Ig-Like Transcript 3 Regulates Expression of Proinflammatory Cytokines and Migration of Activated T Cells

This information is current as Chih-Chao Chang, Zhuoru Liu, George Vlad, Haiyan Qin, of September 26, 2021. Xugang Qiao, Donna M. Mancini, Charles C. Marboe, Raffaello Cortesini and Nicole Suciu-Foca J Immunol 2009; 182:5208-5216; ; doi: 10.4049/jimmunol.0804048

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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,† 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 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␣, IL-1␤, and IL-6 and type I IFN. Downloaded from 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-␥ and IL-17A both in MLC and in culture with autologous DC pulsed with CMV protein. ILT3 signaling results in inhibition of NF-␬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. The Journal of Immunology, 2009, 182: 5208–5216. http://www.jimmunol.org/ mmunoglobulin-like transcript 3 (ILT3)3 is an immune in- of Ag-specific CD8ϩ Ts both in vitro (7) and in vivo, inducing hibitory receptor that belongs to a family of molecules that tolerance to allogeneic human tissue in SCID mice, which have I contain extracellular Ig-like domains. ILT3 is selectively ex- been humanized by injection of PBMC (6, 8). pressed on provisional myeloid APC such as monocytes, macro- Similar to other inhibitory members of the ILT family, ILT3 phages, and dendritic cells (DC) (1, 2), as well as on nonprofes- displays a cytoplasmic tail containing ITIM. Immunoblotting with sional APC, such as endothelial cells (3). a phospho-tyrosine Ab showed a marked decrease of protein ty- The extracellular domain of ILT3 binds to T cells, shaping their rosine phosphorylation levels in monocytes treated with mAbs to functional development. We have previously shown that APC, ILT3 and HLA class II or Fc␥RIII receptors on the surface of which overexpress ILT3, become tolerogenic, inducing T cell an- myeloid cells (1, 7). This effect is attributable to the recruitment of by guest on September 26, 2021 ergy and differentiation of T suppressor cells (Ts) (4, 5). Further- the inhibitory phosphatase Src homology region 2 domain-con- more, upon direct interaction with APC Ag-specific CD8ϩ Ts taining phosphatase (SHP)-1 to the ITIM and suppression of Ca2ϩ “tolerize” these APC inducing the up-regulation of ILT3 and mobilization. down-regulation of costimulatory molecules on the cell surface of The mechanism(s) by which ILT3 modulates immune responses APC. More recently, we showed that soluble ILT3 (sILT3) can be is largely unknown. We previously reported that suppression of detected in serum from cancer patients and that it is produced by NF-␬B activation and low expression of costimulatory molecules CD68ϩ tumor-associated macrophages (6), contributing to the im- account at least in part for the tolerogenic phenotype of ILT3- pairment of patients’ immune reactivity. Recombinant sILT3-Fc, transduced myeloid (KG1) tumor cells (4). Experimental data like membrane-bound ILT3, induces Th anergy and differentiation show that addition of a blocking anti-ILT3 Ab to cocultures of T cells and DC increases the T cell production of IFN-␥ and other cytokines (7, 9), suggesting a cytokine regulatory component of *Department of Pathology and †Department of Medicine, Columbia University, New ILT3-mediated suppression. To identify genes/pathways that are York, NY 10032 regulated by ILT3 and to better understand the role of ILT3 in Received for publication December 3, 2008. Accepted for publication February physiologically normal, nonmalignant DC, we designed a series of 13, 2009. adenoviral vectors, which efficiently infect monocytes or DC si- The costs of publication of this article were defrayed in part by the payment of page lencing the expression of ILT3 via the production of small inter- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. fering RNA (siRNA). Using this system, we have identified some 1 This work was supported by grants from the Juvenile Diabetes Research Foundation previously unknown functions of the ILT3 molecule such as its (1-2008-550) and the Interuniversitary Organ Transplantation Consortium (Rome, capacity to regulate cytokine responses of APC (including the syn- Italy). thesis of chemoattractants, which ultimately regulate T cell acti- 2 Address correspondence and reprint requests to Dr. Nicole Suciu-Foca, Columbia vation) and their maturation and functional differentiation. University, Department of Pathology, 