IFN- -rich environment programs dendritic cells toward silencing of cytotoxic immune responses

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1 Article IFN- -rich environment programs dendritic cells toward silencing of cytotoxic immune responses Urban Švajger,*,,1 Nataša Obermajer,, and Matjaž Jeras, *Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia; Celica, Biomedical Center, Ljubljana, Slovenia; Departments of Pharmaceutical Biology and Clinical Chemistry, Faculty of Pharmacy, Aškerčeva 7, Ljubljana, Slovenia; and Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia RECEIVED NOVEMBER 20, 2012; REVISED JULY 12, 2013; ACCEPTED JULY 18, DOI: /jlb ABSTRACT Lately, there is increasing evidence that emphasizes the regulatory functions of IFN-, which serve as negative-feedback mechanisms after, e.g., pathogen clearance, to prevent unnecessary tissue destruction. Inflammatory processes involving Th1 and cytotoxic responses are characterized by high, local IFN- concentrations, followed by resolution and immune silencing. Although this is a well-known course of events, extensive attempts to address potential differential effects of IFN- in the manner of its availability (quantitatively) in the environment do not exist. We demonstrate that high doses of IFN- do not induce DC maturation and activation but instead, induce specific regulatory characteristics in DCs. Considering their phenotype, high doses of IFN- extensively induce the expression of ILT-4 and HLA-G inhibitory molecules. Interestingly, the well-known priming effect of IFN- for IL-12p70 production is lost at these conditions, and the DC cytokine profile is switched toward an increased IL-10/IL-12p70 ratio upon subsequent stimulation with CD40L. Furthermore, such DCs are capable of silencing cellular immune responses and activation of cytotoxic CD8 T lymphocytes, resulting in reduced cell proliferation and down-regulation of granzyme B expression. Additionally, we find that in this manner, immune regulation mediated by IFN- is not mainly a result of increased enzymatic activity of IDO in DCs but rather, a result of HLA-G signaling, which can be reversed by blocking mab. Altogether, our results identify a novel mechanism by which a Th1-like environment programs the functional status of DCs to silence ongoing cytotoxic responses to prevent unwanted tissue destruction and inflammation. J. Leukoc. Biol. 95: 33 46; Abbreviations: 1-MT 1-methyl-L-tryptophan, -high/low DC high/low IFN- concentrations, A 450/592 nm absorbance at 450/592 nm, BDCA blood DC antigen, CD40L CD40 ligand, DC-SIGN DC-specific ICAM-3-grabbing nonintegrin, FasL Fas ligand, idc immature DC, ILT Ig-like transcript, mdc mature DC, MFI mean fluorescence intensity, MoDC monocyte-derived DC, mydc myeloid DC, rh recombinant human Introduction Inflammatory states caused by viral or intracellular pathogens, as well as cases of autoimmune diseases and graft-versus-hostdisease reactions, are characterized by strong IFN- signature and Th1 effectors, which then promote the activation of CD8 CTL responses. IFN- is a major driving force of these responses affecting practically all involved cellular types of innate and adaptive immunity. During the early phases of an immune response, NK cells represent an important source of IFN-. When stimulated, they migrate to secondary LNs, where they provide initial levels of IFN- necessary for differentiation of naive T cells into Th1 effectors [1]. Later, Th1 cells as well as CTLs become the most important sources of IFN- production in greater quantities [2]. Activated Th1 cells are capable of producing large quantities of IFN- (as much as 350 ng/ml, equivalent to 35,000 U/ml) [3, 4]. It is therefore likely that various immune cells, such as APCs, can encounter similar concentrations in vivo. In addition, it has been demonstrated that even B cells and nonlymphoid cells, such as macrophages and DCs, can produce IFN- to augment the inflammatory response [5, 6]. As local concentrations of IFN- increase during a particular immune process, it is also known that excessive production of Th1-type cytokines, including IFN-, ifnot properly controlled, leads to tissue destruction, characteristic for autoimmune diseases [2]. The control of Th1-mediated responses is therefore a crucial feedback mechanism necessary for resolving aggressive inflammatory states after they are no longer required, e.g., after pathogen clearance. Paradoxically, it has been shown on many occasions that in addition to its central, proinflammatory role, IFN- acts in ways to suppress responses of the same immune components it once stimulated toward an effector state. Indeed, over the last years, a large body of evidence has accumulated, demonstrating a protective role of IFN- in autoimmune disease models. Gene knockout studies revealed that autoimmune arthritis develops faster and in a more severe form in mice lacking IFN- or IFN- R [7, 8]. 1. Correspondence: Blood Transfusion Center of Slovenia, Slajmerjeva 6, 1000 Ljubljana, Slovenia. urban.svajger@ztm.si /14/ Society for Leukocyte Biology Volume 95, January 2014 Journal of Leukocyte Biology 33

2 Most interestingly, recovery of animals with adjuvant-induced arthritis was found to correlate with the highest levels of IFN- secreted from antigen-stimulated T cells [9]. The protective role of IFN- has also been described in studies of experimental autoimmune encephalitis [10], experimental autoimmune uveoretinitis [11], experimental autoimmune myasthenia gravis [12], and experimental autoimmune tyhroiditis [13]. So far, the main mechanisms behind these immunoregulatory roles of IFN- have included its ability to promote activation and function of regulatory T cells [14] and to APCs, particularly the DCs. Characterized by R. M. Steinman et al. [15], more than one decade ago, the DCs are known as professional APCs with outstanding functional plasticity. Depending on their microenvironment, they can adopt an immunogenic or immunosuppressive functional state. IFN- -induced modulation of DCs is mostly known in terms of being a priming agent, delivering a second activation signal (besides TLR agonists or CD40 ligation) to allow for extensive DC maturation and high IL-12p70 production, which lead to effective cytotoxic responses [16]. However, IFN- -induced signaling in the absence of danger signals has not been studied to the same extent. Nevertheless, it is known that IFN- can contribute to DC-mediated immunosuppression via induction of tryptophancatabolizing enzyme IDO, inos, and HO-1 [14]. As the pleiotropism of IFN- is clearly a result of a singlecytokine acting in different environments and time-frames of an immune