of April 12, 2018.This information is current as

Activated T CellsofProinflammatory Cytokines and Migration

Ig-Like Transcript 3 Regulates Expression of

Raffaello Cortesini and Nicole Suciu-FocaXugang Qiao, Donna M. Mancini, Charles C. Marboe, Chih-Chao Chang, Zhuoru Liu, George Vlad, Haiyan Qin,

http://www.jimmunol.org/content/182/9/5208doi: 10.4049/jimmunol.0804048

2009; 182:5208-5216; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/182/9/5208.full#ref-list-1

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Ig-Like Transcript 3 Regulates Expression of ProinflammatoryCytokines and Migration of Activated T Cells1

Chih-Chao Chang,* Zhuoru Liu,* George Vlad,* Haiyan Qin,* Xugang Qiao,*Donna M. Mancini,† Charles C. Marboe,* Raffaello Cortesini,* and Nicole Suciu-Foca2*

Ig-like transcript 3 (ILT3), an inhibitory receptor expressed by APC is involved in functional shaping of T cell responses towarda tolerant state. We have previously demonstrated that membrane (m) and soluble (s) ILT3 induce allogeneic tolerance to humanislet cells in humanized NOD/SCID mice. Recombinant sILT3 induces the differentiation of CD8� T suppressor cells both in vivoand in vitro. To better understand the molecular mechanisms by which ILT3 suppresses immune responses, we have generatedILT3 knockdown (ILT3KD) dendritic cells (DC) and analyzed the phenotypic and functional characteristics of these cells. In thisstudy, we report that silencing of ILT3 expression in DC (ILT3KD DC) increases TLR responsiveness to their specific ligands asreflected in increased synthesis and secretion of proinflammatory cytokines such as IL-1�, IL-1�, and IL-6 and type I IFN.ILT3KD-DC also secretes more CXCL10 and CXCL11 chemokines in response to TLR ligation, thus accelerating T cell migrationin diffusion chamber experiments. ILT3KD-DC elicit increased T cell proliferation and synthesis of proinflammatory cytokinesIFN-� and IL-17A both in MLC and in culture with autologous DC pulsed with CMV protein. ILT3 signaling results in inhibitionof NF-�B and, to a lesser extent, MAPK p38 pathways in DC. Our results suggest that ILT3 plays a critical role in the in controlof inflammation. The Journal of Immunology, 2009, 182: 5208–5216.

I mmunoglobulin-like transcript 3 (ILT3)3 is an immune in-hibitory receptor that belongs to a family of molecules thatcontain extracellular Ig-like domains. ILT3 is selectively ex-

pressed on provisional myeloid APC such as monocytes, macro-phages, and dendritic cells (DC) (1, 2), as well as on nonprofes-sional APC, such as endothelial cells (3).

The extracellular domain of ILT3 binds to T cells, shaping theirfunctional development. We have previously shown that APC,which overexpress ILT3, become tolerogenic, inducing T cell an-ergy and differentiation of T suppressor cells (Ts) (4, 5). Further-more, upon direct interaction with APC Ag-specific CD8� Ts“tolerize” these APC inducing the up-regulation of ILT3 anddown-regulation of costimulatory molecules on the cell surface ofAPC. More recently, we showed that soluble ILT3 (sILT3) can bedetected in serum from cancer patients and that it is produced byCD68� tumor-associated macrophages (6), contributing to the im-pairment of patients’ immune reactivity. Recombinant sILT3-Fc,like membrane-bound ILT3, induces Th anergy and differentiation

of Ag-specific CD8� Ts both in vitro (7) and in vivo, inducingtolerance to allogeneic human tissue in SCID mice, which havebeen humanized by injection of PBMC (6, 8).

Similar to other inhibitory members of the ILT family, ILT3displays a cytoplasmic tail containing ITIM. Immunoblotting witha phospho-tyrosine Ab showed a marked decrease of protein ty-rosine phosphorylation levels in monocytes treated with mAbs toILT3 and HLA class II or Fc�RIII receptors on the surface ofmyeloid cells (1, 7). This effect is attributable to the recruitment ofthe inhibitory phosphatase Src homology region 2 domain-con-taining phosphatase (SHP)-1 to the ITIM and suppression of Ca2�

mobilization.The mechanism(s) by which ILT3 modulates immune responses

is largely unknown. We previously reported that suppression ofNF-�B activation and low expression of costimulatory moleculesaccount at least in part for the tolerogenic phenotype of ILT3-transduced myeloid (KG1) tumor cells (4). Experimental datashow that addition of a blocking anti-ILT3 Ab to cocultures of Tcells and DC increases the T cell production of IFN-� and othercytokines (7, 9), suggesting a cytokine regulatory component ofILT3-mediated suppression. To identify genes/pathways that areregulated by ILT3 and to better understand the role of ILT3 inphysiologically normal, nonmalignant DC, we designed a series ofadenoviral vectors, which efficiently infect monocytes or DC si-lencing the expression of ILT3 via the production of small inter-fering RNA (siRNA). Using this system, we have identified somepreviously unknown functions of the ILT3 molecule such as itscapacity to regulate cytokine responses of APC (including the syn-thesis of chemoattractants, which ultimately regulate T cell acti-vation) and their maturation and functional differentiation.