630 West 168th Street, P&S 14-401, New York, NY 10032. E-mail address: [email protected] Materials and Methods 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-di- Abs, cytokines, and reagents ␣␤ ␬ ␣␤ oxygenase; IKK ,I B kinase ; p, phosphorylated; polyI:C, polyinosinic-poly- ␬ cytidylic acid; SHP, Src homology region 2 domain-containing phosphatase; sILT3, Purified Abs to NF- B and MAPK pathway proteins were purchased from soluble ILT3; siRNA, small interfering RNA; Ts, T suppressor cell. Cell Signaling Technology. Polyclonal anti-ILT3 Abs were purchased from R&D Systems and anti-␤-actin from Santa Cruz Biotechnology. All flow Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 cytometry conjugated Abs were purchased from BD Biosciences, except www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804048 The Journal of Immunology 5209

IL17A-PE (eBioscience) and ILT3-PC5 (Beckman Coulter). CMV proteins m1), IL8 (Hs00174103_m1), ILT3/LILRB4 (Hs00429000_m1), CD40 (Grade 2 Ag) were obtained from Microbix Biosystems. (Hs00386848_m1), CD14 (Hs00169122_g1), CD80 (Hs00175478_m1), CD86 (Hs00199349_m1), Indo (Hs00158027_m1), ICAM1 (Hs00164932_m1), CD68 Generation of monocyte-derived DC (Hs00154355_m1), CXCL10 (Hs00171138_m1), CXCL11 (Hs00171042_m1), and GAPDH (436317E). Data were collected and analyzed with 7300 SDS 1.31 Peripheral blood samples were purchased from the New York Blood Cen- Software (Applied Biosystems). The relative amount of gene expression was cal- ter. Monocytes were obtained from mononuclear cells by plastic adher- ⌬ culated by 2- Ct, where ⌬Ct ϭ [Ct(gene) Ϫ Ct(CD68)], and Ct is the “crossing ence. DC were generated by culturing monocytes in 6-well plates for 7 threshold” value returned by the PCR instrument for every gene amplification. The days with GM-CSF and IL-4 (R&D Systems), as described previously (4). myeloid-specific marker CD68 selectively expressed by macrophages was used Half of the culture medium was replaced with fresh medium at 2-day for normalization of gene expression data because it is not affected by ILT3KD or intervals. Cultured cells were further purified to Ͼ90% homogeneity by ϩ ϩ LPS treatment. negative selection of contaminating lymphocytes using CD2 and CD19 Dynal magnetic beads (Invitrogen) on day 7. The differentiation of CD14ϪCD11chighCD83lowCD86highHLA-DRϩ immature DC was con- Immunoprecipitation and Western blotting firmed by flow cytometric analysis. DC were lysed in radioimmunoprecipitation assay buffer (Upstate) con- Construction of siRNA ILT3 vectors and knockdown of ILT3 taining both phosphatase inhibitor mixtures I and II (Sigma-Aldrich) and proteinase inhibitors (Roche Applied Science) for 20 min on ice. After a Adenoviral RNAi Expression System (Invitrogen) was used to generate brief centrifugation, equal amounts (20–30 ␮g) of total cell lysate were siRNAILT3 directed against ILT3 expression by targeting two separate re- loaded on 10% precast NuPAGE gels (Invitrogen) and transferred to a gions of the ILT3 mRNA. Two double-stranded 19-mer corresponding to polyvinylidene difluoride membrane. Immunoblotting was performed with the ILT3 nucleotide sequences 281–299 (5Ј-GAC AGG AGC CTA CAG various primary and HRP-conjugated secondary Abs and detected by TAA A-3Ј) and 351–369 (5Ј-GGA GAT ACC GCT GTT ACT A-3Ј) were chemiluminescence (SuperSignal West Pico kit; Pierce) as described pre- cloned separately into an U6 RNA entry vector (Invitrogen), according to viously (6). Expression of proteins was quantitated by NIH ImageJ Soft- Downloaded from the manufacturer’s design. Vectors containing an U6 RNA polymerase ware. Expression of ␤-actin was used to normalize the protein content promoter and the ILT3 siRNA sequences were subsequently recombined between lanes. For detection of ILT3 interacting protein complexes, DC with the pAd/Block-it DEST vector (Invitrogen) to create the final desti- cells were first treated with the phosphatase inhibitor bpV(phen) (Calbio- nation adenoviral vectors, pAd-RNAiILT3–281 and pAd-RNAiILT3–351. All chem) for 20 min, followed by lysis in a 1% Nonidet P-40 buffer containing constructs were verified by sequencing from both ends. To generate RNAi proteinase and phosphatase inhibitors. Supernatants were collected after a recombinant adenoviruses, ILT3 siRNA adenoviral vectors were trans- brief sonication and centrifugation and incubated with 5 ␮g of goat anti- fected into 293A cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). ILT3 polyclonal Ab (R&D Systems) or goat