TY - JOUR
AU - Maraskovsky, Eugene
AB - Abstract Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte‐derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)‐induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up‐regulated the expression of HLA‐DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen‐uptake function, increased the levels of IL‐12p35 mRNA, and prolonged surface expression of peptide–MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co‐factors for CD40L‐mediated function. Here, they enhanced CD40L‐mediated IL‐6, IL‐10 and IL‐12p70 secretion. Furthermore, when combined with IL‐1β and/or IL‐4, IFN‐α2a type I IFN increased CD40L‐mediated IL‐12p70 production by 2‐ to 3‐fold, and biased the IL‐12 p40/p70 ratio towards the IFN‐γ inducing p70 heterodimer, this correlating with higher levels of IFN‐γ secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL‐1β at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L‐mediated DC activation. cellular activation, cellular differentiation, cytokines, dendritic cells, human Introduction Type I IFN (α and β) mediate early defense against viral infections by acting on cells of both the innate and adaptive immune system. IFN have direct antiviral activity which rapidly clears viruses from infected cells (1,2). Furthermore, virally infected cells are triggered by IFN to die by apoptosis, whilst non‐infected neighboring cells become resistant to viral infection in the presence of IFN (3). Furthermore, reports of increased autoimmune phenomena in patients treated with IFN‐α in the absence of viral infections demonstrate that this cytokine family can alter the presentation of auto‐antigens in vivo (4–6). Finally, the use of type I IFN in the setting of immunotherapy of infectious disease (7,8) and cancer (9,10) has shown some promise with respect to enhancement of cytotoxic T lymphocyte (CTL) activity in vivo. We previously demonstrated that type I IFN also induce maturation of human CD34+‐derived dendritic cells (DC) (11), and others have demonstrated this role of IFN in murine DC (12), human monocytes (13) and blood DC (14)—emphasizing a direct role for type I IFN in the maturation of DC and their precursors. A major source of type I IFN following viral infection is the IFN‐producing cell (IPC) (also plasmacytoid T cell or monocyte), an IL‐3R+ precursor DC subset found in the blood and inflamed lymphoid organs. IPC when infected with virus secrete IFN‐α and induce T cells to secrete IFN‐γ (15–18). In this way, type I IFN bridge the innate and the adaptive immune system. Monocytes can also be transformed into DC when cultured in granulocyte macrophage colony stimulating factor (GM‐CSF) and IL‐4 for 5–7 days (19). These DC are considered immature based on phenotype and function, and are distinct to their monocyte precursors (19). Although Santini and colleagues have examined the effect of type I IFN when added to monocytes at the initiation phase of monocyte‐derived DC (MoDC) cultures (13), the effect of type I IFN upon the function of bone fide, immature MoDC has not, as yet, been reported. Although MoDC represent only one facet of the heterogeneous DC family which may not necessarily represent all tissue‐derived DC, they serve as a useful model system from which to examine DC functional development. The study of cytokines and cytokine receptors has been significantly enhanced by the use of murine models, which have uncovered several regulatory features of these molecules in vivo. However, type I IFN appear to exert different biologic effects on the adaptive immune system of humans as compared to mice. Two major differences between murine and human type I and II IFN have been characterized at the level of signal transduction. First, the induction of Th1 and Th2 cell differentiation with regard to Stat4 phosphorylation and expression of the IL‐12 receptor β2 (IL‐12Rβ2) subunit is a function of type I IFN only in humans (20–24). Secondly the induction of IL‐12 secretion with regard to IFN consensus sequence binding protein is differentially influenced by human and murine IFN (25–27). These differences are reflected by the differential capacity of type I IFN to regulate IL‐12 secretion in humans but not in mice (28–32). This suggests that mouse models may not completely reveal the full spectrum of biologic effects of type I IFN that occur within the human system. CD40L‐induced IL‐12 secretion by human DC is greatly enhanced by the type II IFN, IFN‐γ (33). However, the effect of IFN‐α on CD40 ligand (CD40L)‐mediated IL‐12p70 secretion by MoDC has not yet been reported. It was recently suggested that type I IFN inhibit CD40L‐induced IL‐12p40 secretion by MoDC and reduce DC‐mediated IFN‐γ secretion by T cells (31,32). The present study investigates the effects of type I and II IFN on human MoDC activated with CD40L. IFN‐α2a strongly enhanced CD40L‐mediated IL‐6, IL‐10 and IL‐12p70 secretion, albeit less efficiently than IFN‐γ. Maximal CD40L‐mediated IL‐12p70 production was observed when either IFN‐α2a or IFN‐γ were combined with IL‐4 and/or IL‐1β. These increased levels of IL‐12p70 resulted in a concomitant increase in IFN‐γ levels produced by allogeneic T cell subpopulations and NK cells in the mixed leukocyte reaction (MLR). Our results suggest that CD40L signaling is enhanced by several T cell‐dependent and ‐independent factors, and that type I IFN can enhance IL‐12p70 levels by DC and IFN‐γ secretion by T cells. Methods Media DC were cultured in RPMI 1640 (Trace Biosciences, Melbourne, Australia) supplemented with 20mM HEPES, 60 mg/l penicillin G, 12.6 mg/l streptomycin, 2 mM l‐glutamine, 1% non‐essential amino acids and 10% heat‐inactivated FCS (CSL, Melbourne, Australia) in a 5% CO2 incubator. MLR were performed in IMDM (Gibco/Life Technologies, NY) and 5% pooled normal human serum (gift of the Victorian Tissue Typing Service, Royal Melbourne Hospital, Australia) in a 10% CO2 incubator. mAb, ELISA kits and cytokines Flow cytometric analysis of cells was performed using the following mAb: FITC–conjugated IgG1 isotype control; phycoerythrin (PE)–conjugated IgG1 isotype control; anti‐CD80, anti‐HLA‐DR (Becton Dickinson, San Jose, CA); anti‐HLA‐A,B,C; anti‐CD86, anti‐CD56, anti‐CCR7 and anti‐mouse IgM–biotin (BD‐PharMingen, San Diego, CA); FITC–conjugated sheep anti‐mouse mAb (Silenus, Miami, FL); anti‐CD83 (Immunotech, Beckman Coulter, Gladesville, Australia). Cytokine (Opteia) ELISA kits for IL‐1β, IL‐6, IL‐10 and IL‐12p70 were purchased from PharMingen/Becton Dickinson (San Jose, CA). Capture and horseradish peroxidase (HRP)‐conjugated detection antibodies for IFN‐γ ELISAs were a kind gift from CSL. The following cytokines were added to DC cultures: rhTNF‐α (10 ng/ml) (R & D Systems, Minneapolis, MN), rhGM‐CSF (40 ng/ml) (Schering‐Plough, Sydney, Australia), rhIL‐4 (500 U/ml) (Schering‐Plough, Kenilworth, NJ) and rhIL‐1β (1–2 ng/ml) (R&D Systems, Minneapolis, MN), IFN‐α2a was titrated between 10 and 1000 