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The Journal of Immunology, 2009, 182, 5208 -5216
Copyright © 2009 by The American Association of Immunologists, Inc.
doi:10.4049/jimmunol.0804048
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Ig-Like Transcript 3 Regulates Expression of Proinflammatory Cytokines and Migration of Activated T Cells1

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

* Department of Pathology and {dagger} Department of Medicine, Columbia University, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ig-like transcript 3 (ILT3), an inhibitory receptor expressed by APC is involved in functional shaping of T cell responses toward a tolerant state. We have previously demonstrated that membrane (m) and soluble (s) ILT3 induce allogeneic tolerance to human islet cells in humanized NOD/SCID mice. Recombinant sILT3 induces the differentiation of CD8+ T suppressor cells both in vivo and in vitro. To better understand the molecular mechanisms by which ILT3 suppresses immune responses, we have generated ILT3 knockdown (ILT3KD) dendritic cells (DC) and analyzed the phenotypic and functional characteristics of these cells. In this study, we report that silencing of ILT3 expression in DC (ILT3KD DC) increases TLR responsiveness to their specific ligands as reflected in increased synthesis and secretion of proinflammatory cytokines such as IL-1{alpha}, IL-1β, 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 migration in diffusion chamber experiments. ILT3KD-DC elicit increased T cell proliferation and synthesis of proinflammatory cytokines IFN-{gamma} and IL-17A both in MLC and in culture with autologous DC pulsed with CMV protein. ILT3 signaling results in inhibition of NF-{kappa}B and, to a lesser extent, MAPK p38 pathways in DC. Our results suggest that ILT3 plays a critical role in the in control of inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunoglobulin-like transcript 3 (ILT3)3 is an immune inhibitory receptor that belongs to a family of molecules that contain extracellular Ig-like domains. ILT3 is selectively expressed on provisional myeloid APC such as monocytes, macrophages, and dendritic cells (DC) (1, 2), as well as on nonprofessional APC, such as endothelial cells (3).

The extracellular domain of ILT3 binds to T cells, shaping their functional development. We have previously shown that APC, which overexpress ILT3, become tolerogenic, inducing T cell anergy and differentiation of T suppressor cells (Ts) (4, 5). Furthermore, upon direct interaction with APC Ag-specific CD8+ Ts "tolerize" these APC inducing the up-regulation of ILT3 and down-regulation of costimulatory molecules on the cell surface of APC. More recently, we showed that soluble ILT3 (sILT3) can be detected in serum from cancer patients and that it is produced by CD68+ tumor-associated macrophages (6), contributing to the impairment 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, inducing tolerance to allogeneic human tissue in SCID mice, which have been humanized by injection of PBMC (6, 8).

Similar to other inhibitory members of the ILT family, ILT3 displays a cytoplasmic tail containing ITIM. Immunoblotting with a phospho-tyrosine Ab showed a marked decrease of protein tyrosine phosphorylation levels in monocytes treated with mAbs to ILT3 and HLA class II or Fc{gamma}RIII receptors on the surface of myeloid cells (1, 7). This effect is attributable to the recruitment of the inhibitory phosphatase Src homology region 2 domain-containing 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 of NF-{kappa}B activation and low expression of costimulatory molecules account at least in part for the tolerogenic phenotype of ILT3-transduced myeloid (KG1) tumor cells (4). Experimental data show that addition of a blocking anti-ILT3 Ab to cocultures of T cells and DC increases the T cell production of IFN-{gamma} and other cytokines (7, 9), suggesting a cytokine regulatory component of ILT3-mediated suppression. To identify genes/pathways that are regulated by ILT3 and to better understand the role of ILT3 in physiologically normal, nonmalignant DC, we designed a series of adenoviral vectors, which efficiently infect monocytes or DC silencing the expression of ILT3 via the production of small interfering RNA (siRNA). Using this system, we have identified some previously unknown functions of the ILT3 molecule such as its capacity to regulate cytokine responses of APC (including the synthesis of chemoattractants, which ultimately regulate T cell activation) and their maturation and functional differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs, cytokines, and reagents

