Toll-Like Receptor Ligands Induce Expression of the Costimulatory Molecule CD155 on Antigen-Presenting Cells

Genotoxic stress and RAS induce the expression of CD155, a ligand for the immune receptors DNAM-1, CD96 and TIGIT. Here we show that antigen-presenting cells upregulate CD155 expression in response to Toll-like receptor activation. Induction of CD155 by Toll-like receptors depended on MYD88, TRIF and NF-κB. In addition, IRF3, but not IRF7, modulated CD155 upregulation in response to TLR3 signals. Immunization of CD155-deficient mice with OVA and the TLR9 agonist CpG resulted in increased OVA-specific IgG2a/c titers when compared to wild type mice. Splenocytes of immunized CD155-deficient mice secreted lower levels of IL-4 and fewer IL-4 and GATA-3 expressing CD4+ T cells were present in the spleen of Cd155−/− mice. Our data suggest that CD155 regulates Th2 differentiation. Targeting of CD155 in immunization protocols using peptides may represent a promising new approach to boost protective humoral immunity in viral vaccines.


Introduction
Toll-like receptors (TLRs) are key sensors of the innate immune system that recognize conserved microbial domains known as pathogen-associated molecular patterns (PAMPs) such as LPS, flagellin and double-stranded RNA [1,2]. Upon activation, TLRs recruit the proximal adapter molecule myeloid differentiation factor 88 (MYD88) and/or the Toll/IL-1 receptor domaincontaining adaptor inducing IFN-b (TRIF) and activate various downstream pathways including mitogen-activated protein kinases (MAPKs), nuclear factor-kB (NF-kB) and interferon regulatory factors (IRFs). Most TLRs depend on MYD88 for their functions, whereas TLR3 requires TRIF for its activity and TLR4 activates both MYD88-dependent and TRIF-dependent responses. TLRs promote the function of antigen-presenting cells (APCs) by enhancing their antigen-presenting activity, cytokine production and expression of costimulatory molecules [3].
Activation of naïve CD4 + T cells by APCs leads to their differentiation into different subsets depending on the costimulatory signals and cytokines expressed by the APCs. T helper (T h ) 1 cells preferentially produce IFN-c and stimulate B cells to produce IgG2a/c antibodies, while T h 2 cells secrete IL-4, IL-5 and IL-13 that are critical for IgE production [4]. It was reported that TLR4 and TLR9 agonists induce the secretion of proin-flammatory cytokines such as IL-12 and IL-18, which support T h 1 cell differentiation, while TLR2-induced expression of ICOSL was shown to promote T h 2 differentiation [5,6,7]. However, the TLRinduced costimulatory signals involved in the regulation of T h 1 and T h 2 differentiation are not well characterized.
CD155, also known as Necl-5/Tage4/poliovirus receptor, is an immunoglobulin-like cell adhesion molecule and a member of the nectin-like family [8,9,10]. It is poorly expressed on normal cells, but its expression levels are upregulated on tumor cells and activated human dendritic cells (DCs) [11,12]. Ras activation and genotoxic stress have been shown to induce CD155 expression [13,14]. However, it is not known how CD155 expression is regulated on APCs. CD155 binds to several receptors including leukocyte adhesion-molecule DNAM-1/CD226, CD96/Tactile and TIGIT/VSTM/WUCAM [15,16,17]. DNAM-1 is expressed on most immune cells [18] and is upregulated on activated T h 1 cells and T h 2 cells [19,20]. In contrast, CD96 and TIGIT have a more restrictive expression pattern. CD96 is expressed by NK cells, T cells and activated B cells [21]. TIGIT is absent from naïve T cells, but is expressed on activated and memory CD4 + T cells and regulatory T cells [16]. Recognition of CD155 by DNAM-1 or CD96 renders tumor cells sensitive to NK cells and CD8 + T cell-mediated cytotoxicity [22,23]. DNAM-1 was also shown to be important for CD8 + T cell activation by non-professional APCs [24]. TIGIT inhibits T cell activation by inducing secretion of IL-10 and inhibiting the expression of pro-inflammatory cytokines such as IL-12 [16,25].
Here we show that CD155 expression is significantly induced on APCs by all tested TLR agonists. CD155 upregulation by TLR signals depended on MYD88 or TRIF-mediated NF-kB activation. In addition, IRF3, but not IRF7, modulated CD155 upregulation by TLR3 agonists. A role for CD155 in regulating the T h 2 polarization in response to TLR agonsists was suggested by increased antigen-specific IgG2a/c isotype titers and lower levels of IL-4 and fewer IL-4 + and GATA-3 + CD4 + T cells in the spleen of CD155-deficient mice. Hence, blocking of CD155 may be a promising novel approach to enhance the efficacy of vaccines that require strong T h 1 responses.