response, not enough research has been focused on such simple characteristics of an immunological event as quantitative properties of an acting cytokine. So far, to our knowledge, only one study addressed the quantitative aspect of IFN- signaling, describing its negative effects on T cell trafficking at extremely low doses (0.1 1 U/ml). It was demonstrated recently that Th1-mediated inflammation is indeed selflimiting, where IFN- accelerates inflammation in the early stage and mediates the process of self-limitation in late stages [17]. Although the suggested mechanisms included IFN- -induced up-regulation of inos and production of NO, questions exist regarding whether this is the only mechanism and how different cell types are affected. In this study, we show that when DCs are exposed to high concentrations of IFN- (resembling peak Th1-like conditions), they acquire a specific phenotype with low expression of costimulatory molecules and extensive expression of surface HLA-G and ILT-4 molecules. Such DCs possess an extremely low allostimulatory capacity, do not induce Th1 polarization from naive CD4 T cells, and silence CTL activation via HLA-G. MATERIALS AND METHODS Cell culture and isolation Buffy coats from venous blood of normal, healthy volunteers were obtained by the Blood Transfusion Centre of Slovenia, according to institutional guidelines. PBMCs were isolated using Lympholyte-H (Cedarlane Laboratories, Ontario, Canada). The cells were washed twice with DPBS, counted, and used as a source for immunomagnetic isolation of CD14-positive cells (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of monocytes was always 90%, determined by flow cytometry. These were cultured in RPMI-1640 (Cambrex) medium supplemented with 10% FBS, gentamicin (50 g/ml; Gibco, Paisley, UK), and 800 U/ml rhgm-csf and 1000 U/ml rhil-4 (both PeproTech EC, London, UK). On Day 2, one-half of the medium was exchanged with starting quantities of rhgm-csf (800 U/ml) and rhil-4 (1000 U/ml). After 5 days nonadherent idcs were harvested and characterized by flow cytometry, as CD1a hi, DC-SIGN hi, CD80 low, CD83, CD86 low, and HLA-DR low. To study the effects of IFN- treatment, DCs were stimulated with 5 U/ml, 50 U/ml, 500 U/ml, or 5000 U/ml rhifn- (PeproTech EC) for h, depending on the experiment. To obtain fully mdcs for controls, cells were activated with LPS (20 ng/ml) or a combination of IFN- (500 U/ml) and LPS (20 ng/ml). For experiments in which the effects of low and high IFN- concentrations were compared, 50 U/ml IFN- was used for low and 5000 U/ml IFN- for high concentration, as a result of phenotypic and basic functional characterization of variously treated DCs in early studies. DCs treated with low IFN- concentration are thus designated -low DCs, and those treated with -high DCs. Primary BDCA-1 mydcs were isolated from human buffy coats using the Miltenyi Biotec CD1c (BDCA-1) DC isolation kit (Miltenyi Biotec GmbH). This is a two-step isolation procedure, first, involving depletion of CD20 cells, followed by positive selection of BDCA-1 DCs. The purity of obtained mydcs was always 90%. Afterward, we cultured mydcs with GM-CSF alone (800 U/ml) or with the addition of LPS (20 ng/ml) or IFN- (50 or 5000 U/ml) for 48 h. T cells were purified from human buffy coats. Whole CD4 T cells were obtained by positive selection using CD4 microbeads (Miltenyi Biotec GmbH). The purity of CD4 cells was always 95%, as determined by flow cytometry. Naive CD4 CD45RA T cells were isolated using the naive CD4 T cell isolation kit from Miltenyi Biotec GmbH, strictly following the manufacturer s protocol. The purity of isolated naive CD4 T cells was always 98%. Whole CD8 T cells were obtained by positive selection using CD8 microbeads (Miltenyi Biotec GmbH). Naive CD8 CD45RA CD45RO T cells were isolated using the CD8 T cell isolation kit in combination with CD45RO microbeads (Miltenyi Biotec GmbH), resulting in a uniform population of CD8 CD45RA CD45RO cells. The purity of naive CD8 T cells was always 98%. Phenotypic characterization and endocytosis studies For flow cytometry analysis of DC phenotype, we used the following mab: FITC-labeled anti-cd1a (HI149), anti-cd14 (HCD14), anti-cd40 (HB14), anti-cd80 (2D10), anti-cd83 (HB15e), and anti-hla-a/b/c (W6/32); Alexa Fluor 488-labeled anti-ccr7 (G043H7), anti-cd8 (SK1), and anti-cd86 (IT2.2); R-PE-labeled anti-hla-g (87G), anti-hla-dr (L243), anti-cd209 (9E9A8), and anti-ilt-2 (GHI/75); PE/Cy5-labeled anti-cd8 (HIT8a); and APC-labeled anti-cd4 (OKT4; all from BioLegend, San Diego, CA, USA); and FITC-labeled anti-ilt-4 (287219; R&D Systems, Minneapolis, MN, USA). FITC-IgG1 and R-PE-IgG2a cocktail was used for isotype control (BioLegend). idcs, mdcs, or IFN- -treated DCs (as described above) were harvested and collected by centrifugation. Antibody was added, and the cells were incubated for 15 min in the dark, then washed twice, and resuspended in 2% PHA. Samples were analyzed on a FACSCalibur system (Becton Dickinson, San Diego, Ca, USA). Data were analyzed with the CellQuest software (BD Biosciences, San Jose, CA, USA). Endocytosis was monitored by flow cytometry after incubation of DCs with FITC-dextran, on ice or at room temperature for 1 h. The cells were then washed twice with DPBS and resuspended in 2% PFA. Apoptosis experiments We used human peripheral-blood monocytes for differentiation of idcs using GM-CSF (800 U/ml) and IL-4 (1000 U/ml). The DCs were left untreated, matured for 2 days with LPS (20 ng/ml), or stimulated with a low (50 U/ml) or high (5000 U/ml) dose of IFN-. To evaluate the cytotoxic effects of high-dose IFN-, we cultured DCs, as described, for h. Afterward, we analyzed the percentage of dead and early apoptotic cells by Annexin-V FITC conjugate and PI staining. To evaluate apoptosis in DC-T cell cocultures, we prestained the DCs with a cell-tracking agent, CFSE 34 Journal of Leukocyte Biology Volume 95, January