Purified Abs to NF-{kappa}B and MAPK pathway proteins were purchased from Cell Signaling Technology. Polyclonal anti-ILT3 Abs were purchased from R&D Systems and anti-β-actin from Santa Cruz Biotechnology. All flow cytometry conjugated Abs were purchased from BD Biosciences, except 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 Center. Monocytes were obtained from mononuclear cells by plastic adherence. DC were generated by culturing monocytes in 6-well plates for 7 days 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-day intervals. Cultured cells were further purified to >90% homogeneity by negative selection of contaminating lymphocytes using CD2+ and CD19+ Dynal magnetic beads (Invitrogen) on day 7. The differentiation of CD14–CD11chighCD83lowCD86highHLA-DR+ immature DC was confirmed by flow cytometric analysis.

Construction of siRNA ILT3 vectors and knockdown of ILT3

Adenoviral RNAi Expression System (Invitrogen) was used to generate siRNAILT3 directed against ILT3 expression by targeting two separate regions of the ILT3 mRNA. Two double-stranded 19-mer corresponding to the ILT3 nucleotide sequences 281–299 (5'-GAC AGG AGC CTA CAG TAA A-3') and 351–369 (5'-GGA GAT ACC GCT GTT ACT A-3') were cloned separately into an U6 RNA entry vector (Invitrogen), according to the manufacturer’s design. Vectors containing an U6 RNA polymerase promoter and the ILT3 siRNA sequences were subsequently recombined with the pAd/Block-it DEST vector (Invitrogen) to create the final destination adenoviral vectors, pAd-RNAiILT3–281 and pAd-RNAiILT3–351. All constructs were verified by sequencing from both ends. To generate RNAi recombinant adenoviruses, ILT3 siRNA adenoviral vectors were transfected into 293A cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). Viral stocks were amplified at least twice by reinfecting 293A cells and filtered through a 0.45 µM cellulose membrane filter before use. A recombinant adenovirus, pAd-RNAicon, containing only the U6 RNA polymerase promoter (without ILT3 RNAi sequences) was similarly generated and was used as control throughout the study.

A two-step adenoviral infection protocol was used for efficient ILT3 knockdown (ILT3KD). Monocytes were first infected with recombinant adenoviruses pAd-RNAiILT3–281 on the first day of the 7-day culture with GM-CSF/IL-4, adding viral stocks to the medium at a 1:5 (v/v) ratio. These cells 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 vector pAd-RNAicon for infections. Surface expression of ILT3 was monitored by flow cytometry using an anti-ILT3 mAb (Beckman Coulter). The schematic structure of adenoviral constructs and efficiency of ILT3 siRNA transduction are shown in Fig. 1. Immature DCs were used 9–11 days after infection.


Figure 1
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  		FIGURE 1. A, Schematic structures of adenoviral constructs used for silencing of ILT3. Abbreviations are as follows: ITR, inverted terminal repeat; U6P, U6 polymerase promoter; PoIIITerm, polymerase III terminator; and dsOligos, insertion regions for ILT3 siRNA oligos. B, Analysis of the level of expression of ILT3 in DC infected with either pAd-control or pAd-ILT3/KD vectors by flow cytometry and RT-PCR. DC treated with IL-10 (5 ng/ml) and IFN-{alpha} (1000 U/ml) for induction of ILT3 were used as positive control.

 
TLR ligand treatments of DC

Ultrapure LPS of E. coli K12 strain, flagellin, and Pam3CSK4 were obtained from InvivoGen. Polyinosinic-polycytidylic acid (polyI:C) was purchased from Sigma-Aldrich. The NF-{kappa}B inhibitor Bay11–7082, the MAPK p38 inhibitor SB203580 and its inactive form SB202474 were purchased from CalBiochem. ILT3KD- and ctrl-DC were treated with various TLR ligands 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) were used at 2 µg/ml and TLR5 (flagellin) at 1 µg/ml. NF-{kappa}B and MAPKp38 pathway inhibitors were used at 10 µM. The supernatants were tested using the Proteome Profiler Array (R&D Systems), according to the manufacturer’s instructions. A pulse-chase type experiment was conducted to measure cytokine transcription and mRNA stability following TLR activation of DC. LPS (100 ng/ml) was used to stimulate ILT3KD- and ctrl-DC for 1 h, then transcription was blocked with actinomycin D (1 µg/ml; Sigma Aldrich). DC were lysed for PCR analysis at 1-h intervals following actinomycin treatment.

