Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions

Molecular Immunology 54 (2013) 355–367

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Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions夽 Gang Chen a,∗,1 , Sandip Panicker a,1 , Kai-Yeung Lau b , Subramaniam Apparsundaram a , Vaishali A. Patel a , Shiow-Ling Chen a , Rothschild Soto a , Jimmy K.C. Jung c , Palanikumar Ravindran b , Dayne Okuhara d , Gary Bohnert d , Qinglin Che d , Patricia E. Rao d , John D. Allard c , Laura Badi c , Hans-Marcus Bitter b , Philip A. Nunn a , Satwant K. Narula a , Julie A. DeMartino a a

Inflammation Discovery, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA RNA Biomarker Technology, Translational Research Science, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA c BEDA, Translational Research Science, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA d Synta Pharmaceutical Corp., 45 Hartwell Avenue, Lexington, MA 02421, USA b

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Article history: Received 15 August 2012 Received in revised form 7 December 2012 Accepted 14 December 2012 Available online 26 January 2013 Keywords: CRAC T cell Gene expression

a b s t r a c t Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3 -dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction The activation of T lymphocytes through the TCR triggers a series of signaling events that leads to an increase of intracellular calcium (Ca2+ ), activation of the NFAT pathway, subsequent cytokine production and ultimately T cell proliferation (Altman et al., 1990). The rise in intracellular Ca2+ is a result of store-operated Ca2+ entry (SOCE), a mechanism that triggers Ca2+ influx following depletion of Ca2+ from intracellular stores such as the endoplasmic reticulum (ER). SOCE in T lymphocytes is mediated by the direct interaction of

Abbreviations: SOCE, store operated calcium entry; CRAC, Calcium Release Activated Calcium; GSEA, Gene Set Enrichment Analysis. 夽 This work was solely supported by Hoffmann-La Roche Inc. ∗ Corresponding author. Tel.: +1 973 235 2197; fax: +1 973 235 5430. E-mail address: [email protected] (G. Chen). 1 These two authors contribute equally. 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.12.011

the Ca2+ sensor STIM1 on the ER membrane with the Orai subunits of the CRAC channel on the plasma membrane (Feske et al., 2006; Lioudyno et al., 2008; Roos et al., 2005; Vig et al., 2006; Zhang et al., 2005). Three homologous subunits, Orai1, Orai2 and Orai3, have each been shown to function as homomeric channels in recombinant systems, but can also form heteromultimeric assemblies (Lis et al., 2007). While the subunit composition of the native CRAC channel is not fully understood, functional Orai1 and STIM1 are essential for CRAC channel activity in T lymphocytes (Feske et al., 2006; Lioudyno et al., 2008; Ohga et al., 2008; Roos et al., 2005). A subset of human severe combined immune deficiency (SCID) patients display impaired Ca2+ -NFAT pathways and defective SOCE (Feske et al., 2000a,b, 2001, 2005; Vig et al., 2006). Recent studies have demonstrated that loss-of-function mutations in Orai1 or STIM1 genes are responsible for SOCE defects in these patients (Bergmeier et al., 2009; Byun et al., 2010; Feske, 2010; Feske et al., 2012; McCarl et al., 2009; Picard et al., 2009). Human T cells also express various K+ channels, including a calcium-activated K+

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channel (KCa3.1) and a voltage-gated K+ channel (Kv1.3), both of which have been shown to co-localize with the TCR-CD3 complex in lipid rafts upon TCR activation (Fanger et al., 2001; Grgic et al., 2009; Nicolaou et al., 2007; Panyi et al., 2003, 2004a). Activation of both K+ channels is tightly regulated and critically regulates CRAC current. K+ efflux mediated by these two channels is responsible for maintaining a hyperpolarized membrane potential which provides a driving force for continuous Ca2+ influx through the CRAC channel (Panyi et al., 2004b; Wulff et al., 2003). Given the critical role of T lymphocytes in human autoimmune diseases, blockade of the Ca2+ –calcineurin–NFAT pathway has long been considered an attractive therapeutic approach in patients with autoimmune disorders or in transplant recipients. Calcineurin inhibitors, such as Cyclosporin A (CsA) and FK506, are potent immunosuppressants of the ICRAC -NFAT pathway and have been widely used to prevent transplant rejection and treat autoimmune diseases. However, their usage is limited due in part to the expression of calcineurin outside of the immune system, a consequence of which may lead to significant nephrotoxicity (Platz et al., 1995). As the dominant Ca2+ channel in human T, B and mast cells, CRAC has been proposed as another attractive therapeutic target for immune modulation in this clinically validated pathway. Based on the phenotype of the SCID patients, inhibition of CRAC may avoid the kidney and liver toxicities associated with calcineurin blockade, but proof of this awaits the development of a suitable, specific, small molecule inhibitor of the CRAC channel. The potential of a CRAC inhibitor for control of autoimmunity has been demonstrated using Synta 66 to potently block cytokine production by IBD lamina propria mononuclear cells (LPMC) and intestinal biopsy tissue samples stimulated in vitro (Di Sabatino et al., 2009; Ng et al., 2008). Another CRAC inhibitor, YM-58483 (also known as BTP2) has been shown to be efficacious in blocking IL-2 production, NFAT activation, antigen-induced asthmatic response and Th2 cytokine production (Ishikawa et al., 2003; Yoshino et al., 2007). Later studies with YM-58483 however found it had pleiotropic effects on multiple TRP channels found on T cells in addition to the CRAC channel (He et al., 2005; Takezawa et al., 2006; Zitt et al., 2004), making the results from these studies difficult to interpret. Thus, development of a CRAC inhibitor more potent than Synta 66 and more selective than YM-58483 is required to dissect the role the CRAC current plays in human T cell functions (Ng et al., 2008). The aim of this study is to characterize a novel CRAC current 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2inhibitor, yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959). Using electrophysiological measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3 dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1. We also compared the effects of RO2959, YM-58483, and CsA on resting human CD4+ T cell functions including cytokine production, proliferation, Ca2+ influx and gene transcription downstream of TCR activation. We show that RO2959 is a potent blocker of the CRAC current and inhibits T cell effector functions comparably to YM-58483. Evidence from all lines of investigation suggests the CRAC channel plays a dominant role in mediating SOCE and regulating T effector functions in human CD4+ T cells.

