Published online before print October 2, 2003
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* Dipartimento di Scienze e Tecnologie Biomediche and
Scienze Precliniche, Cattedra di Immunologia, Università degli Studi di Milano, Italy
1Correspondence: Dipartimento di Scienze e Tecnologie Biomediche, Cattedra di Immunologia, Università degli Studi di Milano, L.I.T.A., via Fratelli Cervi 93, 20090 Segrate (MI), Italy. E-mail: silvia.dellabella{at}unimi.it
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(IFN–DCs). We found that IFN- induces DC differentiation more efficiently than IL-4, yielding similar numbers of DCs in a shorter time and that this differentiation persists upon removal of cytokines. Although IFN–DCs had a more mature immunophenotype than IL-4–DCs, showing higher expression of CD80, CD86, and CD83, they still preserved comparable endocytic and phagocytic capacities and responsiveness to maturation stimuli. IFN–DCs had strong antigen-presenting capacity, inducing intense proliferation of T cells to alloantigens or influenza virus. Moreover, IFN–DCs produced lower levels of IL-12p70 and higher levels of IFN-, IL-4, and IL-10 than IL-4–DCs. As a consequence of this different pattern of cytokine secretion, IFN–DCs induced T cells to produce type 1 (IFN-
) and type 2 (IL-4 and IL-10) cytokines, and as expected, IL-4–DCs induced only Th1 differentiation. As immune responses with extreme Th1 bias are considered inadequate for the induction of optimal, systemic antitumor immunity, the ability of IFN–DCs to promote more balanced cytokine responses may suggest the advisability to consider these cells in the development of future, DC-based immunotherapy trials.
Key Words: antigen presenting cells • cytokines • Th1/Th2
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and IL-1, by lipopolysaccharide (LPS) and by CD40 ligand (CD40L). Although DCs obtained after this standard two-step treatment (IL-4–DCs) are endowed with potent antigen-presenting and T cell-activating abilities, recent studies revealed that their functional repertoire may be incomplete. In fact, important functions of DCs, such as migration from the injection site and the ability to induce T helper cell type 2 (Th2) differentiation, appear to be impaired [6
, 7
]. These defects have been ascribed to the inhibitory effects of high levels of IL-4, continuously present in the culture, on the eicosanoid metabolism [8
]. DCs generated from human monocytes with IL-4 replaced by type 1 interferon (IFN–DCs) may represent a promising alternative to IL-4–DCs. In fact, it has been demonstrated that culture of human monocytes with GM-CSF and IFN- rapidly generates mature DCs endowed with potent APC functions in vitro and in vivo [9
, 10
]. As large amounts of type 1 IFNs are locally produced by specific cell types in response to viral, bacterial, and protozoan infections [11
, 12
], it is reasonable to assume that the activation of the IFN system can represent the early, important mechanism involved in the maturation/induction of DCs in response to invading pathogens and possibly to tumors [9
]; therefore, exposure of monocytes to type 1 IFNs, rather than IL-4, may represent a more physiological way to obtain DCs in vitro. Furthermore, replacement of IL-4 with type 1 IFN may overcome the functional defects of IL-4–DCs, as suggested by the finding that DCs obtained in the presence of GM-CSF and IFN- are endowed with a strong, migratory response to specific chemokines [13
].
In view of a possible role of IFN–DCs in therapeutic trials, in the present study, we characterized in detail DCs generated from human monocytes cultured with GM-CSF and IFN- in comparison with IL-4–DCs. Our results clearly confirm that IFN–DCs are generated more rapidly than IL-4–DCs and are endowed with excellent APC function. Moreover, we could demonstrate that unlike IL-4–DCs, IFN–DCs have the capacity to induce the production of type 1 and type 2 cytokines by T lymphocytes. This was related to their ability to promote T cell differentiation toward Th1 cells, which expressed type 1 cytokines only, and Th0-like cells, which concomitantly expressed type 1 and type 2 cytokines. All together, our results suggest that IFN–DCs may deserve further consideration in the development of future DC-based immunotherapy trials.