U/ml, and used in all experiments at a final concentration of 1000 U/ml (Roferon‐A; Roche, Sydney, Australia), IFN‐γ (PeproTech, Rocky, NJ), and CD40L trimer (1 µg/ml final concentration) was a gift from Immunex (Seattle, WA). MoDC Peripheral blood mononuclear cells (PBMC) were obtained from buffy coat preparations of normal donors from the Red Cross Blood Bank (Melbourne, Australia) and used to produce MoDC. CD14+ monocytes (5 × 105) were affinity purified using the MACS CD14 isolation kit (Miltenyi Biotech, Sunnyvale, CA) and cultured in 1 ml RPMI, 10% FCS, GM‐CSF (40 ng/ml) and IL‐4 (500 U/ml) in 24‐well plates. By day 7, MoDC represented >90% of cultured cells. On day 7, all wells were pooled and re‐adjusted to a concentration of 1 × 105 DC/ml. Maturation inducing factors were added on day 7, and cells and supernatants were harvested on day 10 for functional assessment. All cytokines examined for their ability to stimulate DC functional maturation in the present study (e.g. IFN‐α2a, IFN‐γ, CD40L and IL‐1β) were thoroughly tested out in dose titration analyses and the concentrations used in the figures represent those found to be optimal. Measurement of antigen uptake MoDC were harvested after culture in maturation inducing conditions. Following incubation with 1 mg/ml FITC–dextran (44 and 260 kDa) (Sigma, St Louis MO) for 30–60 min at 0 or 37°C. Cells were washed 3 times in PBS/5% FCS and then incubated with PE–anti‐CD11c. FITC–dextran uptake was quantified as mean fluorescence intensity on gated CD11c+ cells. Non‐specific FITC signal was assessed by incubating MoDC with FITC–dextran at 0°C. Phagocytosis was assessed by incubating cells with 1 mg/ml PE–latex beads (Sigma, St Louis, MO) for 90 min at 37°C. In some conditions, cells were pre‐treated with 10 µM cytochalasin D (Sigma) for 30 min at 37°C to depolymerize actin. To verify the flow cytometry‐based FITC signal represented internalized dextran or beads, cells were analyzed by epifluorescence and phase‐contrast microscopy. Measurement of cell migration Lower chambers of transwell plates (8.0 µm pore size) (Costar, Corning, NY) were filled with 500 µl IMDM/5% human serum (HS) with or without chemokines: CCL21 (MIP‐3β) (3–300 ng/ml) or CCL19 (6Ckine) (5–250 ng/ml). MoDC (1–2 × 104) were added in 50 µl IMDM/5% HS into the upper chamber. After 2 h, cells in the lower chambers were harvested, concentrated to 50 µl volumes in Eppendorf tubes and counted microscopically with a hemocytometer. Each stimulation condition was performed in replicate wells. RNA isolation and cDNA synthesis Total RNA was isolated from dendritic cells using a RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In brief, cells were lysed and homogenized in lysis buffer containing guanidine isothiocyanate and β‐mercaptoethanol. Seventy percent ethanol was added to the samples, and the RNA immobilized on spin columns and eluted in RNase‐free water. Total RNA (0.16µg) was used to synthesize cDNA using 1 µg random hexamers (Promega, Madison, WI), 1 mM dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 2 U RNase inhibitor (Promega), 5 mM MgCl2 (Applied Biosystems, Foster City, CA), 1 × PCR Buffer (Applied Biosystems) and 2 U MMLV reverse transcriptase (Life Technologies, Rockville, MD) in a 20 µl reaction, for 60 min at 42°C. The enzyme was inactivated at 95°C for 5 min. Then 1 µl of the resulting 20 µl cDNA was used for real‐time PCR quantitation. Quantitative real‐time PCR Gene expression levels were quantitated using the ABI Prism 7700 sequence detection system (Applied Biosystem, Foster City, CA). Pre‐developed assay reagents (PDAR) for IL‐12p35 and IL‐12p40 were obtained from Applied Biosystems, and used in multiplex reactions with 18S rRNA PDAR (Applied Biosystems) for normalization. PCR reactions were set up in 96‐well plates (25 µl/reaction) according to the manufacturer’s instructions and analyzed using the SDS program version 1.7. Relative expression was calculated using the ΔCt method and is expressed relative to a calibrator, in this case the GM‐CSF/IL‐4 DC control. ΔCt = Ctgene – Ct18S ΔΔCtsample = ΔCtsample – ΔCtGI Relative expression = 2–ΔΔCtsample T cell purification and MLR Allogeneic CD2+ T lymphocytes were obtained by rosetting PBMC with AET‐treated sheep red blood cells. T cells were further fractionated using anti‐CD4 and anti‐CD8 MACS beads (Miltenyi Biotech), and were 88–95% pure. These purified CD4+ or CD8+ T cells contained <5% CD56+ cells. Negatively selected CD4– and CD8– cells were separated into CD45RA+ and CD45RA– cells using MACS beads (Miltenyi). NK cells were purified from unfractionated peripheral blood lymphocytes (PBL) using anti‐CD56 in combination with rat anti‐murine IgG MACS beads (Miltenyi) and were 85–93% pure. Immature MoDC were cultured in round‐bottomed 96‐well plates in triplicate at various cell numbers with 1 × 105 allogeneic PBMC or purified T cell subsets or NK cells for 5 days in RPMI with 10% HS. After 5 days, 200 µl of supernatants was harvested and fresh medium containing 1 µCi/well [3H]thymidine (DuPont, Sydney, MA) was added for 8 h. Cells were transferred onto a glass fiber filter (Wallac, Turku, Finland) and [3H]thymidine incorporation was measured using an NXT TopCount Betaplate scintillation counter (Packard, Meriden, CT). Cytokine ELISAs Cytokine secretion by stimulated MoDC or by allogeneic T cells was measured by cytokine ELISAs. IL‐1β, IL‐6, IL‐10 and IL‐12p70 ELISAs were performed on supernatants of MoDC and MLR according to the manufacturer’s instructions using Maxisorp plates (Nunc, Roskilde, Denmark). The HRP substrate was TMB peroxidase (KPL, Gaithersburg, MD); the color reaction was terminated by adding 100 µl ortho‐phosphoric acid (1 M). IL‐12p40 was measured using anti‐IL‐12p40 and biotinylated anti‐IL‐12p40 (C8.3; PharMingen, San Diego, CA) as capture and detection antibodies on PVC microtiter plates (Dynatech, Chantilly, VA). rhIL‐12p40 (PharMingen) was used as the standard. The streptavidin–HRP conjugate was purchased from CSL. Wells were developed using a substrate solution of 548 mg/ml ABTS (Sigma Aldrich, Castle Hill, Australia), with 0.001% hydrogen peroxide (Ajax Chemicals, Auburn, Australia) in 0.1 M citric acid, pH4.2. Plates were read in a Thermomax microplate reader (BioMediq, Melbourne, Australia). IFN‐γ ELIspot assays ELIspot assays were performed in order to quantify antigen‐presenting cell–target cell interactions. Briefly, Millipore Multiscreen Plates (Millipore, Molsheim, France) were coated with anti‐IFN‐γ antibody (5 µg/ml, in 0.1M NaHCO3 buffer, pH8.3, 2 h at room temperature) and blocked for 1 h at room temperature with PBS/10% FCS. Peptide‐pulsed target cells (5000) were co‐incubated overnight with 15,000 CTL. After lysing cells with H2O for 30 min, the secondary HRP‐labeled, anti‐IFN‐γ antibody was added for 2 h (10 µg/ml, PBS/3% FCS/0.05% Tween 20). Spots were developed with AEC in acetate buffer for 8 min, washed under tap water and