RNA extraction, cDNA synthesis, and real-time PCR

Total RNA was extracted from 1 to 10 x 105 purified cell suspensions using the Absolute RNA kit (Stratagene). First-strand cDNA was synthesized using oligo dT primers with Superscript III First Strand kit (Invitrogen). Real-time quantitative RT-PCR was performed on a 7300 Real Time PCR instrument (Applied Biosystems) in 50-µl reactions using 1 µl 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 1.31 Software (Applied Biosystems). The relative amount of gene expression was calculated by 2-{Delta}Ct, where {Delta}Ct = [Ct(gene) – Ct(CD68)], and Ct is the "crossing threshold" value returned by the PCR instrument for every gene amplification. The myeloid-specific marker CD68 selectively expressed by macrophages was used for normalization of gene expression data because it is not affected by ILT3KD or LPS treatment.

Immunoprecipitation and Western blotting

DC were lysed in radioimmunoprecipitation assay buffer (Upstate) containing both phosphatase inhibitor mixtures I and II (Sigma-Aldrich) and proteinase inhibitors (Roche Applied Science) for 20 min on ice. After a brief centrifugation, equal amounts (20–30 µg) of total cell lysate were loaded on 10% precast NuPAGE gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane. Immunoblotting was performed with various primary and HRP-conjugated secondary Abs and detected by chemiluminescence (SuperSignal West Pico kit; Pierce) as described previously (6). Expression of proteins was quantitated by NIH ImageJ Software. Expression of β-actin was used to normalize the protein content between lanes. For detection of ILT3 interacting protein complexes, DC cells were first treated with the phosphatase inhibitor bpV(phen) (Calbiochem) for 20 min, followed by lysis in a 1% Nonidet P-40 buffer containing proteinase and phosphatase inhibitors. Supernatants were collected after a brief sonication and centrifugation and incubated with 5 µg of goat anti-ILT3 polyclonal Ab (R&D Systems) or goat IgG (Sigma-Aldrich) for 16 h followed with 20 µl of protein A/G-agarose for 1 h. After extensive washing, protein A/G agarose was transferred to a polyvinylidene difluoride membrane probed sequentially with anti-SHP-1, anti-SHP-2, SHIP-1, and SHIP-2 (Cell Signaling Technology) and anti-ILT3 Abs and analyzed by a chemiluminescence as described above.

Flow cytometry and cytokine detection

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

T cell proliferation assays

Human CD3+ T cells were isolated from mononuclear cell populations using a Pan T cell isolation kit (Miltenyi Biotec). Immature ctrl-DCs or ILT3KD-DC were irradiated (3000 rad) and used as stimulators. Primary MLC were performed in a 96-well culture plate using T cells (5 x 104 cells/well) stimulated for 6 days with allogeneic DC or with autologous DC at various responder to stimulator ratios (100:1–400:1). For T cell responses to CMV Ags, CD3+ T cells (5 x 104 cells/well) were incubated for 5 days with Ctrl- or ILT3KD-transfected autologous DC (1 x 104 cells/well) in cultures containing various concentrations (2.5 and 5 µg/ml) of CMV proteins (grade 2 Ag; Microbix Biosystems). Tritiated [3H]TdR was added to the cultures over the final 18 h of incubation. [3H]TdR incorporation 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 activation plates (BD Biosciences) in the presence of 2 µg/ml anti-CD28 mAb (BD Bioscience). A total of 2 x 104 of these cells were added to the upper chamber of a 24-well Transwell plate (pore size, 5 µm; Corning Costar), whereas DC supernatants (0.5 ml) or chemokines were added to the lower chamber. After 2-h incubation at 37°C, the contents of the lower chamber were collected by low-speed centrifugation (250g) and counted directly under a light microscope. Each experiment was performed in duplicate. Values are given as percentage of cells that migrated.