2. Materials and methods 2.1. Reagents and antibodies Mitomycin C was purchased from SIGMA (St. Louis, MO). CyclosporinA (CsA) was purchased from Fluka. 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959)

is a selective CRAC current inhibitor synthesized by Synta Pharmaceutical Corp. (Lexington, MA). YM-58483 was synthesized at Roche as described previously (Djuric et al., 2000). CFSE, Fura2AM, probenecid, pluronic acid were purchased from Invitrogen. Charybdotoxin (ChTx) was obtained from Bachem (Torrance, CA). Purified anti-human CD3 (clone Hit3a, or UCHT1), anti-CD28 (clone CD28.2), CD4 PE-Cy7 were purchased from BD Biosciences (San Jose, CA). Anti-CD28 (clone ANC20.1/5D10) was purchased from Ancell (Bayport, MN). Biotin anti-human CD3 clone HIT3a was purchased from Biolegend. Bioplex and Flexsetcytokine kits were from Bio-Rad Laboratories. Human IL-2 AlphaLISA kit was purchased from PerkinElmer. All human subjects gave informed consent. The use of blood or PBMC for the current study was approved by the Institutional Review Board at Hoffmann-La Roche Inc. 2.2. Cell line generation Stim1 cDNA sequence was purchased from Invitrogen (Grand Island, NY) and an N-terminal Myc-Myc epitope tag was added before inserting into pcDNATM 4/TO. The Stim1 vector was transfected into T-RExTM -CHO cells and selected in culture media containing 200 ␮g/ml zeocin and 10 ␮g/ml blasticidin for 3 weeks. Stim1 expression was induced by incubating the cells overnight with 1 ␮g/ml doxycycline (Sigma–Aldrich; St. Louis, MO) and confirmed by immunofluorescence. Orai1, Orai2 and Orai3 cDNA sequences were purchased from Invitrogen and an N-terminal HAHA tag was added before inserting into pcDNA3.1. The Orai1, Orai2 or Orai3 vectors were then transfected into the T-RExTM -CHO cells stably expressing STIM1 and selected in culture media containing 500 ␮g/ml geneticin, 200 ␮g/ml zeocin, and 10 ␮g/ml blasticidin for 2 weeks. Clones were isolated and expanded by serial dilution. The co-expression of Orai1, Orai2 or Orai3 and Stim1 were confirmed by immunofluorescence. A single clone was selected for Orai1/Stim1, Orai2/Stim1 and Orai3/Stim1 for all electrophysiological experiments. Cells were maintained in F12 media supplemented with 10% FBS, 100 IU/ml penicillin and 100 ␮g/ml streptomycin. Unless noted, all tissue culture media, antibiotics and cloning vectors were purchased from Invitrogen. Overnight induction of STIM1 with tetracycline was required to obtain CRAC currents using these cell lines. 2.3. Automated patch clamp electrophysiology and concentration-response methods RBL-2H3 cells were cultured in DMEM + 10% FBS at 37 ◦ C, 5% CO2 . On the day before recording, a T225 flask ∼70–80% confluent was split 1:6 into daughter T225 flasks, generating six identical flasks plated at the same concentration and confluency. Only flasks from the same mother flask were used for an IC50 determination. On the day of recording, cells in a daughter flask (∼40% confluent) were washed with PBS, harvested using Detachin (Genlantis), spun down at 300 × g for 5 min, then resuspended in 7 mL of CHO-SFM II (Invitrogen) + 20 mM HEPES containing either control (0.1% DMSO) or a given concentration of test compound. Cells were incubated in the presence of the compound for ∼½–1 h at RT prior to running on an automated multielectrode patch clamp system (QPatch, Sophion Inc.). Following compound incubation, cells were placed in the cell station on the QPatch and were subjected to whole cell patch clamp using QPlates (single hole, QPlate 16 Large). Average hole resistances ranged between 1.7 and 2.2 M. Only cells that attained G resistances were analyzed (on average, ∼70% of wells attained G resistances). Extracellular recording solution (saline) consisted of (in mM): 140 NaCl, 2.8 KCl, 2 CaCl2 , 2 MgCl2 , 10 CsCl, 10 Glucose (pH = 7.4, NaOH, ∼345 mOsm). Intracellular recording solution consisted of (in mM): 145 CsGlutamate, 8 NaCl, 3 MgCl2 ,

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10 HEPES, 10 EGTA (pH = 7.2, CsOH; ∼330 mOsm) and 20 ␮M IP3 added fresh on the day of recording. Upon attaining whole cell configuration, cells were subjected to the following voltage pulse (VP): −100 mV (25 ms), ramp from −100 mV to +100 mV (100 ms). Cells were held at 0 mV during the intersweep interval (6.15 s). Series resistance and membrane capacitance were routinely compensated. ICRAC was allowed to develop for 15 VPs in the presence of saline (or in the case of compound treated cells, in saline plus compound), and then allowed to stabilize for another 15 VPs following a second application of saline (or saline plus compound) – see Fig. 1B. Then 50 ␮M 2-APB was applied to define the magnitude of ICRAC (or remaining ICRAC in the case of compound treated cells). The amplitude of the CRAC current at −100 mV during the first 2–3 sweeps, when the CRAC current is inactive, was averaged and subtracted from −100 mV values for each sweep in the experiment to subtract out leak current. The analysis was performed using QPatch software.

2.4. Manual electrophysiology and concentration-response methods Stim1 expression was induced in Orai1/Stim1 cells by incubation overnight with 1 ␮g/ml doxycycline. Cells were then removed with Trypsin/EDTA (Invitrogen) and maintained in CHO-SFM media (Invitrogen) supplemented with 20 mM HEPES and 1 ␮g/ml Trypsin inhibitor (Invitrogen). A small aliquot of the cells was transferred to a RC-22 (Warner Instruments; Hamden, CT) recording chamber and perfused with the following external buffer (in mM): 130 NaCl, 5.4 KCl, 1 MgCl2 , 1 CaCl2 , 10 CsCl, 5.5 Glucose and 10 HEPES (pH 7.4). The patch pipettes had resistances between 1.7 and 2.3 M and contained the following internal solution (in mM): 90 CsGlutamate, 8 NaCl, 20 CsCl, 3 MgCl2 , 10 CsBAPTA (Invitrogen), 10 HEPES (pH 7.2). 20 ␮M IP3 (Invitrogen) was added fresh on the day of recording. Whole cell patch clamp experiments were performed using a standard voltage clamp configuration at room temperature using an EPC10 amplifier (Heka; Lambrecht, Germany) and Patchmaster software (Heka, Bellmore, NY). Immediately after obtaining the whole-cell configuration, cells were voltage clamped at 0 mV and 50 ms ramps from −100 to +100 mV were generated every 2 s for the first 140 s. After the first 140 s, the perfusion buffer was switched to an external buffer containing a fixed concentration of compound for the next 500–650 s. During this time, voltage ramps were generated every 5 s. Off-line analysis was performed using Fitmaster software (Heka). The first 2–3 sweeps, when the CRAC current is inactive, were averaged and subtracted from each subsequent sweep in the experiment. The current values at −80 mV were used to measure ICRAC . Percent inhibition = 100 × [(ICRAC70 − ICRAC200 )/ICRAC70 ], where ICRAC70 is the maximum ICRAC current between sweeps 68–70 (136–140 s) and ICRAC200 is the inhibited ICRAC current at sweeps 198–200 (773–780 s). Unless noted, all tissue culture media and reagents were purchased from standard vendors.

2.5. Off-target screening To establish specificity of RO2959 for the CRAC channel the compound was tested at 10 ␮M (or 3 ␮M when solubility in the assay buffer was limiting) for inhibition of the following ion channels using electrophysiology: hNav1.5, hKvLQT1/hminK, hKv4.3, hKv1.5, hKir2.1, hHCN4, hCav1.2, hERG, TRPC1 (ChanTest, Cleveland, OH), TRPM2 and TRPM4. TRPM2 and TRPM4 assays were performed at Synta using the native channels of Jurkat cells. In addition RO2959 was tested for inhibition of binding to 80 cellular receptors, ion channels and transporters at Cerep (Poitiers, France).