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2b (1000 U/ml; Schering-Plough, Milano, Italy). The amount of IFN- was established on the basis of preliminary, dose-response experiments, indicating that 1000 U/ml was the minimum amount required to differentiate efficient DCs. One-third of the medium (including all supplements) was exchanged every 2 days. After 3 and 5 days of culture, nonadherent and loosely adherent cells were collected and used for subsequent analysis. Where indicated, DCs were further stimulated by addition of LPS serotype 055:B5 (1 µg/ml; Sigma Chemicals Co., St. Louis, MO), polyriboinosinic-polyribocytidylic acid [poly(I:C); 12.5 µg/ml; Sigma Chemicals Co.), soluble rhCD40L (10 ng/ml+50 ng/ml cross-linking enhancer; Alexis Biochemicals, Lausen, Switzerland), or monocyte-conditioned medium (MCM; 25% v/v). After 48 h, the cells were collected and used for subsequent analysis; culture supernatants were collected and assayed for cytokine levels. For IL-4 and IFN- detection, DCs were washed extensively and cultured without any cytokine for an additional 24 h. For polymerase chain reaction (PCR) analysis, DCs incubated with the above-mentioned stimuli or with influenza virus (FLU; prepared as described previously [14
]) were collected after 12 or 24 h as indicated. MCM was prepared as described [15
], by incubating monocytes obtained by 2-h plastic adhesion with Staphyloccus aureus Cowan strain I (SACS) (1/10,000; Calbiochem, La Jolla, CA); after 24 h, the cell-free supernatants were filtered and stored at –20°C. CD45RA+CD4+ naïve T lymphocytes were purified from monocyte-depleted PBMCs by isolation of CD4+ T cells by negative selection with a Miltenyi Biotec human CD4+ T cell isolation kit, followed by isolation of CD45RA+ cells from the CD4+ T cells by positive selection using CD45RA MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
Immunophenotypic analysis
DCs, washed and suspended in phosphate-buffered saline (PBS), supplemented with 1% bovine serum albumin, were incubated with different fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridinin chlorophyll protein-, or TriColor-conjugated monoclonal antibodies (mAb) for 30 min at 4°C. The following commercial mAb were used: anti-CD3, -CD4, -CD11c, -CD14, -CD19, and -human leukocyte antigen (HLA)-DR (Caltag, Burlingame, CA); anti-CD1a, -CD40, -CD80, -CD83, -CD86, and -HLA-A,B,C (Becton Dickinson, San Jose, CA); anti-CD123 (Miltenyi Biotec); and anti-CD209 (R&D Systems, Minneapolis, MN). Cells were collected and analyzed using a FACScan (Becton Dickinson) flow cytometer. Data analysis was performed by CellQuest software (Becton Dickinson). Cells were electronically gated according to light-scatter properties to exclude cell debris and contaminating lymphocytes.
Mannose receptor-mediated endocytosis
FITC–dextran (molecular weight, 70 kDa; Molecular Probes, Eugene, OR) was used to assess cell endocytosis, as described by Sallusto et al. [16
]. Briefly, 1 x 105 cells were incubated with 1 mg/ml FITC–dextran at 37°C or 0°C for 60 min. Uptake was stopped by adding ice-cold PBS followed by extensive washes in a refrigerated centrifuge. Samples were then subjected to flow cytometry. The level of antigen uptake by DCs was assessed on the FITC channel and expressed as the difference in mean fluorescence intensity (
MFI) between the test (37°C) and control (4°C) tubes for each sample.
Phagocytosis of apoptotic cells
Monocyte-depleted PBMCs were labeled with 0.5 µM chloro-methyl-fluorescein-diacetate (CMFDA; Molecular Probes) for 30 min at 37°C and were washed extensively [17
]. Cell apoptosis was induced by 24-h treatment with 10 µM H2O2 [18
]. Cell apoptosis was assessed by morphology and staining with two DNA-binding dyes, as described by Echaniz et al. [19
] with minor modifications. Briefly, cells were stained with a mixture of propidium iodide (PI; 130 nM) plus acridine orange (AO; 50 nM) for 5 min in the dark and were analyzed by flow cytometry. PI is excluded from live cells but enters cells that have damaged plasma membrane. AO is a metachromatic dye taken up by lysosomes of live cells and recognizes apoptotic cells via their discrete decrease in fluorescence. The labeled apoptotic cells were subsequently cocultured with HLA-DR-labeled, allogeneic DCs at a 1:1 ratio, selected from preliminary experiments. After 2 h, the cells were washed and treated with 0.05% trypsin/0.02% EDTA for 5 min to disrupt cell–cell binding [20
]. Phagocytosis was quantified by flow cytometry as the percentage of double-positive cells (CMFDA+/HLA-DR+). Negative controls were performed at 0°C.
Allogeneic and autologous T cell proliferation assay
To test their direct allostimulatory activity, DCs (15x103) were cocultured in 96-well round-bottom plates with allogeneic, monocyte-depleted PBMCs in triplicate for 5 days. A stimulator/responder ratio of 1:20 was used on the basis of preliminary experiments (not shown). To test the ability of DCs to present alloantigens through the indirect pathway of alloantigen presentation, DCs (15x103) that had previously engulfed apoptotic, allogeneic cells were cocultured with autologous, monocyte-depleted PBMCs (3x105) in triplicate for 5 days. To test the ability of DCs to present soluble antigens, DCs were incubated with or without FLU at 37°C for 24 h. After extensive washes, 1.5 x 104 FLU-pulsed or unpulsed DCs were cocultured with 3 x 105 autologous, monocyte-depleted PBMCs in 96-well round-bottom microtiter plates in triplicate for 7 days. This stimulator/responder ratio was chosen on the basis of preliminary experiments (not shown).
In all cases, 5-bromo-2'-deoxyuridine (BrdU; 20 µm; Sigma Chemicals Co.) was added in each well during the last 6 h of culture, and lymphocyte proliferation was assessed by flow cytometry as BrdU incorporation by CD4+ lymphocytes, as described by Toba et al. [21 ]. Results were expressed as percentage of proliferating (BrdU+) lymphocytes. Supernatants were collected for determination of cytokine levels.