TLR Agonists Upregulate CD155 Expression on Mouse and Human APCs
To test if TLR agonists induce CD155 expression on different APCs, we treated the TLR-responsive murine macrophage-like cell line RAW264.7 with agonists for various TLRs [26]. CD155 expression was upregulated by all tested TLR agonists with the exception of flagellin, a TLR5 agonist ( Figure 1A). Flagellintreated RAW264.7 cells also failed to secrete IL-6, a cytokine produced in response to TLR5 activation suggesting that RAW267.4 cells are unresponsive to TLR5 stimulation in agreement with earlier reports (data not shown) [27].
To study the effect of TLR agonists on CD155 expression in primary mouse APCs such as macrophages, DCs and B cells, we established bone marrow-derived macrophages (BMDM), bone marrow-derived DCs (BMDC) and splenic B cell cultures. Similar to RAW264.7 cells, primary BMDMs and BMDCs upregulated CD155 expression in response to all tested TLR agonists. BMDCs also upregulated CD155 expression in presence of flagellin ( Figures 1A and 1B). In contrast to BMDMs and BMDCs, CD155 expression was only induced by TLR7, 8 and 9 agonists in B cells, despite the ability of all tested TLR agonists to activate B cells as indicated by the upregulation of the TLR responsive gene CD40 (data not shown). Upregulation of CD155 by TLR agonists was delayed in B cells when compared to BMDMs and BMDCs ( Figure  S1). In summary our data show that various TLR agonists can induce CD155 expression in APCs, while CD155 upregulation in B cells is restricted to MYD88-dependent TLRs expressed in the endosome. We observed no upregulation of CD155 in nonhematopoietic cells in response to different TLR agonists (data not shown).
Previous studies suggested that CD155 expression is enhanced on activated human DCs [12]. To study if TLR agonists induce CD155 expression on human DCs, we established monocytederived DCs (MoDCs) from human peripheral blood mononuclear cells. Treatment of MoDCs with agonists for TLR3 and TLR4, which are commonly expressed by MoDCs, induced upregulation of CD155 expression indicating that TLRs induce CD155 expression on human and mouse DCs ( Figure 1C) [2].
To test the regulation of CD155 expression by TLR agonists in vivo, wild type (WT) mice were immunized with LPS, Poly I:C, Pam3CSK4, CpG or saline. Splenic DCs and macrophages of immunized mice upregulated CD155 expression in response to all tested TLRs agonists, but not saline ( Figure 1D). MYD88 and TRIF   TLR signals are initiated by the adaptor proteins MYD88 and/  or TRIF depending on the TLR [26,28,29,30]. To gain better insight into the role of MYD88 and TRIF in the upregulation of CD155 expression in response to various TLR agonists, we treated Myd88and Trif-deficient BMDCs with different TLR agonists. Induction of CD155 expression in response to TLR agonists that specifically activate MYD88, such as Pam3CSK4 and CpG was abrogated in Myd88 2/2 , but not Trif 2/2 BMDCs (Figures 2A  and S2A). Similarly, agonists such as Poly I:C that signal exclusively through TRIF failed to upregulate CD155 expression in Trif 2/2 , but not Myd88 2/2 BMDCs (Figures 2B and S2B). Stimulation of Myd88 2/2 or Trif 2/2 BMDCs with LPS, a TLR4 agonist that activates both MYD88-and TRIF-dependent pathways, resulted in impaired CD155 induction when compared to WT BMDCs (Figures 2 and S2). These data support the conclusion that MYD88-or TRIF-initiated signals are sufficient to induce CD155 expression.