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2.6. [3 H] Thymidine incorporation (MLR) Freshly isolated PBMC from healthy donors were either used as responders, where the cells were pre-treated with various concentrations of inhibitors (as indicated in the figure legends), or stimulators where the cells were pre-treated with 0.05 mg/ml Mitomycin C for 20 min. Stimulator cells were washed twice with RPMI 1640 and mixed with responder PBMCs at a 1:1 ratio, totaling 2 × 105 cells/well in quadruplicate in a 96-well U-bottom tissue culture plates (Falcon). Cells were cultured in complete RPMI 1640 (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 IU/ml penicillin-streptomycin, 0.01 M HEPES and 55 ␮M ␤-ME [Invitrogen]) for 4 days at 37 ◦ C in 5% CO2 . Cultures were pulsed with 0.5 ␮Ci [3 H]-thymidine (PerkinElmer) during the last 6 h of culture. Cells were harvested on filter plates using a 96-well plate harvester (PerkinElmer), and the incorporation of [3 H]-thymidine was measured as cpm in a Topcount Microplate scintillation counter (PerkinElmer). Stimulator cells alone or responder cells alone were set up in parallel as controls. 2.7. CFSE staining Freshly isolated PBMC were obtained from healthy donors using AccuSPIN tubes (SIGMA) according to the manufacturer’s protocol. The isolated cells were suspended at 1 × 107 cells/ml in PBS and labeled with 0.5 ␮M of CFSE (Molecular Probes, Invitrogen) for 7 min at room temperature in the dark and washed with ample volumes of complete RPMI 1640. Cells were then pre-treated with various concentrations of compounds as indicated in the figures for 30 min at 37 ◦ C in 5% CO2 . Cells were then plated at 1 × 106 cell/ml in 24-well plates (Falcon) pre-coated with anti-human CD3/CD28 and cultured in complete RPMI 1640 at 37 ◦ C in 5% CO2 for 4 days in the continued presence of inhibitors. CD4+ T cells were labeled with mouse anti-human CD4 PE-Cy7. The dilution of CFSE was measured by an LSR II flow cytometer (BD biosciences). FACS data were analyzed using FlowJo software (Tree Star) and proliferation of CD4+ T cell populations presented as the histograms. 2.8. Gene transcription profile analysis Freshly isolated PBMC from healthy donors were treated with CRAC current or calcineurin inhibitors indicated in Fig. 5 for 30 min. Cells were then transferred to a flat-bottom 96-well culture plate (Falcon) at 2 × 105 cells/well and stimulated with plate-bound anti-CD3/CD28 for 24 h in the continued presence of inhibitors. Cells were harvested and cell pellets frozen at −80 ◦ C until RNA was isolated by Perfect Pure RNA Cell Kit (5Prime, CA). After RNA quality was assessed, 100 ng of total RNA was hybridized to Affymetrix Human U133 plus 2 Genechips (Affymetrix, Santa Clara, CA) according to manufacturer’s protocol. A total of 25,602 probe sets passed the QC and curation process, corresponding to 13,445 genes. Genes differentially expressed after anti-CD3/CD28 stimulation were identified with a fold change (FC) cutoff of >2 and p-value cutoff <0.05. p-Value adjustment was not performed since the number of samples was small (n = 4 donors). Modulation of stimulated genes by the inhibitors was measured using Percent Modulation (PMOD ) defined as: PMOD =

log(RatioStim vs Stim+blocker ) × 100 log(RatioStim vs Unstim )

where RatioStim vs Stim+blocker is the expression ratio between stimulation alone and stimulation in the presence of inhibitors, and RatioStim vs Unstim is the expression ratio between stimulation and un-stimulated control. Stimulated genes that were being modulated by inhibitors were identified using a PMOD cutoff of ≥20% and a p-value cutoff of <0.05. Pathway analysis of

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Fig. 1. Inhibition of ICRAC by RO2959. (A) Sample raw traces showing ICRAC following the second saline application (black trace) and subsequently, 50 ␮M 2-APB block (red trace). Shown is an example of a control cell (no compound). See Methods for voltage sweep protocol. (B) Current vs. time plot of the control cell shown in (A). Time = 0 is break-in; arrows 1 and 2 depict saline addition, arrow 3 is application of 50 ␮M 2-APB. Current values plotted are leak subtracted (see Methods). (C) Concentration response curve for RO2959 on ICRAC in RBL-2H3 cells, providing an IC50 = 402 ± 129 nM (n = 66 cells). (D) Whole cell patch clamp recordings of T-RExTM -CHO cells expressing human

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anti-CD3/CD28 stimulation and inhibition by inhibitors were performed using GSEA (Mootha et al., 2003; Subramanian et al., 2005) on a collection of pathways from public domains (Reactome http://www.reactome.org/download/, NCI Nature Pathway Interaction Database http://pid.nci.nih.gov/ and The Cancer Cell Map http://cancer.cellmap.org, full list of pathways see Supplemental Table 2). The pathways were clustered based on Jaccard Index which measures the overlap of genes between pathways. A pathway is considered significantly up- or down-regulated if the FDR q-value calculated by GSEA is <0.25. A score is calculated for each pathway by taking the negative log of the FDR q-value multiplied by the direction of regulation. A mean score is calculated for each pathway cluster and normalized across all inhibitors. Expression levels of individual genes were analyzed by ANOVA followed by post hoc analysis.

2.9. PBMC culture, cytokine and apoptosis analysis PBMC were prepared from heparinized blood by separation on a Ficoll gradient. PBMC were pre-treated with CRAC current inhibitors or CsA at various concentrations as indicated in the figures for 30 min. Flat-bottom 96-well tissue culture plates (Falcon) were coated with 5 ␮g/ml anti-CD3 (Hit3a, Becton Dickinson, San Jose, CA) and 1 ␮g/ml anti-CD28 (CD28.2, Becton Dickinson, San Jose, CA) in PBS for 2 h at 37 ◦ C. After coating, the plates were washed twice with PBS before adding the cells. Cells were plated at 2 × 105 cells/well. Cell culture supernatant was harvested at 24 h. Supernatant IL-2 concentration was determined by AlphaLISA assay (PerkinElmer). In parallel, multiple cytokine (including IFN␥, TNF-␣, IL-10, IL-13) levels in the culture supernatant harvested at 48 h were determined by Bioplex multiplex cytokine assay (BioRad Laboratories) per manufacturer’s instructions. Measurements were performed on Bio-plex 200 system (Bio-Rad Laboratories). Data were analyzed by GraphPad Prism 5. The apoptosis of stimulated cells treated with RO2959 or DMSO was evaluated by Annexin V/PI staining on CD4+ T cell and non-CD4 subsets on day 3 upon stimulation with plate-bound anti-CD3/CD28. The cells were first stained with CD4 PE-Cy7, and then stained with Annexin V-Alexa Fluor 488 (Molecular Probes) and PI (BD Bioscience) using the manufacturer’s suggested conditions. Cell samples were analyzed on an LSR II flow cytometer (BD Biosciences). FACS data were analyzed using FlowJo software (Tree Star). The apoptosis of CD4+ T cell populations is presented as % of Annexin V+ PI− in the dot plot. The effects of RO2959 on naïve and memory T cell subset cytokine production were further examined in purified CD454RO+ and CD45RA+ T cells. PBMC were prepared from heparinized blood by separation on a Ficoll gradient. CD45RA+ or CD45RO+ T cells were isolated by positive selection using magnetic beads and an AutoMACS Pro separator (Miltenyi Biotec, Cambridge, MA). Tissue culture plates were coated with 5 ␮g/ml anti-CD3 (UCHT1, Becton Dickinson, San Jose, CA) + 5 ␮g/ml anti-CD28 (Ancell, Bayport, MN) in PBS for 2 h at 37 ◦ C. After coating, plates were washed twice with PBS prior to the addition of cells. Diluted compounds were added to coated tissue culture wells followed immediately by isolated CD45RA+ or CD45RO+ cells diluted in RPMI 1640 + 10% FCS + penicillin and streptomycin at 2 × 106 cells/ml. Supernatants from tissue culture wells were sampled after 48 h and cytokines

