Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;72:939-945.)
© 2002 by Society for Leukocyte Biology

Differential regulation of dendritic cell function by the immunomodulatory drug thalidomide

Mohamad Mohty*, Anne-Marie Stoppa{dagger}, Didier Blaise{dagger}, Daniel Isnardon*, Jean-Albert Gastaut{dagger}, Daniel Olive*,{ddagger} and Béatrice Gaugler*,{ddagger}

* Laboratoire d’Immunologie des Tumeurs and
{dagger} Département d’Hématologie, Institut Paoli-Calmettes, Université de la Méditerranée, Marseille, France; and
{ddagger} Institut National de la Santé et de la Recherche Médicale (INSERM) U119, Marseille, France

Correspondence: Dr. Béatrice Gaugler, Institut de Cancérologie et d’Immunologie de Marseille (IFR57), INSERM U119, Laboratoire d’Immunologie des Tumeurs, Institut Paoli-Calmettes, 232 Bd. Ste Marguerite, 13273 Marseille Cedex 09, France. E-mail: gauglerb{at}marseille.fnclcc.fr or mohtym{at}marseille.fnclcc.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thalidomide (Thal) was shown to be a potent immunomodulating agent. Because of their central role in controlling immunity, we investigated the effects of Thal on monocyte-derived dendritic cells (Mo-DC). The addition of 10 µg/ml or 20 µg/ml Thal from the beginning of monocyte culture with granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 did not block Mo-DC differentiation. Moreover, Thal alone could not induce Mo-DC maturation. However, Thal exerted a modulation of Mo-DC functional properties. At 10 µg/ml, Thal modified the allostimulatory capacity of DC little, whereas a dose of 20 µg/ml up-regulated this capacity (P=.05) and increased IL-12p70 production in a dose-dependent manner between 10 and 20 µg/ml (P=.001). Mo-DC generated with 10 µg/ml Thal were poor stimulators of T helper cell type 1 (Th1) responses (P=.01), but 20 µg/ml was able to strengthen Th1 responses (P=.03). Also, Thal induced a significant reduction of IL-10 production in response to the maturation-inducing stimulus CD40L. Similarly, tumor necrosis factor {alpha} production was significantly decreased when Mo-DC were exposed to 10 µg/ml Thal, and a dose of 20 µg/ml did not induce any significant changes. The effects of Thal in vitro on the secretion of IL-12p70 and strengthening of Th1 responses might contribute to the antitumor effects of Thal. Thus, DC appear to be potential targets for the immunomodulatory capacity of Thal, defining a new mechanism of action of this drug.

Key Words: antigen-presenting cells • immune response • IL-12 • immunotherapy • cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thalidomide (Thal) was shown to be a potent immunomodulating agent with a broad spectrum of immunologic effects. Beside its efficiency in the treatment of erythema nodosum leprosum, it has potential therapeutic applications that span a wide spectrum of diseases, including malignancies, especially multiple myeloma (MM) [1 ]. Given its broad spectrum of activities, Thal may be acting in several ways. Thal may inhibit tumor growth and survival through free radical-mediated, oxidative DNA damage that has been shown to play a role in the teratogenicity of Thal [2 ]. Thal can also modulate adhesive interactions [3 ] and thereby may alter tumor cell growth, survival, and drug resistance. Thal was also shown to interfere with the secretion of cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}), a possible mechanism for the anti-inflammatory effects of Thal therapy [4 ]. Moreover, Thal could exert an important antiangiogenic activity [5 ]. Finally, Thal was shown to provide a costimulatory signal for CD8+ T cell proliferation and lymphokine production [6 ].

The aim of our work was to study the effects of Thal on human dendritic cells (DC). Being the most potent antigen presenting cells in vitro and in vivo, DC play a key role in the initiation of the immune response and are considered promising targets for immunotherapy [7 ]. In vitro-differentiated DC show functional and phenotypic characteristics of immature DC and can be further differentiated in vitro into mature DC with TNF-{alpha}, lipopolysaccharides, or CD40L [7 ]. Thus, for immunotherapeutic applications, it appears crucial to identify factors that might affect the differentiation, maturation, and function of DC. Because of its wide immunomodulatory properties, DC might be potential targets for Thal. Therefore, we assayed the ability of Thal to influence the differentiation of DC from circulating peripheral blood monocytes. Here, we show that in vitro, Thal can affect DC function. Thal did not block the granulocyte macrophage-colony stimulating factor (GM-CSF) plus interleukin (IL)-4-driven differentiation of monocytes into DC, but Thal exerted a modulation of the stimulatory capacity of monocyte-derived DC (Mo-DC) and their cytokine secretion profile.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Blood samples
Peripheral blood mononuclear cells (PBMC) from healthy donors (Regional Transfusion Center, Marseille, France) were isolated on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradients prior to cryo-preservation.