IgG (Sigma-Aldrich) for 16 h http://www.jimmunol.org/ Viral stocks were amplified at least twice by reinfecting 293A cells and followed with 20 ␮l of protein A/G-agarose for 1 h. After extensive wash- filtered through a 0.45 ␮M cellulose membrane filter before use. A recom- ing, protein A/G agarose was transferred to a polyvinylidene difluoride binant adenovirus, pAd-RNAicon, containing only the U6 RNA polymerase membrane probed sequentially with anti-SHP-1, anti-SHP-2, SHIP-1, and promoter (without ILT3 RNAi sequences) was similarly generated and was SHIP-2 (Cell Signaling Technology) and anti-ILT3 Abs and analyzed by a used as control throughout the study. chemiluminescence as described above. A two-step adenoviral infection protocol was used for efficient ILT3 knockdown (ILT3KD). Monocytes were first infected with recombinant Flow cytometry and cytokine detection 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 CD3ϩ T cells were stimulated with allogeneic ctrl-DC or ILT3KD-DC at cells were reinfected with the second ILT3KD virus, pAd-RNAiILT3–351, 1:10 (stimulator:responder) ratio for 5 days. Alloactivated T cells were then on day 3 at the same viral stock to medium ratio. In parallel, control DC stimulated with 1 ␮g/ml ionomycin and 100 ng/ml PMA (Sigma-Aldrich) by guest on September 26, 2021 (ctrl-DC) were generated by the same protocol using the empty vector for 5 h. Brefeldin A (10 ␮g/ml; BD Biosciences) was added for the final 3 h pAd-RNAicon for infections. Surface expression of ILT3 was monitored by of culture. Cells were fixed and permeablized using the Fix&Perm kit flow cytometry using an anti-ILT3 mAb (Beckman Coulter). The sche- (Invitrogen) and were incubated with anti-IL17A-PE (eBioscience) and matic structure of adenoviral constructs and efficiency of ILT3 siRNA anti-IFN-␥ (BD Biosciences). Cell surface molecules were analyzed by transduction are shown in Fig. 1. Immature DCs were used 9–11 days after flow cytometry as described previously (4). Cytokines IL-1␤, IL-6, and infection. IFN-␣ in supernatants of cultured cells were tested using cytokine beads array kits (BD Biosciences), according to the manufacturer’s instructions. TLR ligand treatments of DC Data was acquired and analyzed on a FACSCalibur instrument (BD Bio- sciences) using six-parameter acquisition. 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 MAPK T cell proliferation assays p38 inhibitor SB203580 and its inactive form SB202474 were purchased ϩ Human CD3 T cells were isolated from mononuclear cell populations from CalBiochem. ILT3KD- and ctrl-DC were treated with various TLR using a Pan T cell isolation kit (Miltenyi Biotec). Immature ctrl-DCs or ligands overnight (18 h). LPS was used in a wide range of concentrations ILT3KD-DC were irradiated (3000 rad) and used as stimulators. Primary (3–100 ng/ml). Antagonists of TLR1/2 (synthetic tripalmitoyl lipopeptide, MLC were performed in a 96-well culture plate using T cells (5 ϫ 104 Pam3CSK), and TLR3 (synthetic double-stranded RNA, poly I:C) were cells/well) stimulated for 6 days with allogeneic DC or with autologous DC used at 2 ␮g/ml and TLR5 (flagellin) at 1 ␮g/ml. NF-␬B and MAPKp38 at various responder to stimulator ratios (100:1–400:1). For T cell re- pathway inhibitors were used at 10 ␮M. The supernatants were tested using ϩ sponses to CMV Ags, CD3 T cells (5 ϫ 104 cells/well) were incubated the Proteome Profiler Array (R&D Systems), according to the manufac- for 5 days with Ctrl- or ILT3KD-transfected autologous DC (1 ϫ 104 turer’s instructions. A pulse-chase type experiment was conducted to mea- cells/well) in cultures containing various concentrations (2.5 and 5 ␮g/ml) sure cytokine transcription and mRNA stability following TLR activation of CMV proteins (grade 2 Ag; Microbix Biosystems). Tritiated [3H]TdR of DC. LPS (100 ng/ml) was used to stimulate ILT3KD- and ctrl-DC for was added to the cultures over the final 18 h of incubation. [3H]TdR in- 1 h, then transcription was blocked with actinomycin D (1 ␮g/ml; Sigma corporation was measured using an LKB 1250 Betaplate counter Aldrich). DC were lysed for PCR analysis at 1-h intervals following acti- (PerkinElmer). Mean cpm of triplicate cultures and the SE were calculated. nomycin treatment. RNA extraction, cDNA synthesis, and real-time PCR T cell migration assays Total RNA was extracted from 1 to 10 ϫ 105 purified cell suspensions Purified T cells were stimulated for 3 days on anti-CD3 T cell activation using the Absolute RNA kit (Stratagene). First-strand