Statistical analysis

Data from multiple experiments were expressed as mean ± SEM. The two-tailed, paired Student’s t test was performed to compare two or more mean values. A value of p < 0.05 was considered statistically significant and is indicated by *. A value of p < 0.01 was considered statistically very significant and is indicated by **, whereas p > 0.05 (denoted as #) was considered insignificant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Modulation of LPS inducible cytokines induction by ILT3

To determine the role of ILT3, monocyte-derived immature DC were transfected with ILT3 siRNA (ILT3KD-DC), whereas ctrl-DC were infected in parallel cultures with an empty vector (Fig. 1A). Flow cytometry showed reduced surface expression of ILT3 in ILT3KD cells. The ILT3 mean fluorescence intensity was 56 in ILT3KD-DC compared with 205 in empty vector-transfected control cells and 769 in ILT3high DC, in which ILT3 up-regulation was induced by IFN-{alpha}/IL-10 treatment, as described previously (10) (Fig. 1B). Quantitative RT-PCR showed that gene-specific knockdown suppressed ILT3 mRNA expression by up to 90% (p < 0.01; Fig. 1B). Examination of immature, ctrl-, and ILT3KD-DC by quantitative RT-PCR or flow cytometry for expression of myeloid lineage markers (CD68, CD14), costimulatory molecules (CD40, CD80, CD86), cytokines (IFN-{gamma}, IL-1{alpha}&β, IL-8, IL-10, IL-12β, TNF-{alpha}), and adhesion molecules (ICAM-1) showed no significant differences between ILT3KD-DC and nonactivated control DC (Fig. 2A and data not shown).


Figure 2
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  		FIGURE 2. Modulation of LPS-inducible gene expression in ILT3KD DC. A, Expression of the myeloid marker CD68 measured by real-time PCR (and normalized by GAPDH) in ctrl-DC, ILT3KD-DC, and lymphocytes. B, Effect of silencing ILT3 on IL-1β and IL-6 mRNA induction by various concentrations 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. Results are representative of three to five independent experiments. D, IL-1β and IL-6 expression in supernatants from ctrl- or ILT3KD-DC treated overnight with 100 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 three independent experiments ± SEM. (*, p < 0.05; **, p < 0.01.)

 
To explore the possibility that ILT3 plays an inhibitory role only upon activation of DC, ctrl-DC, or ILT3KD-DC were treated with ultrapure LPS (E. coli K12 strain) for 18 h. Ultrapure LPS is known to specifically activate DC via TLR4, affecting the expression of various genes, including proinflammatory cytokines, costimulatory, and other molecules (11). The transcription level of a small group of proinflammatory cytokines (IL-1{alpha}, IL-1β, IL-6) was consistently 2- to 3-fold higher (*, p < 0.05) in LPS-activated ILT3KD-DC than in ctrl-DC (Fig. 2, B and C). This ILT3KD-mediated enhancement of cytokine responses occurred at LPS concentrations ranging from 3 to 100 ng/ml (Fig. 2B). Enhanced expression of proinflammatory cytokine mRNA in ILT3KD-DC also occurred at the protein level as shown by cytometric bead analysis of soluble proteins in the culture supernatants of LPS-treated DC (Fig. 2D).

Other LPS-induced genes involved in inflammation such as IL-8, IL-12β, indoleamine-pyrrole 2,3-dioxygenase (INDO), costimulatory (CD40, CD86) molecules and type I (IFN-{alpha}1) and type II (IFN-{gamma}) showed no change (Fig. 2C). Addition of the transcriptional inhibitor actinomycin D to LPS-activated ILT3KD- and ctrl-DC rapidly suppressed the IL-1β and IL-6 mRNA levels. The half-life of IL-1β mRNA was 51 ± 6 min in both ILT3KD- and ctrl-DC, while that of IL-6 was 82 ± 12 min (data not shown), indicating that ILT3 affects the transcription of these cytokines but not the mRNA stability.