TLR Agonists-induced CD155 Expression Depends on NF-kB
Activation of MYD88 and TRIF by TLR agonists leads to the activation of NF-kB [28]. To test the role of NF-kB in regulating CD155 induction, RAW264.7 cells were treated with the NF-kB inhibitor, BMS-345541, prior to stimulation with LPS [31]. Pretreatment of RAW264.7 cells with BMS-345541 at concentrations equal or higher than the published IC 50 inhibited CD155 upregulation in response to LPS stimulation ( Figures 3A, S3A and S3B). Similar to RAW264.7 cells, pretreatment of BMDMs with BMS-345541 impaired CD155 upregulation in response to various TLR agonists suggesting that MYD88-and TRIF-induced CD155 expression critically depended on NF-kB ( Figures 3B  and S3C). Furthermore, induction of CD155 and IL-6 expression was impaired in R848-stimulated RAW264.7 cells overexpressing the IkBa-super repressor when compared to cells expressing a control plasmid ( Figures 3C and 3D). IkBa-super repressor expressing RAW264.7 cells also showed a modest reduction in CD155 upregulation in response to Pam3CSK4 and CpG stimulation when compared to cells expressing the control plasmid.
To address the question if NF-kB activation was sufficient to induce CD155 expression, we treated RAW264.7 cells with TNFa, a well-characterized activator of NF-kB [32]. Although TNFa treatment induced the upregulation of CD40, a known NF-kB target gene, it failed to upregulate CD155 expression ( Figures 3E and S3D) suggesting that NF-kB is necessary, but not sufficient to induce CD155 expression in response to TLR agonists.

CD155 Upregulation in Response to TLR Agonists does not Depend on MAPKs
TLR agonists also activate MAPK signaling pathways [1]. We therefore tested the contribution of p38 MAPK in regulating CD155 expression. Chemical inhibition of p38 MAPK activity by SB203580 did not impair the upregulation of CD155 expression on RAW264.7 cells by LPS, Poly I:C or Pam3CSK4 ( Figure 4A). Similarly, inhibition of p42/p44 MAPKs by PD98059 or simultaneous blocking of p38 and p42/44 MAPKs did not affect the ability of RAW264.7 cells to induce CD155 expression ( Figures 4B and 4C), but inhibited LPS-induced phosphorylation of p38 and p42/44 MAPK (data not shown). Overall our data

IRF3 Modulates TLR3-mediated Induction of CD155 Expression
The transcription factors IRF3 and IRF7 are important downstream regulators of the TRIF and MYD88 signaling pathways [33]. IRF3 is activated by TRIF-mediated signals in response to TLR3 and TLR4 agonists while recruitment of MYD88 by TLR7, 8, and 9 leads to the activation of IRF7 [34].
To study the role of IRF3 in CD155 expression, we analyzed the ability of Irf3 2/2 BMDCs to upregulate CD155 expression in response to Poly I:C and LPS. CD155 upregulation was impaired in Irf3 2/2 BMDCs in response to Poly I:C while upregulation in response to LPS was not affected when compared to WT BMDCs, suggesting that IRF3 is not essential for CD155 upregulation by TLR agonists that also activate MYD88 ( Figures 5A and S4A). IRF3 is phosphorylated and activated by the TANK-binding kinase 1 (TBK1) and/or IKK-related kinase epsilon (IKKe) in response to Poly I:C [35,36]. To investigate the role of TBK1, we used Tbk1 2/2 BMDCs on a Tnf 2/2 background as the lethal phenotype of Tbk1 2/2 mice can be rescued by the absence of TNF [37]. Lack of TBK1 had no effect on the ability of BMDCs to upregulate CD155 expression in response to LPS, Poly I:C or the MYD88-dependent TLR agonist CpG indicating that IKKe may function redundantly with TBK1 in the activation of IRF3 and induction of CD155 expression ( Figures 5B and S4B).
To test the contribution of IRF7 in CD155 upregulation by TLR agonists, we treated Irf7 2/2 BMDCs with different TLR agonists. CD155 upregulation was not impaired in Irf7 2/2 BMDCs in response to TLR agonists that recruit MYD88 and/ or TRIF indicating that IRF7 was not critical for CD155 upregulation ( Figures 5C and S4C).