air‐dried. To assess the numbers of IFN‐γ‐spots objectively, a video camera (TK‐1280E; Zeiss, Jena, Germany) and VideoPro software (Olympus, Sydney, Australia) were used. Image analysis included: (i) subtraction of background, (ii) increase of contrast, (iii) definition of positive spots according to grey values, (iv) binary functions (clear and centers), (v) definition of area with the Draw and Fill functions, and (vi) analysis of the field. Background images were taken from wells containing CTL alone. Target cells pulsed with an irrelevant peptide (GILGFVFTL, influenza matrix) were used as negative controls for peptide‐specific cytokine release. Statistical analysis Data from replicate wells (n > 3) or experiments using separate donors (n > 3) are represented as the means ± SEM. Paired Student’s t‐tests were preformed for determination of significance analysis using the Microsoft Excel software package (Microsoft Office 98). Results Effect of type I and II IFN on DC maturation Purified CD14+ monocytes can be induced to differentiate into immature MoDC when cultured in GM‐CSF and IL‐4 for 7 days (19). These immature MoDC express low to negligible levels of the maturation markers CD25, CD80, CD83 and CD86, and low to intermediate levels of surface HLA‐I and ‐II. Immature MoDC are phenotypically and functionally distinct from their monocyte precursors (19). Phenotypic maturation of DC can be induced by pathogen‐derived signals, inflammatory cytokines or CD40L (12,31,34–38). CD40L up‐regulated the surface expression of CD25, CD80, CD83, and HLA‐I and ‐II on MoDC above that seen on immature MoDC (Fig. 1A–C). In this regard, CD40L up‐regulated the level of all markers homogeneously as shown for CD86 in Fig. 1(A). In contrast, IFN‐α2a and IFN‐γ alone were less efficient than CD40L at up‐regulation of surface markers resulting in heterogeneous levels of CD86 on the surface of the MoDC (Fig. 1A). This incomplete induction of surface marker expression by IFN‐α2a and IFN‐γ alone was also seen for CD25 and CD80, but not for HLA‐I and ‐II (data not shown). Finally, IFN‐α2a and IFN‐γ did not substantially induce CD83 expression (Fig. 1B and C). Up‐regulation of the above surface markers was induced as early as 18 h, reaching maximal levels by 48 h (data not shown). Although the addition of either IFN‐α2a or IFN‐γ to CD40L could further up‐regulate the level of expression of CD25, CD80, and HLA‐I and ‐II (but not CD83 and CD86) above that seen with CD40L alone, there was great donor to donor variability (Fig. 1B and C). However, within individual donor experiments, type I and II IFN consistently enhanced (albeit <2‐fold) CD40L‐mediated up‐regulation of maturation markers above that seen with CD40L alone. Effect of type I IFN on DC antigen‐uptake capacity DC capture a variety of antigens using several different mechanisms [reviewed in (40)]. Particulate antigens, such as microbes and apoptotic cells, can be internalized and degraded via the actin‐dependent process of phagocytosis. Soluble antigens, such as proteins, can be internalized and degraded by actin‐dependent macropinocytosis or clathrin‐dependent endocytosis (including non‐selective fluid‐phase endocytosis and receptor‐mediated endocytosis). We examined endocytosis and phagocytosis of MoDC matured with IFN‐α2a and compared this to CD40L or combinations of CD40L and IFN‐α2a. Analysis of MoDC by FACS revealed that, as expected, immature MoDC were maximally capable of internalizing soluble dextran (260 kDa) and phagocytosing 1‐µm latex particles (Fig. 2A and B). Maturation with IFN‐α2a reduced the MoDC capacity to ingest FITC–dextran (260 kDa) by 50% and PE–latex (1 µm) by 40% respectively. However, only CD40L or CD40L + IFN‐α2a maximally reduced the ability of MoDC to ingest these particulates, suggesting that maturation with IFN‐α2a alone partially induced MoDC functional maturation. This intermediate effect of IFN‐α2a is consistent with its ability to weakly up‐regulate the expression of some surface markers (e.g. CD80, CD86, and HLA‐I and ‐II) but not others (e.g. CD83). Effect of type I IFN on DC migratory capacity Maturation of MoDC is a coordinated process that not only results in the reduction of antigen‐uptake capacity, but also the up‐regulated expression of chemokine receptors and acquisition of migratory capacity towards chemokines. Maturing DC up‐regulate CCR7 which allows them to migrate to CCL19 (6Ckine) and CCL21 (MIP‐3β), chemokines which direct DC to draining lymph nodes (41). We have recently identified that the acquisition of migratory function by immature MoDC can be facilitated by exposure to the pro‐inflammatory factor combination of tumor necrosis factor (TNF)‐α, IFN‐α2a and prostaglandin (PG) E2, and is exquisitely dependent upon the presence of PGE2 (Luft et al., manuscript in preparation). Examining the role of IFN‐α2a in inducing MoDC migratory capacity, Fig. 2(C) shows that MoDC matured with IFN‐α2a only weakly up‐regulated the expression of CCR7, whereas CCR7 expression was clearly induced when IFN‐α2a was used in combination with TNF‐α and PGE2 (Fig. 2C). As with IFN‐α2a, IFN‐γ alone was a poor inducer of CCR7 expression (data not shown). The low CCR7 expression induced by IFN‐α2a was insufficient to allow DC to migrate towards the CCR7 ligand, CCL21 (MIP‐3β), whereas the high levels of CCR7 induced by the combination of IFN‐α2a, TNF‐α and PGE2 resulted in efficient migratory capacity (Fig. 2D). Finally, although the combination of TNF‐α and PGE2 induced migratory capacity, this was suboptimal as compared to the combination of IFN‐α2a, TNF‐α and PGE2, suggesting that IFN‐α2a augmented the induction of migratory function by TNF‐α and PGE2. These data indicate that although type I IFN alone can induce incomplete phenotypic maturation of immature MoDC, this is not sufficient to induce complete MoDC functional maturation (i.e. decreased antigen uptake and increased migration towards chemokines). Effect of type I and II IFN on CD40L‐mediated secretion of bioactive IL‐12p70 by unwashed MoDC CD40L not only induces the phenotypic maturation of DC, but can also induce DC cytokine secretion. We, and others, have shown that this effect is dependent upon soluble factors present in the DC‐conditioned medium (MoDC‐CM) such as IL‐4 (35–37) and IL‐1β (38,39). Furthermore, murine IL‐12p40 molecules can form homodimers, which antagonize the effects of bioactive IL‐12p70 (42). Although this potential regulatory role of IL‐12p40 homodimers requires further study in the human system, the ratio of p70/p40 may more accurately reflect the IFN‐γ‐inducing potential of these IL‐12 forms. We therefore examined whether IFN‐α2a and IFN‐γ could augment CD40L‐mediated IL‐12p70 secretion and whether they affected the IL‐12 p70/p40 ratio. DC culture supernatants were harvested following 3 days stimulation with various combinations of stimuli, and IL‐12p70 and p40 ELISAs were performed. Neither IFN‐α2a nor IFN‐γ alone induced IL‐12p70 