Cell lines
Murine L cells transfected with human CD40L were kindly provided by Schering-Plough (Laboratory for Immunological Research, Dardilly, France) and were used after a 75 Gy irradiation.

Cell separation and DC generation
CD14+ monocytes were immunomagnetically purified with CD14 monoclonal antibody (mAb)-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of the CD14+ cells by flow cytometry analysis was always >98%. Purified monocytes were cultured in RPMI-1640 medium containing 10% fetal calf serum (BioWhittaker, Verviers, Belgium) at 0.5 x 106/ml in the presence of 100 ng/ml GM-CSF (kind gift of Novartis, Berne, Switzerland) and 20 ng/ml IL-4 (kind gift of Schering-Plough Research Institute, Kenilworth, NJ) for Mo-DC generation. Thal (CN Biosciences UK Ltd., Nottingham) was dissolved in dimethyl sulfoxide (DMSO) to give a stock solution of 20 mg/ml, which was kept for up to 1 week. For Mo-DC generation, monocytes were cultured with appropriate cytokines (control Mo-DC) and with Thal (Thal-Mo-DC) at the concentrations indicated hereinafter. On day 5, final maturation of Mo-DC and Thal-Mo-DC was induced by adding 75 Gy-irradiated CD40L-transfected L cells (ratio, 1/10). The medium was replenished with cytokines every 3 days.

Flow cytometry analysis
The following mAb were used for flow cytometry: anti- CD1a, CD14, CD40, CD54, CD58, CD80, CD83, human leukocyte antigen (HLA)-DR, HLA-ABC, isotypic controls, mouse immunoglobulin G (IgG)1, mouse IgG2a, and mouse IgG2b from Beckman-Coulter (Marseille, France). CD86 and CCR-5 were purchased from Pharmingen (San Diego, CA). All mAb were used as fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, cyanin-5-, allophycocyanin (APC)-conjugated mAb. Samples were analyzed using a FACSCalibur (BD Biosciences, Le Pont de Claix, France). Data for at least 10 x 103 cells/sample were acquired and analyzed using CellQuest software (BD Biosciences).

Primary mixed lymphocyte reaction (MLR)
CD4+/CD45RA+ naive T cells were purified by negative selection of adult blood PBMC using goat anti-mouse Ig-coated magnetic beads (Beckman-Coulter) incubated with mAb against CD8, CD14, CD56 (D. Olive, INSERM U119, Marseille, France), CD19 (Diaclone, Besançon, France), and CD45RO (Beckman-Coulter). Purity was superior to 95% as controlled by fluorescein-activated cell sorter analysis. Graded numbers of stimulating cells were cultured in triplicate with 105 allogeneic, naive T cells in 96-well flat-bottom plates (Costar, Corning, NY). Proliferation of T cells was monitored by measuring methyl-[3H]-thymidine (1 µCi/well; Amersham, Little Chalfont, UK) incorporation during the last 16 h of a 6-day culture. Thymidine uptake was counted on a gas-phase ß-counter (Matrix 9600, Packard, Downers Grove, IL).

Cytokine production assay and intracellular analysis
Supernatants of DC cultures were harvested after 2 days of maturation with CD40L. IL-10, TNF-{alpha}, and IL-12p70 concentrations were measured using specific enzyme-linked immunosorbent assay (ELISA) OptEIA sets purchased from Pharmingen. When allogeneic, naive CD4+/CD45RA+ T cells were cocultured with mature Mo-DC or Mo-DC generated in the presence of Thal, cells were harvested after 6 days and replated in 48-well culture plates at 5 x 105 cells/well in medium containing 25 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Quentin Fallavier, France), 1 µg/ml ionomycin (Sigma Chemical Co.), and 10 µg/ml brefeldin A (BFA; Sigma Chemical Co.) for 5 h. For intracellular cytokine production analysis, anti-IL-4-FITC-, anti-IL-10-PE-, anti-interferon-{gamma} (IFN-{gamma})-APC-, and FITC/PE/APC-conjugated isotypic mAb (Pharmingen) were used according to the manufacturer’s instruction. Cells were collected, washed, fixed, and permeabilized using the CytoStain kit (Pharmingen) and were stained with 0.5 µg/test of cytokine-specific mAb.