Cytokine measurements
IL-4, IL-10, IL-12p70, and IFN- release in the supernatants was measured by specific sandwich enzyme-linked immunosorbent assay (ELISA), by use of commercially available pairs of mAb (Endogen, Woburn, MA). IL-12p40 was quantified by use of specific 2/4A1 and 4D6 Ab (generous gift of Dr. Panina-Bordignon, Roche Milano Ricerche, Milan, Italy). IFN- was measured with an IFN- ELISA kit (Bender, MedSystems, Vienna, Austria), used according to the manufacturer’s instructions.
Intracellular cytokine expression was assessed in T lymphocytes. DCs, unloaded or loaded with apoptotic, allogeneic cells, FLU, or keyhole limpet hemocyanin (KLH; 100 µg/ml for 3 h, on the basis of preliminary experiments; Sigma Chemicals Co.) were cocultured with allogeneic or autologous, monocyte-depleted PBMCs. In some experiments, CD45RA+ CD4+ naïve T cells were used. For inhibition experiments, T cells were preincubated with various doses of neutralizing mAb against IL-4 (PharMingen, San Diego, CA) for 30 min, and the inhibitor was left in culture for the entire stimulation period, as described by Geginat et al. [22
]. After the indicated days of DC–T coculture, T cells were reactivated with phorbol 12-myristate 13-acetate (PMA; 25 ng/ml) plus ionomycin (1 µg/ml) for 5 h. Brefeldin A (BFA; 10 µg/ml; Sigma Chemicals Co.) was added during the last 4 h to accumulate most of the cytokine in the Golgi complex. Cells were labeled with CD4 mAb, fixed, and permeabilized using Fix and Perm (Caltag), according to the manufacturer’s instructions, and were then labeled with mAb against cytokine IFN-, IL-4, or IL-10.
Quantification of mRNA specific for IL-4 and IFN-
Total RNAs were extracted from the cells with RNAzolTM B (Duotech, Friendswood, TX), and 1 µg RNA was reverse-transcribed into cDNA in 20 µl final volume.
To compare IL-4 and IFN- mRNA expression in the different conditions of stimulation, it was essential to use equivalent amounts of substrate cDNA. So, we normalized all samples for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA content by competitive PCR (TaKaRa, Otsu, Japan) as described previously [23
].
IL-4 and IFN- mRNA expression was quantified using a quantitative, competitive PCR kit (TaKaRa). The results were expressed as IL-4/GAPDH and IFN-/GAPDH ratios.
Statistical analysis
Statistical analysis was performed with SPSS 11 software (SPSS Inc., Chicago, IL). Comparisons of samples to establish the statistical significance of difference were determined by the two-tailed Mann-Whitney rank sum test for independent samples. The paired Wilcoxon test was also used when indicated. Values of P< 0.05 were considered significant.
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leads within 3 days to loss of plastic adherence associated with cellular aggregation in large-cell clusters and appearance of typical DC morphology, as assessed by fluorescence microscopy after AO staining. At the same time, a large part of monocytes treated with GM-CSF plus IL-4 were still firmly adherent to the plastic surface; as expected, they became floating, nonadherent cells with typical DC features at 5 days. IFN–DCs at 3 days were morphologically similar to IL-4–DCs at 5 days, with lobulated nuclei and numerous fine cytoplasmic projections, consistent with a DC phenotype. The percentage of cells with DC morphology as well as the percentage of viable cells (trypan blue exclusion test) were calculated after 3 and 5 days of culture. Cell viability was >90% in all experiments. The yield of viable IFN–DCs at 3 days, expressed as percentage of the number of monocytes plated on day 0 (mean±SD: 80±6%, range 73–90), was similar to the yield of viable IL-4–DCs at 5 days (78±4%, range 71–86). Contaminating cells in the IL-4–DC cultures were: T cells (CD3), <1%; B cells (CD19), 6 ± 1%; natural killer (NK) cells (CD56), 5 ± 1%; and monocytes (CD14), 7 ± 1%. In the IFN–DCs cultures, contaminating cells were: T cells, <1%; B cells, 4 ± 2%; NK cells, 5 ± 1%; and monocytes, 6 ± 2%. The immunophenotypic analysis of IFN–DCs compared with IL-4–DCs at 3 and 5 days confirmed that IFN–DCs are well differentiated after 3 days of culture, and IL-4–DCs still express CD14 on a relevant percentage of cells (13±3.3%). Therefore, comparison between the two types of DCs was thereafter performed between 3-day IFN–DCs and 5-day IL-4–DCs. IFN–DCs presented a more mature phenotype characterized by a significantly higher expression of the costimulatory molecules CD80 (82.2±4.5% vs. 58.3±8.6, P<0.001) and CD86 (87.2±8.1% vs. 59.2±6.1, P<0.001) and of the maturation marker CD83 (14.8±2.6% vs. 5.3±1.4, P<0.001), although they had a lower expression of CD40 than IL-4–DCs (22.3±2.9% vs. 66.7±3.8, P=0.002). IFN–DCs also differed from IL-4–DCs, as they displayed markedly reduced expression of CD1a (6.7±1.6% vs. 87.7±3.7, P<0.001) and CD209 [DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (SIGN), 14.2±3.4% vs. 96.8±0.8, P=0.001]. Similar results were obtained when data were expressed as MFI instead of percentage of positive cells (data not shown). Moreover, although the totality of IFN–DCs and IL-4–DCs expressed the adhesion molecule CD11c, only IFN–DCs coexpressed CD123 (IL-3R) on a relevant percentage of cells (range, 15.3–27.4%). As shown in Figure 1A
, this was related to a different ability of IL-4 and IFN- to down-regulate CD123, which is expressed on most freshly isolated monocytes. To assess whether CD123 expression could identify functionally different subpopulations within IFN–DCs, we compared the expression of CD80, CD86, CD40, and CD83 on CD123+ and CD123–cells and found no differences (data not shown). Finally, we evaluated the persistence of the differentiation process promoted by cytokine treatment of monocytes and observed that upon cytokine removal, up to 3 days, IFN–DCs retained a DC morphology and immunophenotype, and IL-4–DCs readhered to culture plates and reacquired monocyte features (Table 1
).
