1 Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, MGH-East, Building 149-5102, 13th Street, Boston, 02129, USA
2 Center for Immunology and Inflammatory Diseases and Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA
Correspondence to: M. Sykes; E-mail: megan.sykes{at}tbrc.mgh.harvard.edu
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Abstract |
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Keywords: dendritic cells, human, prostaglandin E2, T helper 1, T helper 2
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Introduction |
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Prostaglandin E2 (PGE2), an arachidonic acid metabolite (11), is a common inflammatory mediator mainly produced by APCs. PGE2 acts as an immunomodulator (12, 13). It has been established that PGE2 induces the terminal maturation of monocyte-derived dendritic cells (MoDCs) and increases the expression of MHC and co-stimulatory molecules, particularly when added to tumor necrosis factor (TNF) or a maturation cocktail containing TNF, IL-1ß and IL-6 (1417). MoDCs matured in the presence of PGE2 enhance the proliferative response of allogeneic T cells (5, 9, 14, 1618). The level of production of the heterodimer IL-12p70 by DCs is the major determinant of their ability to promote Th1 differentiation (19). The bioactive IL-12p70 is composed of two subunits, p35 and p40. The p35 subunit, which is produced in lower amounts than p40, determines the amount of secreted IL-12p70 (20). In contrast, p40 homodimers, and to a lesser extent p40 monomers, have been reported to inhibit the function of IL-12p70 by an antagonistic action on the IL-12R (21, 22). The effects of PGE2 on IL-12p70 production and T cell differentiation by human MoDCs are controversial (9, 14, 17, 18, 2327) and remain to be clarified.
The trafficking of immature DCs to sites of inflammation and of mature DCs to the T cell area of secondary lymphoid organs is regulated by the expression, at the transcriptional level, of different chemokines and chemokine receptors (3, 28, 29). Immature DCs express inflammatory chemokines (CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1ß, CCL5/RANTES and CCL20/MIP-3
) and chemokine receptors that bind to inflammatory chemokines (CCR1, CCR2, CCR5, CCR6 and CXCR1). On maturation, DCs down-regulate the inflammatory chemokines and their receptors and up-regulate constitutive chemokines such as CXCL10/IP-10, CCL17/TARC, CCL18/PARC, CCL19/MIP-3ß, CCL22/MDC and the chemokine receptor CCR7, ligand of the lymph node-derived chemokines CCL19/MIP-3ß and CCL21/SLC. PGE2 has been shown to up-regulate CCR7 and CXCR4 on mature MoDCs and increase their migration to CCL19/MIP-3ß and CCL21/SLC (17, 26). However, the up-regulation of CCR7 mRNA expression by PGE2 and TNF-matured MoDCs was not confirmed in another study (25). Moreover, the effects of PGE2 on chemokine production by MoDCs are unknown. Therefore, additional studies on the effect of PGE2 on chemokine and chemokine receptor expression by MoDCs are required.
Accumulating evidence suggests that, in addition to their stimulatory properties, several subsets of human DCs can promote peripheral T cell tolerance by inducing the differentiation of T cells with regulatory properties, including CD4+CD25+ T cells and T cells that produce immunosuppressive cytokines such as IL-10 and transforming growth factor-ß. These subsets of human DCs include MoDCs at the immature stage of differentiation or after modulation by immunoregulatory cytokines (IL-10), drugs [1,25-dihydroxyvitamin D(3)] or CD8+CD28 T cells (3034). In contrast, DCs activated by microbial induction of the Toll-like receptor pathway can block the suppressive effect of CD4+CD25+ T cells, at least partially through the production of IL-6 (35). Whether or not PGE2-matured MoDCs influence the suppressive effects of CD4+CD25+ T cells has not yet been investigated.
In this study, we have further explored the characteristics of MoDCs matured in the presence of PGE2. We have analyzed their expression of chemokines and chemokine receptors and cytokine production following several stimuli, as well as their effects on the inhibitory functions of regulatory T cells.