Statistical analysis
The significance of differences between the indicated values was assessed by two-tailed Student’s t-test for paired and unpaired data; a P value of 0.05 or less was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Mo-DC in the presence of Thal
To investigate the effect of Thal on DC differentiation from human monocytes, we cultured purified CD14+ monocytes with GM-CSF, IL-4 (Mo-DC), and various concentrations of Thal (Thal-Mo-DC). Thal (5 µg/ml) did not induce any significant changes on Mo-DC (data not shown), and more than 30 µg/ml affected cell recovery. Although some Mo-DC could be generated, more than a 90% decrease in cell number was demonstrated as compared with a control group. Thus, 10 µg/ml and 20 µg/ml were selected and used in all of our subsequent experiments for phenotypic and functional characterization. We characterized Thal10-Mo-DC and Thal20-Mo-DC (10 or 20 µg/ml Thal added at the beginning of monocytes culture) in comparison with untreated Mo-DC. The addition of 10 µg/ml or 20 µg/ml Thal from the beginning of Mo-DC differentiation from monocytes did not induce significant morphological changes as compared with normal, mature Mo-DC (data not shown). On the phenotypic level, the presence of Thal at the beginning of culture did not impair Mo-DC differentiation. The monocytic marker CD14 was down-regulated, and CD1a, a lineage marker of DC, was expressed on Mo-DC, Thal10-Mo-DC, and Thal20-Mo-DC. We next analyzed the expression of the adhesion molecules (CD54 and CD58), major histocompatibility molecules (class I and HLA-DR), costimulatory molecules (CD80 and CD86), chemokine receptor CCR-5 (a marker of immature Mo-DC), and the maturation marker CD83. Except for CD54, which was up-regulated, Thal10-Mo-DC and Thal20-Mo-DC showed a similar expression profile of these molecules as compared with Mo-DC. Moreover, Thal did not induce the expression of CD83, which is classically found on mature Mo-DC (Fig. 1 ). Thus, Thal alone could not induce Mo-DC maturation. Therefore, we examined Thal-treated Mo-DC to undergo maturation after culture for 5 days followed by CD40L for 2 days. Phenotypic analysis indicated that Thal10-Mo-DC and Thal20-Mo-DC could acquire the expression of CD83 and express the costimulatory molecules CD80 and CD86. The expression of CD40, CD58, HLA-DR, and especially CD54 was up-regulated (Fig. 2 ).



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  		Figure 1. Thal does not inhibit Mo-DC differentiation. Immunomagnetically selected CD14+ monocytes were cultured for 5 days with GM-CSF and IL-4 (immature Mo-DC), in the absence or presence of 10 or 20 µg/ml Thal. Open histograms show the background staining with isotype-control mAb, and solid histograms represent specific staining of the indicated cell surface markers. The % of CD1a+/CD14- DC and the mean fluorescence intensity (MFI) are provided for this representative experiment out of three independent experiments.

 


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  		Figure 2. Mo-DC and Thal-Mo-DC triggered through CD40 can achieve an activated phenotype. After 5 days of culture, immature Mo-DC, immature Thal10-Mo-DC, and immature Thal20-Mo-DC (exposed to Thal at the beginning of monocyte culture only) were activated with irradiated murine L cells transfected with human CD40L for 2 days. Open histograms show the background staining with isotype-controls mAb, and solid histograms represent specific staining of the indicated cell surface markers. The % of CD83+ DC and the MFI are provided for this representative experiment out of five independent experiments.