TLR Agonists Increase CD155 mRNA Levels in a NF-kBdependent Manner
Our data supported the possibility that CD155 expression is directly regulated by TLR-mediated activation of the transcription factor NF-kB. To gain better insight into potential transcriptional regulation of CD155 in response to TLR agonists, we measured mRNA levels by quantitative real-time RT-PCR upon LPS stimulation ( Figure 6A). The induction of CD155 mRNA levels by LPS followed the biphasic expression pattern typical of NF-kB target genes [1]. The increase of CD155 mRNA levels in response to LPS stimulation was significantly impaired by pretreating cells with the NF-kB inhibitor BMS-345541 at concentrations above the published IC 50 ( Figure 6B). Furthermore, pretreatment of RAW264.7 cells with the transcriptional inhibitor actinomycin D abrogated upregulation ( Figure 6C). In contrast to the mRNA level, CD155 protein levels steadily increased after 3 to 5 hours of TLR agonist treatment and CD155 reached maximal expression after 18 hours of treatment ( Figures 6D and S5). Pretreatment of RAW264.7 cells with the protein synthesis inhibitor cycloheximide prevented induction of CD155 expression in response to LPS ( Figure 6C). In summary our data suggest that TLR

CpG-induced CD155 Expression Modulates Antigenspecific IgG2a/c Titers
TLRs regulate the generation of the adaptive immune responses by modulating the induction of accessory signals such as costimulatory molecules and cytokines [2]. The TLR9 agonist CpG has been successfully used as an adjuvant to boost immunity against influenza virus, measles virus and hepatitis B surface antigen [38]. The ability of CpG to enhance the immune response critically depended on MYD88 signaling in B cells [39,40,41]. Furthermore, CD155 expression on B cells was shown to be important for the activation of CD8 + T cells [42]. To test the role of CD155 in the adjuvant effect of CpG, Cd155 2/2 and WT mice were immunized with CpG together with OVA or OVA alone. 21 days after immunization, OVA-specific serum IgG levels were measured by ELISA. Cd155 2/2 mice immunized with CpG and OVA produced significantly higher titers of OVA-specific IgG2a/ c antibodies than WT mice ( Figures 7A and S7). IgG2a/c titers were analyzed as CD155-deficient mice were on a mixed genetic background and some mouse strains express IgG2c instead of IgG2a [43,44]. We observed no significant differences in the levels of OVA-specific total IgG or other IgG isotypes in Cd155 2/2 mice when compared to WT mice ( Figures 7B, 7C, S6A, S6B and S7). In further support for a role of CD155 in CpG-mediated adjuvant effects, splenic B cells of OVA and CpG immunized mice expressed significantly higher amounts of CD155 when compared to OVA only-injected mice ( Figure 7D). IgG2a/c isotype switching is associated with T h 1 responses. Costimulatory molecules have been shown to promote T h 2 immunity and to test the possibility that CD155 modulated IgG2a/c titers through the polarization of naïve CD4 + T cells, we determined if the CD155-binding receptors DNAM-1 and TIGIT are expressed on naïve CD4 + T cells of WT and Cd155 2/2 mice. CD4 + T cells with a naïve phenotype (CD4 + CD25 2 CD44 low ) expressed DNAM-1, but not TIGIT. DNAM-1 expression was significantly higher on CD4 + T cells of Cd155 2/2 mice when compared to WT mice ( Figures 7E, S6C and S6D). Cd155 2/2 splenocytes of OVA and CpG-immunized mice secreted significantly lower levels of IL-4, a cytokine shown to drive T h 2 differentiation, when compared to WT splenocytes ( Figure 7F) [45]. Furthermore, spleens of OVA and CpG immunized Cd155 2/2 mice harbored significantly less IL-4 and GATA-3 expressing CD4 + T cells when compared to WT mice (Figures 7G and 7H). In contrast, the percentage of IFN-c and T-Bet expressing CD4 + T cells was similar between Cd155 2/2 and WT mice ( Figures S6E, S6F and S6G). In summary, our data suggest that CpG-induced CD155 expression suppresses IgG2a/c isotype switching by regulating T h 2 polarization of naïve CD4 + T cells.