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Methods |
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Isolation of human PBMC, monocytes, CD4+CD25+ and CD4+CD25 T cells
All subjects donating blood for these experiments were healthy volunteers of both sexes, aged 2550 years. PBMCs were isolated by standard density gradient centrifugation (400 x g) on Ficoll Histopaque® (Sigma). Monocytes and CD4+ T cells were isolated from PBMCs by negative selection using, respectively, the Monocyte Isolation Kit II and the CD4+ T Cell Isolation Kit II, purchased from Miltenyi Biotec (Auburn, CA, USA), according to the manufacturer's instructions. CD4+CD25+ and CD4+CD25 T cells were then separated from negatively selected CD4+ T cells using anti-CD25 microbeads (Miltenyi). The purities of the purified populations were >90% for monocytes and >80% for the CD4+CD25+ and CD4+CD25 cell populations (data not shown) based on the surface expression of CD14 and CD4 plus CD25, respectively.
Generation of human MoDCs
Immature MoDCs were generated by culturing monocytes (1.5 x 106 ml1) in the medium described above with rhGM-CSF (1000 U ml1) and rhIL-4 (700 U ml1) at 37°C in 7% CO2. RhTNF (1000 U ml1), with or without PGE2 (3 µg ml1), was added on Day 5 for a period of 348 h. In some experiments, immature MoDCs were pulsed with chicken IgG (50 µg ml1) or TT (10 µg ml1) on Days 1 and 4 of culture. All subsequent assays were performed after removal of GM-CSF, IL-4, TNF, PGE2, soluble chicken IgG and TT by extensive washing.
Proliferation assay using carboxyl fluorescein succinimidyl ester labeling
Freshly isolated autologous PBMCs (107 cells ml1) were labeled in PBS with 2.5 µM carboxyl fluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA) for 15 min at 37°C in 7% CO2. Labeling was quenched with cold RPMI 1640 containing 2% FCS (Sigma). CFSE-labeled PBMCs were washed in MLR media and added (106 per well) to 105 per well irradiated (30 Gy) non-pulsed, chicken IgG-pulsed or TT-pulsed autologous MoDCs matured in the presence or absence of PGE2 in a 48-well plate. Unstimulated CFSE-labeled PBMCs were plated as controls. Some non-CFSE-labeled and CFSE-labeled PBMCs (2 x 106 cells ml1) were stimulated with 2 µg ml1 of PHA (GIBCO) and used as positive controls. Cells were incubated at 37°C in 7% CO2 for 47 days.
Measurement of proliferation and cytokine production of responding T cells by flow cytometry
T cell responses in cell cultures performed with CFSE-labeled responding cells were analyzed after 47 days of culture. Concomitant analysis of CFSE, T cell-surface markers and intracellular cytokine staining was performed directly or following a 6-h re-stimulation with 105 thawed MoDCs previously pulsed with the same antigen as that used in the stimulation phase before freezing in human AB serum with 10% dimethyl sulfoxide (both from Sigma). Control PHA blasts were stimulated for the last 4 h of culture with 100 nM phorbol myristate acetate and 1 µg ml1 ionomycin (both from Sigma). Brefeldin A (GolgiStop, PharMingen) (2 µg ml1) was added to each well for the last 4 h of incubation. Cells were then harvested and stained with PerCP-conjugated anti-CD3 (PharMingen) and allophycocyanin-conjugated anti-CD4 or CD8 (PharMingen) in FACS buffer for 30 min at 4°C in a 96-well round-bottom plate. Mouse IgG1peridininchlorophyll protein complex (PerCP) and mouse IgG1allophycocyanin were used as isotype controls. In some experiments, cells were then washed twice in PBS and incubated in 100 µl Cytofix/Cytoperm solution (PharMingen) for 20 min in the dark, at room temperature. Fixed and permeabilized cells were washed twice with PermWash buffer (PharMingen) and stained with purified mouse anti-human IFN-, TNF, IL-4, granzyme A and perforin (PharMingen) in the corresponding wells for 10 min at 4°C. PE-conjugated anti-IFN-
, TNF, IL-4 granzyme A and perforin (PharMingen) were added to the respective wells for 30 min at 4°C. Rat IgG2aPE was used as a negative control. Cells were then washed in PermWash buffer and re-suspended in 100 µl FACS buffer. Alternatively, cells were washed in FACS buffer after the first step of surface staining and labeled with 0.5 µg of 7-amino-actinomycin-D (7-AAD) (Sigma) immediately before FCM analysis. A minimum of 30 000 events were acquired on a FACS Calibur flow cytometer and analyzed using Winlist software (Verity Software House, Topsham, ME, USA).
