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T Cell Activation Triggers Reversible Inosine-5′-Monophosphate Dehydrogenase Assembly Krisna C

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T Cell Activation Triggers Reversible Inosine-5′-Monophosphate Dehydrogenase Assembly Krisna C © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs223289. doi:10.1242/jcs.223289 SHORT REPORT T cell activation triggers reversible inosine-5′-monophosphate dehydrogenase assembly Krisna C. Duong-Ly1, Yin-Ming Kuo2,*, Matthew C. Johnson3, Joy M. Cote2, Justin M. Kollman3, Jonathan Soboloff4, Glenn F. Rall5, Andrew J. Andrews2 and Jeffrey R. Peterson1,‡ ABSTRACT levels in MPA-treated T cells rescues their proliferation (Quemeneur T cell-mediated adaptive immunity requires naïve, unstimulated T et al., 2003), demonstrating that production of guanine nucleotides cells to transition from a quiescent metabolic state into a highly is the primary role of IMPDH in T cell activation. Intriguingly, proliferative state upon T cell receptor engagement. This complex mTOR also regulates guanine nucleotide synthesis (Ben-Sahra process depends on transcriptional changes mediated by et al., 2016; Valvezan et al., 2017). 2+ Ca2+-dependent NFAT signaling, mTOR-mediated signaling and An early event in T cell activation is an increase in cytosolic Ca . 2+ increased activity of the guanine nucleotide biosynthetic inosine- TCR-triggered release of endoplasmic reticulum (ER) Ca is 5′-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 sensed by stromal interaction molecule 1 (STIM1), a protein that 2+ and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve mediates store-operated Ca entry and is critical for sustained 2+ as potent immunosuppressants. Unexpectedly, we discovered that all cytosolic Ca (Hogan et al., 2003; Gwack et al., 2007; Oh-Hora 2+ three pathways converge to promote the assembly of IMPDH protein et al., 2008; Srikanth and Gwack, 2013). Cytosolic Ca activates into micron-scale macromolecular filamentous structures in response calcineurin, which dephosphorylates nuclear factor of activated to T cell activation. Assembly is post-transcriptionally controlled by T cells (NFAT) leading to nuclear import. Indeed, calcineurin mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH inhibitors are potent immunosuppressants (Martinez-Martinez and assembly and catalytic activity were negatively regulated by guanine Redondo, 2004). Importantly, the mTOR, IMPDH and STIM1/ nucleotide levels, suggesting a negative feedback loop that limits calcineurin pathways are thought to mediate the immune response biosynthesis of guanine nucleotides. Filamentous IMPDH may be via non-overlapping mechanisms. Here, we present evidence that more resistant to this inhibition, facilitating accumulation of the higher these pathways converge to promote the TCR-mediated assembly of GTP levels required for T cell proliferation. a macromolecular structure containing IMPDH. IMPDH is the rate-limiting enzyme in guanine nucleotide KEY WORDS: IMP dehydrogenase, Metabolism, T cell activation biosynthesis and is dramatically upregulated by T cell activation (Dayton et al., 1994). IMPDH assembles into micron-scale structures INTRODUCTION in cultured cells, generally in response to pharmacological T cell receptor (TCR) stimulation initiates events leading to T cell perturbations (Ji et al., 2006; Calise et al., 2014, 2016; Carcamo activation, proliferation and differentiation. This core process of et al.,2014; Keppeke et al., 2015; Zhao et al., 2015). The physiological adaptive immunity is associated with dramatic metabolic alterations relevance of these filamentous assemblies is unknown, although required for T cell proliferation (MacIver et al., 2013; Buck et al., they are reported to be catalytically active (Chang et al., 2015; Zhao 2015; Almeida et al., 2016; Bantug et al., 2018). Indeed, several et al., 2015; Anthony et al., 2017). We report that IMPDH assembles immunosuppressive agents target these metabolic dependencies. into filaments in T cells both in vivo and ex vivo in response to TCR For example, inhibitors targeting mechanistic target of rapamycin engagement. Unexpectedly, we found that assembly, but not (mTOR) and an inhibitor of the inosine-5′-monophosphate (IMP) upregulated expression, of IMPDH was dependent on STIM1 and dehydrogenase 1 and 2 (IMPDH1 and IMPDH2, hereafter IMPDH) mTOR. Thus, IMPDH regulation is a common thread linking the enzymes mycophenolic acid (MPA) are used to suppress organ pathways targeted by three major classes of immunosuppressive transplant rejection, although how they function is not fully drugs, suggesting that IMPDH assembly serves an essential function understood (Allison and Eugui, 1996; Thomson et al., 2009; in T cell activation by supporting guanine nucleotide production. Nguyen le et al., 2015). However, restoring guanine nucleotide RESULTS AND DISCUSSION TCR stimulation promotes IMPDH assembly in 1 Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. T lymphocytes 2Cancer Epigenetics Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. 