Discussion
Our study shows that CD155 expression is upregulated in response to various TLR agonists on APCs. Using APCs deficient in MYD88 or TRIF, we found that MYD88 and TRIF-initiated pathways were sufficient to induce CD155 expression and when activated together exhibited an additive effect on CD155 upregulation. Chemical and genetic inhibition of NF-kB sup- ported the conclusion that NF-kB was critical for MYD88/TRIFmediated CD155 upregulation. In contrast, IRF3 was not essential for TLR-mediated CD155 induction, but modulated CD155 expression in response to TLR3 agonists that activate TRIF. Our findings are in accordance with Andersen et al. who recently classified CD155 as an IRF3 augmented gene [46]. Other IRFs may contribute to CD155 induction as several TLR agonists activate IRF5 and IRF7, although our data do not support a role for IRF7 [47]. Coregulation of genes by NF-kB and IRFs has been found for the transcriptional regulation of other cell adhesion proteins such as ICAM-1 and VCAM-1 [48]. Preliminary analysis of the CD155 promotor indicated potential NF-kB and IRF transcription factor binding sites in close proximity to the transcription start site of CD155 (data not shown). It has previously been reported that CD155 contains AP-1 transcription factor binding sites in its promoter [14]. However, chemical inhibition of MAPK pathways that lead to AP-1 activation, did not abrogate CD155 upregulation suggesting that AP-1 is not required for CD155 induction in response to TLR agonists. Future studies will be needed to address the role of these potential binding sites  effector cytokines [2]. Upon stimulation of naïve T h cells by cognate antigen presented on APCs, T h cells differentiate into different subsets. Two important T h cells subsets are T h 1 cells, which produce IFN-c and promote immunity against intracellular pathogens and T h 2 cells, which secrete IL-4, IL-5 and IL-13 and generate immunity against extracelluar parasites. TLRs are critically involved in the early T h cell fate decision. MYD88deficient mice were found to be impaired in the activation of antigen-specific T h 1, but not T h 2 cell responses [49]. Furthermore, stimulation of APCs with the MYD88 activating TLR9 agonist CpG induced the secretion of T h 1-associated cytokines and the expression of co-stimulatory molecules on APCs [50,51]. Administration of CpG and antigen directs antibody production by murine B cells to T h 1-associated immunoglobulin isotypes, such as IgG2 and IgG3, while suppressing IgG1 and IgE-associated T h 2 isotypes [52]. In contrast, signals by CD28 and other costimulatory molecules preferentially promote T h 2 differentiation [53,54]. Interestingly, CD155 plays an important role in T h 1-type cellular immunity by promoting IFN-c production by NK cells and CD8 + T cell activation by non-professional APCs such as B cells [12,42]. DNAM-1, a receptor that binds CD155, was reported to be upregulated on T h 1 cells and to regulate their effector functions [19]. We found that Cd155 2/2 mice immunized with CpG and OVA produced higher titers of T h 1-associated isotypes IgG2a/c in comparison to WT littermates suggesting that CpG-induced CD155 expression suppresses T h 1 cell differentiation. Consistent with this possibility, splenocytes of immunized Cd155 2/2 mice produced significantly less IL-4. Furthermore, a lower percentage of CD4 + T cells expressed intracellular IL-4 and the T h 2 promoting transcription factor GATA-3. As IFN-c and IL-12p70 levels were similar between WT and Cd155 2/2 splenocytes our data indicate that CD155 does not regulate differentiation of T h 2 cells, but not T h 1 cells (Figures S6E and  S6H). It is unlikely that CD155 mediates these effects by binding to TIGIT as CD4 + T cells with a naïve phenotype did not express detectable levels of TIGIT at the cells surface and TIGIT is expressed at similar levels on T h 1 and T h 2 subsets (Figures 7E  and S6D) [16]. Furthermore, our data do not suport a role for CpG-induced CD155 expression in the differentiation of regulatory T cells as splenocytes of immunized Cd155 2/2 and WT mice with OVA and CpG induced similar amounts of IL-10, a cytokine produced by regulatory T cells ( Figure S6I). In summary, our data suggest that CpG-induced expression of CD155 on B cells and possibly other APCs modulates the humoral immune response by polarizing naïve CD4 + T cells towards a T h 2 phenotype.

Ethics Statement
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. Mice C57BL/6 mice were purchased from the Centre for Animal Resources at the National University of Singapore. Irf3 2/2 mice on a C57BL/6 background were purchased from the RIKEN Bioresource Centre (Japan). Irf7 2/2 mice on a C57BL/6 background were generously provided by Dr. F. Ginhoux (Singapore Immunology Network, Singapore) [55]. The Cd155 2/2 mice were generated as described in Abe et al. [56]. The genetic background of the Cd155 2/2 mice was 129/Sv (50%), C57BL/6 (25%) and DBA (25%). Offsprings of Cd155 2/2 mice6C57BL/6 mice breedings were used for all experiments.