Modulation of other TLR responses by ILT3

Ligation of various pattern recognition receptors such as TLR is known to result in production of inflammatory cytokines (reviewed in Ref. 12). We tested the capacity of ILT3– (ILT3KD-DC) and ILT3+ (ctrl-DC) to produce IL-1{alpha}, IL-1β, IL-6, IL-12β, TNF-{alpha}, INDO, and both type I (IFN-{alpha}1) and type II (IFN-{gamma}) IFNs in response to ligation of TLRs. We choose antagonists of TLR1/2 (synthetic tripalmitoyl lipopeptide, Pam3CSK), TLR3 (synthetic double-stranded RNA, polyI:C), and TLR5 (flagellin), which are known to activate monocyte-derived DC (13). As shown in Fig. 3A, these TLR ligands varied with respect to their capacity to induce the transcription of these inflammatory cytokines with polyI:C triggering the strongest inflammatory responses. ILT3 silencing in ILT3KD-DC resulted consistently in a 1.5- to 3-fold higher transcriptional induction of IL-1{alpha}, IL-1β, and IL-6 by all forms of TLR ligands (Fig. 3A). In addition, the lack of ILT3 expression in ILT3 KD-DC was accompanied by enhanced transcription of IL-12β and TNF-{alpha} mRNA upon ligation of TLR3 (polyI:C) but not of TLR1/2 (Pam3CSK4) or TLR5 (flagellin). Expression levels of both type I IFN (IFN-{alpha}1) and type II IFN (IFN-{gamma}) were also significantly induced by ILT3 silencing (3- to 5-fold, p < 0.01, and 2- to 3-fold, p < 0.05, respectively; Fig. 3A). Analysis of IL-1β and IL-6 at the protein level confirmed the results obtained by analysis of mRNA expression (Fig. 3B). These data indicate that DC responsiveness to various pathogens/foreign Ags provided by a variety forms of TLR ligands is modulated by ILT3.


Figure 3
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  		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 ({square} and {blacksquare}, respectively) treated with various TLR ligands. Data from four independent experiments are expressed as the mean ± SEM. B, Cytokine bead array detection of IL-1β and IL-6 in supernatants of Ctrl- or ILT3KD-DC treated with various TLR ligands. Data are represented as mean ± SEM of three independent experiments (*, p < 0.05; **, p < 0.01; #, p > 0.05).

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

We used a cytokine array system (Proteome Profiler Array; R&D Systems) to study the soluble factors released from untreated or activated ILT3KD-DC into culture medium. Silencing of ILT3 in ILT3KD-DC had minimal effect on secretion of soluble factors in resting DC. As expected, ligation of TLR4 by treatment of DC with LPS triggers the production of numerous pro-inflammatory cytokines and chemokines. Knockdown of ILT3 further potentiates the induction of several of these genes, including complement 5a, CXCL10, CXCL11, MIF, MIP-1a, and MIP-1β for an additional ≥1.5 fold (Fig. 4A). RT-PCR analysis of ILT3KD-DC and ctrl-DC treated with various concentrations (3–100 ng/ml) of LPS showed that ILT3KD-DC generated a CXCL10 and CXCL11 response 2- to 3-fold stronger at all the concentrations tested (Fig. 4B).


Figure 4
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  		FIGURE 4. Expression of chemotactic factors by ctrl-DC and ILT3KD-DC. A, Proteome Profiler Array analysis of supernatants ctrl- or ILT3KD-DC treated 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 after normalizations of values by the positive control. B, RT-PCR analysis of CXCL10 and CXCL11 transcription in ctrl-DC and ILT3KD-DC after treatment with various concentrations (0–100 ng/ml) LPS. The mean from three independent experiments and SEM are indicated. C, Comparison of T cell chemoattractant properties of supernatants from LPS-activated ctrl- and ILT3KD-DC. Activated T cells were added into the upper chambers of a Transwell plate (5 µM pore size), and supernatants from ctrl-DC or ILT3KD-DC were added to the lower chambers. Results are expressed as mean ± SEM of the total numbers of migrated T cells after 2 h (*, p < 0.05).