cDNA was syn- plates (BD Biosciences) in the presence of 2 ␮g/ml anti-CD28 mAb (BD thesized using oligo dT primers with Superscript III First Strand kit Bioscience). A total of 2 ϫ 104 of these cells were added to the upper (Invitrogen). Real-time quantitative RT-PCR was performed on a 7300 chamber of a 24-well Transwell plate (pore size, 5 ␮m; Corning Costar), Real Time PCR instrument (Applied Biosystems) in 50-␮l reactions whereas DC supernatants (0.5 ml) or chemokines were added to the lower using 1 ␮l of cDNA. The following qPCR probes (Applied Biosystems) chamber. After 2-h incubation at 37°C, the contents of the lower chamber were used: IL1A (Hs00174092_m1), IL1B (Hs00174097_m1), IL6 were collected by low-speed centrifugation (250g) and counted directly (Hs00174131_m1), IL10 (Hs00174086_m1), TNF (Hs00174128_m1), under a light microscope. Each experiment was performed in duplicate. IFNG (Hs00174143_m1), IFNA1 (Hs00256882_s1), IL12B (Hs01011518_ Values are given as percentage of cells that migrated. 5210 ILT3 KNOCKDOWN AND INFLAMMATION

pression 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-12␤, indoleamine-pyrrole 2,3-dioxygenase (INDO), co- stimulatory (CD40, CD86) molecules and type I (IFN-␣1) and type II (IFN-␥) showed no change (Fig. 2C). Addition of the transcrip- tional inhibitor actinomycin D to LPS-activated ILT3KD- and ctrl-DC rapidly suppressed the IL-1␤ and IL-6 mRNA levels. The half-life of IL-1␤ mRNA was 51 Ϯ 6 min in both ILT3KD- and ctrl-DC, while that of IL-6 was 82 Ϯ 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 FIGURE 1. A, Schematic structures of adenoviral constructs used for known to result in production of inflammatory cytokines (reviewed Ϫ silencing of ILT3. Abbreviations are as follows: ITR, inverted terminal in Ref. 12). We tested the capacity of ILT3 (ILT3KD-DC) and Downloaded from repeat; U6P, U6 polymerase promoter; PoIIITerm, polymerase III termi- ILT3ϩ (ctrl-DC) to produce IL-1␣, IL-1␤, IL-6, IL-12␤, TNF-␣, nator; and dsOligos, insertion regions for ILT3 siRNA oligos. B, Analysis INDO, and both type I (IFN-␣1) and type II (IFN-␥) IFNs in re- of the level of expression of ILT3 in DC infected with either pAd-control sponse to ligation of TLRs. We choose antagonists of TLR1/2 or pAd-ILT3/KD vectors by flow cytometry and RT-PCR. DC treated with (synthetic tripalmitoyl lipopeptide, Pam3CSK), TLR3 (synthetic IL-10 (5 ng/ml) and IFN-␣ (1000 U/ml) for induction of ILT3 were used as positive control. double-stranded RNA, polyI:C), and TLR5 (flagellin), which are

known to activate monocyte-derived DC (13). As shown in Fig. http://www.jimmunol.org/ 3A, these TLR ligands varied with respect to their capacity to Statistical analysis induce the transcription of these inflammatory cytokines with polyI:C triggering the strongest inflammatory responses. ILT3 si- Ϯ Data from multiple experiments were expressed as mean SEM. The lencing in ILT3KD-DC resulted consistently in a 1.5- to 3-fold two-tailed, paired Student’s t test was performed to compare two or more ␣ ␤ mean values. A value of p Ͻ 0.05 was considered statistically significant higher transcriptional induction of IL-1 , IL-1 , and IL-6 by all and is indicated by *. A value of p Ͻ 0.01 was considered statistically very forms of TLR ligands (Fig. 3A). In addition, the lack of ILT3 significant and is indicated by **, whereas p Ͼ 0.05 (denoted as #) was expression in ILT3 KD-DC was accompanied by enhanced tran- considered insignificant. scription of IL-12␤ and TNF-␣ mRNA upon ligation of TLR3 (polyI:C) but not of TLR1/2 (Pam3CSK4) or TLR5 (flagellin). by guest on September 26, 2021 Results Expression levels of both type I IFN (IFN-␣1) and type II IFN Modulation of LPS inducible cytokines induction by ILT3 (IFN-␥) were also significantly induced by ILT3 silencing (3- to To determine the role of ILT3, monocyte-derived immature DC 5-fold, p Ͻ 0.01, and 2- to 3-fold, p Ͻ 0.05, respectively; Fig. 3A). were transfected with ILT3 siRNA (ILT3KD-DC), whereas Analysis of IL-1␤ and IL-6 at the protein level confirmed the re- ctrl-DC were infected in parallel cultures with an empty vector sults obtained by analysis of mRNA expression (Fig. 3B). These (Fig. 1A). Flow cytometry showed reduced surface expression of data indicate that DC responsiveness to various pathogens/foreign ILT3 in ILT3KD cells. The ILT3 mean fluorescence intensity was Ags provided by a variety forms of TLR ligands is modulated 56 in ILT3KD-DC compared with 205 in empty vector-transfected by ILT3. control