Measurement of surface antigen expression by flow cytometry (FCM)
Cell-surface staining was performed on 5 x 104 to 2 x 105 cells per sample. FITC-conjugated anti-CD3 and -CD19 and PE-labeled anti-CD56 and -CD14 (PharMingen) were used to evaluate the purity of monocytes, while anti-CD4FITC and anti-CD25PE mAbs (both purchased from PharMingen) were used to determine the purity of the CD4+CD25+ and CD4+CD25 populations. PE-conjugated anti-CCR5, CCR7 and CXCR4 mAbs (all purchased from PharMingen) were used to phenotype mature MoDCs. Non-reactive control mAbs HOPCFITC (mouse IgG2a) and rat IgG2aPE (prepared in our laboratory) were used as isotype controls. Exclusion of dead cells was performed by propidium iodide (PI) staining and gating on PI-negative cells. For most stains, 10 000 viable cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA, USA) and further analysis was performed using Windows Multiple Document Interface(WinMDI) software (Scripps Research Institute, La Jolla, CA, USA).
RNA isolation and cDNA synthesis
Total RNA was extracted using the RNeasy kit according to the manufacturer's protocol (Qiagen, Valencia, CA, USA). Briefly, after DNase I (Invitrogen) treatment, 1 µg of total RNA from each sample was used as template for the reverse transcription (RT) reaction. One hundred microliters of cDNA was synthesized using oligo(dT)15, random hexamers and multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA, USA). All samples were reverse transcribed under the same conditions (25°C for 10 min and 48°C for 30 min) and from the same RT master mix to minimize differences in RT efficiency.
Quantitative real-time reverse transcriptionPCR
Primer sequences for quantitative PCR (QPCR) were previously described (36). The 25-µl QPCR mixture contains 2 µl of cDNA, 12.5 µl of 2x SYBR Green master mix (Stratagene, La Jolla, CA, USA) and 250 nmol of sense and anti-sense primer. The reaction conditions were as follows: 50°C for 2 min, 95°C for 10 min and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Emitted fluorescence for each reaction was measured during the annealing/extension phase, and amplification plots were analyzed using the MX4000 software version 3.0 (Stratagene). A series of standards was prepared by performing 10-fold serial dilutions of full-length cDNAs in the range of 20 million copies to 2 copies per QPCR. Subsequent analysis was performed on the data output from the MX4000 software using Excel XP (Microsoft, Redmond, WA, USA). The threshold cycle, i.e. the cycle number at which the amount of amplified gene of interest reaches threshold fluorescence, was determined by using the adaptive baseline algorithm in the MX4000 analysis software. This algorithm sets the threshold cycle at 10 standard deviations (SDs) above the background fluorescence between cycles 5 through 9. Quantity values (i.e. copies) for gene expression were generated by comparison of the fluorescence generated by each sample with standard curves of known quantities. Next, the calculated number of copies was divided by the number of copies of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In addition, we saw no significant changes in the QPCR results when the data were normalized using another constitutively active gene, ß2-microglobulin.
Cytokine assays
Cytokine production by immature MoDCs was analyzed on supernatants collected on Day 5 of culture and stored at 80°C. Two days later, the same MoDCs, matured in the presence or absence of PGE2, were extensively washed in MLR media. Mature MoDCs (5 x 105) were then incubated with media alone or with 1000 IU ml1 of rh IFN- alone or with 1.25 x 105 CD40-L-transfected NIH3T3 cells (referred to as L-cells) with or without 1000 IU ml1 of rh IFN-
in 1 ml of MLR media, in a 48-well plate at 37°C in 7% CO2. In addition, control wells containing 1.25 x 105 L-cells with or without 1000 IU ml1 of rh IFN-
were plated in the same conditions. After 48 h of stimulation, supernatants were collected and stored at 80°C. Cytokine concentrations were measured using specific ELISA kits for human IL-12p40, IL-12p70 and IL-10 purchased from R&D Systems, according to the manufacturer's instructions.