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Figure 1. Immunophenotypic characterization. (A) The expression of CD123 (IL-3R) on monocytes is differently regulated by IL-4 and IFN-. Monocytes incubated in medium alone or with GM-CSF plus IL-4 (IL-4–DCs) or IFN- (IFN–DCs) were analyzed by flow cytometry for the expression of CD123 at the indicated times of culture. Density plots from one representative of three independent experiments are shown. CD123, expressed on the totality of freshly isolated monocytes, was rapidly down-regulated in the presence of GM-CSF and IL-4. On the contrary, in the presence of IFN-, CD123 was down-regulated slower, and its expression was preserved on a relevant percentage of differentiated IFN–DCs. (B) DC maturation in response to different stimuli. Five-day IL-4–DCs (open bars) and 3-day IFN–DCs (shaded bars) were incubated with LPS, poly(I:C), MCM, or CD40L, as indicated. After an additional 2 days of culture, cells were collected and subjected to flow cytometry. The expression of CD80, CD86, CD40, and CD83 is presented as mean ± SD of MFI from 10 independent experiments. IFN–DCs responded as well as IL-4–DCs to all the stimulators. The surface expression of CD40 was significantly lower on IFN–DCs than on IL-4–DCs (Mann-Whitney test for independent samples: *, P<0.03; **, P<0.01).
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Table 1. Effects of Cytokine Removal on DC Immunophenotype
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Antigen-capture capacity of DCs
The capacity of DCs to take up antigens was measured in two systems using FITC–dextran, as an indicator of mannose-receptor (MR)-mediated endocytosis, and CMFDA-labeled apoptotic cells, as an indicator of phagocytosis. In each case, IFN–DCs were as efficient as IL-4–DCs in capturing antigens. In fact, similar levels of FITC-dextran uptake were presented by IL-4–DCs (MFI: 133.2±15.7, mean±SD of seven independent experiments) and IFN–DCs (MFI: 118±15.3). As expected, MCM-induced maturation of both types of DCs was associated with a reduction of endocytosis (MFI lowered to 13.6±0.6 and 16.9±1.6, respectively). Phagocytosis of CMFDA-labeled apoptotic cells (>70% dead cells as determined by PI and AO staining), assessed by flow cytometry as the percentage of HLA-DR-labeled DCs that expressed the green dye CMFDA and confirmed by the increased granularity of the double-positive population at forward-scatter/side-scatter analysis, was similar in IL-4–DCs (HLA-DR+/CMFDA+ DCs: 70.1±8.2%, mean±SD of six independent experiments) and IFN–DCs (80.3±6.7), thus confirming the excellent antigen-capture capacity of IL-4–DCs and IFN–DCs. Again, MCM-induced maturation of both types of DCs was associated with a reduction of their phagocytic capacity (% of CMFDA+ DCs lowered to 6.2±1.3 and 4.8±0.7, respectively).
Lymphocyte proliferation induced by DCs
To evaluate the allostimulatory properties of IFN–DCs compared with IL-4–DCs, we evaluated the direct and indirect pathways of alloantigen presentation. As shown in Figure 2A
, despite their more mature phenotype, IFN–DCs presented a direct allostimulatory activity similar to IL-4–DCs, assessed in mixed leukocyte reaction as BrdU incorporation by allogeneic, monocyte-depleted PBMCs. As expected, maturation of DCs induced by LPS, poly(I:C), MCM, or CD40L significantly increased the allostimulatory activity of IL-4–DCs and IFN–DCs (Wilcoxon test, P<0.03; Fig. 2A
). IFN–DCs were also as efficient as IL-4–DCs in their ability to present alloantigens through the indirect pathway of alloantigen presentation: following phagocytosis of apoptotic, allogeneic cells, IL-4–DCs and IFN–DCs similarly induced the proliferation of autologous CD4+ lymphocytes, measured as BrdU incorporation (Fig. 2B)
.