Materials and MethodsAbs, cytokines, and reagents

Purified Abs to NF-�B and MAPK pathway proteins were purchased fromCell Signaling Technology. Polyclonal anti-ILT3 Abs were purchased fromR&D Systems and anti-�-actin from Santa Cruz Biotechnology. All flowcytometry conjugated Abs were purchased from BD Biosciences, except

*Department of Pathology and †Department of Medicine, Columbia University, NewYork, NY 10032

Received for publication December 3, 2008. Accepted for publication February13, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Juvenile Diabetes Research Foundation(1-2008-550) and the Interuniversitary Organ Transplantation Consortium (Rome,Italy).2 Address correspondence and reprint requests to Dr. Nicole Suciu-Foca, ColumbiaUniversity, Department of Pathology, 630 West 168th Street, P&S 14-401, NewYork, NY 10032. E-mail address: ns20@columbia.edu3 Abbreviations used in this paper: ILT3, Ig-like transcript 3; ctrl-DC, control DC;DC, dendritic cell; ILT3KD, ILT3 knockdown; INDO, indoleamine-pyrrole 2,3-di-oxygenase; IKK��, I�B kinase ��; p, phosphorylated; polyI:C, polyinosinic-poly-cytidylic acid; SHP, Src homology region 2 domain-containing phosphatase; sILT3,soluble ILT3; siRNA, small interfering RNA; Ts, T suppressor cell.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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IL17A-PE (eBioscience) and ILT3-PC5 (Beckman Coulter). CMV proteins(Grade 2 Ag) were obtained from Microbix Biosystems.

Generation of monocyte-derived DC

Peripheral blood samples were purchased from the New York Blood Cen-ter. Monocytes were obtained from mononuclear cells by plastic adher-ence. DC were generated by culturing monocytes in 6-well plates for 7days with GM-CSF and IL-4 (R&D Systems), as described previously (4).Half of the culture medium was replaced with fresh medium at 2-dayintervals. Cultured cells were further purified to �90% homogeneity bynegative selection of contaminating lymphocytes using CD2� and CD19�

Dynal magnetic beads (Invitrogen) on day 7. The differentiation ofCD14�CD11chighCD83lowCD86highHLA-DR� immature DC was con-firmed by flow cytometric analysis.

Construction of siRNA ILT3 vectors and knockdown of ILT3

Adenoviral RNAi Expression System (Invitrogen) was used to generatesiRNAILT3 directed against ILT3 expression by targeting two separate re-gions of the ILT3 mRNA. Two double-stranded 19-mer corresponding tothe ILT3 nucleotide sequences 281–299 (5�-GAC AGG AGC CTA CAGTAA A-3�) and 351–369 (5�-GGA GAT ACC GCT GTT ACT A-3�) werecloned separately into an U6 RNA entry vector (Invitrogen), according tothe manufacturer’s design. Vectors containing an U6 RNA polymerasepromoter and the ILT3 siRNA sequences were subsequently recombinedwith the pAd/Block-it DEST vector (Invitrogen) to create the final desti-nation adenoviral vectors, pAd-RNAiILT3–281 and pAd-RNAiILT3–351. Allconstructs were verified by sequencing from both ends. To generate RNAirecombinant adenoviruses, ILT3 siRNA adenoviral vectors were trans-fected into 293A cells (Invitrogen) using Lipofectamine 2000 (Invitrogen).Viral stocks were amplified at least twice by reinfecting 293A cells andfiltered through a 0.45 �M cellulose membrane filter before use. A recom-binant adenovirus, pAd-RNAicon, containing only the U6 RNA polymerasepromoter (without ILT3 RNAi sequences) was similarly generated and wasused as control throughout the study.

A two-step adenoviral infection protocol was used for efficient ILT3knockdown (ILT3KD). Monocytes were first infected with recombinantadenoviruses pAd-RNAiILT3–281 on the first day of the 7-day culture withGM-CSF/IL-4, adding viral stocks to the medium at a 1:5 (v/v) ratio. Thesecells were reinfected with the second ILT3KD virus, pAd-RNAiILT3–351,on day 3 at the same viral stock to medium ratio. In parallel, control DC(ctrl-DC) were generated by the same protocol using the empty vectorpAd-RNAicon for infections. Surface expression of ILT3 was monitored byflow cytometry using an anti-ILT3 mAb (Beckman Coulter). The sche-matic structure of adenoviral constructs and efficiency of ILT3 siRNAtransduction are shown in Fig. 1. Immature DCs were used 9–11 days afterinfection.

