Mice were housed and bred in pathogen free conditions in strict compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines at the National University of Singapore, in accordance with the National Advisory Committee for Laboratory Animal Research (NACLAR) Guidelines (Guidelines on the Care and Use of Animals for Scientific Purposes). The protocol was approved by Institutional Animal Care and Use Committee of the National University of Singapore (Protocol Number: 041/08) and steps were taken to minimize suffering. All research involving human samples was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the National University of Singapore Institutional Review Board (NUS-IRB), Singapore. Peripheral blood samples were obtained with written informed consent from human volunteers.
C57BL/6 mice were purchased from the Centre for Animal Resources at the National University of Singapore. Irf3−/−
mice on a C57BL/6 background were purchased from the RIKEN Bioresource Centre (Japan). Irf7−/−
mice on a C57BL/6 background were generously provided by Dr. F. Ginhoux (Singapore Immunology Network, Singapore) 
. The Cd155−/−
mice were generated as described in Abe et al. 
. The genetic background of the Cd155−/−
mice was 129/Sv (50%), C57BL/6 (25%) and DBA (25%). Offsprings of Cd155−/−
mice×C57BL/6 mice breedings were used for all experiments.
The mouse monocytic leukemic macrophage cell line RAW264.7 (TIB-71, ATCC, USA) was cultured in RPMI 1640 medium (Invitrogen, Singapore) supplemented with 5% heat inactivated FCS (Invitrogen, Singapore), 20 mM HEPES (HyClone, USA), 1.4 µM L-glutamine (Sigma, Singapore), 20 U/mL penicillin-streptomycin (Invitrogen, Singapore), 5.2 mg/ml gentamicin sulphate (Invitrogen, Singapore) and 1.82
µl/ml β-mercaptoethanol (Sigma, Singapore). Bone marrow from Myd88−/−
mice was generously provided by Dr. S. Biswas (Singapore Immunology Network, Singapore) 
bone marrow was kindly provided by Dr. K. Ishii (Osaka University, Japan) 
, Myd88−/, Trif−/−
BMDCs were generated as described by Inaba et al 
. Bone marrow derived macrophages (BMDMs) were generated and cultured as described before 
. Human PBMCs of healthy volunteers were isolated by Ficoll-Hypaque (Sigma, Singapore) gradient centrifugation. Monocytes were purified using the MACS CD14 isolation kit (Miltenyi Biotech, Singapore) and were subsequently cultured in 6 well dishes (1.5–2×106
cells/ml) in the presence of 800 U/ml of GM-CSF and 500 U/ml of IL-4 (eBioscience, USA) to generate monocyte-derived dendritic cells (MoDCs). Mouse B cells were purified from spleen using the mouse EasySep CD19 positive selection kit (STEMCELL Technologies, Singapore). Splenic B cells were cultured in 10% FCS supplemented RPMI 1640 medium (Invitrogen, Singapore). All cells were grown at 37°C in a humidified 5% CO2
incubator (Thermo Scientific, Singapore).
All TLR ligands were obtained from Invivogen (USA). Human MoDCs were treated for 48 hrs as follows: 1 µg/ml LPS and 25 µg/ml Poly I:C. RAW264.7 cells, mouse BMDCs, BMDMs and B cells were treated with 1 µg/ml LPS, 25 µg/ml Poly I:C, 40 ng/ml Pam3CSK4, 1 µM CPG ODN 1668 and 1 µg/ml R848 for 24 hrs or 48 hrs in the case of B cells. BMDCs were stimulated with 100 ng/ml flagellin derived from S. typhimurium for 24 hrs. RAW264.7 cells were treated with 20 ng/ml of TNFα (eBioscience, USA) for 24 hrs.
For analysis of CD155 expression in vivo, C57BL/6 mice were immunized with saline, 100 µg LPS, 100 µg Poly I:C, 50 µg Pam3CSK4 and 50 µg CpG ODN 1668 intraperitoneally (i.p.). 18 hrs later splenocytes were harvested and stained for CD155 expression.