 
Because supernatants from activated ILT3KD cells showed increased amounts of CXCL10 and CXCL11, we evaluated their ability to attract immune effectors cells, such as activated CXCR3+ T cells, in Transwell assays. Supernatants from LPS-activated ILT3KD-DC and ctrl-DC, but not supernatants from untreated DC, induced the transmigration of activated T cells (Fig. 4C). Transmigration of activated T cells in response to supernatants from LPS-activated ILT3KD-DC was significantly increased (p < 0.05) when compared with transmigration in response to supernatants from ctrl-DC that were similarly treated (Fig. 4C).

ILT3KD-DC elicit increased T cell proliferate responses

To determine whether ILT3KD increases the stimulatory capacity of DC, we tested in parallel the capacity of ILT3KD-DC and ctrl-DC from the same donor to stimulate the proliferation of allogeneic T cells. As shown in Fig. 5, ILT3KD-DC induced significantly stronger (p < 0.05 at 1:200 ratio and p < 0.001 at 1:400 ratio, respectively) T cell proliferation compared with ctrl-DC at 1:200–400 stimulator to responder cells ratios. Similar results were obtained in the experiment in which T cells were primed to autologous ILT3KD-DC or ctrl-DC in cultures containing CMV protein. ILT3KD-DC induced significantly stronger (p < 0.05 at 5 µg and p < 0.01 at 2.5 µg protein, respectively) T cell responses to CMV Ags at concentrations ranging from 2.5 to 5 µg/ml (Fig. 5A).


Figure 5
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  		FIGURE 5. The effect of ILT3KD on priming T cells responses. A, CD3+ T cell proliferative responses to allogeneic ctrl-DC and ILT3KD-DC or autologous stimulators presenting CMV Ags. Proliferation of cells was determined by [3H]thymidine incorporation. Results were expressed as mean ± SEM of four independent experiments. *, p < 0.05; **, p < 0.01. B, Increased intracellular expression of IFN-{gamma} and IL-17A by CD3+ T cells cocultured with ILT3KD DC for 5 days. A representative result of flow cytometric analysis of T cells gated out from T-DC cocultures is shown in B. C, Results obtained from three independent experiments show 3- to 7-fold expansion of Th1 and Th17 populations following T cell coculture with allogeneic ILT3KD-DC vs ctrl-DC. *, p < 0.05.

 
Flow cytometric analysis of the frequency of Th1 and Th17 cells in 5-day cultures allostimulated with ILT3KD-DC or ctrl-DC showed that knockdown of ILT3 elicited an increase in the size of the T cell populations producing IFN-{gamma} (from 1.0 to 6.7%) and IL-17 (from 0.3 to 1.5%) (Fig. 5B). Three repeat experiments show a consistent 3- to 7-fold increase in the size of IFN-{gamma} (p < 0.05) and IL-17A (p < 0.05) secreting T cell populations induced by ILT3KD-DC vs ctrl-DC (Fig. 5C).

ILT3 regulates the NF-{kappa}B and MAPKp38 kinase pathways

To better understand how ILT3 silencing enhances DC response to danger signals and identify the signaling pathways involved we used specific inhibitors for MAPK p38, SB203580, or NF-{kappa}B, and Bay11-7082. Addition of Bay11-7082 or SB203580 (but not of its inactive analog SB202474) to LPS-activated DC blocked IL-1β expression, both at the mRNA (data not shown) and soluble protein level (>60%; Fig. 6A). To determine whether expression of ILT3 affects the phosphorylation of MAPK and I{kappa}B kinases, LPS-treated ILT3KD-DC and ctrl-DC were subjected to immunoblot analyses using various Abs that recognize the total (T) or phosphorylated (p) forms of MAPKp38, I{kappa}B-{alpha}, and its regulator, I{kappa}B kinase {alpha}β (IKK{alpha}β). In both types of cells, we found that phosphorylation of IKK{alpha}β and MAPKp38 was induced by LPS in a time-dependent manner with a peak at 30 min after treatment. However, at the 3-h time point the resynthesis and degradation of I{kappa}B-{alpha} had reached an equilibrium state as indicated by others (14, 15, 16). More p-IKB{alpha} (2.0x), p-IKK{alpha}β (1.7x), and p-MAPKp38 (1.3x) was detectable in LPS-treated ILT3KD-DC than in control DC (Fig. 6B). Phosphorylation of MAPKp42/p44, JNK, and NF-{kappa}Bp65 (RelA), on the other hand, were unchanged (data not shown). The total amounts of each of the respective proteins were also unchanged. This result supports the notion that both NF-{kappa}B and MAPKp38 pathways are required for LPS activation and that signaling is affected by ILT3 expression.