 
Functional properties of Mo-DC generated in the presence of Thal
To investigate the function of Thal-Mo-DC as stimulators of naive CD4+ T cells, their ability to stimulate an allogeneic MLR was compared with that of normal immature and mature Mo-DC. Thal10-Mo-DC and Thal20-Mo-DC matured with CD40L were used to stimulate naive CD4+CD45RA+ T cells from an unrelated donor at different stimulator/responder ratios. Mature Thal20-Mo-DC were found to be the most potent stimulators of allogeneic MLR. The stimulating activity of Thal20-Mo-DC was always higher than that of untreated, mature Mo-DC (P=.05). In contrast, the stimulatory activity of mature Thal10-Mo-DC, although not statistically significant, was lower than that of normal mature Mo-DC. Immature Mo-DC induced the lowest proliferation of naïve, allogeneic CD4+ T cells (Fig. 3 ). We next examined the profile of primary, allogeneic T cell responses induced by normal and Thal-treated Mo-DC. Naive CD4+CD45RA+ T cells isolated from human peripheral blood were cocultured for 6 days with mature Mo-DC, Thal10-Mo-DC, and Thal20-Mo-DC. The cultured cells were counted and restimulated with PMA and ionomycin for 5 h for single-cell cytokine analysis by flow cytometry. T cells originally cultured with mature Thal20-Mo-DC secreted the largest amounts of IFN-{gamma} as compared with untreated, mature Mo-DC but little or undetectable IL-10 and IL-4 (Fig. 4A and 4B , and data not shown). T cells originally cultured with mature Thal10-Mo-DC secreted the lowest amounts of IFN-{gamma} (Fig. 4A and 4B , and data not shown). This polarization profile suggested a potent T helper cell type 1 (Th1) response associated with Thal20-Mo-DC, whereas Thal10-Mo-DC induced a weak Th1 response.



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  		Figure 3. Thal modulate T cell allostimulatory capacities of mature Mo-DC. CD4+/CD45RA+ naive T cells were purified by negative selection of adult blood PBMC. Graded numbers of irradiated, stimulating cells were cultured in triplicate with 105 allogeneic, naive T cells in 96-well flat-bottom plates. Proliferation of T cells was monitored by measuring methyl-[3H]-thymidine incorporation during the last 16 h of a 6-day culture. The mean results obtained from four independent experiments are indicated; *, P = .05 for mature Thal20-Mo-DC versus normal, mature Mo-DC.

 


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  		Figure 4. Polarization capacity of mature Mo-DC generated in the absence or presence of Thal. (A) Naive CD4+CD45RA+ T cells sorted as described in Materials and Methods (purity >95%) were cocultured for 6 days with mature Mo-DC, mature Thal10-Mo-DC, and mature Thal20-Mo-DC. The cultured cells were counted and restimulated with PMA and ionomycin for 5 h in the presence of BFA. IFN-{gamma} and IL-10 cytokines were measured by intracellular staining. (B) % of IFN-{gamma}-secreting cells measured by intracellular staining. Results are represented as the mean and standard deviation obtained from four independent experiments. *, P = .01 for mature Thal10-Mo-DC versus normal, mature Mo-DC; **, P = .03 for mature Thal20-Mo-DC versus normal, mature Mo-DC.

 
Thal affects cytokine production by Mo-DC
In view of the fact that Thal influenced functional properties of Mo-DC, we investigated the capacity of Thal to interfere with cytokine production by Mo-DC. After 2 days of exposition to CD40L, supernatants were quantified for IL-10, IL-12p70, and TNF-{alpha}. Thal10-Mo-DC and Thal20-Mo-DC showed a significant reduction of IL-10 production in response to the maturation-inducing stimulus CD40L (Fig. 5A ). TNF-{alpha} production was significantly decreased when Mo-DC were exposed to 10 µg/ml Thal, and a dose of 20 µg/ml Thal did not induce any significant changes in comparison with normal, mature Mo-DC (Fig. 5B) . In contrast, IL-12p70 production was significantly increased when Mo-DC were exposed to 20 µg/ml Thal, and a dose of 10 µg/ml Thal did not induce any significant changes in comparison with normal, mature Mo-DC (Fig. 5C) . Immature Mo-DC cultured with DMSO alone (same volume as used for Thal preparation) did not show secretion of detectable levels of IL-12p70. Similarly, the addition of DMSO without Thal to mature Mo-DC activated with CD40L did not show any significant difference in IL-12p70 secretion, thus excluding a possible interference of DMSO used to dissolve Thal in the variations of IL-12p70 levels observed above (Fig. 5D) .