3Department of Biochemistry, University of Washington, Seattle, WA 98195, Murine splenic T cells were isolated and activated using antibodies USA. 4Fels Institute for Cancer Research and Molecular Biology, Temple University against the TCR co-receptors CD3ε and CD28 (Fig. 1A). Strikingly, School of Medicine, Philadelphia, PA 19140, USA. 5Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. IMPDH assembled into linear assemblies and toroids in the vast *Present address: Department of Radiology, University of Pennsylvania School of majority of T cells within 24 h (Fig. 1A,B). Refinement of the T cell Medicine, Philadelphia, PA 19104, USA. population into CD4+ and CD8+ subsets by fluorescence-activated ‡Author for correspondence ([email protected]) cell sorting (FACS) revealed IMPDH filaments in both subsets (Fig. S1A). Filament assembly was accompanied by a dramatic K.C.D., 0000-0002-4593-8145; Y.-M.K., 0000-0002-3036-1902; J.M.K., 0000- increase in IMPDH protein levels (Fig. 1C,D) demonstrating that 0002-0350-5827; J.S., 0000-0001-5192-1297; G.F.R., 0000-0003-2717-3238; A.J.A., 0000-0002-2529-3714; J.R.P., 0000-0002-0604-718X increased IMPDH expression and filament assembly are direct downstream consequences of TCR activation and establishing a Received 1 August 2018; Accepted 6 August 2018 system to analyze these processes in vitro. Journal of Cell Science 1 SHORT REPORT Journal of Cell Science (2018) 131, jcs223289. doi:10.1242/jcs.223289 Fig. 1. Ex vivo TCR stimulation promotes IMPDH protein expression and filament assembly. (A) Immunofluorescence images of IMPDH (green) in murine splenic T cells either stimulated overnight with anti-CD3ε and anti-CD28 antibodies or left unstimulated. Nuclei were stained with DAPI (blue). (B) Quantification of the mean±s.e.m. proportion of cells containing IMPDH filaments from three biological replicates (n=100). (C) Western blot of IMPDH protein expression (representative of eight biological replicates). GAPDH was used as a loading control. (D) FACS quantification of IMPDH expression in T cells (representative of three biological replicates). (E) Immunocompetent C57BL/6 mice were infected intraperitoneally with LCMV-Armstrong virus. At the indicated times post-infection, splenic T cells were immunostained for IMPDH. White arrows highlight IMPDH filaments. (F) Western blot of IMPDH protein expression in total splenic T cells isolated from uninfected mice or from mice 7 days after infection. Scale bars: 5 µm. recognizing LCMV antigens become activated and proliferate. Following the resolution of infection, ∼95% of activated T cells undergo apoptosis and surviving memory T cells confer protection against future LCMV infection (Murali-Krishna et al., 1998). We infected mice with LCMV for 7 days, a time of peak anti-viral CD8+ T cell cytotoxicity (Hassett et al., 2000; Knipe and Howley, 2013), and isolated splenic T cells. Immunostaining revealed IMPDH filaments that were absent in cells from uninfected mice (Fig. 1E). Western blotting revealed a 3-fold increase in IMPDH protein levels in total splenic T cells from LCMV-challenged versus control mice (Fig. 1F). To ask whether IMPDH filaments persist in memory T cells, CD69+ T cells (representing a mixed population of both memory T cells and activated T cells) were isolated by FACS at 30 days post-infection. No IMPDH filaments were observed in these CD69+ T cells (Fig. S1B), demonstrating that the transient IMPDH filament assembly during initial activation does not persist in quiescent memory cells. STIM1 and mTOR regulate IMPDH filament assembly To elucidate signaling mechanisms controlling IMPDH assembly, we compared IMPDH filament formation and expression in splenic T cells isolated from mice with a T cell-specific knockout of STIM1 (STIM1fl/fl/Cd4-Cre) and control mice (STIM1fl/fl) (Oh-Hora et al., 2008). There was a strong reduction of IMPDH filament formation in STIM1-deficient cells compared to control (Fig. 2A,B). STIM1 loss had only moderate effects on IMPDH protein expression, indicating that STIM1 is not primarily responsible for IMPDH upregulation (Fig. 2C); this demonstrates that IMPDH assembly is not a simple consequence of IMPDH upregulation, implying that assembly relies on a post-translational Ca2+-dependent regulatory mechanism. Interestingly, IMPDH expression and assembly occurred normally in T cells isolated from STIM2-deficient mice (Fig. S2), demonstrating a critical role for STIM1 specifically in IMPDH filament assembly. mTOR is a master regulator of diverse metabolic pathways during T cell activation (Chi, 2012; MacIver et al., 2013).
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