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

 
We then immunoprecipitated lysates from bpV(phen)-treated cells (ILT3KD-DC and ctrl-DC) with a goat anti-ILT3 Ab (or goat IgG isotype control) and examined the immunoprecipitates by immunoblotting with an anti-phospho-tyrosine (p-Tyr) Ab (Fig. 6C). Our results indicate that the anti-ILT3 Ab specifically pulls down several Tyr-phosphorylated proteins, in addition to p-Tyr-ILT3, from ctrl-DC lysates but not from that of ILT3KD-DC, supporting the notion that phospho-ILT3 interacts with other p-Tyr proteins. Probing the membrane with various phosphatase-specific Abs known to interact with ITIM indicates that SHP-1 and SHIP-1 were present in protein complexes immunoprecipitated by anti-ILT3. Other phosphatases, such as SHP-2, and SHIP-2, which may interact with ITIM domains of inhibitory receptors (17, 18), did not appear to interact with ILT3. Immunoprecipitation with IgG did not yield any ILT3 interacting proteins (data not shown). Taken together with Fig. 6B, these data indicate that the NF-{kappa}B and, to a lesser extent, the MAPKp38 pathways are negatively regulated by ILT3 signaling through interaction with SHP-1 and/or SHIP-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In previous studies, we demonstrated that ILT3 is a crucial inhibitory molecule whose expression affects the function of the APC and that of the T cells with which they interact (3, 4, 6, 7, 8, 10, 19). Although the function of this molecule has been well documented, the mechanisms by which ILT3 operate remains elusive. The present study attempts to expand the mechanistic understanding of ILT3-driven suppression.

TLR are a type of pattern recognition receptors that recognize molecules that are broadly shared by pathogens but distinguishable from host molecules (13, 20, 21, 22). TLR play an important role in innate immunity, and by signaling the presence of pathogens, they trigger inflammation and the recruitment of adaptive immune response to the affected microevironment. If unchecked, the self-amplification of TLR signaling can lead to inflammatory/autoimmune disease (reviewed in Ref. 20). We show here that overactive inflammation is accompanied by a more vigorous proinflammatory cytokine response by ILT3KD-DC when compared with ctrl-DC, which express physiological levels of ILT3 in response to "danger" signals relayed through a variety of TLR. From a signaling perspective, our results also show that ILT3 recruits SHP-1 and/or SHIP-1 to restrain the APC’s (LPS-triggered) activation pathways which rely on NF-{kappa}B and, to a lesser extent, MAPKp38. Taken together, these findings provide direct evidence that the physiological concentration of ILT3 on APC may work as a "check and balance" for overactive immune responses by interacting with inhibitory phosphatases SHP-1 and/or SHIP-1 and dampening NF-{kappa}B and MAPKp38 activity.