cells and 769 in ILT3high DC, in which ILT3 up-regulation was induced by IFN-␣/IL-10 treatment, as described previously ILT3KD-DC produce increased amounts of T cell (10) (Fig. 1B). Quantitative RT-PCR showed that gene-specific chemoattractants, CXCL10 and CXCL11 knockdown suppressed ILT3 mRNA expression by up to 90% We used a cytokine array system (Proteome Profiler Array; R&D ( p Ͻ 0.01; Fig. 1B). Examination of immature, ctrl-, and Systems) to study the soluble factors released from untreated or ILT3KD-DC by quantitative RT-PCR or flow cytometry for ex- activated ILT3KD-DC into culture medium. Silencing of ILT3 in pression of myeloid lineage markers (CD68, CD14), costimulatory ILT3KD-DC had minimal effect on secretion of soluble factors in molecules (CD40, CD80, CD86), cytokines (IFN-␥, IL-1␣&␤, resting DC. As expected, ligation of TLR4 by treatment of DC IL-8, IL-10, IL-12␤, TNF-␣), and adhesion molecules (ICAM-1) with LPS triggers the production of numerous pro-inflammatory showed no significant differences between ILT3KD-DC and non- cytokines and chemokines. Knockdown of ILT3 further potentiates activated control DC (Fig. 2A and data not shown). the induction of several of these genes, including complement 5a, To explore the possibility that ILT3 plays an inhibitory role only CXCL10, CXCL11, MIF, MIP-1a, and MIP-1␤ for an additional upon activation of DC, ctrl-DC, or ILT3KD-DC were treated with Ն1.5 fold (Fig. 4A). RT-PCR analysis of ILT3KD-DC and ctrl-DC ultrapure LPS (E. coli K12 strain) for 18 h. Ultrapure LPS is treated with various concentrations (3–100 ng/ml) of LPS showed known to specifically activate DC via TLR4, affecting the expres- that ILT3KD-DC generated a CXCL10 and CXCL11 response 2- sion of various genes, including proinflammatory cytokines, co- to 3-fold stronger at all the concentrations tested (Fig. 4B). stimulatory, and other molecules (11). The transcription level of a Because supernatants from activated ILT3KD cells showed in- small group of proinflammatory cytokines (IL-1␣, IL-1␤, IL-6) creased amounts of CXCL10 and CXCL11, we evaluated their was consistently 2- to 3-fold higher (*, p Ͻ 0.05) in LPS-activated ability to attract immune effectors cells, such as activated ILT3KD-DC than in ctrl-DC (Fig. 2, B and C). This ILT3KD- CXCR3ϩ T cells, in Transwell assays. Supernatants from LPS- mediated enhancement of cytokine responses occurred at LPS con- activated ILT3KD-DC and ctrl-DC, but not supernatants from un- centrations ranging from 3 to 100 ng/ml (Fig. 2B). Enhanced ex- treated DC, induced the transmigration of activated T cells (Fig. The Journal of Immunology 5211 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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-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. Results are representative of three to five independent experiments. D, IL-1␤ 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 (.p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) .independent experiments Ϯ SEM

4C). Transmigration of activated T cells in response to superna- 0.05 at 5 ␮g and p Ͻ 0.01 at 2.5 ␮g protein, respectively) T cell tants from LPS-activated ILT3KD-DC was significantly increased responses to CMV Ags at concentrations ranging from 2.5 to 5 ( p Ͻ 0.05) when compared with transmigration in response to ␮g/ml (Fig. 5A). supernatants from ctrl-DC that were similarly treated (Fig. 4C). Flow cytometric analysis of the frequency of Th1 and Th17 cells in 5-day cultures allostimulated with ILT3KD-DC or ctrl-DC ILT3KD-DC elicit increased T cell proliferate responses showed that knockdown of ILT3 elicited an increase in the size of To determine whether ILT3KD increases the stimulatory capacity the T cell populations producing IFN-␥ (from 1.0 to 6.7%) and of DC, we tested in parallel the capacity of ILT3KD-DC and IL-17 (from 0.3 to 1.5%) (Fig. 5B). Three repeat experiments show ctrl-DC from the same donor to stimulate the proliferation of al- a consistent 3- to 7-fold increase in the size of IFN-␥ ( p Ͻ 0.05) logeneic T cells. As shown in Fig. 5, ILT3KD-DC induced sig- and IL-17A ( p Ͻ 0.05) secreting T cell populations induced by nificantly stronger ( p Ͻ 0.05 at 1:200 ratio and p Ͻ 0.001 at 1:400 ILT3KD-DC vs ctrl-DC (Fig. 5C). ratio, respectively) T cell proliferation compared with ctrl-DC at ␬ 1:200–400 stimulator to responder cells ratios. Similar results ILT3 regulates the NF- B and MAPKp38 kinase pathways were obtained in the experiment in which T cells were primed To