T cell proliferation assays
Irradiated (30 Gy) non-pulsed MoDCs (5 x 103 per well), matured in the presence or absence of PGE2, were added to freshly isolated allogeneic CD4+CD25 T cells (5 x 104 per well) with varying numbers of CD4+CD25+ T cells from the same allogeneic donor (ratio CD4+CD25/CD4+CD25+: 1:1 to 16:1) in MLR medium, in duplicate, in flat-bottom 96-well plates at 37°C in 7% CO2. Cultures were pulsed with 1 µCi (0.037 MBq) [3H]thymidine ([3H]TdR) (Amersham Pharmacia Biotech, Piscataway, NJ, USA) 5 days after incubation and harvested 16 h later. [3H]TdR uptake was counted on a Betaplate ß counter (Wallac, Gaithersburg, MD, USA). Results are expressed as mean ± SD of counts per minute (c.p.m.) incorporated in the duplicate samples or as percent inhibition of proliferation [100 x (1 mean c.p.m. with the addition of CD4+CD25+ cells/mean c.p.m. in the absence of CD4+CD25+ T cells)].
Statistical analysis
Differences between means of results obtained with MoDCs matured in the presence of PGE2 versus MoDCs matured without PGE2 were tested using the Student's t-test by Microsoft Excel software. A P-value <0.05 was considered to be significant.
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Results |
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Results on Days 5 and 6 (data not shown) and Day 7 (Fig. 1A) demonstrated increased anti-TT proliferative responses compared with responses to MoDC alone or those pulsed with chicken IgG for both CD4+ and CD8+ T cells. Of note, the percentage of CD4+ and CD8+ T cells dividing in response to chicken IgG-pulsed MoDCs was twice as high as that responding to non-pulsed MoDCs. The percentages of CD4+ and CD8+ T cells dividing in response to TT, MoDC alone or chicken IgG were increased 3- to 6-fold by stimulation with MoDCs matured in the presence compared with the absence of PGE2. Stimulation with PGE2-matured MoDCs was associated with increased responding CD4+ and CD8+ T cell precursor frequencies, as calculated with the CFSE dilution technique described by Lyons (37) (data not shown). Increased proliferative responses with MoDCs matured in the presence of PGE2 were confirmed by [3H]TdR incorporation assays (data not shown).
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PGE2-matured MoDCs induce the differentiation of Th1 cytokine-producing T cells in response to exogenous protein antigens
We next evaluated the effect of maturation of MoDCs with PGE2 on the development of specific Th1 T cell responses to exogenous protein antigens. CFSE-labeled PBMCs were stimulated with irradiated non-pulsed, chicken IgG-pulsed or TT-pulsed autologous MoDCs, matured with or without PGE2, for 57 days. Cells were re-stimulated on Day 5, 6 or 7 with the initially used source of pulsed MoDCs that had been kept frozen before staining for CD3, CD4 or CD8 and intracellular IFN-, TNF, IL-4, granzyme A and perforin. Cytokine production was assessed simultaneously with T cell proliferation by flow cytometric analysis of CFSE and intracellular cytokine, granzyme A or perforin staining of gated CD3+CD4+ or CD3+CD8+ T cells.
Levels of expression of these molecules by non-dividing MoDC-stimulated T cells were similar, regardless of whether MoDCs were unpulsed, pulsed with chicken IgG or pulsed with TT (<1% of non-dividing CD4+ or CD8+ T cells expressed cytokines or perforin and 3050% of non-dividing CD8 T cells expressed granzyme A, Fig. 2A and data not shown). Thus, further analysis was focused on the production of these molecules by dividing T cells.
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PGE2 enhances the maturation of MoDCs
We next assessed the effect of PGE2 on the acquisition of MoDC maturation characteristics. In concordance with published results (18, 20), we observed that MoDCs matured with TNF and PGE2 expressed higher levels of MHC class I and II, co-stimulatory molecules (CD80 and CD86) and the maturation marker CD83 than MoDCs matured with TNF alone (data not shown). We also analyzed the expression of chemokine receptors that have been associated with the maturation of MoDCs. We used QPCR to examine the mRNA expression of CCR5, CXCR4 and CCR7 in immature MoDCs before and at several time points following the induction of maturation with or without PGE2. These results were compared by flow cytometry to analyze surface expression of these chemokine receptors on MoDCs matured for 48 h. Compared with non-PGE2-matured MoDCs, those matured with PGE2 showed more rapid down-regulation of CCR5 and increased up-regulation of CXCR4 and CCR7 at the mRNA level (Fig. 3A). In accordance with these results, surface expression of CCR5 was decreased, while surface expression of CXCR4 was increased on MoDCs matured with PGE2 compared with those matured with TNF alone. However, no increase in surface expression of CCR7 was detected on PGE2-matured MoDCs at 48 h (Fig. 3B).