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Figure 2. Induction of lymphocyte proliferation by DCs. The antigen-presenting function of 5-day IL-4–DCs and 3-day IFN–DCs was compared in three different experimental models. (A) To test their direct allostimulatory activity, DCs, unstimulated or stimulated with LPS, poly(I:C), MCM, or CD40L, were cultured with allogeneic, monocyte-depleted PBMCs at a stimulator/responder ratio of 1:20 for 5 days. (B) To test their ability to present alloantigens through the indirect pathway of alloantigen presentation, DCs were allowed to engulf apoptotic (apo), allogeneic cells, extensively washed, and then cultured for 5 days with autologous, monocyte-depleted PBMCs. (C) To test their ability to present soluble antigens, DCs were incubated with FLU for 24 h, extensively washed, and cocultured with autologous, monocyte-depleted PBMCs for 7 days. In all cases, BrdU was added during the last 6 h of culture, and lymphocyte proliferation was assessed by flow cytometry as percentage of CD4+ lymphocytes that had incorporated BrdU. (A and B) Results are expressed as mean ± SD of 10 and eight independent experiments, respectively; in both models, similar levels of lymphocyte proliferation were induced by IFN–DCs (shaded bars) and IL-4–DCs (open bars). (C) Proliferating lymphocytes were identified in density plots as CD4+/BrdU+ double-positive cells. Control cultures included monocyte-depleted PBMCs alone (left column), cocultured with unpulsed DCs (middle column), or antigen-specific proliferation assessed in cultures with FLU-pulsed DCs (right column). Upper row, IL-4–DCs; lower row, IFN–DCs. The numbers represent the percentage of cells in each quadrant. One representative of five independent experiments is shown. IFN–DCs were as efficient as IL-4–DCs in inducing antigen-specific lymphocyte proliferation.
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Cytokine production by DCs
DC-derived cytokines were measured by ELISA in the supernatants of DC cultures, unstimulated or upon stimulation with LPS, poly(I:C), MCM, or CD40L. Preliminary kinetic experiments indicated that the levels of all the analyzed cytokines were maximal in IL-4–DCs and IFN–DCs at 48 h in response to all the stimuli used; only the amount of IL-10 in the supernatants of IFN–DCs peaked after 24 h of culture, although it was still detectable after 48 h of culture with any stimulator (Fig. 3
). Therefore, all DC-derived cytokines were subsequently measured at 48 h. As shown in Figure 4A
, the pattern of cytokine production of IL-4–DCs and IFN–DCs differed in many aspects. IFN–DCs produced lower levels of IL-12p70 and IFN- and higher levels of IL-12p40 than IL-4–DCs, regardless of the stimulus considered. Also, IFN–DCs produced higher levels of IL-10 in most of the culture conditions; the difference in IL-10 production between IL-4–DCs and IFN–DCs could have been partially undervalued indeed because of the kinetics of this cytokine. Moreover, only IFN–DCs secreted sizeable amounts of IL-4 and IFN-. The different production of IL-4 may not be ascribed to contaminating T lymphocytes, as contamination by these cells was similarly negligible in IL-4–DCs and IFN–DCs. To further assess the production of IL-4 and IFN-, DCs were analyzed for the expression of specific mRNA after incubation with the above-mentioned stimuli or with FLU. The expression of IL-4 mRNA was measured after 12 h of stimulation and that of IFN- mRNA, after 24 h on the basis of preliminary kinetic experiments (data not shown). As shown in Figure 4B
, the production of IL-4 and IFN- by IFN–DCs was confirmed at the RNA level in response to all the stimuli considered. Minimal amounts of mRNA specific for IL-4 were also expressed by stimulated IL-4–DCs.
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Figure 3. Kinetics of the production of cytokines by DCs. Five-day IL-4–DCs and 3-day IFN–DCs were incubated for the indicated time in culture medium alone ( ) or added with LPS ( ), poly(I:C) (•), MCM ( ), or CD40L ( ), and the released cytokines were measured in the supernatants by ELISA. One representative of two independent experiments is shown.
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Figure 4. Production of cytokines by IFN–DCs as compared with IL-4–DCs. (A) DCs were incubated for 2 days with or without LPS, poly(I:C), MCM, or CD40L, and the released cytokines were measured in the supernatants by ELISA. Results are expressed as mean ± SD of six independent experiments. IFN–DCs (shaded bars) secreted lower levels of IL-12p70 and IFN- and higher levels of IL-12p40 and IL-10 than IL-4–DCs (open bars). Furthermore, only IFN–DCs secreted sizeable amounts of IL-4 and IFN-. (Mann-Whitney test, IFN–DCs compared with IL-4–DCs: *, P<0.05; **, P<0.01.) (B) Expression of mRNA specific for IL-4 and IFN- by IFN–DCs (shaded bars) as compared with IL-4–DCs (open bars). DCs were incubated with or without LPS, poly(I:C), MCM, CD40L, or FLU, and their expression of IL-4 mRNA and IFN- mRNA was quantified by competitive PCR after 12 or 24 h of stimulation, respectively. Results are expressed as IL-4/GAPDH and IFN-/GAPDH ratios. One representative of two independent experiments is shown. The production of IL-4 and IFN- by IFN–DCs was confirmed at the RNA level in response to all the stimuli considered. Minimal amounts of mRNA specific for IL-4 were also expressed by stimulated IL-4–DCs.