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measured using a Flex-set multiplex bead array read on an LSRII flow cytometer equipped with a high throughput sampler (Becton Dickinson).

2.10. Calcium imaging Purified human T cells (>90% purity; 100,000 cells/200 ␮l) were incubated for 30 min at 37 ◦ C with 1 ␮M fura-2 acetoxymethyl ester (Molecular Probes) in the presence of 250 ␮M probenecid in culture medium. Cells were then incubated with 2.5 ␮g/ml biotinylated anti-CD3 antibody for 15 min at 37 ◦ C. Cells were washed, resuspended, allowed to adhere to poly-l-lysine-coated glass coverslips, and then mounted in the recording chamber (Warner instruments). The fura-2 loaded/anti-CD3 antibody coated cells were perfused with HEPES-buffered saline containing (in mM): 155 NaCl, 4.5 KCl, 1 MgCl2 , 2 CaCl2 and 10 d-glucose, and 0.05% BSA. For TCR activation, cells were stimulated by cross-linking of surface-bound biotinylated anti-CD3 with 2.5 ␮g/ml of streptavidin (Pierce). Fura2 was excited at 340 nm and 380 nm using a Lamda 10-2 driven DG-4 (Sutter Instruments) Argon light source and filter systems. Emission was monitored with Andor CCD camera (IDEC Biosystems), connected to a Nikon TE300 microscope. The 340 and 380 nm images were acquired every 5 s. Our setup permits the acquisition of full frame images of 640 × 480 pixels size at a resolution of 0.379 ␮m2 for 1 pixel (395 × 295␮m for full frame) with an oilimmersion 40×/0.80 W objective. Changes in fluorescence were quantified using Image workbench (IDEC). Ca2+ calibration studies using Ca2+ /EDTA solutions yielded linear change in fluorescence ratio from 0.2 to 2 units when free Ca2+ concentration was changed from 200 nM to 2 ␮M.

2.11. FLIPR on human CD4+ T cells Purified human CD4+ T cells isolated from whole blood (RosetteSep, Stemcell Technologies) were stimulated for 48 h using anti-CD3/CD28 coated beads (Invitrogen). Cells were then loaded with the fluorescent calcium indicator Fluo4 AM (Molecular Probes) and plated at ∼100,000 cells/well in HBSS containing 0.5 mM CaCl2 in a PDL coated 96-well plate (BD Biosciences). 8-Point concentration response curves for RO2959 and YM-58483 were generated by pre-incubating each well with a given concentration of compound in the presence of 10 ␮M thapsigargin and 0.5 mM EGTA 30 min prior to running the plate on the FLIPR. Baseline measurements of calcium fluorescence were taken for 10 s, after which 2 mM CaCl2 was added on line. Eight minutes after calcium addition, Ionomycin (5 ␮M) was added at the end of the experiment as a dye loading control. Fluorescence measurements were sampled at 1 Hz. Concentration response curves are plotted as a function of compound concentration versus the peak calcium fluorescence value (baseline subtracted) during the 2 mM CaCl2 addition.

2.12. Data analysis All cytokine and cell proliferation data were analyzed with GraphPad PRISM 5.0.

Fig. 1 (Continued). Orai1/Stim1 showing the current traces for the first 120 s after establishing whole-cell recording. Traces are 6 s apart. Arrows point to the 6 s and 120 s (darkest trace) trace. Current traces were generated by voltage ramps as depicted below the trace (see Methods for details). (E) Current vs. time plot for typical ICRAC experiments. The normalized ICRAC response (±STD) at −80 mV for a cell perfused with either 0.1% DMSO (green circles) or 200 nM RO2959 (red squares) as indicated by the solid line below the data points. (F) The inhibition of ICRAC (±STD) by different concentrations of RO2959 for human Orai1/Stim1, Orai2/Stim1 or Orai3/Stim1 expressed in CHO cells. (G) Raw FLIPR traces showing a concentration dependent inhibition by RO2959 of thapsigargin-mediated SOCE in activated CD4+ T cells. RLU, relative light units. Arrow denotes online CaCl2 addition. (H) Concentration response curve of RO2959 (IC50 = 265 ± 16 nM, Hill = −1.9) and YM-58483 (190 ± 11 nM, Hill = −2.2, n = 3 donors) inhibition of SOCE in CD4+ T cells.