RAW264.7 cells transduced with IκBα dominant-negative mutant (Addgene, USA) or empty vector plasmid were treated as indicated above in 6 well dishes at a density of 2×105 cells in each well. Cells were used for FACS and culture supernatants tested for IL-6 by ELISA (eBioscience, USA) according to the manufacturer’s instructions.
To analyze the kinetics of CD155 expression, 2×106 RAW264.7 cells were stimulated with 1 µg/ml LPS for different times. At indicated time, cells were harvested and CD155 expression was assessed by real-time RT-PCR and flow cytometric analysis. In order to test whether CD155 upregulation requires de novo transcription or translation, 1×106 RAW264.7 cells in a 10 cm dish were treated with 5 µg/ml actinomycin D (Sigma, Singapore) or 50 µg/ml cycloheximide (Calbiochem, Singapore) for 1 hr, followed by treatment with 1 µg/ml LPS for 5 hrs.
For pharmacological inhibition of NF-κB or MAPK signaling, 1×106 BMDMs or 2×106 RAW264.7 cells were seeded in 6-well plates and 10 cm dishes respectively. The next day cells were pretreated with 1 µM or 4 µM or 10 µM BMS-345541 or 20 µM SB203580 or 20 µM PD98059 (Calbiochem, Singapore) for 1 hr, followed by stimulation with TLR agonists at doses indicated above for an additional 24 hr. For real time and western blot assays of NF-κB inhibitor treated cells, the TLR ligand stimulation time was shortened to 5 hrs.
The pBABE-puro-IκBα (Addgene plasmid 15291) and empty pBABE-puro plasmids were obtained from Addgene 
. Transduction of cells was performed as described before 
. 48 hrs after transduction, cells were selected with 5 µg/ml puromycin (Sigma, Singapore) for at least 7 days.
PE anti-human CD155, APC anti-mouse CD155, Alexa-fluor 647 anti-mouse TIGIT, PE anti-mouse CD40, PB anti-mouse CD11c, PE anti-mouse F4/80, PB anti-mouse CD19, FITC anti-mouse CD3, PB anti-mouse CD4, APC anti-mouse CD8, PB anti-mouse CD44, FITC anti-mouse CD25, PE anti-mouse CD80 and FITC anti-mouse CD86 antibodies were purchased from eBioscience (USA). DNAM-1 and TIGIT expression on naïve T cells was studied using spleens harvested from 8 weeks old WT and Cd155−/− mice. Data was acquired using a CyAn ADP analyzer (Beckman Coulter, Singapore) and FlowJo software version 8.8.7 (TreeStar, USA). All plots show cells gated for viable cells, as determined by live-cell gating by forward scatter, side scatter, and lack of propidium iodide (Sigma, Singapore) uptake; viability of cells for all experiments was >80%.
Intracelluar IFN-γ and IL-4 staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences, Singapore) following the manufacturer’s instructions. For staining of T-Bet and GATA-3, Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) was used. Splenocytes were harvested from OVA and CpG immunized WT and Cd155−/− mice on day 21 after immunization. 5×106 splenocytes were cultured in 1 ml of medium for 24 hours. 20 ng/ml PMA and 500 ng/ml Ionomycin (Invitrogen, Singapore) was added to the culture for the last 6 hours together with 1 µl/ml each of GogliPlug and GolgiStop (BD Biosciences, Singapore). Cells were first stained with APC-Cy7 conjugated Fixable Viability dye (eBioscience, USA) followed by staining with FITC anti-mouse CD3 and PB anti-mouse CD4 antibodies. Cells were then fixed and permeabilized using the components provided in the BD Cytofix/Cytoperm kit and stained with APC anti-mouse IFN-γ (BD Biosciences, Singapore) and PE anti-mouse IL-4 (BD Biosciences, Singapore) antibodies or fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set and stained with PE conjugated T-Bet (eBioscience, USA) and APC conjugated GATA-3 antibodies (eBioscience, USA). Data was acquired using a CyAn ADP analyzer (Beckman Coulter, Singapore) and analyzed using FlowJo software version 8.8.7 (TreeStar, USA).