better understand how ILT3 silencing enhances DC response to to autologous ILT3KD-DC or ctrl-DC in cultures containing danger signals and identify the signaling pathways involved we CMV protein. ILT3KD-DC induced significantly stronger ( p Ͻ used specific inhibitors for MAPK p38, SB203580, or NF-␬B, and 5212 ILT3 KNOCKDOWN AND INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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 (Ⅺ and f, respectively) treated with various TLR ligands. Data from four independent experiments are expressed as the mean Ϯ SEM. B, Cytokine bead array detection of IL-1␤ and IL-6 in supernatants of Ctrl- or ILT3KD-DC treated with various TLR ligands. Data are represented as mean Ϯ SEM of three .(p Ͻ 0.01; #, p Ͼ 0.05 ,ءء ;p Ͻ 0.05 ,ء) independent experiments

Bay11-7082. Addition of Bay11-7082 or SB203580 (but not of its 16). More p-IKB␣ (2.0ϫ), p-IKK␣␤ (1.7ϫ), and p-MAPKp38 inactive analog SB202474) to LPS-activated DC blocked IL-1␤ (1.3ϫ) was detectable in LPS-treated ILT3KD-DC than in control expression, both at the mRNA (data not shown) and soluble pro- DC (Fig. 6B). Phosphorylation of MAPKp42/p44, JNK, and NF- tein level (Ͼ60%; Fig. 6A). To determine whether expression of ␬Bp65 (RelA), on the other hand, were unchanged (data not ILT3 affects the phosphorylation of MAPK and I␬B kinases, LPS- shown). The total amounts of each of the respective proteins were treated ILT3KD-DC and ctrl-DC were subjected to immunoblot also unchanged. This result supports the notion that both NF-␬B analyses using various Abs that recognize the total (T) or phos- and MAPKp38 pathways are required for LPS activation and that phorylated (p) forms of MAPKp38, I␬B-␣, and its regulator, I␬B signaling is affected by ILT3 expression. kinase ␣␤ (IKK␣␤). In both types of cells, we found that phos- We then immunoprecipitated lysates from bpV(phen)-treated phorylation of IKK␣␤ and MAPKp38 was induced by LPS in a cells (ILT3KD-DC and ctrl-DC) with a goat anti-ILT3 Ab (or goat time-dependent manner with a peak at 30 min after treatment. IgG isotype control) and examined the immunoprecipitates by im- However, at the 3-h time point the resynthesis and degradation of munoblotting with an anti-phospho-tyrosine (p-Tyr) Ab (Fig. 6C). I␬B-␣ had reached an equilibrium state as indicated by others (14– Our results indicate that the anti-ILT3 Ab specifically pulls down The Journal of Immunology 5213 Downloaded from

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 http://www.jimmunol.org/ with 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 Transwell plate (5 ␮M pore size), and supernatants from ctrl-DC or ILT3KD-DC were added to the lower chambers. Results are expressed as mean Ϯ SEM of the .(p Ͻ 0.05 ,ء)total numbers of migrated T cells after2h several Tyr-phosphorylated proteins, in addition to p-Tyr-ILT3, Probing the membrane with various phosphatase-specific Abs from ctrl-DC lysates but not from that of ILT3KD-DC, supporting known to interact with ITIM indicates that SHP-1 and SHIP-1 the notion that phospho-ILT3 interacts with other p-Tyr proteins. were present in protein complexes immunoprecipitated by anti-ILT3. by guest on September 26, 2021

FIGURE 5. The effect of ILT3KD on priming T cells responses. A, CD3ϩ T cell proliferative responses to allogeneic ctrl-DC and ILT3KD- DC or autologous stimulators present- ing CMV Ags. Proliferation of cells was determined by [3H]thymidine in- corporation. Results were expressed as mean Ϯ SEM of four independent p Ͻ ,ءء ;p Ͻ 0.05 ,ء .experiments 0.01. B, Increased intracellular ex- pression of IFN-␥ and IL-17A by CD3ϩ T cells cocultured with ILT3KD DC for 5 days. A represen- tative result of flow cytometric anal- ysis of T cells gated out from T-DC cocultures is shown in B. C, Results obtained from three independent ex- periments show 3- to 7-fold expan- sion of Th1 and Th17 populations following T cell coculture with allo- geneic ILT3KD-DC vs ctrl-DC. .p Ͻ 0.05 ,ء 5214 ILT3 KNOCKDOWN AND INFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 6. A, Suppression of IL-1␤ induction by MAPKp38 and NF-␬B pathway inhibitors in ctrl-DC and ILT3KD-DC. Supernatants of variously treated 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 and ILT3KD-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 various by guest on September 26, 2021 Abs. Quantitation of the total (T) and phosphorylated (p) protein fractions was performed by normalizing results to the ␤-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.