TLR ligand treatments of DC

Ultrapure LPS of E. coli K12 strain, flagellin, and Pam3CSK4 were ob-tained from InvivoGen. Polyinosinic-polycytidylic acid (polyI:C) was pur-chased from Sigma-Aldrich. The NF-�B inhibitor Bay11–7082, the MAPKp38 inhibitor SB203580 and its inactive form SB202474 were purchasedfrom CalBiochem. ILT3KD- and ctrl-DC were treated with various TLRligands overnight (18 h). LPS was used in a wide range of concentrations(3–100 ng/ml). Antagonists of TLR1/2 (synthetic tripalmitoyl lipopeptide,Pam3CSK), and TLR3 (synthetic double-stranded RNA, poly I:C) wereused at 2 �g/ml and TLR5 (flagellin) at 1 �g/ml. NF-�B and MAPKp38pathway inhibitors were used at 10 �M. The supernatants were tested usingthe Proteome Profiler Array (R&D Systems), according to the manufac-turer’s instructions. A pulse-chase type experiment was conducted to mea-sure cytokine transcription and mRNA stability following TLR activationof DC. LPS (100 ng/ml) was used to stimulate ILT3KD- and ctrl-DC for1 h, then transcription was blocked with actinomycin D (1 �g/ml; SigmaAldrich). DC were lysed for PCR analysis at 1-h intervals following acti-nomycin treatment.

RNA extraction, cDNA synthesis, and real-time PCR

Total RNA was extracted from 1 to 10 � 105 purified cell suspensionsusing the Absolute RNA kit (Stratagene). First-strand cDNA was syn-thesized using oligo dT primers with Superscript III First Strand kit(Invitrogen). Real-time quantitative RT-PCR was performed on a 7300Real Time PCR instrument (Applied Biosystems) in 50-�l reactionsusing 1 �l of cDNA. The following qPCR probes (Applied Biosystems)were used: IL1A (Hs00174092_m1), IL1B (Hs00174097_m1), IL6(Hs00174131_m1), IL10 (Hs00174086_m1), TNF (Hs00174128_m1),IFNG (Hs00174143_m1), IFNA1 (Hs00256882_s1), IL12B (Hs01011518_

m1), IL8 (Hs00174103_m1), ILT3/LILRB4 (Hs00429000_m1), CD40(Hs00386848_m1), CD14 (Hs00169122_g1), CD80 (Hs00175478_m1), CD86(Hs00199349_m1), Indo (Hs00158027_m1), ICAM1 (Hs00164932_m1), CD68(Hs00154355_m1), CXCL10 (Hs00171138_m1), CXCL11 (Hs00171042_m1),and GAPDH (436317E). Data were collected and analyzed with 7300 SDS 1.31Software (Applied Biosystems). The relative amount of gene expression was cal-culated by 2-�Ct, where �Ct � [Ct(gene) � Ct(CD68)], and Ct is the “crossingthreshold” value returned by the PCR instrument for every gene amplification. Themyeloid-specific marker CD68 selectively expressed by macrophages was usedfor normalization of gene expression data because it is not affected by ILT3KD orLPS treatment.

Immunoprecipitation and Western blotting

DC were lysed in radioimmunoprecipitation assay buffer (Upstate) con-taining both phosphatase inhibitor mixtures I and II (Sigma-Aldrich) andproteinase inhibitors (Roche Applied Science) for 20 min on ice. After abrief centrifugation, equal amounts (20–30 �g) of total cell lysate wereloaded on 10% precast NuPAGE gels (Invitrogen) and transferred to apolyvinylidene difluoride membrane. Immunoblotting was performed withvarious primary and HRP-conjugated secondary Abs and detected bychemiluminescence (SuperSignal West Pico kit; Pierce) as described pre-viously (6). Expression of proteins was quantitated by NIH ImageJ Soft-ware. Expression of �-actin was used to normalize the protein contentbetween lanes. For detection of ILT3 interacting protein complexes, DCcells were first treated with the phosphatase inhibitor bpV(phen) (Calbio-chem) for 20 min, followed by lysis in a 1% Nonidet P-40 buffer containingproteinase and phosphatase inhibitors. Supernatants were collected after abrief sonication and centrifugation and incubated with 5 �g of goat anti-ILT3 polyclonal Ab (R&D Systems) or goat IgG (Sigma-Aldrich) for 16 hfollowed with 20 �l of protein A/G-agarose for 1 h. After extensive wash-ing, protein A/G agarose was transferred to a polyvinylidene difluoridemembrane probed sequentially with anti-SHP-1, anti-SHP-2, SHIP-1, andSHIP-2 (Cell Signaling Technology) and anti-ILT3 Abs and analyzed by achemiluminescence as described above.

