* Laboratoire d’Immunologie des Tumeurs and
Département d’Hématologie, Institut Paoli-Calmettes, Université de la Méditerranée, Marseille, France; and
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
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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
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(TNF-), 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-, 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.
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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-, 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-
(IFN-)-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.
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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.
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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- (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- and IL-10 cytokines were measured by intracellular staining. (B) % of IFN--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.
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. 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- 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- 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-: *, 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-, and three independent experiments for IL-12p70 with DMSO.
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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.
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production by monocytes [4
, 8
, 9
], effects on IL-2, IL-6, IL-12, and IFN- [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-, and IFN-. 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-. The level of IFN- 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- [4
, 9
]. However, it has also been found to increase plasma TNF- 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- 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- 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- 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.
Received April 14, 2002; revised July 26, 2002; accepted July 30, 2002.
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