Other phosphatases, such as SHP-2, and SHIP-2, which may in- a more vigorous proinflammatory cytokine response by teract with ITIM domains of inhibitory receptors (17, 18), did not ILT3KD-DC when compared with ctrl-DC, which express appear to interact with ILT3. Immunoprecipitation with IgG did physiological levels of ILT3 in response to “danger” signals not yield any ILT3 interacting proteins (data not shown). Taken relayed through a variety of TLR. From a signaling perspective, together with Fig. 6B, these data indicate that the NF-␬B and, to a our results also show that ILT3 recruits SHP-1 and/or SHIP-1 to lesser extent, the MAPKp38 pathways are negatively regulated by restrain the APC’s (LPS-triggered) activation pathways which ILT3 signaling through interaction with SHP-1 and/or SHIP-1. rely on NF-␬B and, to a lesser extent, MAPKp38. Taken to- gether, these findings provide direct evidence that the physio- Discussion logical concentration of ILT3 on APC may work as a “check In previous studies, we demonstrated that ILT3 is a crucial inhib- and balance” for overactive immune responses by interacting itory molecule whose expression affects the function of the APC with inhibitory phosphatases SHP-1 and/or SHIP-1 and damp- and that of the T cells with which they interact (3, 4, 6–8, 10, 19). ening NF-␬B and MAPKp38 activity. Although the function of this molecule has been well documented, Our results therefore are consistent with observation that the mechanisms by which ILT3 operate remains elusive. The SHP-1 mutant mice (mev/mev) demonstrate higher NF-␬B and present study attempts to expand the mechanistic understanding of MAPp38 activities (23) and are hypersensitive to LPS and ILT3-driven suppression. pathogenic challenges (23–25). There is a notable difference, TLR are a type of pattern recognition receptors that recognize however, between ILT3KD-DC and SHP-1 mutant (mev/mev) molecules that are broadly shared by pathogens but distinguish- mice, with respect to induction of type I IFN following TLR able from host molecules (13, 20–22). TLR play an important ligation. Although our results indicate that silencing ILT3-en- role in innate immunity, and by signaling the presence of patho- hanced type I IFN, IFN-␣1, mRNA production in DC, mutation gens, they trigger inflammation and the recruitment of adaptive on Shp-1 (mev/mev) in mice decreases the synthesis of IFN-␤ immune response to the affected microevironment. If un- after LPS treatments (23). This discrepancy may suggest that checked, the self-amplification of TLR signaling can lead to the negative signaling delivered by ILT3 does not entirely rely inflammatory/autoimmune disease (reviewed in Ref. 20). We on SHP-1 and other signaling molecules, such as SHIP-1 (Fig. show here that overactive inflammation is accompanied by 6C), may also contribute to the ILT3KD phenotype. A recent The Journal of Immunology 5215 study has implicated SHIP-1 in preventing TLR ligand induc- Disclosures tion of type I IFN synthesis in mice (26). The authors have no financial conflicts of interest. Binding of CXCL10 and CXCL11 to their receptor, CXCR3, induces various cellular responses, most notably the attraction Th1 References cells and promotion Th1 cell maturation (reviewed in Refs. 27 and 1. Cella, M., C. Dohring, J. Samaridis, M. Dessing, M. Brockhaus, 28). Dysregulation of CXCR3 and its ligand expression has been A. Lanzavecchia, and M. Colonna. 1997. A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen implicated in various types of diseases, such as multiple sclerosis processing. J. Exp. Med. 185: 1743–1751. (29) and type I diabetes (30). Our results showed that T cells re- 2. Brown, D., J. Trowsdale, and R. Allen. 