Flow cytometry and cytokine detection

CD3� T cells were stimulated with allogeneic ctrl-DC or ILT3KD-DC at1:10 (stimulator:responder) ratio for 5 days. Alloactivated T cells were thenstimulated with 1 �g/ml ionomycin and 100 ng/ml PMA (Sigma-Aldrich)for 5 h. Brefeldin A (10 �g/ml; BD Biosciences) was added for the final 3 hof culture. Cells were fixed and permeablized using the Fix&Perm kit(Invitrogen) and were incubated with anti-IL17A-PE (eBioscience) andanti-IFN-� (BD Biosciences). Cell surface molecules were analyzed byflow cytometry as described previously (4). Cytokines IL-1�, IL-6, andIFN-� in supernatants of cultured cells were tested using cytokine beadsarray kits (BD Biosciences), according to the manufacturer’s instructions.Data was acquired and analyzed on a FACSCalibur instrument (BD Bio-sciences) using six-parameter acquisition.

T cell proliferation assays

Human CD3� T cells were isolated from mononuclear cell populationsusing a Pan T cell isolation kit (Miltenyi Biotec). Immature ctrl-DCs orILT3KD-DC were irradiated (3000 rad) and used as stimulators. PrimaryMLC were performed in a 96-well culture plate using T cells (5 � 104

cells/well) stimulated for 6 days with allogeneic DC or with autologous DCat various responder to stimulator ratios (100:1–400:1). For T cell re-sponses to CMV Ags, CD3� T cells (5 � 104 cells/well) were incubatedfor 5 days with Ctrl- or ILT3KD-transfected autologous DC (1 � 104

cells/well) in cultures containing various concentrations (2.5 and 5 �g/ml)of CMV proteins (grade 2 Ag; Microbix Biosystems). Tritiated [3H]TdRwas added to the cultures over the final 18 h of incubation. [3H]TdR in-corporation was measured using an LKB 1250 Betaplate counter(PerkinElmer). Mean cpm of triplicate cultures and the SE were calculated.

T cell migration assays

Purified T cells were stimulated for 3 days on anti-CD3 T cell activationplates (BD Biosciences) in the presence of 2 �g/ml anti-CD28 mAb (BDBioscience). A total of 2 � 104 of these cells were added to the upperchamber of a 24-well Transwell plate (pore size, 5 �m; Corning Costar),whereas DC supernatants (0.5 ml) or chemokines were added to the lowerchamber. After 2-h incubation at 37°C, the contents of the lower chamberwere collected by low-speed centrifugation (250g) and counted directlyunder a light microscope. Each experiment was performed in duplicate.Values are given as percentage of cells that migrated.

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Statistical analysis

Data from multiple experiments were expressed as mean � SEM. Thetwo-tailed, paired Student’s t test was performed to compare two or moremean values. A value of p 0.05 was considered statistically significantand is indicated by *. A value of p 0.01 was considered statistically verysignificant and is indicated by **, whereas p � 0.05 (denoted as #) wasconsidered insignificant.

ResultsModulation of LPS inducible cytokines induction by ILT3

To determine the role of ILT3, monocyte-derived immature DCwere transfected with ILT3 siRNA (ILT3KD-DC), whereasctrl-DC were infected in parallel cultures with an empty vector(Fig. 1A). Flow cytometry showed reduced surface expression ofILT3 in ILT3KD cells. The ILT3 mean fluorescence intensity was56 in ILT3KD-DC compared with 205 in empty vector-transfectedcontrol cells and 769 in ILT3high DC, in which ILT3 up-regulationwas induced by IFN-�/IL-10 treatment, as described previously(10) (Fig. 1B). Quantitative RT-PCR showed that gene-specificknockdown suppressed ILT3 mRNA expression by up to 90%( p 0.01; Fig. 1B). Examination of immature, ctrl-, andILT3KD-DC by quantitative RT-PCR or flow cytometry for ex-pression of myeloid lineage markers (CD68, CD14), costimulatorymolecules (CD40, CD80, CD86), cytokines (IFN-�, IL-1�&�,IL-8, IL-10, IL-12�, TNF-�), and adhesion molecules (ICAM-1)showed no significant differences between ILT3KD-DC and non-activated control DC (Fig. 2A and data not shown).

To explore the possibility that ILT3 plays an inhibitory role onlyupon activation of DC, ctrl-DC, or ILT3KD-DC were treated withultrapure LPS (E. coli K12 strain) for 18 h. Ultrapure LPS isknown to specifically activate DC via TLR4, affecting the expres-sion of various genes, including proinflammatory cytokines, co-stimulatory, and other molecules (11). The transcription level of asmall group of proinflammatory cytokines (IL-1�, IL-1�, IL-6)was consistently 2- to 3-fold higher (*, p 0.05) in LPS-activatedILT3KD-DC than in ctrl-DC (Fig. 2, B and C). This ILT3KD-mediated enhancement of cytokine responses occurred at LPS con-centrations ranging from 3 to 100 ng/ml (Fig. 2B). Enhanced ex-

pression of proinflammatory cytokine mRNA in ILT3KD-DC alsooccurred at the protein level as shown by cytometric bead analysisof soluble proteins in the culture supernatants of LPS-treated DC(Fig. 2D).

