Figure 6. Effects of long-term mitochondrial drug treatments on viability and activation of T cells. (A) Ex vivo purified whole lymph node cells (B6) were treated for 24 hrs with titrated concentra- tions of the respective mitochondrial antagonists. Viabilities of the T cells in the culture were determined based on P.I. exclusion and compared with that of the T cells treated with DMSO alone (1% V/V). (B) Shown are P.I. staining and sizes (forward scatter, FSC) of 2C T cells analyzed after culture for 24 hrs with the peptide-loaded LdB7-1ICAM-1 pMVs in the presence of DMSO alone or titrated concentrations of Deguelin as indicated. Percentages of live (gated P.I.-low) cells and their mean FSC are denoted inside plots; percentage of live cells/mean FSC. (C) Purified CFSE-labeled CD8+ 2C T cells were cultured with P1A or QL9-loaded LdB7-1ICAM-1 pMVs in the presence of the DMSO alone or titrated concentrations of Deguelin as indicated for up to 3 days. Extents of cell proliferation judged by CFSE-dilution were analyzed daily by flow cytometry. Data obtained after treatments with other mitochondrial antagonists are also shown in Figure S3. doi:10.1371/journal.pone.0007738.g006 Mitochondria and TCR Signaling treatment, as shown in Figure S3, was the exception in that a population of surviving 2C T cells formed the blast to a greater extent and underwent several rounds of cell division.). Significant improvements in cell viability and blast formation were found when 2C T cells were cultured for 24 hours with the respective mitochondrial antagonists at concentrations near the IC50s for the pMV-absorption (Figure 6B and Figure S3). The effects of the drug treatments were still apparent, however, so that only 50–55% of 2C T cells survived after the culture and the average sizes of the surviving cells were still considerably smaller than that of the DMSO-treated cells. Different from the 2C T cells surviving the 24 hour treatments at concentrations near the IC80s for the pMV absorption, a significant population of 2C T cells surviving the treatments at concentrations near the IC50s entered cell division cycle to proliferate. Nonetheless, the cell cycle progression was greatly impeded; thus the drug-treated cells underwent one cell division at most after 2 days of the culture while the DMSO-treated cells underwent up to three cell divisions. When the cells were examined after three days of the culture, the DMSO-treated cells underwent more than six cell divisions almost completely diluting out the CFSE while the drug-treated cells underwent less than four cell divisions (Figure 6C and Figure S3). When 2C T cells were cultured with QL9-loaded LdB7- 1ICAM-1 pMVs with the respective mitochondrial antagonists at concentrations 2–3 fold lower than the IC50s for the pMV- absorption, viabilities of 2C T cells increased further to the level comparable to that of the DMSO-treated cells (Figure 6B and C and Figure S3). In contrast, blast formation of 2C T cells was still significantly affected by the drug treatments, except for Antimycin and FCCP treatments. In parallel, proliferation of 2C T cells was also found to be impeded by those treatments. The effects of Deguelin and Atpenin were particularly noticeable; thus 2C T cells treated with each of those compounds underwent only 1 and 3–4 cell divisions after 2 and 3 days of the culture, respectively. Discussion While 2C T cell absorption of LdB7-1ICAM-1 pMVs proceeds via complex mechanisms, the assay for measuring extents of the pMV-absorption is simple, expeditious and statistically accurate. In addition, considering that primary resting T cells isolated from lymph nodes of TCR Tg mice along with physiological ligands expressed on biological membrane are used, results obtained using the assay may hold superior physiological relevance. Those features make the assay system ideal for systematic investigation of cellular/molecular mechanisms underlying the pMV-absorp- tion, namely structural basis for TCR/pMHC and LFA-1/ICAM- 1 interactions and ‘inside-out’ signaling for TCR-mediated LFA-1 activation, via chemical genetics approach [15]. Applying the assay, we had established a cell-based flow cytometric HTS platform and screened a small molecule library to validate the system [15]. One of the intriguing findings from the pilot-scale HTS campaign was that various mitochondrial antagonists with distinct mechanisms of action ubiquitously inhibited the pMV-absorption without compromising the viability of 2C T cells (Figure 1). The acute inhibition of the pMV absorption by the mitochondrial antagonists suggested an unconventional role of mitochondria of regulating receptor- mediated membrane-proximal signaling cascades, e.g., TCR- mediated ‘inside-out’ signaling. One may argue that the mitochondrial antagonists inhibit the pMV-absorption by depleting the pool of ATP and thereby suppressing activities of signaling molecules using ATP as a substrate, e.g., kinases. According to results from the HPLC PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7738