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MoDCs matured with PGE2 do not impair the suppressive effects of CD4+CD25+ T cells on the proliferation of allogeneic CD4+CD25 T cells
Since MoDCs matured with PGE2 enhanced IL-6 mRNA production, which has been shown to inhibit the suppressive effects of CD4+CD25+ T cells (35), we next asked whether PGE2-matured MoDCs could inhibit the suppressive effects of such regulatory T cells on the proliferation of alloreactive CD4+CD25 T cells. Proliferative responses of allogeneic CD4+CD25 T cells stimulated by irradiated MoDCs matured with TNF and PGE2 in the absence or presence of decreasing numbers of CD4+CD25+ T cells were analyzed by incorporation of [3H]TdR after 5 days of culture and compared with those stimulated by MoDCs matured with TNF alone. Although PGE2-matured MoDCs induced higher proliferative responses of allogeneic purified CD4+CD25 T cells than control MoDCs, these responses were reduced, in a dose-dependent fashion, by CD4+CD25+ T cells in the presence of both types of MoDCs (Fig. 6A). No significant difference in the degree of suppression by CD4+CD25+ cells was detected following stimulation with MoDCs matured with or without PGE2 (Fig. 6B).
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Discussion |
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As reported by others (5, 9, 1820), we observed that PGE2-matured MoDCs enhanced T cell proliferative responses to alloantigens (data not shown). In addition, we provide the first demonstration that MoDC maturation in PGE2 enhances CD4+ and CD8+ T cell responsiveness to exogenous protein antigens, including recall antigens (TT) and neoantigens (chicken IgG). Both more rapid and increased recruitment of antigen-specific CD4+ and CD8+ precursor T cells was achieved with PGE2-matured MoDC, and this was not due to increased activation-induced death of T cells activated by MoDCs matured without PGE2. The mechanisms involved in the improved responses to recall and neoantigens to CD8 cells remain to be determined. These might be due to enhanced cross-presentation of exogenous antigens by class I molecules or increased CD4+ T cell- or NK-mediated help to CD8+ T cells in the presence of MoDCs matured with PGE2. Regardless of the mechanism, this observation has important implications for the generation of tumor antigen-specific CTLs.
The polarization of T cells toward Th1 or Th2 responses is essential in determining the final outcome of immune responses (41). The differentiation of T cells toward Th1 or Th2 by human DCs was initially reported to be determined by the subset of DCs: myeloid DCs (DC1), now known to be MoDCs, were reported to induce Th1 responses, while lymphoid/plasmacytoid DCs (DC2) induce Th2 responses (42). More recently, however, MoDCs inducing Th2 responses have been described (43) and the concept that Th polarization promoted by DCs depends on their culture conditions and activation signals has emerged (44, 45). Our studies demonstrate that PGE2-matured MoDCs induce the differentiation of Th1, antigen-specific, CD4+ and CD8+ T cells. Indeed, antigen-activated CD4+ T cells displayed a phenotype of Th1 cells, producing IFN- (and also TNF), with no IL-4. TT-specific CD8+ T cells did not produce very high levels of IFN-
, but did express molecules associated with cytotoxic effector differentiation. Consistent with several other reports (16, 19, 20, 2527, 46), similar results were obtained when we explored alloantigen-specific T cell responses (data not shown). However, our results contrast with those of Kalinski and colleagues (17, 44, 47, 48), who showed that MoDCs matured with PGE2 induced the differentiation of Th2 cytokine-producing T cell responses. The differing results cannot be readily explained by differences in culture conditions or activation signals, as these were similar to those in our studies and those of other groups with results consistent with our own. The discrepancy might best be explained by the type of T cell response explored and the signals used to induce cytokine production by activated T cells. Kalinski and colleagues studied naive T cell responses to superantigens after two stimulations with autologous MoDCs and explored the cytokine production of T cells activated non-specifically via CD3 and CD28 mAbs. We explored responses of naive and/or memory T cells to TT protein antigens and analyzed the cytokine production of activated T cells after a short 6-h restimulation with MoDC presenting the same antigen. This approach addressed the cytokine responses of T cells activated by specific antigens, which may be closer to the in vivo situation of cancer patients, in whom the objective of vaccination with MoDCs-bearing tumor antigens is to break the tolerance of patient T cells against the tumor and achieve tumor antigen-specific cytotoxic responses.