Other LPS-induced genes involved in inflammation such asIL-8, IL-12�, indoleamine-pyrrole 2,3-dioxygenase (INDO), co-stimulatory (CD40, CD86) molecules and type I (IFN-�1) and typeII (IFN-�) showed no change (Fig. 2C). Addition of the transcrip-tional inhibitor actinomycin D to LPS-activated ILT3KD- andctrl-DC rapidly suppressed the IL-1� and IL-6 mRNA levels. Thehalf-life of IL-1� mRNA was 51 � 6 min in both ILT3KD- andctrl-DC, while that of IL-6 was 82 � 12 min (data not shown),indicating that ILT3 affects the transcription of these cytokines butnot the mRNA stability.

Modulation of other TLR responses by ILT3

Ligation of various pattern recognition receptors such as TLR isknown to result in production of inflammatory cytokines (reviewedin Ref. 12). We tested the capacity of ILT3� (ILT3KD-DC) andILT3� (ctrl-DC) to produce IL-1�, IL-1�, IL-6, IL-12�, TNF-�,INDO, and both type I (IFN-�1) and type II (IFN-�) IFNs in re-sponse to ligation of TLRs. We choose antagonists of TLR1/2(synthetic tripalmitoyl lipopeptide, Pam3CSK), TLR3 (syntheticdouble-stranded RNA, polyI:C), and TLR5 (flagellin), which areknown to activate monocyte-derived DC (13). As shown in Fig.3A, these TLR ligands varied with respect to their capacity toinduce the transcription of these inflammatory cytokines withpolyI:C triggering the strongest inflammatory responses. ILT3 si-lencing in ILT3KD-DC resulted consistently in a 1.5- to 3-foldhigher transcriptional induction of IL-1�, IL-1�, and IL-6 by allforms of TLR ligands (Fig. 3A). In addition, the lack of ILT3expression in ILT3 KD-DC was accompanied by enhanced tran-scription of IL-12� and TNF-� mRNA upon ligation of TLR3(polyI:C) but not of TLR1/2 (Pam3CSK4) or TLR5 (flagellin).Expression levels of both type I IFN (IFN-�1) and type II IFN(IFN-�) were also significantly induced by ILT3 silencing (3- to5-fold, p 0.01, and 2- to 3-fold, p 0.05, respectively; Fig. 3A).Analysis of IL-1� and IL-6 at the protein level confirmed the re-sults obtained by analysis of mRNA expression (Fig. 3B). Thesedata indicate that DC responsiveness to various pathogens/foreignAgs provided by a variety forms of TLR ligands is modulatedby ILT3.

ILT3KD-DC produce increased amounts of T cellchemoattractants, CXCL10 and CXCL11

We used a cytokine array system (Proteome Profiler Array; R&DSystems) to study the soluble factors released from untreated oractivated ILT3KD-DC into culture medium. Silencing of ILT3 inILT3KD-DC had minimal effect on secretion of soluble factors inresting DC. As expected, ligation of TLR4 by treatment of DCwith LPS triggers the production of numerous pro-inflammatorycytokines and chemokines. Knockdown of ILT3 further potentiatesthe induction of several of these genes, including complement 5a,CXCL10, CXCL11, MIF, MIP-1a, and MIP-1� for an additional�1.5 fold (Fig. 4A). RT-PCR analysis of ILT3KD-DC and ctrl-DCtreated with various concentrations (3–100 ng/ml) of LPS showedthat ILT3KD-DC generated a CXCL10 and CXCL11 response 2-to 3-fold stronger at all the concentrations tested (Fig. 4B).

Because supernatants from activated ILT3KD cells showed in-creased amounts of CXCL10 and CXCL11, we evaluated theirability to attract immune effectors cells, such as activatedCXCR3� T cells, in Transwell assays. Supernatants from LPS-activated ILT3KD-DC and ctrl-DC, but not supernatants from un-treated DC, induced the transmigration of activated T cells (Fig.

FIGURE 1. A, Schematic structures of adenoviral constructs used forsilencing of ILT3. Abbreviations are as follows: ITR, inverted terminalrepeat; U6P, U6 polymerase promoter; PoIIITerm, polymerase III termi-nator; and dsOligos, insertion regions for ILT3 siRNA oligos. B, Analysisof the level of expression of ILT3 in DC infected with either pAd-controlor pAd-ILT3/KD vectors by flow cytometry and RT-PCR. DC treated withIL-10 (5 ng/ml) and IFN-� (1000 U/ml) for induction of ILT3 were usedas positive control.

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4C). Transmigration of activated T cells in response to superna-tants from LPS-activated ILT3KD-DC was significantly increased( p 0.05) when compared with transmigration in response tosupernatants from ctrl-DC that were similarly treated (Fig. 4C).