3 Švajger et al. Regulation of DC function by high-dose IFN- (BioLegend). After 6 days, we stained the cells with PI. The analysis was performed by flow cytometry, and we determined the percentage of dead DCs (CFSE,PI ) and dead T cells (CFSE,PI ). For apoptosis experiments of activated, cytotoxic CD8 cells, we first prepared the T cells by culturing naive CD8 CD45RA CD45RO T cells with CD3/CD28 Dynabeads (2.5 l/ml; Invitrogen Dynal AS, Oslo, Norway) for 5 6 days in 96-well plates, cells/well. After 5 days, the activated CD8 lymphocytes were used in coculture experiments with differentially treated DCs. Parallel cocultures were performed with neutralization antibodies against HLA-G (Exbio, Prague, Czech Republic) and FasL (Antibodies Online, Atlanta, GA, USA). Both antibodies were used at 10 g/ml. Mouse polyclonal IgG was used as control. After 48 h of coculture, the percentage of dead and apoptotic CD8 cells was determined by Annexin-V FITC conjugate and PI staining. Gating of CD8 cells was performed by prestaining with anti-cd8 PE/Cy5-conjugated antibody (BioLegend). Flow cytometry was performed using a FACSCalibur system (Becton Dickinson). Quantification of cytokine production The BD human CBA (BD Biosciences) was used to assay the protein levels of IL-10 and IL-12p70 in the cell culture supernatant, according to the manufacturer s protocol. Briefly, DCs were treated variously for 48 h, as described above. Cells were centrifuged, and cell culture supernatants were used for further analysis. In an appropriate assay tube, 50 l capture beads, 50 l detection reagent, and 50 l sample (five- or tenfold dilution) were mixed and incubated for 3 h at room temperature and protected from light. Samples were then washed with 1 ml wash buffer and centrifuged at 3000 rpm for 5 min. The supernatant was aspirated carefully and discarded from each assay tube. The bead pellet was resuspended by adding 300 l wash buffer. Flow cytometry was performed using a FACSCalibur system (Becton Dickinson). A standard curve was prepared by serial dilutions of standards and used for determination of cytokine concentrations in supernatants. ELISA analysis of IL-12p70 and IL-10 (IL-10 and IL-12p70 Deluxe sets, both from BioLegend), by in vitro-generated DCs, was performed. To mimic the interaction with CD40L-expressing Th cells, CD40L-transfected J558 cells (University of Birmingham, UK) were added [18]. Briefly, the DCs were treated or not with 5000 U/ml IFN- for 48 h. Afterward, the cells were restimulated for 24 h with CD40L-expressing J588 cells. Cells ( and )/well were used for DCs and J558 cells, respectively, in 200 l medium. At the end of stimulation, supernatants were analyzed for levels of cytokines and the results obtained by measuring A 450 nm. Confocal immunofluorescence microscopy idcs ( ) on Day 5 of differentiation, DCs matured for 2 days with 800 U/ml rhgm-csf and 20 ng/ml LPS, or DCs treated with 50 U/ml or 5000 U/ml IFN- for 2 days were centrifuged onto glass coverslides with cytospin (CytoFuge) for 6 min at 1000 rpm. Before labeling, cells were allowed to recover for 15 min and then fixed with 4% PFA for 45 min and permeabilized by 0.1% Triton X-100 in PBS, ph 7.4, for 10 min. Nonspecific staining was blocked with 3% BSA in PBS, ph 7.4, for 1 h. Podosomes and dendrites were counted after labeling of actin with phalloidin tetramethylrhodamine B isothiocyanate conjugate (Fluka, Germany; 500 ng/ml) for 30 min at room temperature. For the NF- B activation assay, nontreated DCs or DCs treated with 50 U/ml or 5000 U/ml IFN- or 20 ng/ml LPS for 1.5 h were centrifuged onto glass coverslides with cytospin (CytoFuge) for 6 min at 1000 rpm, fixed with 4% PFA for 45 min, and permeabilized by 0.1% Triton X-100 in PBS, ph 7.4, for 10 min. Nonspecific staining was blocked with 3% BSA in PBS, ph 7.4, for 1 h. NF- Bp65 was labeled with Alexa Fluor 488-conjugated antibody (F-6; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1.5 h in blocking buffer and cells then washed with PBS. ProLong antifade kit (Molecular Probes, Carlsbad, CA, USA) was used to mount coverslips on glass slides. Circular regions of interest (area 10 m 2 ) were selected in nucleus and cytoplasm and activation of NF- B determined as the ratio of MFI of nucleus and cytoplasm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Alexa Fluor 488 or phalloidin tetramethylrhodamine B isothiocyanate conjugate was excited with an argon (488 nm) or He/Ne (543 nm) laser, and emission was filtered using narrow band nm and long-pass 560 nm filters, respectively. Images were analyzed using Carl Zeiss LSM image software 3.0. Allogeneic T cell proliferation Control idcs and mdcs, together with DCs treated with various concentrations of IFN- for 48 h, were washed twice in DPBS and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO, USA). Purified, whole CD4 T cells were used as responders. The assays were carried out in 96-well plates, with a total volume of 200 l/well. In each well, we seeded T cells with ,2 10 3,or of variously treated DCs. In a parallel experiment, we seeded T cells with DCs and added 20 U/ml IL-2 (PeproTech) on the 2nd day. After 4 days, the wells were pulsed with 1 Ci/well 3 H-thymidine (Perkin Elmer, Boston, MA, USA) and proliferation measured by its incorporation after 18 h by liquid-scintillation counting. Suppression of effector CD8 T cell generation Naive CD8 CD45RA CD45RO T cells were stimulated with CD3/CD28 Dynabeads (2.5 l/ml; Invitrogen Dynal AS) in the presence of control idcs, mdcs, and DCs treated with various concentrations of IFN- for 48 h. Parallel cocultures were performed with neutralizing anti-hla-g antibody (Exbio) and 1-MT (Sigma-Aldrich), an IDO inhibitor, at 100 M. Mouse polyclonal was used for control. CFSE staining of CD8 T cells (Invitrogen Dynal AS) was performed, according to the manufacturer s instructions. On Days 4 5, expanded CD8 T cells were analyzed for the granzyme B expression and proliferation [19]. Whereas in the preliminary experiments, we tested the impact of DCs on CTL development at the DC:T cell ratios of 1:2, 1:5, 1:10, and 1:20, in the subsequent standard experiments, we used a 1:10 ratio, determined to be optimal ( and cells were used for DCs and responder T cells, respectively). T cell migration assay Transwells (Corning Costar, Corning, NY, USA), with 5 m polycarbonate filters (5 m pore size), were used. CD4 T lymphocytes were washed with PBS and resuspended in complete RPMI-1640 medium. T lymphocytes ( ) were added to upper compartments in 100 l medium. The lower compartments were filled with 600 l DC conditioned medium (diluted one-half with complete RPMI-1640 medium). The plate was incubated for 3 h at 37 C and 5% CO 2. Afterward, transwells were lifted, and media with migrated cells from the lower compartment were transferred separately to Eppendorf tubes and centrifuged at 2000 rpm for 5 min. Supernatants were discarded, and cells resuspended in 150 l medium and transferred to a 96-well culture plate. MTS reagent (20 l) was added to each well and incubated for an additional 4hat37 C and 5% CO 2, with A 592 nm. Migration was recorded as the percentage of cells that migrated through the filter. Intracellular cytokine staining Cytokine secretion profiles for CD4 T cells at the single-cell level were obtained by staining the cells intracellularly and carrying out the analysis by flow cytometry. Nontreated idcs, mdcs, and DCs, treated with 50 U/ml or 5000 U/ml IFN-, were cocultured with allogeneic, freshly isolated, naive CD4 T cells for 10 days in complete medium (RPMI 1640 containing 10% FBS). In parallel experiments, neutralizing anti-ilt-2 [20] (10 g/ml) and anti-ilt-4 [21] (10 g/ml; both from BioLegend; Clones GHI/75 and 42D1, respectively) antibodies were added to cultures to block the interactions between HLA-G and ILT-2/4. Mouse polyclonal was used for control cocultures. After 10 days, the cells were collected, washed twice with DPBS, and rested overnight in complete medium. Cytokine secretion was then induced by the addition of 50 ng/ml PMA and 500 ng/ml ionomycin. Af- Volume 95, January 2014 Journal of Leukocyte Biology 35