Total RNA was isolated using the RNeasy kit according to the manufacturer’s instructions (Qiagen, Singapore). 2 µg of total RNA was reverse transcribed using random hexamer primer and M-MLV reverse transcriptase (Promega, Singapore). Each amplification mixture (25 µl) contained 50 ng of reverse transcribed RNA, 0.8 µM forward primer, 0.8 µM reverse primer and 12.5 µl of iTaq SYBR Green Supermix with ROX (Bio-Rad Laboratories, Singapore). PCRs were performed in triplicates using the ABI PRISM 7700 Sequence Detection System from Applied Biosystems. PCR thermocycling parameters were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 15 sec and 72°C for 1 min. All samples were normalized to the signal generated from the housekeeping gene Hprt. The following primers were used: Hprt-5′: tgggaggccatcacattgt; Hprt-3′: gcttttccagtttcactaatgaca; Cd155-5′: cgtgtccatctctggctatg; Cd155-3′ cgtgttcgtgctccagttat. Samples prepared without RNA served as negative control templates. Experiments were analyzed using GraphPad Prism software (Version 5.0a, GraphPad Software, USA).
Cells were lysed in Radio-Immunoprecipitation Assay buffer (RIPA) consisting of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 1% sodium deoxycholate (Sigma). Prior to lysis, protease inhibitor cocktail set III (Merck, Singapore) and phosphatase inhibitor cocktail set V (Merck, Singapore) were added to the lysis buffer according to the manufacturer’s instructions. 35 µg of lysate was loaded on a 10% reducing SDS PAGE gel. Lysate was transferred to a nitrocellulose membrane (Amersham, Singapore) and probed with anti-mouse IκBα antibody (Cell Signaling Technology, USA) followed by HRP-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, USA). Blots were reprobed for tubulin expression using a mouse tubulin specific antibody (Sigma, Singapore) followed by HRP-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch, USA).
20 µg ovalbumin (OVA) (Grade V, Sigma, Singapore) or 20 µg OVA and 50 µg CpG ODN 1668 (Invivogen, USA) was administered i.p. to 7 to 8 week old CD155-deficient mice and age matched littermate controls. Blood samples were taken one day before injection by facial vein bleeding. On day 21 mice were sacrificed and blood samples were obtained by cardiac puncture. Serum was analyzed for OVA-specific antibody titer using indirect ELISA 
. Briefly, 96 well MaxiSorp plates (Nunc, Singapore) were coated overnight at 4°C with 10 µg/ml of OVA (Sigma, Singapore) dissolved in 0.1 M carbonate/bicarbonate buffer at pH 9.6. Plates were blocked with 0.05% PBS-Tween containing 2% BSA (Sigma, Singapore) and 5% goat serum (Invitrogen, Singapore) for 2 hrs at room temperature. After washing, plates were incubated with serially diluted serum samples and incubated overnight at 4°C. OVA-specific IgG, IgG1 and IgG3 levels were determined by incubating plates for 1 hr with peroxidase-conjugated goat anti-mouse IgG, Fcγ
fragment specific, goat anti-mouse IgG, Fcγ
subclass 1 specific or and goat anti-mouse IgG, Fcγ
subclass 3 specific antibodies (Jackson Immunoresearch, USA). To analyze IgG2a/c titers, a 1
1 molar ratio of goat anti-mouse IgG Fcγ
subclass 2a specific and goat anti-mouse IgG Fcγ,
subclass 2c-specific antibodies was added to the plates. After 1 hr, plates were washed and incubated with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate according to the manufacturer’s instructions (eBioscience, USA). Results were analyzed using GraphPad Prism software (Version 5.0a, GraphPad Software, USA).
5×106 spleen cells of OVA and CpG immunized mice were cultured in 1 ml RPMI medium (Invitrogen, Singapore) for 48 hrs after which, the levels of IL-4, IFN-γ, IL-12p70 and IL-10 in the supernatant was measured by ELISA according to the manufacturer’s instructions (eBioscience, USA).