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  		Figure 5. Thal modulates Mo-DC cytokine secretion profile in vitro. Culture supernatants from 106 Thal-exposed or control Mo-DC were harvested after 48 h of maturation with CD40L, and IL-10, IL-12p70, and TNF-{alpha} secretion was analyzed by ELISA. (A) IL-10: *, P = .005 for mature Thal10-Mo-DC versus normal, mature Mo-DC; **, P = .01 for mature Thal20-Mo-DC versus normal, mature Mo-DC. (B) TNF-{alpha}: *, P = .02 for mature Thal10-Mo-DC versus normal, mature Mo-DC; not significant for mature Thal20-Mo-DC versus normal, mature Mo-DC. (C) IL-12p70: *, P = .001 for mature Thal20-Mo-DC versus normal, mature Mo-DC; not significant for mature Thal10-Mo-DC versus normal, mature Mo-DC. (D) IL-12p70: Immature Mo-DC cultured with DMSO alone (same volume as used for Thal preparation) did not show secretion of detectable levels of IL-12p70; also, to exclude a possible interference of DMSO used to dissolve Thal in the variations of IL-12p70 levels, DMSO alone was added from the beginning of culture to mature Mo-DC activated with CD40L; not significant for mature Mo-DC versus control, mature Mo-DC cultured with DMSO. Results are represented as the mean and standard deviation obtained from 10 independent experiments for IL-10 and IL-12p70, five independent experiments for TNF-{alpha}, and three independent experiments for IL-12p70 with DMSO.

 
Dose-dependent modulation of IL-12p70 secretion by Thal
In view of the intriguing impact of Thal on IL-12p70 secretion and because of the major role of IL-12p70 in antitumor immunity and cancer immunotherapy, we also investigated whether Thal influences IL-12p70 secretion by Mo-DC in a dose-dependent manner. A Thal dose less than 5 µg/ml appeared to decrease IL-12p70 secretion by Mo-DC. In contrast, exposure of Mo-DC to Thal doses between 10 and 25 µg/ml induced a progressive and sustained increase of IL-12p70 secretion (Fig. 6 ), thus further confirming that Thal can exert a complex, dose-dependent modulation of IL-12p70 production by Mo-DC.



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  		Figure 6. Dose-dependent modulation of IL-12p70 secretion by Thal. Culture supernatants from 106 control Mo-DC or Mo-DC exposed to progressive doses of Thal were harvested after 48 h of maturation with CD40L, and IL-12p70 secretion was analyzed by ELISA. Representative experiment of three experiments from different donors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we describe a novel, immunomodulatory mechanism of action of Thal that could exert complex effects on Mo-DC. Although Thal did not block the GM-CSF plus IL-4-driven differentiation of monocytes into DC, Thal exerted a modulation of the stimulatory capacity of Mo-DC and their cytokine secretion profile. The impact of Thal on DC has not been addressed before. Previous studies highlighted inhibitory or stimulatory effects of Thal on a wide range of cellular targets. Current data suggest that the action of Thal may be related to several different mechanisms, including suppression of TNF-{alpha} production by monocytes [4 , 8 , 9 ], effects on IL-2, IL-6, IL-12, and IFN-{gamma} [6 , 9 10 11 12 ], down-regulation of selected cell surface adhesion molecules (CD11b), and shifts in the ratio of CD4+ to CD8+ lymphocytes [3 , 10 , 12 ]. Not all these studies are consistent in their findings, and there are several conundrums in the study of Thal. Indeed, in accordance with the findings we describe in this study, a wide variability has already been reported without clear explanations in the relationship between Thal and cytokines such as IL-2, IL-12, TNF-{alpha}, and IFN-{gamma}. Several studies have reported Thal-mediated increases in IL-2 [13 ] and IL-2-related phenomena, such as increased IL-2-mediated T cell proliferation [6 ] as well as elevated production of soluble IL-2 receptors [12 ]. However, there are also investigators who found no association between Thal and IL-2 [14 , 15 ]. Inconsistencies have also been found in the impact of Thal on IFN-{gamma}. The level of IFN-{gamma} was found to be elevated [6 , 12 ], decreased [9 ], or not altered at all by Thal [10 ]. Numerous reports described a Thal-mediated suppression of TNF-{alpha} [4 , 9 ]. However, it has also been found to increase plasma TNF-{alpha} levels in HIV-seropositive patients [16 ]. Finally, the production of IL-12, a critical cytokine in the induction of Th1 and antitumoral responses, was shown to be potently suppressed by Thal [11 ]. However, other studies did not observe any effect of Thal on the level of IL-12 [10 ]. For instance, Moller et al. [11 ] showed that Thal can exert an inhibitory effect on IL-12 secretion in a dose-dependent manner. In their PBMC model, Moller et al. [11 ] used a maximum dose of 3.6 µg/ml. Although the PBMC model used in previous studies cannot be phenotypically or functionally compared with the Mo-DC model, our data are consistent with data from Moller et al. [11 ], where a Thal dose less than 5 µg/ml appeared to decrease IL-12p70 secretion by Mo-DC. In contrast, exposure of Mo-DC to Thal doses between 10 and 25 µg/ml induced a progressive and sustained increase of IL-12p70 secretion, showing that Thal can exert a dose-dependent and complex effect on IL-12p70 production by Mo-DC. It is also possible that changes in the physicochemical conditions being studied in vitro or in vivo may explain the complex action of Thal, but the aforementioned studies did not specifically focus on the impact of Thal dose, which appears from our results to be a major determinant in the balance between immunosuppressive and immunostimulatory properties of Thal. As the absolute bioavailability of Thal is variable from one patient to another and has not yet been well characterized [17 ], one could assume that peak plasma concentrations can be achieved early after administration, and our in vitro observations of the effects of Thal on DC may have an in vivo counterpart. This delicate balance was already illustrated in the treatment of chronic graft versus host disease (GVHD), where Thal has been reported to be an effective agent [18 ]. However, in a prophylaxis setting, Thal demonstrated that not only was it not effective in preventing GVHD, but those patients on Thal developed a higher incidence of GVHD resulting in a higher mortality rate [19 ]. Therefore, the effects of Thal on DC may account for some of its broad spectrum of action.