Our results therefore are consistent with observation that SHP-1 mutant mice (mev/mev) demonstrate higher NF-{kappa}B and MAPp38 activities (23) and are hypersensitive to LPS and pathogenic challenges (23, 24, 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 TLR ligation. Although our results indicate that silencing ILT3-enhanced type I IFN, IFN-{alpha}1, mRNA production in DC, mutation on Shp-1 (mev/mev) in mice decreases the synthesis of IFN-β after LPS treatments (23). This discrepancy may suggest that the negative signaling delivered by ILT3 does not entirely rely on SHP-1 and other signaling molecules, such as SHIP-1 (Fig. 6C), may also contribute to the ILT3KD phenotype. A recent study has implicated SHIP-1 in preventing TLR ligand induction 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 Th1 cells and promotion Th1 cell maturation (reviewed in Refs. 27 and 28). Dysregulation of CXCR3 and its ligand expression has been implicated in various types of diseases, such as multiple sclerosis (29) and type I diabetes (30). Our results showed that T cells respond to the higher levels of CXCL10 and CXCL11 produced by ILT3KD-DC, with increased migration rates toward the chemokine gradient, suggesting a possible regulatory role for ILT3 in controlling the trafficking of inflammatory T cells. Down-regulation of ILT3 can cause excess inflammation and infiltration of T cells in locally affected lesions, leading to destruction of tissue or autoimmune diseases. The importance of ILT3 in heart transplantation as a tolerogenic marker has been documented in our previous studies (3, 31).

Mechanistically, there are still some questions that remain unanswered. For example, although the phosphorylation of I{kappa}B is increased by ILT3 silencing, the total levels of I{kappa}B are not drastically affected. This may be explained by recent findings that Tyr-phosphorylation of I{kappa}B is not always followed by degradation (14, 15, 16, 32). It is also unclear why only a handful of (IL-1{alpha}β, IL-6, IFN type I/II, CXCL10, CXCL11) genes are affected by ILT3KD, despite the fact that many inflammatory cytokine genes are known to be NF-{kappa}B-regulated (33). However, based on the data presented here, we propose that the loss of ILT3 during external stimuli prevents binding of I{kappa}B to the transcription factor p50/p65 in the cytoplasm and partially activates MAPK p38. The heterodimeric p50/p65 complexes subsequently translocate to the nucleus whereas phospho-MAPp38 kinase induces phosphorylation of mitogen- and stress-activated kinase 1 (MSK1) or other histone kinases. This MAPK p38 kinase-dependent activation has been shown to be capable of increasing DNA accessibility for NF-{kappa}B binding at specific promoters in a dose-dependent manner (34). Therefore, ILT3 silencing triggers the concerted action of both of these signaling molecules, and perhaps others, to selectively induce the transcription of some genes involved in inflammation.

Previously, we (3, 4) and others (9) showed that APC, including DC, can be differentiated to a tolerogenic, ILT3high phenotype via cytokine mixtures or interaction with regulatory T cells. ILT3high APC were shown to suppress CD4+ Th cell proliferation and favor the differentiation of CD8+ Ts cells (3). In the current study, we showed that knockdown of ILT3 significantly augments proliferation of T cells primed to such APC. This enhanced T cell proliferative response occurs both upon stimulation with allogenic DC or autologous DC pulsed with CMV Ags. Flow cytometry studies showed expanded Th1 and Th17 populations in response to ILT3KD-DC stimulation. These observations that ILT3 silencing improves not only the Ag presentation capacity but also the T cell recruitment may be clinically relevant. Attempts to use DC-based vaccines to mount a strong Ag-specific immune response against tumor-associated Ags or pathogenic agents, which elude the human immune system, rely on the use of adjuvants. Various TLR ligands have been used with some success as adjuvants in DC-based vaccines against tumor-associated Ags (35, 36, 37, 38). The present findings offer the tantalizing possibility that knockdown of ILT3 could be used as an adjuvant itself to improve the effectiveness of DC based vaccines to generate immunogenic responses against tumor Ags or chronic pathogenic infections.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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). Back

2 Address correspondence and reprint requests to Dr. Nicole Suciu-Foca, Columbia University, Department of Pathology, 630 West 168th Street, P&S 14-401, New York, NY 10032. E-mail address: ns20{at}columbia.edu Back

3 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-dioxygenase; IKK{alpha}β, I{kappa}B kinase {alpha}β; p, phosphorylated; polyI:C, polyinosinic-polycytidylic acid; SHP, Src homology region 2 domain-containing phosphatase; sILT3, soluble ILT3; siRNA, small interfering RNA; Ts, T suppressor cell. Back

Received for publication December 3, 2008. Accepted for publication February 13, 2009.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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