viability of resting lymph node T cells and 2C T cells cultured with the pMVs following longer treatments with the mitochondrial antagonists at the concentrations where the pMV absorption by 2C T cells was affected to measurable extents; i.e., concentrations near the IC80s and IC50s for the pMV absorption (Figure 6). An important finding from the experiments shown in Figure 6 is, as manifested by treatments with Deguelin, that mitochondrial drug treatments at carefully controlled conditions may selectively inhibit activation and proliferation of antigen-stimulated T cells without compromising viability of neighboring resting T cells. While it is yet to be understood how the mitochondrial drug treatments slow the process of T cell activation and proliferation, the retardation of the blast formation by the drug treatments of 2C T cells cultured with the QL9-loaded pMVs appeared in line with the observation that the treatments interfered with dynamic reorganization of 2C T cells upon culture with the pMVs (Figure 5). In order for certain compounds to be used as T cell immune modulators, it is desirable for them to show little toxicity against resting T cells in lymph nodes and spleen at the concentrations where they exert anti-proliferative effects on T cells undergoing activation process. In that regard, the results obtained with Deguelin are particularly encouraging. Here, studies having revealed cancer chemo-preventive effect of Deguelin are worth noting [43]. It is also of interest that chemo-preventive and anti- inflammatory effects of various oriental medicines and herbs are at least in part exerted by ingredients known to have an activity of antagonizing mitochondrial function [44,45,46]. Materials and Methods Ethics Statement All procedures met the guidelines of the National Institutes of Health ‘‘Guide for the Care and Use of Laboratory Animals’’. Animals C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). 2C TCR Tg mice [47] were bred in the vivarium of The Scripps Research Institute (TSRI). Cell Lines and Culture Medium Dros APCs were maintained in Schneider’s medium supple- mented with 10% fetal bovine serum (FBS) plus antibiotics and glutamine as described [48]. Short-term T cell cultures were performed in DMEM medium (Invitrogen) supplemented with 10% heat inactivated (HI) FBS plus antibiotics and glutamine. T cell proliferation assays were performed in RPMI medium supplemented with 10% HI FBS, 10 mM HEPES (pH 7.2), antibiotics, glutamine and 561025 M 2-mercaptoethanol. Peptides, Chemicals and Antibodies QL9 (QLSPFPFDL) and P1A (LPYLGWLVF) [48] peptides were purchased from Invitrogen. Rotenone, Deguelin, Piericidin, Antimycin, Oligomycin, FCCP, Metformin and Phenformin were purchased from Sigma-Aldrich. Atpenin (A5) was purchased from Alexis Biochemicals (Farming- dale, NY). PP2 and Wortmannin were purchased from EMD Bioscience. Carboxyfluorescein succinimidyl ester (CFSE), Indo- 1/AM, Dihydroethidium and Annexine V apoptosis detection kit were purchased from Invitrogen. Anti-CD11a (M17/4), Alexa 488-conjugated Thy-1 (30-H12), PE-conjugated anti-B7-1 (16-10A1) and Alexa 647-conjugated anti-CD8 (53–6.7) were purchased from Biolegend (San Diego, CA). FITC-labeled Phalloidin was purchased from Sigma-Aldrich. Mitochondria and TCR Signaling Anti-CD8 (3.168) mAb was prepared as an ascites form (a gift from Dr. Surh in Department of Immunology at TSRI). Polyclonal antibodies directed to pan-Akt, phospho-Akt (Thr308), phospholi- pase C-c1 (PLC-c1), phospho-PLC-c1 (Tyr783), AMPKa1, phospho-AMPKa1 (Thr172), Acetyl-CoA Carboxylase (ACC) and phospho-ACC (Ser79) were purchased from Cell Signaling Technology. Horse radish peroxidase (HRP)-tagged goat anti- rabbit IgG Ab was purchased from Santa Cruz Biotechnology. Preparation of Dros pMVs PMVs were prepared from Dros APCs expressing Ld alone or Ld plus B7-1 and ICAM-1 as described previously [14,15]. Purification of 2C TCR Tg T Cells 2C T cells were negatively purified from pooled lymph nodes using CD8a+ T cell isolation MACSTM kit (Miltenyi Biotech). If needed, the MACS purified 2C T cells were purified further by panning on anti-CD8 mAb (3.168)-coated plates to isolate CD8+ 2C T cells. Assay for 2C T Cell Absorption of LdB7-1ICAM-1 pMVs The pMV-absorption assays were performed as described (15). Briefly, 45 ml of purified 2C T cells (4.56105) in a pre-warmed culture medium (DMEM medium) were mixed with 5 ml of the peptide-loaded LdB7-1ICAM-1 pMVs (0.5 mg/ml) in round- bottom 96 well plate and cultured for 30 min in a 37uC humidified CO2 incubator. After culture, PE-conjugated anti-B7-1 plus Alexa 647-conjugated anti-CD8 mAbs were added and the plate was incubated on ice for 20 min. The plate was then filled with ice-cold FACS buffer (1X PBS, 1% BSA, 2.5% horse serum, 2 mM EDTA) and spun down for cell washing. The T cells were resuspended in the FACS buffer containing propidium iodide (PI) and immediately analyzed by flow cytometry. For compound testing, 2C T cells were incubated with 0.5 ml of respective compounds in DMSO for 60 min at 37uC prior to culture with the pMVs. Cell Viability Assays Annexin V staining was performed following manufacturer’s manual with purified 2C T cells treated with titrated concentra- tions of respective mitochondrial antagonists for 90 min. Effects of long-term drug treatments on viability of T cells were examined as follows. Ex vivo purified whole body lymph node cells (B6) (16106) in 200 ml of RPMI medium were treated with titrated concentrations of respective mitochondrial antagonists for 24 hrs in a 37uC humidified CO2 incubator. The cells were then stained with Alexa 488-conjugated anti-Thy-1 mAb plus P.I. (5 mg/ml) and analyzed by flow cytometry. HPLC Analysis of Adenine Nucleotides Purified 2C T cells (36106) in 1.5 ml of pre-warmed culture medium (DMEM medium) were treated with titrated concentra- tions of respective mitochondrial antagonists for 80 min at 37uC. Then, the cells were washed and lyzed in 250 ml of 10% ice-cold trichloroacetic acid. The cell lysates were spun down to remove cell debris and the supernatants were neutralized with Freon/n- octylamine mix (3:1). The supernatant-Freon mixtures were spun down to separate the phases. Adenine nucleotides in the aqueous phase were then analyzed by HPLC using anion exchange column (Whatman Partisil 10-SAX column). After injection of 100 ml of sample, adenine nucleotides were eluted by linear gradient of 5 mM ammonium dihydrogen phosphate at pH 3.2 and 750 mM ammonium dihydrogen phosphate at pH 4.0 at a flow rate of 1 mL/min for 50 min [49]. PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e7738