production by MoDC (data not shown). Figure 3(A and B) shows IL‐12p70 and IL‐12p40 measured in the supernatants of seven to 10 experiments. Firstly, CD40L induced 10‐fold higher levels of IL‐12p40 than IL‐12p70. Secondly, IFN‐γ strongly enhanced CD40L‐mediated IL‐12 secretion resulting in 6‐fold higher levels of IL‐12p70 and ∼2‐fold higher levels of IL‐12p40 than CD40L alone. Finally, IFN‐α2a consistently enhanced IL‐12p70 secretion (∼2‐fold) but not IL‐12p40 secretion. Thus, both IFN‐α2a and IFN‐γ consistently increased the IL‐12p70/p40 ratio towards the bioactive IL‐12p70 form. Figure 3(C) shows the means of IL‐12p70/p40 ratios generated from the seven separate donors examined (not the p70/p40 ratio of the means shown in Fig. 3A and B). These results demonstrate that both type I and II IFN not only enhance CD40L‐mediated phenotypic maturation and IL‐12 secretion by immature MoDC, but also bias the production of IL‐12 towards the bioactive IL‐12p70. Effect of type I and II IFN on CD40L‐mediated IL‐12p35 and p40 gene expression Production of IL‐12p70 is dependent on the expression of two genes which form two separate subunits (p35 and p40). The levels of IL‐12p40 appear to be constitutively present in many types of cells, whereas IL‐12p35 requires induction and is the rate‐limiting subunit for the formation of bioactive IL‐12p70. IFN‐γ has been shown to enhance the induction of lipopolysaccharide (LPS)‐mediated IL‐12p35 and p40 expression in monocytes (33). We examined mRNA levels of IL‐12p35 by quantitative real‐time PCR (Taqman) to determine if type I and II IFN on their own were able to up‐regulate the level of expression of p35 mRNA. Relative expression of IL‐12p35 was calculated using the ΔCt method and is expressed relative to a calibrator, in this case the GM‐CSF + IL‐4 immature MoDC condition (see Methods). Figure 3(D) shows that in three different MoDC donors IFN‐α2a alone increased p35 mRNA levels by between 3‐ and 7‐fold, whilst IFN‐γ increased them by between 3‐ and 15‐fold (Fig. 3D). IL‐12p40 mRNA was not significantly increased above that seen in immature MoDC (data not shown). However, these increases in IL‐12p35 by type I and II IFN were not sufficient to induce detectable levels of bioactive IL‐12p70 protein in the culture supernatant (data not shown). Conditions which induced detectable IL‐12p70 protein required the presence of CD40L. Figure 3(E) shows that although CD40L could increase IL‐12p35 mRNA levels (range 60‐ to 850‐fold), the addition of IFN‐α2a increased it 370‐ to 1800‐fold, whilst IFN‐γ increased IL‐12p35 mRNA 2000‐ to 10,000‐fold. Although IFN‐α2a enhanced CD40L‐mediated increase in IL‐12p35 mRNA, it had no detectable effect on enhancing CD40L‐mediated IL‐12p40 mRNA levels (Fig. 3F). This is consistent with the ELISA data of Fig. 3(A–C) showing that IFN‐α2a achieved its increase in IL‐12p70/p40 ratio (Fig. 3C) towards the bioactive IL‐12p70 form by specifically increasing IL‐12p70 (Fig. 3A) whilst having no effect on IL‐12p40 (Fig. 3B). In contrast, IFN‐γ enhanced both IL‐12p35 and p40 mRNA levels above that seen with CD40L alone (Fig. 3E and F). Thus, treatment of DC with either IFN‐α2a or IFN‐γ alone increases the level of IL‐12p35, but does not induce bioactive IL‐12p70, whilst maximal secretion of bioactive IL‐12p70 is only induced when immature MoDC are activated with the combination of CD40L with either IFN‐α2a or IFN‐γ. Synergy of GM‐CSF, IL‐4 and IL‐1β with type I and II IFN to enhance CD40L induced IL‐12p70 secretion Soluble factors present in MoDC‐CM, such as IL‐4, GM‐CSF and IL‐1β, can potentiate CD40L‐mediated IL‐12p70 secretion (35–39). We, therefore, investigated whether the enhancing effect of IFN on CD40L‐mediated IL‐12 secretion, shown in Fig. 2, was dependent upon the presence of these cytokines in the CM of immature MoDC cultures. Figure 4 shows the effect of IFN‐α2a (Fig. 4A) and IFN‐γ (Fig. 4B) on IL‐12p70 secretion when added to MoDC that were washed and re‐cultured in fresh culture medium. The enhancing effects of either IFN‐α2a or IFN‐γ upon CD40L‐mediated IL‐12p70 secretion were suboptimal in the absence of GM‐CSF and IL‐4 or IL‐1β (Fig. 4A and B). Here, IFN‐α2a only enhanced CD40L‐mediated IL‐12p70 secretion 2‐fold (Fig. 4A), whilst IFN‐γ enhanced IL‐12p70 by 40‐fold (Fig. 4B). However, IFN‐α2a was equivalent to IFN‐γ in inducing maximal CD40L‐mediated IL‐12p70 secretion when used in the presence of GM‐CSF and IL‐4 (Fig. 4A and B). Interestingly, IFN‐α2a was less effective than IFN‐γ at enhancing CD40L‐mediated IL‐12p70 secretion in the presence of IL‐1β (Fig. 4A and B). These results confirm previous findings that CD40L is a poor inducer of IL‐12p70 secretion in the absence of additional co‐factors that are present in the MoDC‐CM (35,38,39). They also demonstrate that both type I and II IFN can synergize with other cytokines to enhance CD40L‐mediated IL‐12p70 secretion. Modulation of IL‐1β, IL‐6 and IL‐10 secretion by IFN MoDC secrete a variety of cytokines in response to CD40L activation. In order to further elucidate how type I and II IFN may modulate CD40L activity, we measured the levels of IL‐1β, IL‐6 and IL‐10 secreted by MoDC following stimulation. Figure 5(A) demonstrates that neither IFN‐α2a nor IFN‐γ enhanced CD40L‐induced IL‐1β secretion, indicating that IFN does not regulate CD40L‐mediated IL‐1β production. Examination of CD40L‐mediated IL‐6 and IL‐10 production indicated substantial variation between different MoDC donors (for IL‐6: range 0.1–82.5 ng/ml and for IL‐10: range 164–1924 pg/ml). Therefore, the data shown in Fig. 5(B, C and D) are normalized to represent the levels of IL‐6 or IL‐10 produced relative to those produced in response to CD40L alone (standardized to = 1). Although both IFN‐α2a and IFN‐γ increased CD40L‐mediated IL‐6 secretion 3‐fold (Fig. 5B), they differed in their regulation of IL‐10 secretion (Fig. 5C and D). Consistent with previous findings, the addition of either IL‐1β or GM‐CSF and IL‐4 significantly enhanced CD40L‐mediated IL‐10 secretion over that seen with CD40L alone (Fig. 5C and D) (P < 0.05), indicating that CD40L‐mediated activity requires the presence of cofactors (35–39). Figure 5(C) demonstrates that IFN‐α2a can further enhanced CD40L‐mediated IL‐10 secretion when combined with GM‐CSF and IL‐4 or IL‐1β, whereas Fig. 5(D) shows that IFN‐γ did not. These results demonstrate that the production of different cytokines induced by CD40L are differentially influenced by IFN‐α2a or IFN‐γ. Effect of differentially activated MoDC on IFN‐γ levels secreted by allo‐reactive T lymphocytes and NK cells It has previously been shown that the addition of IFN‐α2a into cultures of DC and pre‐activated T cells induced IL‐10 secretion and inhibited IL‐12 secretion by DC and IFN‐γ secretion by T cells respectively (32). It is unclear from this report, however, whether IFN‐α2a directly inhibited the T cell stimulatory potential of DC or the function of the T cells themselves. We