4 ter 4 h, Brefeldin A (10 g/ml) was added for another 4 h. At the end of stimulation, cells were washed twice, fixed with 4% PHA for 1 h, and permeabilized with 0.1% Triton X-100 solution for 10 min. The permeabilized cells were washed two times with DPBS and incubated for 30 min in PBS containing 3% BSA to prevent nonspecific staining. Then, the cells were stained intracellularly using FITC-labeled anti-ifn-, PE-labeled anti-il-4, and PE-labeled anti-il-10 (all from Invitrogen Dynal AS; Clones B27, MP425D2, and JES3-D97, respectively). Flow cytometry was performed using a FACSCalibur system (Becton Dickinson). Phosflow staining of NF- B p65 The levels of the phosphorylated NF- B p65 subunit were analyzed additionally by phosflow analysis, as described previously [22]. Briefly, DCs treated variously for 20 min were collected after fixation with addition of PFA directly into warm culture medium to ensure rapid freezing of signaling events. Afterward, cells were washed with PBS and permeabilized with ice-cold methanol for 10 min. Cells were then washed twice in PBS, and a solution of 3% BSA in PBS was added to prevent nonspecific staining. A phosphorylated NF- B p65 subunit was stained with AlexaFluor 488-conjugated anti-nf- B p65 mab (BD Phosflow; IgG2b). Conjugated mouse IgG2b was used for isotype control. Results were analyzed on a FACSCalibur system (Becton Dickinson). Neutralization experiments To evaluate whether the inhibitory phenotype inflicted on DCs by IFN- is in any way dependent on the possible release of IL-6 or IL-10 by IFN- treated DCs, we used anti-il-6 (BioLegend; Clone MQ2-13A5) and anti- IL-10 (R&D Systems; Clone 25,209) antibodies with reported blocking activities [23, 24]. DCs were left untreated or were treated with 5000 U/ml IFN- for 48 h in the presence or absence of blocking anti-il-6 (10 g/ml) or anti-il-10 (10 g/ml) antibody. Afterward, the DCs were characterized for the surface expression of CD80, CD86, ILT-4, and HLA-G. RESULTS The effect of high-dose IFN- on general DC characteristics We set out to study basic characteristics that would define -high DCs (5000 U/ml IFN- ) compared with control idcs, mdcs, or -low DCs (50 U/ml IFN- ). The differentiation of DCs from monocytes was performed as described in Materials and Methods. On Day 5, CD1a high, CD14 DCs were left untreated or activated with various doses of IFN- or fully matured using LPS alone or in combination with IFN- (500 U/ml). Morphologically, idcs are known to form podosomes, whereas mdcs tend to form great numbers of dendrites. LPS, particularly when used in combination with IFN- (500 U/ml), caused the majority of DCs to form dendrites (60% and 95% dendrite-forming cells for LPS- and LPS/IFN- -treated DCs, respectively). Surprisingly, dendrite formation was completely absent on DCs stimulated with high IFN- doses (Fig. 1A and B). In addition, -high DCs, even to a greater extent than -low DCs, did not lose the ability to form podosomes, although at a lower percentage than idcs (18%, 24%, and 57% podosome-forming cells for DCs, DCs, and idcs, respectively). We also explored the ability of IFN- to affect endocytosis after idcs were stimulated for 48 h. Endocytotic capability of DCs was measured as mannose receptor-mediated uptake of FITC-labeled dextran. At lower doses, IFN- displayed no effect on the endocytotic capabilities of DCs. At high doses, however, the effect of IFN- became evident as DCs were much less able to endocytose FITC-dextran. Although the endocytotic capability of DCs was lower, it was still approximately fivefold greater than for mdcs (Fig. 1C). NF- B activation was determined as the fluorescence-intensity ratio of nucleus:cytoplasm of DCs labeled with FITC antip65 by means of Phosflow staining, as described previously [22]. For confocal microscopy, DCs were left untreated or stimulated with 50 U/ml or 5000 U/ml IFN- or LPS (20 ng/ ml) for 1.5 h to induce NF- B translocation to the nucleus. Compared with LPS-treated DCs, neither -low DCs nor -high DCs demonstrated any significant NF- B translocation to the nucleus (Fig. 1D). For Phosflow staining, DCs were left untreated or stimulated with 50 U/ml or 5000 U/ml IFN- or LPS (20 ng/ml) for 20 min. The cells were then fixed with PFA and permeabilized with ice-cold methanol and stained for the phosphorylated p65 NF- B subunit (Fig. 1E). Similar to nuclear-translocation analysis, no increase in p65 phosphorylation was seen with -low- or -high DCs compared with nontreated controls. To ensure that immunosuppressive properties of DCs stimulated with IFN- were not a result of extensive DC death, we cultured idcs with various IFN- concentrations for 48 h or 72 h. Afterward, the apoptosis of DCs was measured by annexin and PI staining, as shown in Fig. 1F. An ungated, whole population was analyzed. The highest concentrations of IFN- somewhat increased the percentage of dead cells; however, the majority of DCs remained viable (10.1% and 13.5% dead cells after 48 h and 72 h IFN- stimulation, respectively, and 2.1% and 4.9% dead cells after 48 h and 72 h for idcs, respectively). In our functional studies, the greatest time exposure of DCs to IFN- was 48 h, after which, the cells were washed thoroughly in all cases to ensure no further effects of IFN-. High doses of IFN- do not induce costimulatory molecule expression on DCs We investigated the dose-dependent effects of IFN- alone and on DCs differentiated from human peripheral blood monocytes in the context of expression of various costimulatory molecules and antigen-presenting molecules and CCR7 chemokine receptor. On the 5th day, nonadherent idcs (characterized as CD1a high, CD209 high, CD14, CD80 low, and CD86 ; data not shown) were harvested and left untreated or stimulated for 48 h with LPS; LPS IFN- (500 U/ml); or with 5 U/ml, 50 U/ml, 500 U/ml, and 5000 U/ml IFN- alone to determine the dosedependent effect of IFN- on an array of DC surface markers. Treatment of DCs with LPS or a LPS/IFN- cocktail resulted in strong up-regulation of CD40, CD80, CD86, and HLA-DR, as expected (Fig. 2A). On the other hand, IFN- alone, even at 5000 U/ml, did not induce expression of costimulatory molecules or the expression of CD83 (Fig. 2A). The expression of CD1a on -low DCs and -high DCs was similar to idcs and mdcs, respectively. However, HLA-DR and CCR7 expression was dose-dependently induced by IFN- (Fig. 2B and C). Concentration of 50 U/ml IFN- alone caused significantly greater CCR7 expression than stimulation with LPS or LPS/IFN-. We additionally tested whether IFN- influences the expression 36 Journal of Leukocyte Biology Volume 95, January