Treatments
All TLR ligands were obtained from Invivogen (USA). Human MoDCs were treated for 48 hrs as follows: 1 mg/ml LPS and 25 mg/ml Poly I:C. RAW264.7 cells, mouse BMDCs, BMDMs and B cells were treated with 1 mg/ml LPS, 25 mg/ml Poly I:C, 40 ng/ml Pam3CSK4, 1 mM CPG ODN 1668 and 1 mg/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 TNFa (eBioscience, USA) for 24 hrs.
RAW264.7 cells transduced with IkBa dominant-negative mutant (Addgene, USA) or empty vector plasmid were treated as indicated above in 6 well dishes at a density of 2610 5 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, 2610 6 RAW264.7 cells were stimulated with 1 mg/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, 1610 6 RAW264.7 cells in a 10 cm dish were treated with 5 mg/ml actinomycin D (Sigma, Singapore) or 50 mg/ml cycloheximide (Calbiochem, Singapore) for 1 hr, followed by treatment with 1 mg/ml LPS for 5 hrs.
For pharmacological inhibition of NF-kB or MAPK signaling, 1610 6 BMDMs or 2610 6 RAW264.7 cells were seeded in 6-well plates and 10 cm dishes respectively. The next day cells were pretreated with 1 mM or 4 mM or 10 mM BMS-345541 or 20 mM SB203580 or 20 mM 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-kB inhibitor treated cells, the TLR ligand stimulation time was shortened to 5 hrs.
Intracelluar IFN-c 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 2/2 mice on day 21 after immunization. 5610 6 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 ml/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-c (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).

Real-time PCR
Total RNA was isolated using the RNeasy kit according to the manufacturer's instructions (Qiagen, Singapore). 2 mg of total RNA was reverse transcribed using random hexamer primer and M-MLV reverse transcriptase (Promega, Singapore). Each amplification mixture (25 ml) contained 50 ng of reverse transcribed RNA, 0.8 mM forward primer, 0.8 mM reverse primer and 12.5 ml 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 50uC for 2 min, 95uC for 10 min, and 40 cycles of 95uC for 15 sec and 60uC for 15 sec and 72uC for 1 min. All samples were normalized to the signal generated from the housekeeping gene Hprt. The following primers were used: Hprt-59: tgggaggccatcacattgt; Hprt-39: gcttttccagtttcactaatgaca; Cd155-59: cgtgtccatctctggctatg; Cd155-39 cgtgttcgtgctccagttat. Samples prepared without RNA served as negative control templates. Experiments were analyzed using GraphPad Prism software (Version 5.0a, GraphPad Software, USA).

Western Blotting
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 mg 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 IkBa antibody (Cell Signaling Technology, USA) followed by HRPconjugated 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).
OVA Immunization 20 mg ovalbumin (OVA) (Grade V, Sigma, Singapore) or 20 mg OVA and 50 mg 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 [40]. Briefly, 96 well MaxiSorp plates (Nunc, Singapore) were coated overnight at 4uC with 10 mg/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 4uC. OVA-specific IgG, IgG1 and IgG3 levels were determined by incubating plates for 1 hr with peroxidaseconjugated goat anti-mouse IgG, Fc c fragment specific, goat anti-mouse IgG, Fc c subclass 1 specific or and goat anti-mouse IgG, Fc c subclass 3 specific antibodies (Jackson Immunoresearch, USA). To analyze IgG2a/c titers, a 1:1 molar ratio of goat antimouse IgG Fc c subclass 2a specific and goat anti-mouse IgG Fc c, subclass 2c-specific antibodies was added to the plates. After 1 hr, plates were washed and incubated with 3,39,5,59-Tetramethylbenzidine (TMB) substrate according to the manufacturer's instructions (eBioscience, USA). Results were analyzed using GraphPad Prism software (Version 5.0a, GraphPad Software, USA).
Cytokine ELISA 5610 6 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-c, IL-12p70 and IL-10 in the supernatant was measured by ELISA according to the manufacturer's instructions (eBioscience, USA).