ILT3KD-DC elicit increased T cell proliferate responses

To determine whether ILT3KD increases the stimulatory capacityof DC, we tested in parallel the capacity of ILT3KD-DC andctrl-DC from the same donor to stimulate the proliferation of al-logeneic T cells. As shown in Fig. 5, ILT3KD-DC induced sig-nificantly stronger ( p 0.05 at 1:200 ratio and p 0.001 at 1:400ratio, respectively) T cell proliferation compared with ctrl-DC at1:200 – 400 stimulator to responder cells ratios. Similar resultswere obtained in the experiment in which T cells were primedto autologous ILT3KD-DC or ctrl-DC in cultures containingCMV protein. ILT3KD-DC induced significantly stronger ( p

0.05 at 5 �g and p 0.01 at 2.5 �g protein, respectively) T cellresponses to CMV Ags at concentrations ranging from 2.5 to 5�g/ml (Fig. 5A).

Flow cytometric analysis of the frequency of Th1 and Th17 cellsin 5-day cultures allostimulated with ILT3KD-DC or ctrl-DCshowed that knockdown of ILT3 elicited an increase in the size ofthe T cell populations producing IFN-� (from 1.0 to 6.7%) andIL-17 (from 0.3 to 1.5%) (Fig. 5B). Three repeat experiments showa consistent 3- to 7-fold increase in the size of IFN-� ( p 0.05)and IL-17A ( p 0.05) secreting T cell populations induced byILT3KD-DC vs ctrl-DC (Fig. 5C).

ILT3 regulates the NF-�B and MAPKp38 kinase pathways

To better understand how ILT3 silencing enhances DC response todanger signals and identify the signaling pathways involved weused specific inhibitors for MAPK p38, SB203580, or NF-�B, and

FIGURE 2. Modulation of LPS-inducible gene expression in ILT3KD DC. A, Expression of the myeloid marker CD68 measured by real-time PCR (andnormalized by GAPDH) in ctrl-DC, ILT3KD-DC, and lymphocytes. B, Effect of silencing ILT3 on IL-1� and IL-6 mRNA induction by various concen-trations of LPS. Expression of CD68 was used as normalization control. C, RT-PCR analysis of ctrl vs ILT3KD DC treated with 100 ng/ml LPS. Resultsare representative of three to five independent experiments. D, IL-1� and IL-6 expression in supernatants from ctrl- or ILT3KD-DC treated overnight with100 ng/ml LPS. Expression of soluble forms of IL-1b and IL-6 in supernatants was determined by cytokine bead array and expressed as the mean of threeindependent experiments � SEM. (�, p 0.05; ��, p 0.01.)

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Bay11-7082. Addition of Bay11-7082 or SB203580 (but not of itsinactive analog SB202474) to LPS-activated DC blocked IL-1�expression, both at the mRNA (data not shown) and soluble pro-tein level (�60%; Fig. 6A). To determine whether expression ofILT3 affects the phosphorylation of MAPK and I�B kinases, LPS-treated ILT3KD-DC and ctrl-DC were subjected to immunoblotanalyses using various Abs that recognize the total (T) or phos-phorylated (p) forms of MAPKp38, I�B-�, and its regulator, I�Bkinase �� (IKK��). In both types of cells, we found that phos-phorylation of IKK�� and MAPKp38 was induced by LPS in atime-dependent manner with a peak at 30 min after treatment.However, at the 3-h time point the resynthesis and degradation ofI�B-� had reached an equilibrium state as indicated by others (14–

16). More p-IKB� (2.0�), p-IKK�� (1.7�), and p-MAPKp38(1.3�) was detectable in LPS-treated ILT3KD-DC than in controlDC (Fig. 6B). Phosphorylation of MAPKp42/p44, JNK, and NF-�Bp65 (RelA), on the other hand, were unchanged (data notshown). The total amounts of each of the respective proteins werealso unchanged. This result supports the notion that both NF-�Band MAPKp38 pathways are required for LPS activation and thatsignaling is affected by ILT3 expression.

We then immunoprecipitated lysates from bpV(phen)-treatedcells (ILT3KD-DC and ctrl-DC) with a goat anti-ILT3 Ab (or goatIgG isotype control) and examined the immunoprecipitates by im-munoblotting with an anti-phospho-tyrosine (p-Tyr) Ab (Fig. 6C).Our results indicate that the anti-ILT3 Ab specifically pulls down

FIGURE 3. Modulation of TLR inducible responses of ctrl- or ILT3KD-DC. A, RT-PCR analysis of the cytokine responses of ctrl- or ILT3KD-DC (�and f, respectively) treated with various TLR ligands. Data from four independent experiments are expressed as the mean � SEM. B, Cytokine bead arraydetection of IL-1� and IL-6 in supernatants of Ctrl- or ILT3KD-DC treated with various TLR ligands. Data are represented as mean � SEM of threeindependent experiments (�, p 0.05; ��, p 0.01; #, p � 0.05).