5 Švajger et al. Regulation of DC function by high-dose IFN- Figure 1. General characteristics of -high DCs. (A and B) Fully differentiated CD1a high, CD14 DCs were cultured for 2 more days in the presence of high (5000 U/ml) or low (50 U/ml) IFN- concentrations, matured with a combination of LPS (20 ng/ml) and IFN- (500 U/ml), LPS alone, or left untreated. The cells were then evaluated for their ability to form podosomes (at their immature state) or dendrites (upon maturation) and analyzed by confocal microscopy. Podosomes and dendrites were counted after labeling of cytoskeleton with phalloidin tetramethylrhodamine B isothiocyanate conjugate. The results are shown as confocal images and as the percentage of cells bearing dendrites or podosomes. Shown are mean sd values of three independent experiments. Significance is calculated between individual pairs (treated vs. idcs or LPS-DCs, as depicted), using Student s unpaired t-test. (C) Endocytotic capacity of DCs treated with different concentrations ( U/ml) of IFN- was determined by FITC-dextran uptake. DCs were treated with IFN- for 48 h and afterward, incubated with FITC-dextran at 37 C, along with idcs and activated DCs (LPS, with or without IFN- ) for 1 h. In consideration of unspecific binding, DCs were incubated with FITC-dextran at 4 C and used as controls when performing flow cytometry experiments. Results are expressed as mean sd of three independent experiments. Statistical significance of individually treated cells versus idcs is calculated using Student s unpaired t-test. (D) DCs were left untreated [not treated (n.t.)], activated with LPS, or treated with low (50 U/ml) or high (5000 U/ml) IFN- concentrations for 1.5 h. They were then stained with anti-nf- Bp65 antibody. The translocation of NF- B from cytoplasm to the nucleus was monitored with confocal microscopy and the results expressed as the ratio of MFIs between the nucleus and cytoplasm. Shown are mean sd values of three independent experiments. (E) DCs were left untreated, activated with LPS, or treated with low (50 U/ml) or high (5000 U/ml) IFN- concentrations for 20 min. They were then fixed with PFA and treated with ice-cold methanol and afterward, stained intracellularly with AlexaFluor 488-conjugated anti-nf- B p65 mab. Results are shown as percentage of mean sd of normalized MFI relative to control. Three individual experiments were performed. (F) To determine if the immunosuppressive effect of IFN- is associated with increased apoptosis of DCs, we variously stimulated the DCs for 48 h or 72 h, as described in Materials and Methods. Afterward, the cultures were stained with FITC-Annexin V and PI, and apoptosis was measured by flow cytometry. Shown is one representative experiment out of three performed. ***P HLA class I (A, B, and C). As expected, -high DCs induced the expression of HLA-I, as shown in Fig. 2D ( 1.5-fold compared with idcs). The numbers in panels represent MFI values. Shown is one representative experiment out of four to seven performed. -High DCs greatly induce the expression of inhibitory molecules ILT-4 and HLA-G on DCs We investigated further the effect of various doses of IFN- on the inhibitory surface phenotype of treated DCs. As described in Materials and Methods, nonadherent idcs (characterized as CD1a high, CD209 high, CD14, CD80 low, and CD86 ; data not shown) were harvested and left untreated or stimulated for 48 h with LPS, LPS IFN- (500 U/ml), or 5 U/ml, 50 U/ml, 500 U/ml, and 5000 U/ml IFN- alone to determine the dosedependent effects of IFN- on induction of inhibitory molecules. The ILT-2 and -4, as well as their correspondent receptor HLA-G, have been designated recently with an important immunoregulatory role in the context of bidirectional DC-T cell communication [25 27]. The expression of these molecules on nontreated idcs, as well as fully activated mdcs, was low or absent in our experi- Volume 95, January 2014 Journal of Leukocyte Biology 37

6 Figure 2. Induction of costimulatory molecules is unaffected by high doses of IFN-. Immature MoDCs were treated with various concentrations of IFN- ( U/ml), LPS (20 ng/ml), or LPS and IFN- (500 U/ml) for 48 h, and wide phenotypical analysis of surface markers was performed. (A) DCs were treated with 50 (low) or 5000 (high) U/ml IFN-, LPS, or left untreated and assessed for their expression of CD1a, CD40, CD80, CD83, and CD86. (B) Variously treated DCs, as depicted on the figure, were analyzed for their surface expression of LN homing chemokine receptor CCR7. (C) The expression level of MHC class II molecule HLA-DR was measured on DCs treated with increasing concentrations of IFN-, LPS, or on untreated DCs. (D) The expression of HLA class I (A, B, and C) was measured on DCs treated with low (50 U/ml) or high (5000 U/ml) concentrations of IFN-. Results shown are from one representative experiment out of four to seven performed. Dotted lines represent isotypic controls. Numbers in panels represent the MFI values. ments. Relatively low concentrations of IFN- also failed to result in significant induction of expression (Fig. 3A and B). However, when the DCs were exposed to IFN- concentrations above 500 U/ml, extensive and significant up-regulation of ILT-4 and HLA-G was observed (up to ninefold and 34-fold expression for ILT-4 and HLA-G, respectively, on -high DCs, relative to idcs; Fig. 3B). The expression levels of ILT-2 on DCs were not affected much by IFN- signaling, with levels for -low and -high DCs similar to the control idcs (Fig. 3C). At concentrations above 5000 U/ml IFN-, there was no relevant, further increase in ILT-4 and HLA-G expression (data not shown). For this reason, 5000 U/ml IFN- was the highest concentration used. -High DCs posses low allostimulatory and T cell-attracting characteristics and do not produce increased IL-12p70 quantities upon subsequent CD40 ligation First, we began addressing the functional characteristics of IFN- -treated DCs by evaluating their stimulatory potential in a MLR. The DCs were generated and