Mature DCs are specialized in the activation of T cells because of their high expression of MHC and co-stimulatory molecules, and also because of the acquisition of chemokine receptors allowing their migration to the T cell area of the lymph node and the production of chemokine-attracting T cells. Thus, analysis of these characteristics is important for an understanding of their in vivo T cell stimulatory potential. As previously described (1619), we confirmed that PGE2 increased the expression of MHC class I and II, CD80, CD86 and CD83 molecules on matured MoDCs (data not shown). In addition, PGE2-matured MoDCs completely lost CCR5 expression and showed significantly greater up-regulation of CXCR4 and CCR7 mRNA than MoDC matured without PGE2. While contrasting with the results of Lee et al. (25), our results confirm those of Luft et al. (26) and Scandella et al. (19), who both showed that these phenotypic characteristics are associated with increased migratory capacity.
We also observed that PGE2 regulates the kinetics and the level of chemokine mRNA expression by MoDCs. PGE2 accelerated the down-regulation of the pro-inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1ß and CCL5/RANTES and induced 2- to 4-fold increases in the expression of the constitutively expressed chemokines CCL22/MDC and CCL17/TARC on mature MoDCs. These results consolidate the role of PGE2 in accelerating the maturation of MoDCs and suggest that the greater and more rapid stimulatory capacity of MoDCs matured with PGE2 might be due to an increased ability to attract T cells. Consistent with this hypothesis, we and others have observed increased DCT cell interaction in culture as shown by increased number and size of clumps when DCs were matured in the presence of PGE2 (data not shown) (18). However, in contrast to LPS-matured MoDCs (29, 49), mRNA for chemokines that preferentially attract Th1 T cells, such as CXCL9/MIG, CXCL10/IP-10 and CXCL11/I-TAC (50), was not detectable in MoDCs matured with TNF, regardless of the presence or absence of PGE2. Instead, PGE2-matured MoDCs expressed high levels of CCL22/MDC and CCL17/TARC, which are ligands for CCR4, a chemokine receptor preferentially expressed on Th2 T cells (50, 51). These results might explain the higher Th2 cytokine production by non-specifically activated T cells in the presence of PGE2-matured MoDCs, as reported by Kalinski et al. (17, 44, 47, 48). We and others (16, 19, 20, 2527), however, observed the differentiation of Th1 T cells by MoDCs matured with PGE2. Taken together, these results suggest that CCL17/TARC and CCL22/MDC can attract CCR4-expressing T cells with both Th2 and Th1 potentials and that the polarization of the final T cell response is mainly determined by additional environmental factors and signals.