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3. Results 3.1. In vitro pharmacological characterization of the novel CRAC current inhibitor RO2959 in endogenous and recombinant cell systems We first tested the potency of RO2959 electrophysiologically on endogenous ICRAC in a rat basophilic leukemia cell line (RBL-2H3). This cell line is known to express relatively large CRAC currents (on average ∼40–50 pA at −100 mV) and has been used electrophysiologically to characterize YM-58483 potency (Takezawa et al., 2006). We activated ICRAC by adding 20 ␮M inositol triphosphate (IP3 ) in the intracellular solution and including 10 mM EGTA in the internal solution to prevent stores from refilling. We used the CRAC current inhibitor 2-APB (50 ␮M) to define the amplitude of the CRAC current. In the absence of internal IP3 , CRAC currents (2-APB sensitive) did not develop. To isolate CRAC currents from other known channels expressed on the T cell membrane, cesium and magnesium were added to the intracellular solution to block potassium currents and TRPM7 currents, respectively. Following gigaseal formation and subsequent break-in to attain whole cell configuration, voltage ramps from −100 mV to +100 mV were performed to analyze CRAC currents (Fig. 1A, see Methods for sweep details). Upon attaining the whole cell configuration, ICRAC developed quickly and reached steady state ∼75–90 s following break-in (Fig. 1A and B). In order to study the effects of RO2959 on ICRAC , we pre-incubated cells in the presence of a given concentration of RO2959 for at least a half hour prior to recording, and compared ICRAC amplitudes from RO2959 treated cells with those incubated with vehicle alone (0.1% DMSO). Performing the concentration response curve this way provided the added benefit of ensuring that the compound would have time to come to equilibrium with the channel at lower concentrations prior to current measurements. Following break-in and ICRAC development, 50 ␮M 2-APB (a concentration which blocks ICRAC completely (Takezawa et al., 2006)) was applied to define the remaining CRAC current in the cell (Fig. 1A and B). In this manner, we generated 6point concentration response curves for both RO2959 (shown) and YM-58483 that provided an IC50 of 402 ± 129 nM (Hill = −1.3) and 137 ± 88 nM (Hill = −0.81), respectively on ICRAC (Fig. 1C). The values for the YM-58483 are consistent with those previously reported in the literature. We further assessed the effect of RO2959 on SOCE in TRExTM -CHO cells stably expressing human Orai1, Orai2, or Orai3 and Stim1 using the manual whole cell patch clamp method (see Section 2). We observed that the Orai1 current developed during the first 120 s after whole cell recording was established (Fig. 1D). The inward rectification of Orai1 is apparent in the last trace (darkest trace). The current vs. time plot for the Orai1 current at −80 mV and the effects of 200 nM RO2959 (n = 3) or 0.1% DMSO on it (n = 4) are shown in Fig. 1E. The current was allowed 140 s to fully activate and was then perfused with external buffer containing a given concentration of RO2959 (200 nM in Fig. 1E). ICRAC inhibition was analyzed at 790 s, a time point at which steady state inhibition was achieved with RO2959. Fig. 1F summarizes the inhibition of the Orai1, Orai2, or Orai3 current at different concentrations of RO2959. RO2959 was not tested at concentrations higher than 1 ␮M due to solubility. At 200 nM RO2959, Orai1 was inhibited to a greater extent (88% ± 7.5) compared to Orai3 (32% ± 9.2) and Orai2 (14% ± 9.5). In order to estimate the IC50 for RO2959, the data for Orai1 and Orai3 were fitted to a single-site dose-response curve assuming maximum 100% inhibition (Supplemental Fig. 1). XLfit was used to fit the experimental data to the following formula: % inhibition = 100/(1 + (IC50 /[Drug])N ). The resulting IC50 values for Orai1 and Orai3 were 25 nM (Hill = 0.9) and 530 nM (Hill = 0.8), respectively.

Table 1 Effect of RO2959 on other ion channels. Channel

Tested concentration

Results (% inhibition)

Significant

hNav1.5 (tonic) hNav1.5 (10 Hz) hKvLQT1/hminK hKv4.3 hKv1.5 hKir2.1 hHCN4 hCav1.2 hERG TRPC1 TRPM2 TRPM4 (agonist)

10 ␮M 10 ␮M 3 ␮M 10 ␮M 10 ␮M 10 ␮M 10 ␮M 10 ␮M 3 ␮M 10 ␮M 3 ␮M 10 ␮M

14.0 28.1 14.5 15.4 19.8 11.3 5.0 16.0 <20 2.4 0 1.9

No No No No No No No No No No No No

Lastly, we characterized the effects of RO2959 on store operated calcium entry in human CD4+ T lymphocytes by performing calcium fluorescence measurements on the FLIPR. CD4+ T cells were isolated from whole blood and stimulated for 48 h using antiCD3/CD28 coated beads. A half hour prior to running the cells on the FLIPR, we added the SERCA Ca2+ pump inhibitor thapsigargin in the absence of extracellular calcium to engage SOCE machinery, along with a given concentration of RO2959 to determine its potency on SOCE. Upon calcium addition on line (Fig. 1G, arrow), control cells that were incubated only in the presence of thapsigargin showed a large increase in fluorescence, suggesting that the passive depletion of intracellular stores using thapsigargin was effective in opening channels that allowed for the influx of extracellular calcium (Fig. 1G, DMSO). In cells treated with the CRAC inhibitor RO2959, peak fluorescence was found to be dependent on RO2959 concentration, suggesting that SOCE in activated CD4+ T lymphocytes can be blocked by RO2959. Similarly, the potency of YM-58483 was characterized on SOCE in activated CD4+ T cells. We determined that RO2959 and YM-58483 have IC50 values of 265 ± 16 nM (Hill = −1.94) and 190 ± 11 nM (Hill = −2.2), respectively, on SOCE in human CD4+ T cells (Fig. 1H). These results correlate well with those determined electrophysiologically on RBL-2H3 cells, and taken together, provide evidence that RO2959 is a potent CRAC current inhibitor. To test for selectivity, RO2959 was screened at 10 ␮M when solubility permitted, or 3 ␮M when solubility was limiting, for inhibition of potassium and calcium channels (shown in Table 1). No significant inhibition compared to negative controls was found. In addition, RO2959 was screened in a binding assay against 80 cellular receptors, ion channels and transporters. Significant interaction was only detected with 5-HT2B and the BZD receptors (Supplemental Table 1), neither of which has been reported to be expressed in cells of the lymphoid system. These data lend support to the assertion that the effects of RO2959 on T cell function described in this study are due to its ability to inhibit the CRAC current. 3.2. The effect of RO2959 on TCR activation-mediated Calcium influx on naïve CD4+ T cells While the FLIPR experiments provide evidence that RO2959 can inhibit thapsigargin-mediated SOCE in primary human CD4+ T cells, we wanted to use a more physiologically relevant stimulus to show that RO2959 can inhibit calcium influx mediated by T cell receptor engagement. We therefore performed calcium fluorescence imaging experiments on primary human naïve CD4+ T cells stimulated with a cross linking CD3 antibody to examine the effects of RO2959 on anti-CD3-mediated intracellular Ca2+ fluctuations at the single cell level. Activation of TCR via cross-linking of CD3 in fura-2 loaded vehicle-treated cells produced a gradual increase in intracellular Ca2+ with maximal influx produced around 900 s that was sustained

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Fig. 2. The effect of CRAC inhibitors on TCR-mediated calcium entry in naïve human CD4+ T cells. Representative trace of changes in 340/380 nm fluorescence ratio in fura-2 labeled CD4+ T cells treated with vehicle (A), 3 ␮M RO2959 (B) and 3 ␮M YM-58483 (C). Activation of TCR (arrowhead) was achieved via cross-linking of biotinylated anti-CD3 using streptavidin. Averaged maximal increase of 340/380 nm ratio (D). The data are presented as mean + SEM. Maximal increase of 340/380 nm in individual cells treated with vehicle (n = 87 cells), 3 ␮M RO2959 (n = 65 cells) or 3 ␮M YM-58483 (n = 78 cells). Asterisks represent significant difference (p < 0.05) compared to vehicle control as derived from one-way Analysis of Variance followed by Newman–Keuls post hoc tests.

for over 5 min (Fig. 2A). All the vehicle-treated cells responded, but with varying magnitudes of maximal increases in Ca2+ influx (Fig. 2A). Pretreatment of cells with the CRAC current inhibitors reduced maximal increase in Ca2+ influx (Raw trace see Fig. 2B and C). A saturating concentration of the CRAC current inhibitor RO2959 (3 ␮M) inhibited ∼85% of the maximal increase in Ca2+ influx caused by anti-CD3 stimulation, comparable to the inhibition seen by the previously published CRAC inhibitor YM-58483 (Fig. 2D and E; *p < 0.05 as compared to Vehicle control, derived from one-way Analysis of Variance followed by Newman–Keuls post hoc tests).