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several Tyr-phosphorylated proteins, in addition to p-Tyr-ILT3,from ctrl-DC lysates but not from that of ILT3KD-DC, supportingthe notion that phospho-ILT3 interacts with other p-Tyr proteins.

Probing the membrane with various phosphatase-specific Absknown to interact with ITIM indicates that SHP-1 and SHIP-1were present in protein complexes immunoprecipitated by anti-ILT3.

FIGURE 4. Expression of chemotactic factors by ctrl-DC and ILT3KD-DC. A, Proteome Profiler Array analysis of supernatants ctrl- or ILT3KD-DCtreated or untreated with 100 ng/ml LPS. Genes up-regulated at least 1.5-fold by silencing of ILT3 are indicated. Fold of up-regulation was calculated afternormalizations of values by the positive control. B, RT-PCR analysis of CXCL10 and CXCL11 transcription in ctrl-DC and ILT3KD-DC after treatmentwith various concentrations (0–100 ng/ml) LPS. The mean from three independent experiments and SEM are indicated. C, Comparison of T cell che-moattractant properties of supernatants from LPS-activated ctrl- and ILT3KD-DC. Activated T cells were added into the upper chambers of a Transwellplate (5 �M pore size), and supernatants from ctrl-DC or ILT3KD-DC were added to the lower chambers. Results are expressed as mean � SEM of thetotal numbers of migrated T cells after 2 h (�, p 0.05).

FIGURE 5. The effect of ILT3KDon priming T cells responses. A,CD3� T cell proliferative responsesto allogeneic ctrl-DC and ILT3KD-DC or autologous stimulators present-ing CMV Ags. Proliferation of cellswas determined by [3H]thymidine in-corporation. Results were expressedas mean � SEM of four independentexperiments. �, p 0.05; ��, p 0.01. B, Increased intracellular ex-pression of IFN-� and IL-17A byCD3� T cells cocultured withILT3KD DC for 5 days. A represen-tative result of flow cytometric anal-ysis of T cells gated out from T-DCcocultures is shown in B. C, Resultsobtained from three independent ex-periments show 3- to 7-fold expan-sion of Th1 and Th17 populationsfollowing T cell coculture with allo-geneic ILT3KD-DC vs ctrl-DC.�, p 0.05.

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Other phosphatases, such as SHP-2, and SHIP-2, which may in-teract with ITIM domains of inhibitory receptors (17, 18), did notappear to interact with ILT3. Immunoprecipitation with IgG didnot yield any ILT3 interacting proteins (data not shown). Takentogether with Fig. 6B, these data indicate that the NF-�B and, to alesser extent, the MAPKp38 pathways are negatively regulated byILT3 signaling through interaction with SHP-1 and/or SHIP-1.

DiscussionIn previous studies, we demonstrated that ILT3 is a crucial inhib-itory molecule whose expression affects the function of the APCand that of the T cells with which they interact (3, 4, 6–8, 10, 19).Although the function of this molecule has been well documented,the mechanisms by which ILT3 operate remains elusive. Thepresent study attempts to expand the mechanistic understanding ofILT3-driven suppression.

TLR are a type of pattern recognition receptors that recognizemolecules that are broadly shared by pathogens but distinguish-able from host molecules (13, 20 –22). TLR play an importantrole in innate immunity, and by signaling the presence of patho-gens, they trigger inflammation and the recruitment of adaptiveimmune response to the affected microevironment. If un-checked, the self-amplification of TLR signaling can lead toinflammatory/autoimmune disease (reviewed in Ref. 20). Weshow here that overactive inflammation is accompanied by

a more vigorous proinflammatory cytokine response byILT3KD-DC when compared with ctrl-DC, which expressphysiological levels of ILT3 in response to “danger” signalsrelayed through a variety of TLR. From a signaling perspective,our results also show that ILT3 recruits SHP-1 and/or SHIP-1 torestrain the APC’s (LPS-triggered) activation pathways whichrely on NF-�B and, to a lesser extent, MAPKp38. Taken to-gether, these findings provide direct evidence that the physio-logical concentration of ILT3 on APC may work as a “checkand balance” for overactive immune responses by interactingwith inhibitory phosphatases SHP-1 and/or SHIP-1 and damp-ening NF-�B and MAPKp38 activity.

