cultured with the P1A-loaded pMVs (Figure 5). (Note: Figure 5 shows microscopic images of 2C T cells treated with DMSO alone, Deguelin and Antimycin, respectively, as representative examples. 2C T cells treated with other mitochondrial antagonists also showed the similar pattern.). Of interest, however, 2C T cells treated with the respective mitochondrial antagonists showed relatively even distribution of cortical F-actin along the cell surface compared with 2C T cells treated with DMSO alone. In addition, membrane protrusion and morphological changes, apparent in DMSO-treated 2C T cells, were not evident in mitochondrial drug-treated T cells, indicating that dynamic rearrangement of F-actin following F-actin poly- merization was impaired by the mitochondrial drug treatments (Figure 5). Effects of Long-Term Mitochondrial Drug Treatments on Viability of Resting T Cells TCR signaling is crucial not only for activation/proliferation of T cells encountering cognate foreign antigens but also for survival of resting T cells [31]; TCR signaling for the latter is instigated by interaction of TCR with specific self peptide-MHC complexes. The data that the mitochondrial drug treatments acutely inhibited specific membrane-proximal TCR signaling events prompted us to examine effects of the long-term drug treatments on viability and activation/proliferation of T cells. We first examined their effects on viability of resting primary T cells using ex vivo purified whole lymph node cells (Figure 6A). Treatments (24 hours) of the lymph node cells with the respective mitochondrial antagonists at concentrations near the IC80s for the pMV-absorption (Deguelin: 750 nM, Atpenin: 3000 nM, Anti- mycin: 12.5 nM, Oligomycin: 5 nM, FCCP: 2500 nM) resulted in sizeable decrease in viability of the T cells. Thus, only 20–33% of T cells survived, depending on the mitochondrial antagonist treated, while 67% of the T cells survived when they were treated with DMSO alone. Noticeable increases in viability of the T cells were observed when the lymph node cells were treated at concentrations near the IC50s for the pMV-absorption (Deguelin: 300 nM, Piericidin: 2 Mitochondria and TCR Signaling nM, Atpenin: 1200 nM, Antimycin: 5 nM, Oligomycin: 2 nM, FCCP: 1000 nM). Nevertheless, viabilities of the drug-treated T cells were still lower than that of DMSO-treated cells; approxi- mately 35–50% of the T cells survived after the culture, depending on the mitochondrial antagonist treated (Figure 6A). When the lymph node cells were treated at the concentrations 2–3 fold lower than the IC50s (Deguelin: 120 nM, Piericidin: 0.8 nM, Atpenin: 480 nM, Antimycin: 2 nM, Oligomycin: 0.8 nM, FCCP: 400 nM), viabilities of the T cells improved further to the level comparable to that of the DMSO-treated cells; viabilities of the drug-treated T cells, except for Atpenin and Oligomycin-treated cells, were close to or above 90% of that of the DMSO-treated cells (Figure 6A). Effects of Long-Term Mitochondrial Drug Treatments on Activation and Proliferation of Antigen-Stimulated T Cells 2C T cells undergo robust cell proliferation when they are cultured with nanometric membrane vesicles expressing cognate pMHC (i.e., Ld/QL9 or Ld/p2Ca) plus costimulatory ligands (i.e., B7-1 and ICAM-1); the cell division begins to occur after two days of culture and continues up to three days [13,14]. We examined effects of the mitochondrial drug treatments on the activation (blast formation) and proliferation (CFSE dilution) of 2C T cells by QL9-loaded LdB7-1ICAM-1 pMVs (Figure 6B and C and Figure S3). When the cells were cultured for 24 hours with the pMVs in the presence of the respective mitochondrial antagonists at concen- trations near the IC80s for the pMV-absorption, increased population of 2C T cells died during the culture; approximately 35–45% of the drug-treated T cells survived depending on the mitochondrial drug used in the experiment while about 65% of the DMSO-treated cells survived. Average sizes of the surviving drug- treated 2C T cells were larger than that of 2C T cells cultured with the P1A-loaded pMVs but smaller than that of 2C T cells cultured with QL9-loaded pMVs in the presence of DMSO alone (Figure 6B and S3). The drug treatments completely inhibited the proliferation of 2C T cells; thus, the surviving drug-treated 2C T cells gradually died without cell division (CFSE dilution) as the culture time extended (Figure 6C and Figure S3). (Note: FCCP Figure 5. Impairment of dynamic rearrangement of F-actin by the mitochondrial drug treatments. 2C T cells, pre-treated for 60 min with the respective mitochondrial antagonists (or DMSO alone) at concentrations as in Figure 4 were cultured for 20 min with the peptide-loaded LdB7- 1ICAM-1 pMVs on poly-L-lysine-coated coverslips followed by F-actin staining. Confocal microscopic images of fluorescence F-actin staining and differential microscopic contrast (DIC) of 2C T cells treated with DMSO, Deguelin and Antimycin, respectively, are shown as representative examples. doi:10.1371/journal.pone.0007738.g005 PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7738