therefore examined these possibilities by using MoDC that had been activated with different cytokine combinations for 24 h. DC (1 × 104) were washed following activation and co‐cultured with 1 × 105 allo‐reactive T cells or CD56+ NK cells. Supernatants were harvested after 5 days culture and IFN‐γ secretion measured by ELISA. Figure 5 demonstrates that both IFN‐α2a and IFN‐γ enhanced the capacity of CD40L‐activated MoDC to stimulate IFN‐γ secretion in T cells. This was observed regardless of whether IFN‐α2a or IFN‐γ were added directly to MoDC‐CM (Fig. 6A) or to MoDC that were washed and re‐cultured in fresh culture medium (Fig. 6B). IFN‐γ secretion was enhanced in unsorted PBL, naive and memory CD4+ and CD8+ T cell subsets as well as in NK cell cultures. This suggests that IFN‐α2a and IFN‐γ not only enhance CD40L‐mediated IL‐12p70 production by MoDC, but that these DC in turn induce higher levels of IFN‐γ secretion by different lymphocyte subsets. Finally, MoDC stimulated in the presence of IFN‐α2a or IFN‐γ did not induce IL‐10 production in allo‐reactive T cells (data not shown). In Fig. 6(B), MoDC were washed before addition of activation factors and used as stimulators in the MLR. Both IFN‐α and IFN‐γ enhanced the capacity of MoDC activated with CD40L or CD40L + IL‐1β to stimulate IFN‐γ secretion in allo‐reactive T cells. Interestingly, DC activated with the combination of CD40L and the T cell‐independent factors (IL‐1β + IFN‐α2a) were as potent at inducing IFN‐γ secretion in allo‐reactive T cells as DC activated with CD40L and IFN‐γ (Fig. 6B). However, maximal IFN‐γ was induced in T cells when MoDC were activated with the triple combination of CD40L, IFN‐γ and IL‐1β. These results demonstrate that conditions which induced maximal IL‐12p70 by immature MoDC also induced the highest levels of IFN‐γ by allo‐reactive T cells. This suggests that multiple pro‐inflammatory factors are required for maximal CD40L‐mediated cytokine secretion. Effect of IFN‐α2a on stabilizing MHC I–Melan‐A peptide complexes for prolonged presentation by MoDC to CTL lines We next evaluated whether IFN‐α2a (either alone or in combination with CD40L) enhanced peptide presentation by DC to CTL. Melan‐A was chosen as a model antigen system because of its well‐characterized antigenicity and the fact that multiple HLA‐A2 binding analogs have been identified (43). In particular, it was demonstrated that an A27L substitution in the anchor residue at position 2 [ELAGIGILTV (ELA)] resulted in increased affinity and immunogenicity of the peptide without altering the specificity of the T cells to recognize the native 9mer peptide (43). We have previously demonstrated that the stage of DC maturation influences the efficiency and stability of class I‐restricted peptide presentation, and that exogenously derived peptides are lost more rapidly in immature DC than in mature DC (44). Figure 7 shows that both immature and DC matured with either IFN‐α2a or CD40L or IFN‐α2a + CD40L presented the high‐affinity ELA peptide to Melan‐A‐specific CTL. However, immature MoDC rapidly lost expression of Melan‐A peptide–MHC on their surface within the first 24 h. This is likely due to the recycling and replacement of surface Melan‐A peptide–MHC complexes with irrelevant peptide–MHC complexes (44). However, IFN‐α2a‐matured MoDC maintained their ability to present recognizable Melan‐A–MHC complexes for at least 24 h after pulsing, this being completely lost by 48 h. This suggests that maturation of MoDC with IFN‐α2a alone can prolong the expression of peptide–MHC class I complexes on the cell surface for prolonged presentation to T cells. However, IFN‐α2a was less efficient than CD40L at prolonging peptide presentation on the DC surface since CD40L‐matured MoDC could present peptide to CTL for up to 48 h after pulsing. Finally, the combination of IFN‐α2a + CD40L did not further enhance or prolong peptide presentation beyond that seen with CD40L alone (Fig. 7). Discussion We previously demonstrated that IFN‐α2a can induce the phenotypic maturation of human CD34+‐derived DC (11). This observation has been extended by others demonstrating a role for type I IFN in the maturation of murine DC (12), human monocytes (13) and blood DC (14). These reports all emphasize a direct role for type I IFN in the maturation of DC and their precursors, and alludes to how cytokines, such as IFN, reveal their most potent bioactivities when acting in concert with other cytokines and pro‐inflammatory mediators at sites of infection and inflammation. The present study demonstrates that, like IFN‐γ, IFN‐α2a regulates MoDC maturation and differentiation but that the most potent activities were observed when IFN‐α2a was used in combination with other cytokines (e.g. TNF‐α and PGE2 for migratory function, and CD40L, GM‐CSF, IL‐4 and IL‐1β for cytokine secretion). This conclusion was derived from several observations. First, both type I and II IFN alone were suboptimal at inducing complete MoDC maturation (e.g. phenotype, antigen‐uptake capacity, migration, cytokine secretion and prolongation of surface MHC–peptide complexes). In this regard, the increased expression of CD25, CD80 and CD86 induced by either type I or II IFN was heterogeneous (i.e. incomplete) as compared to CD40L (Fig. 1A). This may reflect that the signal strength provided by IFN was insufficient to induce maximal production and trafficking of de novo synthesized molecules (such as CD25, CD80 and CD86). In contrast, IFN may provide sufficient signal for enhancing the trafficking of pre‐formed molecules (such as HLA‐I and ‐II) to the cell surface. Interestingly, IFN‐α2a did not up‐regulate CD83 and only partially up‐regulated CCR7, the latter being insufficient for the acquisition of migratory capacity towards chemokines. Furthermore, although both IFN‐α2a and IFN‐γ alone increased IL‐12p35 mRNA levels, this was insufficient for the secretion of bioactive IL‐12p70 protein. Second, type I and II IFN showed the most potent effects when used in combination with other stimuli. Here, IFN‐α2a and IFN‐γ augmented CD40L‐mediated phenotypic maturation of MoDC, enhancing surface expression of maturation markers (including CD83) beyond the levels induced by CD40L alone. Furthermore, when combined with TNF‐α and PGE2, IFN‐α2a enhanced migratory capacity of MoDC towards CCL21 chemokine above that seen with TNF‐α and PGE2. Finally, type I and II IFN enhanced CD40L‐induced cytokine secretion (IL‐6, IL‐10 and IL‐12p70) by MoDC, particularly when combined with GM‐CSF and IL‐4 and/or IL‐1β. In attempting to consider these data in a physiological context of DC development and functional maturation, one needs to evaluate them in light of the findings of others. Others have also reported on type I IFN synergizing with other cytokines to enhance cell function. For instance, pretreatment of human monocytes with IFN‐α2a enhances IL‐12p70 secretion induced by Staphylococcus aureus Cowan and LPS (30), and