3.3. RO2959 potently blocks the proliferation of human CD4+ T cells Since it has been well established that CRAC channel activity is essential for human T cell effector functions, we next examined the effects of RO2959 in human T cell proliferation assays in parallel with the calcineurin inhibitors CsA and FK506. We first tested whether RO2959 could block the human MLR, a physiological system commonly used in human T cell functional studies. In cells from healthy donors, we found that RO2959 completely blocked allo-specific T cell proliferation at saturating concentrations (1 ␮M) and potently inhibited T cell proliferation over a wide concentration range in healthy donors (n = 6), with an IC50 of 29 nM (Fig. 3A). CsA, an inhibitor of the phosphatase calcineurin activated downstream of CRAC activation, behaved similarly to RO2959 in blocking the human MLR response with a calculated IC50 of approximately 16 nM (Fig. 3A). Data from this experiment are consistent with our findings in electrophysiological studies and Ca2+ influx studies where RO2959 potently blocked Ca2+ influx in CD4+ T cells stimulated with anti-CD3 and thapsigargin (Figs. 1 and 2). To confirm our findings on the effects of RO2959 on human T cell proliferation, we conducted CFSE staining assays. CFSElabeled human PBMC from healthy donors were treated with the

indicated concentrations of the inhibitors and then stimulated with plate-bound anti-CD3/CD28 for 4 days (Fig. 3B). The proliferation of human CD4+ T cells was analyzed by FACS using CFSE dilution as the readout. Consistent with our MLR experiments, RO2959 produced potent inhibition of CD4+ T cell proliferation, indicated by the significant reduction in the percentage of CFSElow cells by FACS (Fig. 3B and C). CsA and YM-58483 also potently blocked the proliferation of CFSE-labeled CD4+ T cells as expected. CD4+ populations were gated in all FACS analysis. In order to rule out the possibility that the effects of RO2959 on T cell activation are due to cytotoxicity, we conducted Annexin V staining on T cells treated with CRAC current inhibitors or CsA for 3 days. Consistent with our results using CSFE staining, CD4+ T cell proliferation was potently inhibited by RO2959 at 1 ␮M or higher concentrations. The cells appeared to remain in a resting state as judged by forward and side scatter, and the level of apoptosis (determined by % of Annexin V+ PI− ) was the same as controls treated with DMSO alone (Fig. 3D).

3.4. RO2959 potently blocks human T cell cytokine production Next, we examined the effects of RO2959 on T cell cytokine production. Freshly isolated human PBMC from healthy donors were treated with indicated concentrations of the compounds for 30 min and then stimulated with plate-bound anti-CD3/CD28 for 24 and 48 h. The level of IL-2 production at 24 h was then examined by AlphaLISA. Ten other common T cell cytokines in the culture supernatants were tested with a Luminex assay at 48 h. We observed that IL-2 production could be completely inhibited by RO2959 and CsA, which displayed a similar concentration-response as observed in the T cell proliferation study (MLR) (Fig. 4A). The IC50 for RO2959 was 95 ± 63 nM, 45 ± 9 nM for YM58483 and 7 ± 2 nM for CsA (Fig. 4A). These data provide evidence that CRAC current inhibition conferred by RO2959 leads to potent blockade of human T cell IL-2 production. Further, we examined the effects of RO2959 on

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Fig. 3. Inhibition of human CD4+ T cell proliferation by Ca2+ signaling inhibitors. (A) In the mixed lymphocyte reaction, donor PBMC were treated with 0.05 mg/ml of Mitomycin C for 30 min, washed twice with RPMI 1640 and mixed 1:1 with allogeneic responder cells. Serially diluted inhibitors RO2959, CsA and YM-58483 were added to the culture and incubated for 4 days. The DMSO concentration was kept consistent among all the treatment conditions and was used as the negative control. The uptake of [3 H]-thymidine is presented as cpm (data shown are representative of 6 independent donors). (B) CFSE-labeled PBMCs were treated as in (A) and stimulated with plate-bound anti-CD3/CD28 for 4 days. CD4+ T cells were gated and CFSE dilution is illustrated in histograms (data shown are representative of 5 independent donors). (C) The percentage of CFSElow cells within CD4+ gates are summarized. (D) The apoptosis induction by RO2959 in PBMC was evaluated by Annexin V/PI staining. PBMC were stimulated with plate-bound anti-CD3/CD28 for 3 days in the presence of CRAC inhibitors or CsA. CD4+ T cells by were gated and % of Annexin V+ PI− cells was annotated in the dot plots (representative data of 4 donors).

bacterial superantigen (SEB) induced IL-2 production in the same donors. Consistent with a previous report showing that SEB-stimulated T cell activation is dependent on CRAC channel activity (Lioudyno et al., 2008), we observed a similar concentration dependent inhibition of IL-2 production by both RO2959 (IC50 = 84 ± 19 nM) and CsA (IC50 = 23 ± 7 nM) (Fig. 4B). Collectively, our data suggest that RO2959 is a potent inhibitor of human IL-2 production though less potent than CsA (Unpaired t test p < 0.05, n = 6). We also examined several other T cell cytokines, including IFN-␥, IL-6, IL-9, IL-10, IL-17, TNF-␣, from the same donors by Luminex. We observed a similar concentration-dependent inhibition of IFN-␥, IL-6, IL-17 and TNF-␣ production by RO2959, YM58483 and CsA (Fig. 4C). We further examined the effects of RO2959 inhibition of cytokine production from naïve and memory CD4+ T cells. CD45RA+ and CD45RO+ CD4+ T cell subsets were isolated from PBMC and stimulated for 48 h with platebound anti-CD3/CD28 in the presence of RO2959 and CsA. RO2959 potently blocked IL-2 and IFN-␥ production from both naïve (Fig. 4D) and memory T cells (Fig. 4E). In addition, IL-4 and IL-17 production in the memory population were also inhibited by RO2959, although with lower potency (Fig. 4E).

3.5. The effect of RO2959 on TCR-induced gene expression Intracellular Ca2+ signaling in T cells controls diverse cellular functions ranging from gene regulation and cytokine production to cell proliferation (Feske et al., 2001). We therefore examined the effects of CRAC activity blockade on the human T cell gene expression profile in response to TCR activation. Freshly isolated PBMC from healthy donors were stimulated by platebound anti-CD3/CD28 in the presence or absence of the indicated concentrations of inhibitors for 24 h, and total RNA was isolated for gene array analysis. We observed 3921 genes significantly up- or down-regulated 24 h post-stimulation by anti-CD3/CD28. RO2959 significantly modulated 3810 genes (PMOD of at least 20% – see Methods). Bi-clustered heatmap analysis was conducted on 2775 genes commonly modulated by RO2959, CsA and FK506 (Supplemental Fig. 2B). RO2959 potently blocked most TCR activation-induced genes at 1 ␮M, a concentration chosen to ensure complete CRAC current blockade, but devoid of other confounding T cell off-target ion channel inhibition. In addition, mean expression levels of the top 25 up- and down-regulated genes under all treatment conditions are shown in Table 2. Clustered heatmap analysis was conducted on the 50 genes listed in Table 2 (Fig. 5A). In addition, representative genes such as IFN-␥, IL-2RA

