Mitochondria and TCR Signaling Figure 1. Potencies of various mitochondrial antagonists for inhibition of the pMV-absorption and their toxicities against 2C T cells. (A) Chemical structures of mitochondrial antagonists (1: Deguelin, 2: Piericidn, 3: Atpenin, 4: Antimycin, 5: Oligomycin, 6: FCCP, 7: Phenformin, 8: Metformin) are shown. (B) Assays for absorption of LdB7-1ICAM-1 pMVs were performed with 2C T cells pre-treated with titrated concentrations of respective mitochondrial antagonists for 1 hr. IC50 and IC80 were determined with mean fluorescence intensities (MFIs) of B7-1 staining of 2C T cells. Viabilities of 2C T cells were measured after treatment with respective mitochondrial antagonists at their IC80s for 1.5 hrs with propidium iodide (P.I.) and Annexin-V staining, respectively. Shown are viabilities of drug-treated 2C T cells relative to that of DMSO-treated 2C T cells. (C) Histograms represent B7-1 staining of 2C T cells left untreated (purple) or treated with DMSO alone for 1 hr followed by cultured with P1A- (orange) or QL9- loaded LdB7-1ICAM-1 pMVs in the presence (black) or absence (red) of anti-LFA-1 mAb. (D) Histograms represent B7-1 staining of 2C T cells pretreated with DMSO alone (black and red) or with respective mitochondrial antagonists at two separate concentrations (Orange: O., Purple: P.) as indicated and cultured for 30 min with QL9-loaded Ld B7-1ICAM-1 pMVs in the presence (black) or absence (red, orange and purple) of anti-LFA-1 mAb. doi:10.1371/journal.pone.0007738.g001 costimulatory receptor/ligand interactions (i.e., CD28/B7-1 and LFA-1/ICAM-1) also play prominent roles [27,28]. (Note: QL9 peptide forms a complex with Ld and the Ld/QL9 complex interacts with 2C TCR with high affinity [29].). The mitochondrial drug treatments (60 minutes) resulted in ubiquitous inhibition of phosphorylation of both PLC-c1 and Akt occurring upon culture of 2C T cells with QL9-loaded LdB7- 1ICAM-1 pMVs. The inhibition was obvious even after treatments of 2C T cells with the respective mitochondrial antagonists at concentrations near the IC50s (Figure 3A). The same treatments also inhibited the phosphorylation of Akt and PLC-c1 in 2C T cells cultured with pMVs expressing Ld alone in a dose-dependent manner (Figure 3B), assuring their effects on TCR signaling. Entry of extracellular Ca2+ was also attenuated by those treatments (Figure 3C). (Note: In reflection of the role of mitochondria as an intracellular Ca2+ store, the mitochondrial drug treatments themselves, without stimulation by the pMVs, resulted in changes in cytosolic Ca2+ concentration to a certain extent, shown by altered background fluorescence intensities of Indo-1-loaded 2C T cells.) Lack of Inhibition by the Mitochondrial Antagonists of F- Actin Polymerization F-actin content of 2C T cells increases quickly upon culture with QL9-loaded LdB7-1ICAM-1 pMVs. Different from phosphoryla- tion of Akt and PLC-c1 and Ca2+ entry, interaction of 2C TCR PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7738