accelerates their differentiation into MoDC (13). However, there are reports suggesting inhibitory effects of type I IFN on human DC function and IL‐12 secretion (29–32). For instance, the differentiation of monocytes into MoDC in the presence of IFN‐β reduced the capacity of these cells to produce IL‐12p40 in response to CD40L (32). Similarly, when IFN‐α/β was added into cultures of MoDC and pre‐activated T cells, IL‐12p40 secretion by DC was reduced (31). Both these reports demonstrate that reduced IL‐12p40 production by DC after exposure to IFN‐α2a resulted in reduced IFN‐γ secretion by allo‐reactive T cells (31,32). A possible explanation for these contradictory reports lies in a recent study showing that terminally matured DC lose their capacity to secrete cytokines, such as IL‐12p70 [(45) and our own unpublished results]. Since several groups have now shown that type I IFN enhance phenotypic and functional maturation of in vitro generated human DC and their precursors (11,13,14,29,44) as well as murine DC (12), the sequence in which DC and their precursors encounter cytokines may influence their functional development. Hence, if DC are matured by type I IFN prior to being exposed to IL‐12 inducers, such as CD40L or bacterial products, the secretion of IL‐12p70 and, consequently, the capacity of these matured DC to stimulate IFN‐γ secretion in vitro will be reduced. The reduction in IL‐12 (p40 and p70) production by MoDC following culture with type I IFN (29,31,32) is consistent with the premise that these cytokines are also inducers of DC maturation. In contrast, as shown in this study, if immature DC encounter an activating signal, such as CD40L, prior to or simultaneously with IFN‐α2a, IL‐12p70 secretion will be enhanced. This is consistent with the findings of Ebnerr and colleagues who have recently shown that maximal cytokine secretion by immature MoDC is induced when CD40L or pathogen signals are encountered at the onset of maturation (37). It is now evident that activation of DC by soluble CD40L trimers or anti‐CD40 alone does not induce maximal cytokine secretion but requires the presence of co‐factors, such as IFN‐γ (33) or IL‐4 (35–37). In addition, we and others have recently identified the monokine IL‐1β as a potent enhancer of CD40L‐mediated DC activation and cytokine secretion (38,39). The present study extends the group of cytokines which can modulate CD40L activity to include type I IFN. Although IFN‐α2a on its own imposed only minor effects on CD40L‐mediated IL‐12p70 secretion, it significantly augmented the synergistic effects of IL‐1β and IL‐4 (Fig. 4A). Given that cytokines such as IL‐1β (38,39,46,47), IFN‐α2a (15–18,30,46–54) and IFN‐γ (55–59) are rapidly co‐expressed at sites of bacterial and viral infection and/or inflammation prior to the emergence of antigen‐specific T cells, immunological cross‐talk between various cell types maybe essential during the earliest stages of inflammatory responses. Since CD40L can also be expressed on non‐T cells [e.g. inflamed smooth muscle cells, vascular endothelial cells, activated macrophages or activated platelets (60,61)], a powerful, T cell‐independent activation stimulus for immature DC can therefore be provided at the site of inflammation in the presence of IL‐1β, IFN‐α and IFN‐γ. These findings have several implications for immunity. First, the concept of T lymphocytes and pathogens being the main inducers of DC activation during infectious responses may represent an oversimplification. Indeed, CD40L in conjunction with T cell‐derived cytokines (IFN‐γ or IL‐4) is a powerful inducer of IL‐12p70, but this is probably more relevant in the context of pre‐existing memory responses which act to rapidly clear previously encountered infectious agents. Primary responses lack a high‐frequency repertoire of antigen‐specific memory T cells. At the primary infection site, cytokines secreted by many different cell types in response to pathogens, cell death and the release of heat shock proteins will likely act in concert with CD40L expressed by non‐T cells for optimal DC activation. In this scenario, however, complete functional activation would not be restricted to DC presenting pathogen‐derived antigens. Consequently, non‐specific activation of DC expressing self‐antigens in the vicinity of an inflammation may increase the risk of immune mediated pathology or even auto‐immunity. In this regard, type I IFN therapy in humans has been associated with the development of auto‐immune phenomena and reactivation of auto‐immune disease (4–6,62), whilst IL‐1β and CD40L have also been implicated in the pathogenesis of atherosclerosis (63–67). In contrast to the inflammation and pathology associated with primary infections, the rapid reactivation of memory effectors during secondary infections significantly reduces the extent of inflammation at the effector site during pathogen clearance (L. Harrison, unpublished observations). This may also reduce the degree of CD40L expression by non‐T cells as well as the production of pro‐inflammatory cytokines. The reduced probability of non‐specific activation of DC may highlight how vaccination reduces the incidence of auto‐immunity and other immune‐mediated pathologies side effects which are associated with primary infections (68–71). The dual role of type I IFN as maturation factors and factors enhancing CD40L‐mediated DC activation may also require us to re‐evaluate strategies for their clinical use. If immature DC are exposed to IFN‐α2a in combination with other soluble factors such as TNF‐α, the outcome will be a mature DC secreting little IL‐12 when encountering T cells [(37) and own unpublished results]. However, if IFN‐α2a acts on the same immature DC in the context of an inflammatory environment that potentially provides CD40L and other factors such as IL‐1β, the DC will be activated to secrete high levels of IL‐12 and other cytokines. A more complete functional characterization of these two functional stages of DC is now required. Finally, the data in the present manuscript potentially provides important information regarding how best to condition MoDC so as to prolong presentation of immunotherapy‐based vaccines. Although IFN‐α2a alone prolonged the presentation of Melan‐A peptide–MHC class I complexes to antigen‐specific CTL for up to 24 h, this was suboptimal as compared to CD40L, the latter presenting peptide for up to 48 h after peptide pulsing. This demonstrates that the stage of DC maturation influences the efficiency and stability of peptide presentation—mature DC being capable of more prolonged peptide presentation than immature DC. Nonetheless, it is important to establish whether IFN‐α2a injections actually induce or re‐activate immune responses by inducing maturation of antigen‐presenting cells, or whether they directly modulate pre‐existing immune effectors (e.g. anti‐viral or anti‐tumor) at the effector site. The success of both (potentially mutually