Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists Kwangmi Kim.¤, Lin Wang., Inkyu Hwang* Department of Chemistry and Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America Abstract T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane- proximal TCR signaling event(s). Thus, activation of Akt and PLC-c1 and entry of extracellular Ca2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function. Citation: Kim K, Wang L, Hwang I (2009) Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists. PLoS ONE 4(11): e7738. doi:10.1371/journal.pone.0007738 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received September 9, 2009; Accepted October 9, 2009; Published November 10, 2009 Copyright: 2009 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was received from the National Institutes of Health (AI066146). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: inkyu@scripps.edu ¤ Current address: Amorepacific Corporation, R&D Center,Yongin-si, Gyeonggi-do, South Korea . These authors contributed equally to this work. Introduction T cell activation, a series of physiological changes leading to clonal expansion and development of effector functions, com- mences as T cell receptor (TCR) encounters a cognate peptide in the context of major histocompatibility complex (MHC) presented by specialized immune cells called antigen presenting cells (APCs). The interaction of TCR with cognate peptide/MHC complex (pMHC) triggers a host of intracellular signaling cascades leading to cell cycle progression [1]. Despite crucial, TCR/pMHC interaction is generally insufficient for the productive T cell activation, for which accessory (costimulatory) receptor/ligand interactions, typified by CD28/B7-1 and lymphocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1), are required [2,3]. LFA-1, a member of b2 integrin family, plays multiple roles in T cell immunity both as an adhesion and a signaling molecule [4]. Thus, LFA-1/ICAM-1 interaction not only promotes firm and stable T/APC interaction but also triggers signaling cascades for T cell activation. The functional property of LFA-1 is carefully regulated during T cell immunity via transition from low affinity/ avidity state to high affinity/avidity state and vise versa. The functionality of LFA-1 is controlled by a signaling mechanism, called ‘‘inside-out’’ signal, elicited by distinct neighboring receptors such as TCR and chemokine receptors [5,6]. Mitochondria are a powerhouse of cells generating ATP via oxidative phosphorylation/respiratory electron transport system. The rate of ATP production is affected by various environmental factors such as oxygen stress (e.g., hypoxia), nutritional condition and molecules interfering with oxidative phosphorylation [7]. Thus, it is necessary to control the rate of ATP consumption (e.g., anabolic metabolism for cell cycle progression) in response to those environmental changes to preserve the pool of ATP needed for basal cell metabolism. Here, a mechanism for sensing the cellular energy level is indispensible and AMP-activated protein kinase (AMPK) plays the role [8]. Mitochondria also emerge as a center for control over cell signaling. Reactive oxygen species (ROS) (e.g., O22 and H2O2) and reactive nitrogen species (RNS) (e.g., NO) play a role in cell signaling as second messengers [9]. Superoxide (SO), i.e., O22, is produced as a byproduct of oxidative phosphorylation [10]. NO is also produced in mitochondria by mitochondrial NO synthase (mtNOS) [11]. In addition, importance of mitochondria in receptor-mediated extracellular Ca2+ entry has been revealed; PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7738