DCs control T cell responses by the production of cytokines. MoDC-derived IL-12p70 is the major Th1-driving factor (21), while IL-10 inhibits the stimulatory capacity of DCs and impairs DC-mediated Th1 T cell differentiation, promoting Th2 differentiation and T cell anergy (52, 53). We showed that MoDCs matured with TNF, with or without PGE2, produced very low levels of IL-12p70 in the absence of additional stimulation by IFN- and CD40-L, the combination of which led to maximal production of this cytokine. These results are in agreement with other studies showing that only MoDCs matured in the presence of fixed bacteria (Escherichia coli), bacterial products (LPS, Staphylococcus aureus Cowan I) or poly(inosinic acid)poly(cytidylic acid) can produce IL-12p70 without further activation (8, 21, 47), while MoDCs matured under other conditions required either high levels of CD40 cross-linking (54) or the combination of CD40-L and IFN-
to produce optimal levels of IL-12p70 (55). Our studies demonstrated that PGE2 regulates the production by MoDCs of both IL-12p70 and IL-12p40. PGE2 selectively enhanced the production of IL-12p40 by unstimulated or IFN-
-stimulated MoDCs, but reduced IL-12p40 and IL-12p70 production by MoDCs stimulated by CD40-L, with or without IFN-
. Thus, we believe that the previous observation (56) that PGE2 selectively induces the production of IL-12p40 by MoDCs applies only to non-stimulated or IFN-
-stimulated MoDCs. In contrast, when MoDCs were stimulated by CD40-L and IFN-
, which occurs during the interaction between DCs and CD4 Th(57), PGE2-matured MoDCs were able to produce, although at lower levels, both IL-12p70 and IL-12p40. Importantly, the ratio of IL-12p40/IL-12p70 was similar in supernatants from MoDCs matured with or without PGE2. In addition, PGE2 did not influence the production of IL-10 by MoDCs, which was down-regulated during their maturation and maintained at very low levels following stimulation by CD40-L and IFN-
. Together, these data suggest that PGE2-matured MoDCs are likely to produce very low amounts of IL-10 but significant levels of IL-12p70 in response to their interaction with T cells, and can therefore induce Th1-polarized T cell responses. The physiological significance of high production levels of IL-12p40, in particular by unstimulated PGE2-matured MoDCs, is unknown. It has recently been shown that IL-12p40 can associate with IL-23p19 (58). Signaling through IL-23R induces the production of IFN-
and IL-17 by memory T cells (58, 59). Whether the observed effects of PGE2 on the activation of T cells by MoDCs involve IL-23 remains to be determined.
We observed that PGE2 increased the levels of IL-6 mRNA expression in mature MoDCs. Since IL-6 production by DCs has been reported to inhibit the suppressive function of CD4+CD25+ T cells when the DCs are stimulated by the TLR pathway (35), we investigated the effect of PGE2-matured MoDCs on the function of these suppressive T cells. Our results argue against an effect of MoDCs matured with PGE2 on the suppressive activity of CD4+CD25+ T cells.
Another explanation for the lower stimulatory potential of MoDCs matured with TNF alone is that these may be in an intermediate stage of differentiation between immature and fully mature MoDCs, and may maintain the known capacity of immature MoDCs to induce regulatory T cells producing IL-10 (46), as has been reported for TNF-matured mouse bone marrow-derived DCs (60). In contrast, PGE2 may convert TNF-matured MoDCs into fully mature MoDCs that cannot induce such regulatory T cells, as shown by Jonuleit with MoDCs matured with TNF, IL-1ß, IL-6 and PGE2 (46). Further investigations are required to explore this hypothesis.
In conclusion, the addition of PGE2 to TNF to achieve the maturation of human MoDCs induces the generation of migratory-type MoDCs that express high levels of T cell-attracting chemokines, MHC and co-stimulatory molecules, and that produce IL-12p70 but not IL-10 after stimulation with CD40-L and IFN-. These properties are associated with increased stimulatory capacity and the promotion of Th1 antigen-specific T cell responses. Thus, MoDCs matured in the presence of PGE2 display the characteristics of efficient APCs that should be considered as a potential source of APCs in anti-cancer vaccine trials.
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Acknowledgements |
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Abbreviations |
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7-AAD | 7-amino-actinomycin-D |
APC | antigen-presenting cell |
CD40-L | CD40 ligand |
CFSE | carboxyl fluorescein succinimidyl ester |
c.p.m. | counts per minute |
DC | dendritic cell |
FSC | forward scatter |
GAPDH | glyceraldehyde-3-phosphate dehydrogenase |
GM-CSF | granulocyte macrophage cell stem factor |
[3H]TdR | [3H]thymidine |
2ME | 2-mercaptoethanol |
MLR | mixed lymphocyte reaction |
MoDC | monocyte-derived dendritic cell |
PerCP | peridininchlorophyll protein complex |
PGE2 | prostaglandin E2 |
PI | propidium iodide |
QPCR | quantitative PCR |
rh | recombinant human |
RT | reverse transcription |
SD | standard deviation |
SSC | side scatter |
TNF | tumor necrosis factor |
TT | tetanus toxoid |
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Notes |
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Received 23 March 2005, accepted 15 September 2005.
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References |
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