exclusive) responses will likely depend on the immunological context in which these occur. As the tools to manipulate this context become more clearly defined, it will be important to evaluate these parameters in the next generation of IFN‐α based clinical studies. Acknowledgements We would like to thank J. Nielissen and V. Lazarovska for assistance with manuscript preparation. This work was supported by the Sylvia and Charles Viertel Foundation. T. L. is supported by a fellowship from The Anti‐Cancer Council of Victoria, Australia. P. L. and H. H. are supported by a Deutche Krebshilfe fellowship. Abbreviations CD40L—CD40 ligand CM—conditioned medium DC—dendritic cell GM‐CSF—granulocyte macrophage colony stimulating factor HS—human serum IL‐12Rβ2—IL‐12 receptor β2 IPC—IFN‐producing cell LPS—lipopolysaccharide MLR—mixed leukocyte reaction MoDC—monocyte‐derived DC PBL—peripheral blood lymphocyte PBMC—peripheral blood mononuclear cell PDAR—pre‐developed assay reagent PE—phycoerythrin PG—prostaglandin TNF—tumor necrosis factor View largeDownload slide Fig. 1. Role of IFN‐α2a or IFN‐γ upon MoDC phenotype and function. Immature MoDC were prepared by culturing purified CD14+ monocytes for 7 days in GM‐CSF and IL‐4. On day 7, immature MoDC were re‐cultured at 1 × 105/well in their own CM and either CD40L (1 µg/ml) and/or IFN‐α2a (1000 IU/ml) and/or IFN‐γ (1000 IU/ml) were added for 3 days. (A) Surface expression of CD86 was examined by flow cytometry on day 10. Results are shown as one representative experiment out of six separate MoDC donors. Dotted histograms represent isotype‐matched control antibody. Changes in the expression of CD25, CD80, CD83, and HLA‐I and ‐II induced by (B) IFN‐α2a and (C) IFN‐γ. Data are shown as increase in mean fluorescence levels relative to unstimulated, immature MoDC (GM‐CSF + IL‐4, set as 1). Figures represent the means ± SEM of three separate experiments for both IFN‐α2a and IFN‐γ. View largeDownload slide Fig. 1. Role of IFN‐α2a or IFN‐γ upon MoDC phenotype and function. Immature MoDC were prepared by culturing purified CD14+ monocytes for 7 days in GM‐CSF and IL‐4. On day 7, immature MoDC were re‐cultured at 1 × 105/well in their own CM and either CD40L (1 µg/ml) and/or IFN‐α2a (1000 IU/ml) and/or IFN‐γ (1000 IU/ml) were added for 3 days. (A) Surface expression of CD86 was examined by flow cytometry on day 10. Results are shown as one representative experiment out of six separate MoDC donors. Dotted histograms represent isotype‐matched control antibody. Changes in the expression of CD25, CD80, CD83, and HLA‐I and ‐II induced by (B) IFN‐α2a and (C) IFN‐γ. Data are shown as increase in mean fluorescence levels relative to unstimulated, immature MoDC (GM‐CSF + IL‐4, set as 1). Figures represent the means ± SEM of three separate experiments for both IFN‐α2a and IFN‐γ. View largeDownload slide Fig. 2. Effect of IFN‐α2a upon MoDC antigen‐uptake and migratory capacity. Examination of MoDC antigen‐uptake capacity. Immature MoDC and MoDC matured with the indicated cytokine stimuli were incubated with (A) FITC–dextran or (B) PE–latex beads (1 µm) at either 4 or 37°C for 30 min. Cells were examined by flow cytometry to assess internalized FITC or PE as a measure of uptake capacity. The data are presented as the mean fluorescence intensity (MFI) of internalized FITC or PE and represent means ± SEM of four experiments. (C) Analysis of CCR7 expression on immature and mature MoDC. MoDC were stimulated with either IFN‐α2a alone or with IFN‐α2a, TNF‐α and PGE2 for 48 h, and compared to non‐stimulated immature MoDC for CCR7 expression by FACS. Data are representative of three separate experiments. (D) Analysis of migration towards CCL21 (MIP‐3β). Immature MoDC or MoDC were either non‐stimulated or stimulated with either TNF‐α alone or IFN‐α2a alone or combinations of IFN‐α2a, TNF‐α and PGE2 for 48 h, washed, and examined for migratory capacity towards CCL21 (MIP‐3β) chemokine using 8.0‐µm transwell chambers. Data are shown as the means + SEM of triplicate wells and are representative of five separate experiments. An asterisk indicates conditions where statistical significance of P < 0.05 was determined by the paired Student’s t‐test as compared with IFN‐α2a, TNF‐α and PGE2. View largeDownload slide Fig. 2. Effect of IFN‐α2a upon MoDC antigen‐uptake and migratory capacity. Examination of MoDC antigen‐uptake capacity. Immature MoDC and MoDC matured with the indicated cytokine stimuli were incubated with (A) FITC–dextran or (B) PE–latex beads (1 µm) at either 4 or 37°C for 30 min. Cells were examined by flow cytometry to assess internalized FITC or PE as a measure of uptake capacity. The data are presented as the mean fluorescence intensity (MFI) of internalized FITC or PE and represent means ± SEM of four experiments. (C) Analysis of CCR7 expression on immature and mature MoDC. MoDC were stimulated with either IFN‐α2a alone or with IFN‐α2a, TNF‐α and PGE2 for 48 h, and compared to non‐stimulated immature MoDC for CCR7 expression by FACS. Data are representative of three separate experiments. (D) Analysis of migration towards CCL21 (MIP‐3β). Immature MoDC or MoDC were either non‐stimulated or stimulated with either TNF‐α alone or IFN‐α2a alone or combinations of IFN‐α2a, TNF‐α and PGE2 for 48 h, washed, and examined for migratory capacity towards CCL21 (MIP‐3β) chemokine using 8.0‐µm transwell chambers. Data are shown as the means + SEM of triplicate wells and are representative of five separate experiments. An asterisk indicates conditions where statistical significance of P < 0.05 was determined by the paired Student’s t‐test as compared with IFN‐α2a, TNF‐α and PGE2. View largeDownload slide Fig. 3. Modulation of IL‐12 p40 and p70 levels by IFN‐α2a and IFN‐γ. On day 7, immature MoDC were pooled and adjusted to 1 × 105 cells/ml. Cells were activated for 3 days with the indicated cytokine stimuli and cytokine ELISAs were performed on culture supernatant. (A) Secretion of IL‐12p70. (B) Secretion of IL‐12p40. Data represent the means ± SEM of seven different donors. (C) Ratio of IL‐12p70/IL‐12p40 from the seven different donors not from their means ± SEM. *P < 0.05 as compared with CD40L alone. (D and E) Analysis of IL‐12p35 mRNA levels by quantitative real‐time PCR on immature MoDC or MoDC stimulated with the indicated cytokine stimuli for 24 h. (F) Analysis of IL‐12p40 mRNA by quantitative real‐time PCR. Data represents the relative fold increase in mRNA levels as compared to unstimulated, immature MoDC. Shown are individual data from three separate MoDC donors. View largeDownload slide Fig. 3. Modulation of IL‐12 p40 and p70 levels by IFN‐α2a and IFN‐γ. On day 7, immature MoDC were pooled and adjusted to 1 × 105 cells/ml. Cells were activated for 3 days with the indicated cytokine stimuli and cytokine ELISAs were performed on culture supernatant. (A) Secretion of IL‐12p70. (B) Secretion of IL‐12p40. Data represent the means ± SEM of seven different