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Fig. 4. Inhibition of human T cell cytokine production by Ca2+ signaling inhibitors. (A) PBMC were pre-treated for 30 min with the indicated concentration of RO2959, CsA or YM-58483and stimulated with plate-bound anti-CD3/CD28 for 24 h in the continued presence of the blockers. IL-2 concentration in the cell culture supernatant was determined by AlphaLISA (data shown are representative of 6 independent donors). (B) PBMC were stimulated with SEB (50 ng/ml) for 24 h in the presence of RO2959 and CsA (n = 6 donors). IC50 in IL-2 production from 6 donors were summarized and presented as Mean ± SD. * represents p < 0.05 by Unpaired t test when compared to RO2959. (C) Multiplex cytokine analysis on the culture supernatant from PBMC stimulated with plate-bound anti-CD3/CD28 for 48 h (data shown are representative of 4 independent donors). (D) Inhibition of cytokine production from naïve CD4+ T cells (CD45RA+ ) upon anti-CD3/CD28 stimulation (48 h). (E) Inhibition of cytokine production by memory CD4+ T cells (CD45RO+ ).

and IL-17F are also shown in Fig. 5B. Our data clearly showed that RO2959 conferred potent inhibition on the Ca2+ dependent gene expression. In addition, we compared the impact of the RO2959 or CsA on TCR-induced functional pathways using the Gene Set Enrichment Analysis (GSEA) (De Windt et al., 2007; Keller et al., 2007; Subramanian et al., 2005; Zhang et al., 2009). RO2959 significantly inhibited up- or down-regulation of most of the stimulated pathways, including pathways related to cell cycle regulation, DNA replication, NFAT activation and IL-2 signaling, etc. (Fig. 5C). CsA and FK506 inhibited a smaller but similar set of pathways. Consistent with our T cell functional proliferation studies, pathways related to cell proliferation were blocked by RO2959, CsA and FK506, with RO2959 and CsA being most potent. The TNF-␣/NF␬B signaling network and calcineurin dependent NFAT signaling pathway were also inhibited by RO2959, CsA and FK506 as expected.

The potassium channels KCa3.1 and Kv1.3, expressed on the surface of T cells, have also been reported to influence T cell activation and effector functions (Panyi et al., 2004b; Wulff et al., 2003). We therefore also examined the effects of K+ channel blockade on TCR-induced gene expression using TRAM-34 (1 ␮M) and ShK[L5] (10 nM), selective inhibitors of KCa3.1 and Kv1.3, respectively (Beeton et al., 2005; Wulff et al., 2000). The combination of TRAM-34 and ShK[L5], or charybdotoxin (ChTx), a peptide toxin that blocks both K+ channels, were also used. Significance of pathway enrichment measured using GSEA analysis is listed in Supplemental Table 2. Our results indicate that TRAM-34 or ShK[L5] alone showed no significant change in the transcriptional profile of activated T cells. However, cells treated with the combination of TRAM-34 and ShK[L5], or with ChTx alone, had a transcriptional profile that more closely resembled that of cells treated with RO2959. These results strongly suggest that

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Table 2 Expression levels (in log2 scale) in all 5 conditions for top 25 genes up or down regulated by stimulation. Top 25 genes up regulated by stimulation

Top 25 genes down regulated by stimulation

Gene

Unstim

Stim

RO2959

CsA

FK506

Gene

Unstim

Stim

RO2959

CsA

FK506

IFNG ZBED2 IL2RA NDFIP2 TYMS DTL RRM2 IL17F MIR155HG LTA KIAA0101 IL9 CDT1 CDC25A LIF CDC6 CDC20 CXCL5 PBK TNFRSF9 EXO1 SLC27A2 MCM4 LAG3 ZWINT

4.57 4.66 5.20 3.94 4.83 3.57 4.35 3.77 6.55 5.17 4.31 3.21 3.17 2.82 4.71 4.06 4.11 3.16 3.17 5.39 3.80 3.78 4.21 5.26 6.08

12.30 12.09 11.32 9.99 10.67 9.32 10.10 9.13 12.07 10.67 9.62 8.64 8.51 7.92 9.34 8.63 8.67 7.52 7.60 9.96 8.22 8.41 8.50 9.73 10.30

5.67 9.36 6.53 7.58 4.68 3.36 4.44 4.14 8.27 5.56 4.06 3.26 3.20 2.68 4.99 4.13 4.16 5.34 3.24 8.83 3.95 5.09 4.57 5.78 6.18

7.24 9.11 8.27 8.10 6.42 5.07 5.31 5.40 9.43 6.47 5.14 4.45 4.40 3.23 5.32 5.25 4.87 9.37 3.36 8.71 4.62 6.15 5.35 5.49 7.02

9.93 9.85 9.71 8.34 7.87 6.46 6.61 7.08 10.57 7.58 6.90 6.61 6.51 4.75 6.59 6.17 6.09 10.43 4.50 9.16 6.01 7.15 6.50 7.04 8.03

FCN1 MS4A7 CD14 LYZ GAPT S100A8 ADAMDEC1 FAM198B THBS1 CD36 CPVL HNMT RNASE6 RTN1 IRAK3 CD163 MS4A14 MNDA PDGFC TGFBI OLR1 CCR2 S100A12 TLR8 PLA2G7

12.35 12.70 11.47 13.76 10.64 13.07 10.52 9.08 10.85 10.07 9.62 10.34 10.50 10.00 8.97 10.41 10.21 10.50 8.98 12.02 9.50 10.42 9.61 11.06 10.96

4.66 5.27 4.28 7.40 4.24 6.63 4.33 2.87 4.57 3.97 3.50 4.24 4.55 4.08 3.13 4.63 4.52 5.05 3.49 6.54 4.13 5.44 4.30 5.77 5.77

10.81 10.23 8.55 13.51 6.97 12.14 9.01 7.65 8.40 9.00 6.68 8.79 8.28 9.48 6.91 6.05 7.06 9.53 7.36 11.92 9.15 9.25 7.70 10.04 11.45

9.00 9.71 7.19 12.37 6.56 10.97 7.88 5.19 8.52 7.23 5.12 7.54 7.40 8.48 5.91 5.43 7.10 7.94 6.62 10.78 8.25 7.75 7.01 9.01 10.81

7.61 8.37 5.80 10.80 5.42 10.43 6.79 3.93 7.17 5.88 3.91 6.23 6.14 6.03 4.34 5.06 6.18 6.44 4.86 9.62 6.10 6.48 6.81 7.67 9.54

blockade of either K+ channel alone did not inhibit any of the functional pathways, indicating both K+ channels have to be inhibited in order to fully block TCR-mediated signals in total T cells.