Our results therefore are consistent with observation thatSHP-1 mutant mice (mev/mev) demonstrate higher NF-�B andMAPp38 activities (23) and are hypersensitive to LPS andpathogenic challenges (23–25). There is a notable difference,however, between ILT3KD-DC and SHP-1 mutant (mev/mev)mice, with respect to induction of type I IFN following TLRligation. Although our results indicate that silencing ILT3-en-hanced type I IFN, IFN-�1, mRNA production in DC, mutationon Shp-1 (mev/mev) in mice decreases the synthesis of IFN-�after LPS treatments (23). This discrepancy may suggest thatthe negative signaling delivered by ILT3 does not entirely relyon SHP-1 and other signaling molecules, such as SHIP-1 (Fig.6C), may also contribute to the ILT3KD phenotype. A recent

FIGURE 6. A, Suppression of IL-1� induction by MAPKp38 and NF-�B pathway inhibitors in ctrl-DC and ILT3KD-DC. Supernatants of variouslytreated DC were analyzed for IL-1� protein by cytokine bead arrays. B, Modulation of MAPK p38 and I�B pathways by ILT3KD. Both ctrl-DC andILT3KD-DC were treated with 100 �g/ml LPS for time indicated, and an equal amount (20 �g) of protein lysate was analyzed by Western blot using variousAbs. Quantitation of the total (T) and phosphorylated (p) protein fractions was performed by normalizing results to the �-actin expression and wereexpressed in bar-graph form. C, ILT3 immunoprecipitation followed by Western blotting indicates that SHP-1 and SHIP-1 associate with ILT3 in ctrl-DCbut not in ILT3KD-DC. ILT3KD-DC lysate (1/10 input) was used as a positive control; NS denotes a nonspecific band.

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study has implicated SHIP-1 in preventing TLR ligand induc-tion of type I IFN synthesis in mice (26).

Binding of CXCL10 and CXCL11 to their receptor, CXCR3,induces various cellular responses, most notably the attraction Th1cells and promotion Th1 cell maturation (reviewed in Refs. 27 and28). Dysregulation of CXCR3 and its ligand expression has beenimplicated in various types of diseases, such as multiple sclerosis(29) and type I diabetes (30). Our results showed that T cells re-spond to the higher levels of CXCL10 and CXCL11 produced byILT3KD-DC, with increased migration rates toward the chemo-kine gradient, suggesting a possible regulatory role for ILT3 incontrolling the trafficking of inflammatory T cells. Down-regula-tion of ILT3 can cause excess inflammation and infiltration of Tcells in locally affected lesions, leading to destruction of tissue orautoimmune diseases. The importance of ILT3 in heart transplan-tation as a tolerogenic marker has been documented in our previ-ous studies (3, 31).

Mechanistically, there are still some questions that remain un-answered. For example, although the phosphorylation of I�B isincreased by ILT3 silencing, the total levels of I�B are notdrastically affected. This may be explained by recent findingsthat Tyr-phosphorylation of I�B is not always followed by deg-radation (14 –16, 32). It is also unclear why only a handful of(IL-1��, IL-6, IFN type I/II, CXCL10, CXCL11) genes areaffected by ILT3KD, despite the fact that many inflammatorycytokine genes are known to be NF-�B-regulated (33). How-ever, based on the data presented here, we propose that the lossof ILT3 during external stimuli prevents binding of I�B to thetranscription factor p50/p65 in the cytoplasm and partially ac-tivates MAPK p38. The heterodimeric p50/p65 complexessubsequently translocate to the nucleus whereas phospho-MAPp38 kinase induces phosphorylation of mitogen- andstress-activated kinase 1 (MSK1) or other histone kinases. ThisMAPK p38 kinase-dependent activation has been shown to becapable of increasing DNA accessibility for NF-�B binding atspecific promoters in a dose-dependent manner (34). Therefore,ILT3 silencing triggers the concerted action of both of thesesignaling molecules, and perhaps others, to selectively inducethe transcription of some genes involved in inflammation.

Previously, we (3, 4) and others (9) showed that APC, includ-ing DC, can be differentiated to a tolerogenic, ILT3high pheno-type via cytokine mixtures or interaction with regulatory Tcells. ILT3high APC were shown to suppress CD4� Th cell pro-liferation and favor the differentiation of CD8� Ts cells (3). Inthe current study, we showed that knockdown of ILT3 signifi-cantly augments proliferation of T cells primed to such APC.This enhanced T cell proliferative response occurs both uponstimulation with allogenic DC or autologous DC pulsed withCMV Ags. Flow cytometry studies showed expanded Th1 andTh17 populations in response to ILT3KD-DC stimulation.These observations that ILT3 silencing improves not only theAg presentation capacity but also the T cell recruitment may beclinically relevant. Attempts to use DC-based vaccines tomount a strong Ag-specific immune response against tumor-associated Ags or pathogenic agents, which elude the humanimmune system, rely on the use of adjuvants. Various TLRligands have been used with some success as adjuvants in DC-based vaccines against tumor-associated Ags (35–38). Thepresent findings offer the tantalizing possibility that knockdownof ILT3 could be used as an adjuvant itself to improve theeffectiveness of DC based vaccines to generate immunogenicresponses against tumor Ags or chronic pathogenic infections.

DisclosuresThe authors have no financial conflicts of interest.

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