treated as described and used as stimulators in cocultures with allogeneic, whole CD4 T cells at 1:10, 1:50, and 1:250 ratios, respectively (Fig. 4A). For these preliminary, functional experiments, we wanted to analyze the dose-dependency of IFN- treatment and its relevance to DC function. Therefore, the stimulators were treated with increasing doses of IFN- (5, 50, 500, and 5000 U/ml). Increasing concentrations of IFN- dose-dependently and significantly lowered the allostimulatory capacity of DCs, with -high DCs (5000 U/ml IFN- ) possesing approximately fourfold-lower allostimulatory capacity than control idcs (Fig. 4A). We wanted to see whether suppression of T cell proliferation can be reverted by IL-2. For this reason, we added rhil-2 on the 2nd day of the MLR. The effect was similar for all types of stimulators. Although the increase in proliferation was not completely significant, IL-2 increased the proliferation of T cells in all MLR cultures (Fig. 4B). We additionally analyzed the capacity of IFN- -treated DCs to attract responding CD4 T cells. The migrating ability of T cells was evaluated against 48-h supernatants of vari- 38 Journal of Leukocyte Biology Volume 95, January

7 Švajger et al. Regulation of DC function by high-dose IFN- Figure 3. High-dose IFN- causes an extensive up-regulation of ILT-4/HLA-G inhibitory molecules on DCs. Dose-dependent, immunosuppressive effect on inhibitory DC phenotype by IFN- was assessed by using increasing IFN- concentrations ( U/ml) in treating of fully differentiated DCs for 48 h. (A and B) Surface expression of ILT-4 and HLA-G is shown along with statistical analysis. Numbers in panels represent MFIs of the depicted CD marker. (B) Mean sd values of four independent experiments. Statistical significance between individual pairs (as depicted) was calculated using Student s unpaired t-test. (C) DCs treated with low (50 U/ml) or high (5000 U/ml) concentrations of IFN- for 48 h are analyzed for their surface expression of inhibitory molecule ILT-2. Numbers in brackets represent MFI values. Dotted lines represent isotypic controls. Histograms are from one representative experiment out of four independent experiments performed. *P 0.05; **P 0.01; ***P ously treated DCs using a transwell system, as described in Materials and Methods. The migration of T cells was greatest in wells where the supernatant from LPS/IFN- -treated DCs was present in the lower chamber. Interestingly, as lower doses of IFN- caused an up-regulation of T cell migratory levels (particularly, 50 U/ml IFN- ), supernatants from -high DCs cause the lowest levels of T cell migration, significantly lower than supernatants from control idcs (Fig. 4C). We analyzed the capacity of variously treated DCs to produce IL-10 and IL-12p70 (Fig. 4D). IFN-, by itself, has little effect on production of IL-10 and IL-12p70. However, when used as an additional stimulus, IFN- is well-known to highly synergize with TLR agonists or CD40 ligation in production of IL-12p70, as can be seen for LPS/IFN- -treated DCs (Fig. 4C) [28]. As -low and -high DCs, although treated with IFN- alone, most likely interact with CD40L in DC:T cell cocultures, we wanted to observe how pretreatment with high doses of IFN- affects the otherwise-known synergy with CD40 ligation in IL-12p70 production [29]. Interestingly, when DCs were pretreated with 5000 U/ml IFN- for 48 h, they produced increased levels of IL-10 upon CD40 ligation (P 0.05) and decreased levels of IL-12p70 (P ) compared with nontreated, control idcs (Fig. 4E). -High DCs are poor inducers of Th1 effectors from naive CD4 CD45RA T cells As high doses of IFN- clearly affect DCs in a specific manner not observed previously, we wanted to examine further the functional effect of -high DCs in terms of CD4 T cell polarization. We performed coculture experiments with naive CD4 CD45RA T cells, as described in Materials and Methods, using idcs, mdcs, -low DCs, and -high DCs as stimulators. After 10 days of coculture, the resulting CD4 T cell populations were rested overnight and analyzed intracellulary for expression of IL4, IL-10, and IFN- after PMA/ionomycin/ Brefeldin A stimulation (Fig. 5A). Compared with idcs, -high DCs induced similar levels of IFN- -producing T cells and a slightly greater percentage of cells producing IL-10. Although the differences were generally minor, -low DCs were capable of inducing a slightly greater percentage of IFN- -producing T cells. In parallel cocultures, we wanted to assess whether blocking of interactions between HLA-G and ILT-2/4 affects T cell polarization in any way. We used anti-ilt-2/4-neutralizing mab. Of minor interest, although the polarization induced by control idcs was not affected by this blockade, -high DCs did induce somewhat greater levels of IFN- -producing T cell popualtions that also produced less IL-10 (Fig. 5B). Volume 95, January 2014 Journal of Leukocyte Biology 39

8 Figure 4. -High DCs exert an immunosuppressive functionallity and prime DCs for lower production of IL-12p70 upon CD40L stimulation. (A) DCs were fully differentiated from human monocytes using GM-CSF (800 U/ml) and IL-4 (1000 U/ml). CD1a high, DC-SIGN high, CD14 cells were left untreated (GM-CSF only) or treated with various concentrations of IFN- ( U/ml), LPS (20 ng/ml), or LPS in combination with IFN- (500 U/ml) for 48 h and assessed for their functionallity in terms of allostimulatory capacity. The allostimulatory capacity of IFN- -treated DCs was assessed during a 4-day MLR between different numbers of variously treated DCs and whole CD4 allogeneic T cells. On the 4th day, [ 3 H]-thymidine was added to the cultures for 18 h, and proliferation was measured by liquid-scintillation counting. The results are expressed as mean sd of four independent experiments. Statistical significance between pairs, where stimulators was used, was performed using Student s unpaired t-test to compare values of IFN- -treated stimulators with idcs. (B) In a parallel experiment, we added 20 U/ml rhil-2 to MLR cultures on the 2nd day. On the 4th day, [ 3 H]-thymidine was added to the cultures for 18 h, and proliferation was measured by liquid-scintillation counting. Results are expressed as mean sd. Statistical significance between pairs of cultures, treated or not with IL-2, was calculated using Student s unpaired t-test. (C) We assessed the ability of supernatants derived from 48-h cultures of DCs stimulated with various concentrations of IFN- ( U/ml) to cause migration of CD4 T cells in a transwell system. T cells ( ) were added to upper compartments in 100 l medium. The lower chambers were filled with 600 l supernatants from various DC cultures. The number of