4. Discussion In the current study, we report the pharmacological and functional profiles of a novel selective CRAC current inhibitor RO2959. Using the rat RBL-2H3 cell line, we were able to assess the potency of RO2959 on endogenous CRAC current electrophysiologically. Additionally, we characterized the potency of RO2959 on recombinant homomeric human Orai channels on the surface of CHO cells stably expressing either Orai1/Stim1, Orai2/Stim1 or Orai3/Stim1 using a manual patch clamp platform confirming that RO2959 is a potent blocker of SOCE mediated by Orai1/Stim1 channels. While RO2059 was consistently a potent inhibitor of the CRAC current in every studied regard, some variation of the approximate IC50 values obtained using Orai1/Stim-transfected CHO cells versus RBL cells was seen. This may reflect technical differences in the methods used, or may result because relative to transfected channels, the native RBL CRAC channel may not be a homohexamer of Orai1, or may reflect a species-specific binding profile. We also determined the potency of the compound for inhibition of thapsigargin-mediated SOCE in activated human CD4+ T cells, and showed its ability to block TCR-mediated calcium influx in these cells. We further demonstrated that the blockade of the calcium influx produced by RO2959 potently inhibits human T cell effector functions including activation, proliferation, cytokine production and gene expression. These experiments pharmacologically verify that RO2959 is a bona fide CRAC blocker and strongly support the potential applications of CRAC current blockers in the clinic. In more complex MLR experiments, we showed in cells from multiple human donors that RO2959 inhibits T cell proliferation in a concentration-dependent fashion with potency similar to CsA, suggesting a dominant role for the CRAC channel in human TCRmediated cell proliferation. Similar results were obtained with

CD4+ T cells labeled with CFSE, confirming the inhibitory effects of RO2959 on T cell proliferation. A complete blockade of cell division by RO2959 was observed at saturating concentrations (1 ␮M). Consistent with previous reports, YM-58483 also potently blocked T cell proliferation in both MLR reactions and CFSE studies in our hands. Thus, the immunomodulatory effects of targeting the CRAC pathway demonstrated by RO2959 are reinforced by structurally diverse CRAC current inhibitors which demonstrate similar effects. When taken together with the accumulated data from human patients with Orai1 gene mutations, these data suggest a dominant role for Orai1 in CRAC channel activity in T cells (Feske et al., 2006; Lioudyno et al., 2008; Ohga et al., 2008; Roos et al., 2005). These studies confirm that a potent and selective CRAC current inhibitor can mediate strong blockade of human T cell activation and proliferation. In our hands, RO2959 also strongly inhibited human T cell cytokine production. Both anti-CD3 mediated and bacterial superantigen (SEB)-induced IL-2 production was abrogated by RO2959 with similar potency. In addition, Luminex data confirmed that RO2959 not only blocks IL-2 production in T cells, but also potently affects the production of IFN-␥, IL-6, IL-17 and TNF␣. Further, naïve and memory CD4+ T cells were similarly sensitive to CRAC current blockade by RO2959, which potently inhibited anti-CD3/CD28 induced cytokines associated with differentiated Th1, Th2 and Th17 cells, including IL-2, IFN-␥, IL-4 and IL-17. Analysis for cytotoxicity during activation using PI and Annexin V staining confirmed that the cytokine inhibition observed using RO2959 was not due to toxicity. These data strongly suggest that RO2959 is a potent SOCE blocker that exerts significant blockade of human T cell cytokine production from both naïve and differentiated cells, and taken with the proliferation data, indicates that RO2959 can potently inhibit general T cell activation. Our study is also consistent with previous reports that CRAC channel activity is essential for T cell activation independent of the nature of TCR stimulation (Feske et al., 2006; Lioudyno et al., 2008). Our transcriptomic analysis provided further confirmation that the CRAC channel activity is essential for human T cell

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Fig. 5. Gene expression profile of activated human T cells treated with Ca2+ signaling inhibitors. Human PBMC from 4 healthy donors were treated with either 1 ␮M RO2959, 1 ␮M CsA, or 10 nM FK506 for 30 min then stimulated with plate-bound anti-CD3/CD28 for 24 h in the continued presence of the blocker. Gene array analysis was conducted as described in Materials and Methods. (A) Expression profile of the top 50 genes up or down-regulated by TCR stimulation. Red and blue represent genes that are either upregulated or down-regulated, respectively, by anti-CD3/CD28 stimulation. Shade of colors represents level of effect by the compounds. (B) Expression levels of representative genes induced by TCR stimulation. ANOVA followed by post hoc analysis were conducted to determine significance (*** represents p < 0.001 and ** represents p < 0.01). (C) GSEA analysis on complete gene sets. Pathways that were significantly up-regulated or down-regulated were labeled in either yellow or purple, respectively. As for individual pathways, red represents significant inhibition of changes induced by anti-CD3/CD28, while gray represents no significant effect on anti-CD3/CD28 induced effects.

activation. By comparing the gene expression profiles of unstimulated and anti-CD3/CD28 stimulated human PBMCs, we observed that RO2959 completely blocks gene expression associated with multiple pathways induced by TCR activation. Comparison of the genes and pathways inhibited by RO2959, CsA and FK506 confirmed that CRAC current blockade affects most of the pathways blocked by calcineurin inhibitors. More precisely, RO2959 potently blocked most of the functional pathways downstream of TCR activation, with a majority of the inhibited pathways being shared between the CRAC inhibitor and calcineurin inhibitors (CsA and FK506). For instance, blockade of either CRAC current or calcineurin inhibited the up-regulation of cell cycle related pathways, and the up-regulation of the calcineurin-dependent NFAT signaling pathways, confirming that TCR signaling through NFAT can be impeded by blocking either CRAC-mediated Ca2+ influx or downstream at the level of calcineurin. Additionally, using the Gene Set Enrichment Analysis (GSEA), we have also shown that simultaneous blockade of Kv1.3 and KCa3.1 channels with either ChTx or the combination of TRAM-34 and ShK[L5] results in abrogation of most of the functional pathways downstream of TCR activation, similar to the effects of CRAC current or calcineurin inhibition (in supplemental materials). These findings support the hypothesis that one of the functions of the potassium channels on the T cell membrane is to provide the driving force for Ca2+ flow into the cell through CRAC.

Surprisingly, when used independently, neither of the potassium channel blockers strongly affected genes directly associated with TCR activation in our experiments. One potential explanation for this unexpected result could be due to differential expression of either of the K+ channels in effector/memory and central memory T cell subsets, rendering incomplete blockade of total T cell activation in the presence of only one potassium blocker (Beeton et al., 2005, 2006; Ghanshani et al., 2000; Panyi, 2005). We did observe in our study that calcineurin dependent NFAT and NF␬B pathways were significantly inhibited by RO2959, which is consistent with Feske and Rao’s early study on transcriptional profiling of SCID patient T cells (Feske et al., 2001). In summary, we have shown that the novel CRAC current selective blocker RO2959 exerts potent blockade of human TCRmediated calcium influx, cytokine production, cell proliferation and gene expression. Evidence from all lines of investigation consistently suggests CRAC current blockade by a pharmacological agent can inhibit T cell activation and effector functions. Our study thus provides a novel tool for studying the function of CRAC channels under both physiological and pathological conditions. Furthermore, the pharmacokinetic profile of this compound in mice (Supplemental data, Fig. 3) indicates it may be appropriately suitable for xenograft models employing human cells in murine hosts, such as GVHD.

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