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) and type 2 (IL-4 and IL-10) cytokines were measured in the supernatants of monocyte-depleted PBMCs cocultured with allogeneic DCs. As shown in Figure 5A
, IL-4–DCs and IFN–DCs, unstimulated or previously stimulated with LPS, poly(I:C), MCM, or CD40L, induced a significant increase in the levels of IFN- (Wilcoxon test, compared with PBMCs alone: P<0.05); as expected, IFN- amounts were markedly higher when cocultures were performed with previously stimulated DCs. The levels of IFN- induced by stimulated IL-4–DCs and IFN–DCs were comparable. On the contrary, the amounts of IL-10 induced by unstimulated or stimulated IFN–DCs were significantly higher than those induced by IL-4–DCs. Moreover, IL-4, which was abundantly secreted in the cocultures performed with IFN–DCs, was undetectable in the cocultures containing IL-4–DCs. As shown in the same figure, similar results were obtained when monocyte-depleted PBMCs were cocultured with autologous DCs previously pulsed with influenza virus.
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Figure 5. Polarization of T lymphocytes by IFN–DCs as compared with IL-4–DCs. To examine the nature of T cell responses induced by IL-4–DCs and IFN–DCs, (A) monocyte-depleted PBMCs were cultured with allogeneic DCs [unstimulated or previously stimulated with LPS, poly(I:C), MCM, or CD40L as indicated] or autologous DCs (unpulsed or previously pulsed with FLU) for 5 and 7 days, respectively, and T cell-derived cytokines were measured. ELISA quantifed released type 1 (IFN-) and type 2 (IL-4 and IL-10) cytokines in the supernatants. Results are expressed as mean ± SD of five independent experiments. IL-4–DCs (open bars) and IFN–DCs (shaded bars) induced the release of high levels of IFN-, but induction of IL-10 and IL-4 was a preminent prerogative of IFN–DCs. Mann-Whitney test, IFN–DCs compared with IL-4–DCs: *, P < 0.05; ** P < 0.01. (B) Monocyte-depleted PBMCs were cultured alone or with MCM-conditioned allogeneic DCs or with FLU-pulsed autologous DCs. After the indicated days of DC–T coculture, T cells were reactivated with PMA and ionomycin for 5 h, and BFA was added to the culture for the last 4 h before staining to prevent cytokine secretion. Cells were then stained with TriColor-conjugated anti-CD4 mAb, fixed, permeabilized, stained with FITC- or PE-conjugated mAb for intracellular cytokines, and subjected to flow cytometry. One representative of three independent experiments is shown. Gated on CD4+ cells, density plots show the expression of IFN- and IL-10 (upper row) or IL-4 (lower row). Quadrants were set according to the fluorescence intensities of isotype-matched control immunoglobulins with irrelevant specificity. Note that IFN–DCs induce the differentiation of T cells which concomitantly express type 1 and type 2 cytokines, and IL-4–DCs promote Th1 polarization exclusively.
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/IL-4-producing and IFN-/IL-10-producing Th cells have a mixed phenotype resembling Th0 lymphocytes (Fig. 5B)
.
To investigate whether this polarization of T lymphocytes was induced by DCs, also during the priming of primary antigen-specific immune responses, DCs loaded with KLH were cocultured with autologous unfractioned monocyte-depleted PBMCs, or DCs loaded with apoptotic allogeneic cells, FLU, or KLH were cocultured with autologous, magnetic bead-purified CD45RA+ CD4+ naive T lymphocytes. As shown in Figure 6A
and 6B
, the results obtained in all these experimental models confirmed the capacity of IFN–DCs to promote the differentiation of Th1 cells and double-positive IFN-/IL-4-producing and IFN-/IL-10-producing Th lymphocytes, compared with the property of IL-4–DCs to induce the differentiation of Th1 cells only.
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Figure 6. Polarization of T lymphocytes by DCs in primary immune responses. The polarization of T lymphocytes during the priming of antigen-specific immune responses was analyzed by using KLH or performing cultures with CD45RA+CD4+ naïve T cells. (A) Unfractioned, monocyte-depleted PBMCs were cultured with autologous DCs, unpulsed or previously pulsed with KLH, for 7 days. (B) Magnetic bead-purified CD45RA+CD4+ naive T lymphocytes were cultured with autologous DCs, unpulsed or previously pulsed with apoptotic (apo) allogeneic cells, FLU, or KLH for 7 days. In all cases, at the end of DC–T coculture, T cells were reactivated with PMA and ionomycin for 5 h, and intracellular cytokines were evaluated as described in Figure 4B
. One representative of two independent experiments is shown. Gated on CD4+ cells, density plots show the expression of IFN- and IL-10 (upper row) or IL-4 (lower row). Note that also during the priming of primary immune responses, IFN–DCs induce the differentiation of T cells that concomitantly express type 1 and type 2 cytokines, and IL-4–DCs promote Th1 polarization exclusively.
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/IL-4-producing and IFN-/IL-10-producing Th cells, promoting a shift of the Th0-like cells toward IFN--secreting Th1 lymphocytes.