donors. (C) Ratio of IL‐12p70/IL‐12p40 from the seven different donors not from their means ± SEM. *P < 0.05 as compared with CD40L alone. (D and E) Analysis of IL‐12p35 mRNA levels by quantitative real‐time PCR on immature MoDC or MoDC stimulated with the indicated cytokine stimuli for 24 h. (F) Analysis of IL‐12p40 mRNA by quantitative real‐time PCR. Data represents the relative fold increase in mRNA levels as compared to unstimulated, immature MoDC. Shown are individual data from three separate MoDC donors. View largeDownload slide Fig. 4. IFN synergize with IL‐4 and IL‐1β to enhance CD40L‐mediated IL‐12p70 secretion by MoDC. MoDC were washed on day 7 and resuspended in fresh culture medium (RPMI/FCS). IFN‐α2a (A) or IFN‐γ (B) were added to half of the cultures. Cells were activated for 3 days with the indicated cytokine stimuli and cytokine ELISAs were performed on culture supernatant. (A) Modulation of IL‐12p70 secretion by IFN‐α2a. (B) Modulation of IL‐12p70 secretion by IFN‐γ. Results are shown as means ± SEM of four experiments. *P < 0.05 as compared with stimulation without IFN‐α2a or IFN‐γ. View largeDownload slide Fig. 4. IFN synergize with IL‐4 and IL‐1β to enhance CD40L‐mediated IL‐12p70 secretion by MoDC. MoDC were washed on day 7 and resuspended in fresh culture medium (RPMI/FCS). IFN‐α2a (A) or IFN‐γ (B) were added to half of the cultures. Cells were activated for 3 days with the indicated cytokine stimuli and cytokine ELISAs were performed on culture supernatant. (A) Modulation of IL‐12p70 secretion by IFN‐α2a. (B) Modulation of IL‐12p70 secretion by IFN‐γ. Results are shown as means ± SEM of four experiments. *P < 0.05 as compared with stimulation without IFN‐α2a or IFN‐γ. View largeDownload slide Fig. 5. Modulation of IL‐1β, IL‐6 and IL‐10 levels by IFN‐α2a and IFN‐γ. On day 7, MoDC were pooled and adjusted to 105 cells/ml. Cells were activated for 3 days with the indicated cytokine stimuli and cytokine ELISAs were performed on culture supernatant. (A) Secretion of IL‐1β (data are presented as the means ± SEM of three different donors). *P < 0.05 as compared to GM + IL‐4. (B) Secretion of IL‐6 relative to CD40L‐induced IL‐6 levels (set as 1) (data are presented as the means ± SEM of seven different donors). *P < 0.05 compared to CD40L alone. (C) Modulation of IL‐10 by IFN‐α2a. (D) Modulation of IL‐10 by IFN‐γ. Results for (C) and (D) are shown relative to CD40L induced IL‐10 levels (set as 1), (data are presented as the means ± SEM of four different donors). *P < 0.05 as compared with CD40L alone. View largeDownload slide Fig. 5. Modulation of IL‐1β, IL‐6 and IL‐10 levels by IFN‐α2a and IFN‐γ. On day 7, MoDC were pooled and adjusted to 105 cells/ml. Cells were activated for 3 days with the indicated cytokine stimuli and cytokine ELISAs were performed on culture supernatant. (A) Secretion of IL‐1β (data are presented as the means ± SEM of three different donors). *P < 0.05 as compared to GM + IL‐4. (B) Secretion of IL‐6 relative to CD40L‐induced IL‐6 levels (set as 1) (data are presented as the means ± SEM of seven different donors). *P < 0.05 compared to CD40L alone. (C) Modulation of IL‐10 by IFN‐α2a. (D) Modulation of IL‐10 by IFN‐γ. Results for (C) and (D) are shown relative to CD40L induced IL‐10 levels (set as 1), (data are presented as the means ± SEM of four different donors). *P < 0.05 as compared with CD40L alone. View largeDownload slide Fig. 6. Secretion of IFN‐γ by allogeneic T cells stimulated by differentially activated MoDC. MoDC were activated with the indicated cytokine stimuli for 24 h. Cells were then were washed and 104 DC were cultured with 1 × 105 allogeneic T cells. After 5 days, supernatants were harvested and ELISAs were performed for IFN‐γ. (A) IFN‐γ secretion by CD4+ and CD8+ T cell subpopulations and NK cells. CD2+ T cells were first purified using the E‐rosetting technique followed by CD4 or CD8 bead depletion. Purified T cells contained <5% CD56+ cells. T cells were further purified into naive or previously activated T cells using CD45RA beads. MLR were performed with MoDC activated for 3 days by the indicated cytokines in the presence of their own CM. The figure is representative of three separate experiments. (B) IFN‐γ secretion by allo‐reactive T cells. MLR were performed with MoDC, which were washed before activation factors were added. Results are shown relative to IFN‐γ levels induced by CD40L‐activated DC (set as 1). Data are presented as the means ± SEM of three individual experiments. View largeDownload slide Fig. 6. Secretion of IFN‐γ by allogeneic T cells stimulated by differentially activated MoDC. MoDC were activated with the indicated cytokine stimuli for 24 h. Cells were then were washed and 104 DC were cultured with 1 × 105 allogeneic T cells. After 5 days, supernatants were harvested and ELISAs were performed for IFN‐γ. (A) IFN‐γ secretion by CD4+ and CD8+ T cell subpopulations and NK cells. CD2+ T cells were first purified using the E‐rosetting technique followed by CD4 or CD8 bead depletion. Purified T cells contained <5% CD56+ cells. T cells were further purified into naive or previously activated T cells using CD45RA beads. MLR were performed with MoDC activated for 3 days by the indicated cytokines in the presence of their own CM. The figure is representative of three separate experiments. (B) IFN‐γ secretion by allo‐reactive T cells. MLR were performed with MoDC, which were washed before activation factors were added. Results are shown relative to IFN‐γ levels induced by CD40L‐activated DC (set as 1). Data are presented as the means ± SEM of three individual experiments. View largeDownload slide Fig. 7. Time course of Melan‐A peptide presentation by immature and mature MoDC by IFN‐γ ELISpot. Immature MoDC derived from HLA‐A*0201+ donors were matured for 3 days with either IFN‐α2a or CD40L or both, pulsed for 2 h with 10 µg/ml Melan‐A‐modified peptide analog (ELAGIGILTV). Pulsed MoDC were then washed and re‐cultured in their CM for either 48, 24 or 0 h and then tested for their ability to present class I MHC–Melan‐A peptide complexes to a Melan‐A‐specific CTL line by IFN‐γ ELISpot. Numbers of spots are shown as means ± SEM of three replicate wells and data are representative of three separate experiments. View largeDownload slide Fig. 7. Time course of Melan‐A peptide presentation by immature and mature MoDC by IFN‐γ ELISpot. Immature MoDC derived from HLA‐A*0201+ donors were matured for 3 days with either IFN‐α2a or CD40L or both, pulsed for 2 h with 10 µg/ml Melan‐A‐modified peptide analog (ELAGIGILTV). 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TI - IFN‐α enhances CD40 ligand‐mediated activation of immature monocyte‐derived dendritic cells
JF - International Immunology
DO - 10.1093/intimm/14.4.367
DA - 2002-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ifn-enhances-cd40-ligand-mediated-activation-of-immature-monocyte-3t2DDDuR0W
SP - 367
EP - 380
VL - 14
IS - 4
DP - DeepDyve
ER -