Furthermore, the finding that the higher doses of Thal can increase IL-12p70 secretion by mature Mo-DC is of major interest, as the rationale for Thal use in the treatment of cancer is still unclear. Although the antiangiogenic effect and the reduction of TNF-{alpha} levels have been often hypothesized to explain the antineoplastic effects of Thal [20 ], there is some evidence that these mechanisms do not clearly account for the anticancer effects of Thal [21 , 22 ]. It has been demonstrated that the immune balance controlled by cytokines such as IL-10 and IL-12 plays an important role in immune regulation, including antitumor immunity. The Th1 cells that produce IFN-{gamma} have been shown to exert a powerful antitumor effect, whereas a weak Th1 or a Th2 profile may have an opposite effect, that is, down-regulation of innate and acquired antitumor immunity [23 ]. The corollary of these observations is that a Th1 profile may be protective against tumor growth and dissemination. IL-12 is a central regulator in stimulation of Th1 cells [24 ] and induction of protective immunity to a variety of infectious pathogens and malignancies. Also, IL-12 has antitumor effects of its own [25 ] for which it is currently under clinical evaluation.

It has been shown recently that Thal is active against advanced or refractory MM. It can induce marked and durable responses in some patients with MM, including those who relapse after high-dose chemotherapy [26 ]. Of importance, it has been shown that Thal may mediate its anti-MM effect by modulating natural killer (NK) cell number and function [27 ]. The latter is in accordance with our results, as IL-12p70 can exert NK cell proliferation and activation [28 ]. Moreover, it has been reported that MM patients receiving the highest Thal doses are likely to be the best responders to Thal therapy [29 ]. This observation further supports our results where the higher dose of Thal was associated with the highest IL-12p70 level. The effects of Thal on the secretion of IL-12p70 by mature Mo-DC and strengthening of the Th1 response by naive T cells might account, at least in part, for the antitumor effects of Thal. Recently, using an animal model, Dredge et al. [30 ] assessed the ability of a Thal analog to prime a tumor-specific immune response following tumor cell vaccination. In this study, the presence of a Thal analog during the priming phase strongly enhanced antitumor immunity. Protection was associated with tumor-specific production of IFN-{gamma} by CD8+ and CD4+ splenocyte fractions. Coculture of naive splenocytes with anti-CD3 mAb in the presence of a Thal analog increased Th1-type cytokines, demonstrating that Thal and its analogs can prime protective Th1-type responses in vivo [30 ], further supporting the results of our study.

Collectively, this is the first study on the effects of Thal on DC giving new insights into the action of Thal. Although the molecular events leading to the effects of Thal on DC function remain to be resolved, DC appear to be potential targets for Thal. Our study provides a framework for the development and testing of DC/Thal-based immunotherapeutic strategies under stringent biological and cellular monitoring.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Fondation de France (FDF) and from the "Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC), Paris, France (to M. M.). We thank S. Just-Landi and N. Baratier for excellent technical assistance; N. Vey, C. Faucher, and D. Coso (Institut Paoli-Calmettes) for helpful discussions; and S. Guerder, L. Leserman (Centre d’Immunologie Luminy, Marseille), D. Maraninchi (Institut Paoli-Calmettes), and C. Mawas (INSERM U119) for their critical reading of the manuscript.

Received April 14, 2002; revised July 26, 2002; accepted July 30, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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