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Figure 7. Contribution of IL-4 in the polarization of T lymphocytes by IFN–DCs. Monocyte-depleted PBMCs were preincubated with the indicated doses of neutralizing anti-IL-4 mAb for 30 min and then were cultured with MCM-conditioned, allogeneic IFN–DCs for 5 days with anti-IL-4 mAb left in culture for the entire period. At the end of the DC–T coculture, T cells were reactivated with PMA and ionomycin for 5 h, and intracellular cytokines were evaluated as described in Figure 4B
. One representative of two independent experiments is shown. Gated on CD4+ cells, density plots show the expression of IFN- and IL-10 (upper row) or IL-4 (lower row). The numbers represent the percentage of cells in each quadrant. The addition of neutralizing anti-IL-4 mAb induced a dose-dependent reduction of the number of double-positive IFN-/IL-4-producing and IFN-/IL-10-producing Th cells, promoting a shift of the Th0-like cells toward IFN--secreting Th1 lymphocytes.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, combined with GM-CSF alone, does promote DC differentiation. IFN is more efficient than IL-4 in this process, as similar yields of DCs were generated after only 3 days in the presence of IFN compared with 5 days in the presence of IL-4. Moreover, IFN-driven DC differentiation persists upon removal of conditioning cytokines, suggesting that unlike IL-4–DCs, which require the IL-4 step of culture and a further in vitro stimulation by maturation signals to terminally differentiate, fully active IFN–DCs could be generated in a single-step, 3-day culture. Their rapid generation in one-step culture could render IFN–DCs particularly attractive for possible clinical use.
Immunophenotyping at different time points confirmed that IFN–DCs are well differentiated after 3 days of culture; therefore, we next compared the phenotypic and functional properties of 5-day IL-4–DCs with 3-day IFN–DCs. IFN–DCs differed from IL-4–DCs in many aspects. First of all, IFN–DCs appeared more mature than IL-4–DCs, as indicated by their increased surface expression of the costimulatory molecules CD80 and CD86 and the maturation marker CD83. This higher stage of maturation, that is in accordance with the known properties of type 1 IFN to promote DC maturation [13
, 24
, 30
], did not affect the ability of IFN–DCs to internalize antigens by MR-mediated endocytosis, as assessed by FITC-dextran uptake, or by phagocytosis, as assessed by capture of allogeneic, apoptotic cells. Antigen internalization by IFN–DCs was followed by efficient processing and presentation to autologous lymphocytes, as demonstrated by T cell proliferation induced by IFN–DCs, which had previously engulfed apoptotic, allogeneic cells in the model of the indirect pathway of alloantigen presentation [31
, 32
]. The ability of IFN–DCs to present antigens to autologous T lymphocytes was also confirmed when DCs were challenged with influenza virus. These excellent antigen-presenting and T cell-activating properties of IFN–DCs are in agreement with reports from other authors [9
, 11
, 24
]. Moreover, IFN–DCs preserved the ability to further mature upon stimulation with a wide range of maturation stimuli, as assessed by their up-regulation of the activation and maturation markers CD80, CD86, CD40, and CD83 following incubation with microbial components of bacterial (LPS) or viral origin {by means of the synthetic dsRNA poly(I:C), commonly used in models of viral infection [15
]}, inflammatory cytokines (by use of MCM [33
]), or CD40L (which mimicks CD40 ligation mediated by T cells). As expected, DC maturation induced by these different stimuli impaired the ability of IL-4–DCs and IFN–DCs to capture antigens and increased their ability to stimulate T cell proliferation. The surface expression of CD40 was lower on IFN–DCs than on IL-4–DCs, on immature and mature cells. This reduced expression of CD40 did not compromise the ability of IFN–DCs to respond to CD40L stimulation, as assessed by immunophenotyping. It is possible, however, that it partially compromised the ability of these cells to interact with T lymphocytes, as suggested by the finding that although unstimulated IFN–DCs had a more mature phenotype, they did not stimulate more efficiently the proliferation of CD4+ T cells compared with IL-4–DCs. Another difference between IFN–DCs and IL-4–DCs consisted in the expression of the IL-3R (CD123) on a relevant percentage of IFN–DCs, and it was undetectable on IL-4–DCs. This was related to the ability of IFN-, but not IL-4, to partially prevent the down-regulation of CD123 expression on the surface of differentiating monocytes, according to previous observations in a different model [34
]. Although CD123 is usually used to identify peripheral blood plasmacytoid DCs that are considered of lymphoid origin, the myeloid origin of IFN–DCs was suggested by coexpression of the myeloid marker CD11c. Surface expression of CD123 did not identify functionally different DC subpopulations, as indicated by similar regulation of CD80, CD86, CD40, and CD83 on CD123+ and CD123–IFN–DCs. According with previous reports [9
, 10
, 24
, 25
], IFN–DCs lacked the expression of CD1a. Although this molecule is involved in the presentation of lipid antigens to T cells [35
], the lack of CD1a expression did not affect APC capacity of IFN–DCs. It is however possible that as suggested by Ebner et al. [36
], the expression of this molecule may reflect some functional properties of DCs, such as low production of IL-12. Last, IFN–DCs, immature or induced to mature by different stimuli, expressed significantly lower levels of DC-SIGN (CD209) than IL-4–DCs. DC-SIGN is a recently identified DC-specific adhesion receptor belonging to the C-type lectin family, involved in the control of many functions of DCs including initiation and regulation of DC–T cell interactions, migration of DCs from blood into tissues, and endocytosis of pathogens [37
]. However, the lack of DC-SIGN expression on IFN–DCs does not appear associated with impaired, related functions such as the ability of these cells to migrate in vivo [13
] and to activate T lymphocytes. Moreover, DC-SIGN has been described on in vitro differentiated monocyte-derived DCs but is not expressed on peripheral blood DCs [38
, 39
]; thus, the lack of DC-SIGN expression may constitute an important immunophenotypic feature, suggesting that IFN–DCs are more similar to spontaneously differentiated peripheral blood DCs than in vitro monocyte-derived IL-4–DCs.