2004. The LILR family: modulators of innate and adaptive immune pathways in health and disease. Tissue Antigens 64: spond to the higher levels of CXCL10 and CXCL11 produced by 215–225. ILT3KD-DC, with increased migration rates toward the chemo- 3. Manavalan, J. S., S. Kim-Schulze, L. Scotto, A. J. Naiyer, G. Vlad, P. C. Colombo, C. Marboe, D. Mancini, R. Cortesini, and N. Suciu-Foca. 2004. kine gradient, suggesting a possible regulatory role for ILT3 in Alloantigen specific CD8ϩCD28ϪFOXP3ϩ T suppressor cells induce controlling the trafficking of inflammatory T cells. Down-regula- ILT3ϩILT4ϩ tolerogenic endothelial cells, inhibiting alloreactivity. Int. Immu- tion of ILT3 can cause excess inflammation and infiltration of T nol. 16: 1055–1068. 4. Chang, C. C., R. Ciubotariu, J. S. Manavalan, J. Yuan, A. I. Colovai, F. Piazza, cells in locally affected lesions, leading to destruction of tissue or S. Lederman, M. Colonna, R. Cortesini, R. Dalla-Favera, and N. Suciu-Foca. autoimmune diseases. The importance of ILT3 in heart transplan- 2002. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat. Immunol. 3: 237–243. tation as a tolerogenic marker has been documented in our previ- 5. Qin, H., G. Vlad, R. Cortesini, N. Suciu-Foca, and J. S. Manavalan. 2008. CD8ϩ ous studies (3, 31). suppressor and cytotoxic T cells recognize the same human leukocyte antigen-A2 Mechanistically, there are still some questions that remain un- restricted cytomegalovirus peptide. Hum. Immunol. 69: 776–780. 6. Suciu-Foca, N., N. Feirt, Q. Y. Zhang, G. Vlad, Z. Liu, H. Lin, C. C. Chang, Downloaded from answered. For example, although the phosphorylation of I␬Bis E. K. Ho, A. I. Colovai, H. Kaufman, et al. 2007. Soluble Ig-like transcript 3 increased by ILT3 silencing, the total levels of I␬B are not inhibits tumor allograft rejection in humanized SCID mice and T cell responses in cancer patients. J. Immunol. 178: 7432–7441. drastically affected. This may be explained by recent findings 7. Kim-Schulze, S., L. Scotto, G. Vlad, F. Piazza, H. Lin, Z. Liu, R. Cortesini, and that Tyr-phosphorylation of I␬B is not always followed by deg- N. Suciu-Foca. 2006. Recombinant Ig-like transcript 3-Fc modulates T cell re- sponses via induction of Th anergy and differentiation of CD8ϩ T suppressor radation (14–16, 32). It is also unclear why only a handful of cells. J. Immunol. 176: 2790–2798. ␣␤ 8. Vlad, G., V. D. D’Agati, Q. Y. Zhang, Z. Liu, E. K. Ho, T. Mohanakumar,

(IL-1 , IL-6, IFN type I/II, CXCL10, CXCL11) genes are http://www.jimmunol.org/ affected by ILT3KD, despite the fact that many inflammatory M. A. Hardy, R. Cortesini, and N. Suciu-Foca. 2008. Immunoglobulin-like tran- script 3-Fc suppresses T cell responses to allogeneic human islet transplants in cytokine genes are known to be NF-␬B-regulated (33). How- hu-NOD/SCID mice. Diabetes 57: 1878–1886. ever, based on the data presented here, we propose that the loss 9. Penna, G., A. Roncari, S. Amuchastegui, K. C. Daniel, E. Berti, M. Colonna, and L. Adorini. 2005. Expression of the inhibitory receptor ILT3 on dendritic cells is ␬ ϩ ϩ of ILT3 during external stimuli prevents binding of I Btothe dispensable for induction of CD4 Foxp3 regulatory T cells by 1,25-dihy- transcription factor p50/p65 in the cytoplasm and partially ac- droxyvitamin D3. Blood 106: 3490–3497. 10. Manavalan, J. S., P. C. Rossi, G. Vlad, F. Piazza, A. Yarilina, R. Cortesini, tivates MAPK p38. The heterodimeric p50/p65 complexes D. Mancini, and N. Suciu-Foca. 2003. High expression of ILT3 and ILT4 is a subsequently translocate to the nucleus whereas phospho- general feature of tolerogenic dendritic cells. Transpl. Immunol. 11: 245–258. MAPp38 kinase induces phosphorylation of mitogen- and 11. Raetz, C. R., and C. Whitfield. 2002. Lipopolysaccharide endotoxins. Annu. Rev.

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