migrated T cells was determined using a MTT assay, and the results are expressed as the percentage of migrated cells. Results show mean sd values of three independent experiments. Significance between supernatants from idcs and IFN- -treated DCs was determined by Student s unpaired t-test. (D) To determine cytokine production of variously treated DCs, culture supernatants were analyzed for the presence of IL-10 and IL-12p70 after various 48-h stimulations, as described above. The results represent mean sd of three independent experiments. (E) Cytokine secretion upon a second stimulation by CD40L-transfected J558 cells was evaluated in cultures untreated or pretreated with 5000 U/ml IFN- for 48 h. At the end of and additional CD40L 24-h stimulation, supernatants were analyzed for levels of cytokines and the results obtained by measuring A 450 nm. Shown are mean sd values of three independent experiments. Student s unpaired t-test was used to determine significance between individual pairs. *P 0.05; **P Although we did not determine extensive apoptosis of DCs upon high-dose IFN- treatment, we wanted to evaluate whether various treatment of DCs causes increased apoptosis of DCs or T cells during DC-T cell cocultures. We performed separate experiments where DCs were stained with a cell-tracking agent CFSE. In this way, we could analyze the proportion of dead DCs or T cells after PI staining during cocultures. In addition, subsequent DC death could be monitored, in contrast to staining for apoptosis, directly after DC treatment with IFN-. We stained the cells with PI after 6 days of cocultures. We determined the percentage of dead DCs (CFSE,PI ) and dead T cells (CFSE,PI ). As seen in Fig. 5C, the increase of dead DCs or T cells in cultures with -high DCs as stimulators is not extensive and similar to PI staining in other cocultures with -low DCs, mdcs, or idcs. -High DCs affect CD8 cytotoxic T cell activation in an immunosuppressive manner via the HLA-G pathway Although the development of effector CD4 T cells is suppressed by -high DCs, this is clearly not a result of extensive expression of ILT-4 and HLA-G on -high DCs, as shown 40 Journal of Leukocyte Biology Volume 95, January

9 Švajger et al. Regulation of DC function by high-dose IFN- Figure 5. -High DCs are poor inducers of Th1 effector T cells. (A) DCs were generated from human monocytes and used as idc, LPS-activated (mdc), or -low and -high DCs (50 and 5000 U/ml, respectively) in a 10-day allogeneic culture with naive CD4 CD45RA T cells. Afterward, the T cells were collected, washed, rested overnight in complete medium. The next day, the T cells were activated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 h and for an additional 4 h with 10 g/ml Brefeldin A. The cells were then fixed, permeabilized, and stained intracellularly with anti-ifn-, anti-il-4, and anti-il-10, fluorescently conjugated antibodies. Cytokine profiles were measured by flow cytometry. (B) In parallel experiments, the same allogeneic cocultures were performed with the addition of neutralizing anti ( )-ILT-2 and anti- ILT-4 antibodies (both used at 10 g/ml), every 3 days. Mouse polyclonal was used for control. Results shown are representative of three independent experiments. (C) To determine T cell or DC apoptosis after a prolonged time, we prestained DCs with CFSE, performed cocultures with T cells, and stained the cocultures on Day 6 with PI. We determined the percentage of dead cells by flow cytometry analysis (CFSE,PI dead DCs; CFSE,PI dead T cells). Numbers in quadrants represent percentage of cells. Shown is one representative experiment out of three performed. above. We therefore set out to investigate whether the immunosuppression of -high DCs inhibitory surface phenotype could be linked more directly to CD8 cytotoxic T cells. We performed experiments where naive CD8 T cells were activated with anti-cd3/28-activating beads in the presence of idcs, mdcs, -low DCs, or -high DCs or without DCs. After 5 days, the T cell populations were analyzed for their expression of granzyme B and proliferation by analyzing the reduction in fluorescence intensity of CFSE. In Fig. 6A and B, it is seen clearly that in contrast to idcs, mdcs, and -low DCs, -high DCs significantly suppress the activation of CD8 T cells, as can be determined by reduced intracellular staining of granzyme B (approximately twofold reduction vs. idcs, fourfold vs. -low DCs, and 13-fold vs. mdcs). In addition, proliferation, as determined by CFSE staining, was reduced significantly, particularly in cocultures with -high DCs (88 3% for idcs, 94 4% for mdcs, 86 4% for -low DCs, and 56 5% for -high DCs of proliferating cells; Fig. 6C and D). As previously, we wanted to determine to what extent this suppression of cytotoxic responses is mediated by the inhibitory surface phenotype of DCs. We therefore performed parallel experiments where the interaction of HLA-G with its natural ligands was blocked with a neutralizing anti-hla-g mab. Interestingly, whereas the effects in the cases of idcs, mdcs, and -low DCs were less evident and not significant (Fig. 6A D), blockade of HLA-G on -high DCs reversed the suppressive effects significantly on CD8 T cell proliferation and granzyme B expression. To test whether the immunosuppression of -high DCs was also a result of well-known IDO up-regulation by IFN- and consequent tryptophan starvation, we also set parallel experiments with inhibitor of IDO enzymatic activity 1-MT. Surprisingly, inhibition of IDO did not have a significant effect on modulation of cytotoxic responses by -high DCs (Fig. 6B and D). We also tested if the immunosuppressive effect of -high DCs was a result of their ability to induce apoptosis of activated CD8 CTLs. For this purpose, we prepared CTLs by culturing naive CD8 cells with anti-cd3/28-activation beads for 5 days. Afterward, CTLs were cocultured with idc, mdcs, -low DCs, and -high DCs for an additional 48 h. Apoptosis of gated CD8 T cells was determined by Annexin V-FITC and PI staining. Neutralization of effector molecules was preformed by using anti-hla-g mab or anti-fasl-neutralization mab. Coculture of CTLs with variously treated DCs did not Volume 95, January 2014 Journal of Leukocyte Biology 41