Next, we observed that IL-4–DCs and IFN–DCs induced different polarized T cell responses. In fact, both types of DCs induced T cells to produce comparable levels of the type 1 cytokine IFN-, but only IFN–DCs also promoted the production of the type 2 cytokines IL-4 and IL-10. Through analysis of intracellular cytokine expression, we found that this was related to the capacity of IFN–DCs to promote T cell differentiation toward Th1 cells, which expressed type 1 cytokines only, and Th0-like cells, which concomitantly expressed type 1 and type 2 cytokines; on the contrary, IL-4–DCs induced the differentiation of Th1 cells only. This polarization of T lymphocytes was induced by DCs upon presentation of alloantigens or upon activation of primary and secondary antigen-specific responses. Several factors contribute to determine the direction of T cell polarization. The dose of antigen, the strength of antigenic stimulation, and the nature of costimulatory molecules are all known to concur to the balance of Th1 and Th2 responses [40
41
42
] but could not explain the differences between IL-4–DCs and IFN–DCs in our study. In addition, the cytokine microenvironment present during T cell activation plays a key role in commitment toward different Th subsets. In our cultures, the ability of IL-4–DCs to promote T cell production of IFN- was likely supported by bioactive IL-12 (IL-12p70), the best known inducer of Th1 polarization [7
, 43
]. This was not the case for IFN–DCs, whose secretion of IL-12p70 was markedly reduced compared with IL-4–DCs. The induction of Th1 cytokines by these cells was possibly supported by IFN-, which was produced at high levels by IFN–DCs. As IL-12, IFN- is able to promote Th1 polarization through signal transducer and activator of transcription-4 activation [44
]. Also, the different attitude of IL-4–DCs and IFN–DCs to induce the production of type 2 cytokines by T cells could be related to the different pattern of cytokine secretion presented by these two types of DCs. IL-4 is a known inducer of type 2 cytokines, which has been shown to drive the generation of Th0-like cells during priming of antigen-specific, naïve CD4+ lymphocytes [45
]. Moreover, in a murine model, it has been reported that IL-4-secreting DCs up-regulate the production of type 2 cytokines by unprimed CD4+ T lymphocytes in vitro and in vivo [46
]. Therefore, we decided to investigate the contribution of IL-4 to the Th0-like orientation by IFN–DCs. Our finding that, in the presence of neutralizing anti-IL-4 mAb, IFN–DC-driven T cell polarization was skewed toward induction of Th1 lymphocytes strongly suggests that DC-derived IL-4 could represent an important mediator of the differentiation of the double-positive, IFN-/IL-4-producing and IFN-/IL-10-producing Th cells induced by IFN–DCs.
Cell-mediated immune responses, driven by type 1 cytokines, are crucial for detection and elimination of malignant cells. However, it appears that immune responses with extreme Th1 bias, characterized by a lack of type 2 cytokines, may not be suitable for the induction of optimal, systemic antitumor immunity [47
, 48
] and that more balanced immune responses involving type 1 and type 2 cytokines may serve to recruit additional antitumor effector cells and to induce antibodies specific for tumor-associated antigens that would substantially increase the efficacy of the antitumor-immune response. Furthermore, it has been recently reported that the precursors to long-lived memory T cells reside in the IFN--negative fraction, suggesting that in vaccine design it might be advantageous to establish conditions that do not promote full differentiation to Th1 effector cells [49
]. On the basis of these observations, the clinical use of IL-4–DCs, which lack the induction of type 2 cytokines, may appear inadequate. Evidence provided in the present study seems to suggest that, because of their rapid generation, their susceptibility to several classes of DC maturation inducers, their excellent APC function, and their capacity to induce the production of type 1 and type 2 cytokines by T lymphocytes, IFN–DCs deserve to be considered seriously for development of future DC-based immunotherapy trials.
Received April 16, 2003; revised September 1, 2003; accepted September 2, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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induce CD86 (B7.2) expression and accelerate dendritic cell maturation from human peripheral blood mononuclear cells Scand. J. Immunol. 50,499-509[CrossRef][Medline] and -ß inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14+ precursors Blood 96,210-217 enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells Int. Immunol. 14,367-380 J. Immunol. 160,5280-5287This article has been cited by other articles:
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