aristolochic acid. (E) Similar experiment to that in panel A. Inhibitors used were as follows: AACOCF3 (= AA; cPLA2), PACOCF3 (= PA; iPLA2), ONO-RS-082 (= ONO; sPLA2). Agonist used was as follows: 15d-PGJ2 (PPARγ). One exemplary experiment of 2 is shown. Boxes represent average percentage CTL recovery ± SD of 2 experiments. *P < .05, percentage CTL recovery higher comparing Total + 15d-PGJ2 + ONO + AA + PA versus Total + 15d-PGJ2.

PPARγ ligands and PLA2 inhibitors cooperate to reverse M2-mediated CTL suppression. (A) Splenocytes from 5- to 7-week BW-Sp3 progressors were restimulated in vitro with irradiated BW-Sp3(B7-1), and a distinction was made between (1) CTL activity in a 5-day culture without inhibitors (Total). (2) CTL activity in 5-day cultures with inhibitors added at day 2 (Total + inhibitor). Inhibitors used were as follows: SOD (O2–), catalase (H2O2), and neutralizing anti–PD-L2 mAb. (3) CTL activity of nonadherent splenocytes, removed from the adherent fraction after 2 days of culture and restimulated in a fresh plate for another 3 days (Nad). (4) Nad cultures supplemented with inhibitors (Nad + inhibitor). The cytotoxic activity in all cultures was tested using 111In-labeled BW-Sp3(B7-1) cells as targets at a 100:1 effector-target ratio. Results show the average 111In-release ± SD of triplicates. One exemplary experiment is shown, but all inhibitors have been tested in at least 3 independent experiments with comparable results. (B) After 3 days of restimulation, without inhibitors, plastic-adherent cells were recovered from the plate and incubated with 2 μM DHE for 1 hour or 2 μM DCFDA for 30 minutes at 37°C. After washing, cells were labeled with anti-CD11b/PE and analyzed. Histograms represent gated CD11b+ cells without dye (dotted line), or with either DHE or DCFDA (full line) from one experiment. The average percentage of DCFDA-positive (gate M1) ± SD from 3 independent experiments is given. (C) Similar experiment to that in panel A. Inhibitors used were as follows: baicalein (12/15 LOX), GW9662 (PPARγ). Agonists used were as follows: rosiglitazone (PPARγ), 15d-PGJ2 (PPARγ). One exemplary experiment of 4 is shown. Boxes represent average percentage of CTL recovery ± SD of 4 experiments. *P < .05, percentage of CTL recovery higher comparing Total + PPARγ ligands versus Total + baicalein; #P < .05, percentage of CTL recovery higher comparing Total + PPARγ ligands versus Total + GW9662. (D) Similar experiment as panel A. Inhibitor used was as follows: aristolochic acid (PLA2). Agonists used were as follows: rosiglitazone (PPARγ), 15d-PGJ2 (PPARγ). Again, one exemplary experiment of 4 is shown. Boxes represent average percentage of CTL recovery ± SD of 4 experiments.*P < .05, percentage CTL recovery higher comparing Total + aristolochic acid + PPARγ ligands versus Total + PPARγ ligands; #P < .05, percentage CTL recovery higher comparing Total + aristolochic acid + PPARγ ligands versus Total + aristolochic acid. (E) Similar experiment to that in panel A. Inhibitors used were as follows: AACOCF3 (= AA; cPLA2), PACOCF3 (= PA; iPLA2), ONO-RS-082 (= ONO; sPLA2). Agonist used was as follows: 15d-PGJ2 (PPARγ). One exemplary experiment of 2 is shown. Boxes represent average percentage CTL recovery ± SD of 2 experiments. *P < .05, percentage CTL recovery higher comparing Total + 15d-PGJ2 + ONO + AA + PA versus Total + 15d-PGJ2.

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