10 Figure 6. -High DCs silence the activation of CTL responses via HLA-G. (A) The ability of -high DCs to silence the activation of cytotoxic responses was evaluated in a coculture with naive CD8 CD45RA CD45RO T cells, stimulated with CD3/CD28 Dynabeads (2.5 l/ml) for 5 6 days. DCs treated with low (50 U/ml, -low DCs), high (5000 U/ml, -high DCs) IFN- concentrations, as well as idcs and mdcs (matured with LPS IFN- ) were added to CD8 T cell cultures at a 1:10 ratio, as described in Materials and Methods. Stimulation with Dynabeads only was performed as an additional control. In parallel, the same cocultures were performed with the addition of IDO inhibitor 1-MT or the anti-hla-g-neutralizing antibody (10 g/ml). T cells were prestained with CFSE, and on Day 5, the T cells were analyzed for proliferation and granzyme B expression. Results are representative of four independent experiments. (B) Statistical analysis of CTL silencing was performed by comparing differences between individual pairs of 1-MT- or anti-hla-gtreated cocultures with nontreated cocultures with the same DCs as stimulators. Student s unpaired t-test was used. (C) Proliferation of CTLs stimulated with CD3/CD28 Dynabeads and variously treated DCs is depicted in histograms showing comparison with cocultures treated with neutralizing anti-hla-g antibodies. (D) Statistical analysis of CTL proliferation is shown, comparing proliferation of CTLs in nontreated cultures with those treated with neutralizing anti-hla-g antibodies. Student s t-test was used, comparing individual pairs. (E) The ability of variously treated DCs to cause apoptosis of CTLs was assessed. First, CTLs were prepared from naive CD8 CD45RA CD45RO T cells stimulated with CD3/CD28 Dynabeads for 5 6 days. Cocultures with DCs and CTLs were then performed for an additional 48 h. Parallel cocultures were performed with the addition of neutralizing anti-hla-g and anti-fasl antibodies. Mouse polyclonal was used for control. Apoptosis of T cells was performed by staining with Annexin-V FITC conjugate and PI. Gating of CD8 cells was performed by prestaining with anti-cd8 PE/Cy5-conjugated antibody. Results were analyzed by flow cytometry. **P Journal of Leukocyte Biology Volume 95, January

11 Švajger et al. Regulation of DC function by high-dose IFN- result in significant up-regulation of dead apoptotic cells. In this manner, blockade of HLA-G or FasL did not reverse the proapoptotic effect of -high DCs extensively (Fig. 6E). ILT-4/HLA-G expression is not induced by autocrine effects of IL-6 or IL-10 To determine whether the up-regulation of ILT-4 or HLA-G is dependent on possible autocrine actions of cytokines, we stimulated the DCs in the presence of neutralizing anti-il-6 or anti-il-10 antibodies. As seen in Fig. 7A, neutralization of IL-6 or IL-10 did not affect the expression levels of ILT-4 and HLA-G on the DC surface. High doses of IFN- induce a similar inhibitory phenotype on BDCA-1 primary DCs Finally, we wanted to confirm our results with primary mydcs to see whether IFN- exerts a similar inhibitory phenotype at high doses as it does on MoDCs. For this reason, we isolated mydcs from human buffy coats with magnetic isolation and cultured these cells with low (50 U/ml) or high (5000 U/ml) IFN- to compare the effects with previous findings. As shown in Fig. 7B, similar to effects exerted on MoDCs, high doses of IFN- extensively induce the expression of ILT-4 and HLA-G. Compared with low-dose IFN-, this increased induction is approximately twofold for ILT-4 and fivefold for HLA-G. DISCUSSION Evidence presented in this paper highlights the importance of high IFN- concentrations for effective negative regulation of DC programming toward subsequent silencing of cytotoxic T cell responses. The often contradictory effects of IFN- by that, we mean immunostimulation versus immunosuppression seem to be compartmentalized into different time-points of an immune response. This is then linked to changes in the current microenvironment and the presence of various cell types and most importantly, their activation states. We find that in terms of established DCs, not additionally exposed to maturation stimuli, exposure to IFN- concentrations, characteristic of a Th1 response, results in obtainment of a highly inhibitory surface phenotype and their immunosuppressive function. When studying the pleiotropism of cytokines with complex functions, such as IFN-, it is of utmost importance to clearly define the subtype of immune cells and particularly, the state of activation that they are in when acted on by effector molecules. In this manner, Rojas and Krishnan [30] reported recently that IFN- can act in an immunoregulatory fashion on DCs, up-regulating inhibitory molecules and arresting their maturation. However, considering cell type-specific effects, the results of this study are misleading. This is simply a result of the fact that in that particular study, IFN- was added to monocyte cultures at the start of the differentiation process, which is known to result in differentiation of macrophages instead of DCs, even when IL-4 is present [31]. Indeed, when they added IFN- at medium concentrations (500 U/ml) to already established DCs, its immunoregulatory effect was not seen [30]. Therefore, full establishement of DCs from monocytes is essential to evaluate critically the impact of IFN- on DCs themselves. In this manner, the immunosuppressive effect of IFN- on DCs has not yet been well-defined. As concentrations of IFN- above 500 U/ml are rarely used to study its effects on DC function, it was first neccessary to evaluate some general characteristics of such treatment. As seen in Fig. 1A and B, -low DCs (50 U/ml IFN- ) and -high DCs (5000 U/ml IFN- ) displayed an immature morphology with complete absence of dendrites and two- to threefold lower numbers of podosome-forming cells than in idcs. Differences could be seen in the endocytotic capacity, where high Figure 7. Expression of ILT-4 and HLA-G is not dependent on autocrine effects of IL-6 and IL-10. IFN- exerts similar effects on primary mydcs. (A) To determine the effect of possible autocrine IL-6 or IL-10 effects during IFN- stimulation of DCs, we used neutralizing -IL-6 and anti-il-10 antibodies (10 g/ml) during a 48-h stimulation period with 5000 U/ml IFN-. We then measured the expression of CD80, CD86, ILT-4, and HLA-G molecules on DCs in various cultures. Results between individual cultures with neutralizing antibodies were compared with control -high DCs and significance calculated using Student s unpaired t-test. Shown are mean sd of MFI values for depicted surface markers of three independent experiments. (B) We isolated primary human BDCA-1 mydcs, as described in Materials and Methods. We then treated mydcs with a low (50 U/ml) or high (5000 U/ml) dose of IFN- for 48 h. The cells were stained with anti-ilt-4 and -HLA-G, and the expression of inhibitory molecules was analyzed by flow cytometry. Numbers in panel represent MFI values. Shown is one representative experiment out of three performed. Volume 95, January 2014 Journal of Leukocyte Biology 43