mitochondria act as an intracellular Ca2+ buffer prolonging the Ca2+ entry and thereby potentiating downstream signaling cascades [12]. T lymphocytes absorb APC-derived nanometric membrane vesicles, naturally-occurring exosome-like membrane vesicles (eMVs) or artificially prepared plasma membrane-derived mem- brane vesicles (pMVs), via dual receptor/ligand interactions of TCR/pMHC plus LFA-1/ICAM-1[13,14]. Studies using 2C TCR transgenic (Tg) T cells along with pMVs prepared from artificial APCs, Drosophila (Dros) cells expressing Ld (a mouse class I MHC) plus mouse ICAM-1 and B7-1, also have shown that the vesicle absorption requires intracellular signaling events. The signaling mechanism commences as 2C TCR interacts with cognate pMHCs expressed in the pMVs and is thought to be critical for promoting LFA-1 function [15]. Applying those findings, we had established a cell-based flow cytometric high throughput screening (HTS) platform for isolation of small molecules causing inhibition of 2C T cell absorption of LdB7-1ICAM-1 pMVs, which can be exerted by either physical interference of TCR/pMHC or LFA-1/ICAM-1 interaction or disruption of TCR-mediated ‘inside-out’ signaling [15]. As an effort to validate the practicality and effectiveness of the HTS platform, we had carried out a pilot-scale HTS campaign with a commercial library composed of both natural and synthetic molecules whose biological functions are known to differential extents and found that molecules known to antagonize the mitochondrial function (oxidative phosphorylation) inhibited the pMV absorption. In this study, we investigated effects of mitochondrial drug treatments on various membrane proximal TCR signaling events and assessed the potential use of those molecules as immune modulators. Results Inhibition by Various Mitochondrial Antagonists of 2C T cell Absorption of LdB7-1ICAM-1 pMVs Exploiting the simplicity and statistical accuracy of the assay for 2C T cell absorption of LdB7-1ICAM-1 pMVs, we had established a HTS platform for isolation of small molecules interfering with the pMV-absorption and screened a commercial library composed of natural and synthetic molecules whose biological functions are known to differential extents [15]. A intriguing finding from the pilot-scale HTS campaign was that molecules known to antagonize mitochondrial function (i.e., oxidative phosphorylation) were isolated as hits; a group of rotenoids represented by Deguelin (a Complex I inhibitor) [16] and Antimycin (a Complex III inhibitor) [17]. IC50s of Deguelin and Antimycin were determined at 300 and 5 nM, respectively, and toxicities of those molecules against 2C T cells were not detected when measured after the assay by propidium iodide exclusion method. Those results prompted us to examine other mitochondrial antagonists with distinct mechanisms of action; Piericidin (a Complex I inhibitor) [18], Atpenin (a Complex II inhibitor) [19], Oligomycin (a Complex V inhibitor) [20] and FCCP (an H+ decoupling agent) [21]. As summarized in Figure 1, all of those compounds strongly inhibited the pMV-absorption without reveal- ing noticeable toxicity against 2C T cells as measured by either propidium iodide exclusion or Annexin V staining. Flow cytometry data showing the pattern of inhibition by the mitochondrial antagonists of the pMV-absorption are presented in Figure 1D. It is perceived that a class of anti-diabetic drugs, i.e., biguanides such as Metformin and Phenformin, exert their pharmacological activities via interruption of mitochondrial respiratory electron Mitochondria and TCR Signaling transport chain; likely via inhibition of Complex I [22,23]. As other mitochondrial antagonists, both Metformin and Phenformin also inhibited the pMV-absorption (Figure 1). Metabolic Changes Effected by the Mitochondrial Drug Treatments As an effort to understand underlying mechanism(s) for the inhibition by the mitochondrial antagonists of the pMV-absorp- tion by 2C T cells, we first examined metabolic changes resulting from the drug treatments. Effects of the mitochondrial drug treatments on cellular energy balance, defined as ratio of ([AMP] + [ADP]) to entire pool of adenine nucleotides ([AMP] + [ADP] + [ATP]), were examined measuring relative concentrations of respective adenine nucleotides using HPLC. (Note: HPLC elution chromatograms of Deguelin-treated samples are presented as examples in Figure S1.) Treatments (75 minutes) of 2C T cells with the respective mitochondrial antagonists at concentrations near their IC80s for the pMV-absorption (Deguelin: 750 nM, Atpenin: 3000 nM, Antimycin: 12.5 nM, Oligomycin: 5 nM, FCCP: 2500 nM, Phenformin: 2000000 nM) ubiquitously increased the cellular energy balance by decreasing the concentration of ATP and increasing concentrations of ADP and AMP. The maximum change was observed when the cells were treated with Deguelin at 750 nM; as a result, ratio of the concentration ATP to that of total adenine nucleotides dropped from 88% to 63% (Figure 2A). When the cells were treated with the respective drugs at concentrations near the IC50s for the pMV-absorption (Deguelin: 300 nM, Atpenin: 1200 nM, Antimycin: 5 nM, Oligomycin: 2 nM, FCCP: 1000 nM, Phenformin: 80000 nM), however, changes in the cellular energy balance were hardly detected, except for Deguelin and Phenformin treatments. Even in those cases, the ratio of the concentration of ATP to that of total adenine nucleotides dropped only by about 4% and increase in the concentration of AMP was not detected (Figure 2A). The mitochondrial drug treatments resulted in activation of AMPKa1; AMPKa1 is an isotype of AMPK predominantly expressed in T cells [24]. Thus, phosphorylation of AMPKa1 at threonine 172, which is critical for its enzymatic activity [25], was ubiquitously detected in dose-dependent manner after treatments (75 minutes) of 2C T cells with the respective mitochondrial antagonists. The phosphorylation of AMPKa1 was accompanied by the phosphorylation of acetyl-CoA carboxylase (ACC), an enzyme involved in lipid biosynthesis and one of the best known cellular targets of AMPK (Figure 2B) [8]. The mitochondrial drug treatments had effects on intracellular SO level as well (Figure 2C). Notably, however, the drug effects showed disparate and appeared to be related with the target of the molecules; thus, treatments with Complex I and II inhibitors (Deguelin and Atpenin, respectively) resulted in a noticeable increase in the intracellular SO level while treatments with a Complex V inhibitor (Oligomycin) and a H+ decoupling agent (FCCP) resulted in a slight decrease in the SO level. The effects of Antimycin and Phenformin treatments were neutral. Effects of the Mitochondrial Drug Treatments on Activation of Akt and PLC-c1 and Entry of Extracellular Ca2+ Various signaling events proceed rapidly upon contact of 2C T cells with QL9-loaded LdB7-1ICAM-1 pMVs, which include phosphorylation of Akt and phopsholipase C-c1 (PLC-c1) and entry of extracellular Ca2+ [15,26]. While interaction of 2C TCR with Ld/QL9 complex is indispensable for those signaling events, PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7738