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(Journal of Leukocyte Biology. 2001;70:941-949.)
© 2001 by Society for Leukocyte Biology

T-cell-conditioned medium efficiently induces the maturation and function of human dendritic cells

Kazunori Kato*, Yoichi Takaue2 and Hiro Wakasugi*

* Pharmacology Division, National Cancer Center Research Institute, and
{dagger} Department of Medical Oncology, National Cancer Center Hospital, Tokyo, Japan

Correspondence: Kazunori Kato, Ph.D., Section Head, Pharmacology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan. E-mail: kakato{at}gan2.ncc.go.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We present evidence that T-cell-conditioned media (TCCM) can efficiently induce human immature dendritic cells (DC) to express high levels of immune accessory molecules commonly found on mature DC. TCCM prepared from cell-free supernatants of anti-CD3-activated T cells contained several soluble factors including CD40-ligand (sCD40L), TNF-{alpha}, and IFN-{gamma}. In contrast to moderate up-regulation of costimulatory molecules by the addition of individual cytokines or monocyte-conditioned medium, treatment of immature DC with TCCM induced a marked increase in the expression of costimulatory molecules in a dose-dependent manner. The ability of TCCM to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for CD40L, TNF-{alpha}, and IFN-{gamma}, indicating that these factors present in TCCM are mainly implicated in the maturation of DC. Importantly, TCCM-treated DC can produce significantly higher levels of IL-12 and are highly effective stimulators in allogenenic and autologous mixed-lymphocyte reactions. Overall, these findings show that cultivation with TCCM is an efficient approach for the induction of mature DC that should be useful in eliciting antigen-specific immune responses against cancer and viruses.

Key Words: antigen presentation • costimulatory molecules • T lymphocytes • cellular activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) belong to a family of antigen presenting cells (APC) that play a crucial role in the regulation of cellular and humoral immune responses [1 , 2 ]. DC are unique in their ability to recruit naive immune effector cells and to stimulate memory effector cells. This feature has prompted many studies of antigen-loaded DC in the initiation and maintenance of immunity against cancer and infectious diseases [3 4 5 ]. Recent studies revealed that there are at least three different subsets of human DC that differ in phenotype, function, and localization in the microenvironment [6 , 7 ]. It is also known that distinct DC subsets elicit distinct T-helper responses mediated by interferon-{gamma} (IFN-{gamma})-producing Th1 or interleukin (IL)-4-producing Th2 cells [8 , 9 ]. However, much information is derived from the study of DC in vitro because of their rarity in vivo. Previously, methods have been described to differentiate DC from CD14+ peripheral blood monocytes, CD34+ peripheral blood stem cells, or cord blood by in vitro culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. The cultured DC exhibit phenotypic and functional features typical of the immature stages of DC differentiation. These immature DC can efficiently capture specific antigens, soluble antigens, and apoptotic cells via phagocytosis and pinocytosis; however, they express low levels of major histocompatibility complex (MHC) class II molecules and immune accessory molecules. Furthermore, stimulation with immature DC leads to the development of IL-10-producing, nonproliferating T cells [10 ]. To become potent APC, the immature DC need to be activated by stimulation that promotes their maturation and differentiation.

Exposure to appropriate stimulation, such as bacteria, microbial products [lipopolysaccharide (LPS)], or cytokines [tumor necrosis factor {alpha} (TNF-{alpha}; IL-1-ß)], converts immature DC into mature DC that express high levels of MHC molecules, costimulatory molecules (i.e., CD54, CD80, and CD86), and activation/maturation markers (i.e., CD25, CD69, and CD83) [4 , 5 , 11 ]. Previously, Reddy et al. [12 ] revealed that culture of immature DC with monocyte-conditioned medium (MCM) containing TNF-{alpha}, IL-1ß, IL-6, and IFN-{alpha} could promote the maturation of DC. Subsequently, the mature DC interact with T cells, receiving signals that induce their terminal differentiation. A critical surface molecule is the ligand for CD40 (CD40L; CD154), a member of the TNF family [13 ]. This molecule is expressed transiently on CD4+ T cells within 4 h of ligation with T-cell receptor CD3 complex and then is rapidly down-modulated. Expression of CD40L allows activated T cells to stimulate CD40-bearing B cells, monocytes, and DC to express immune accessory molecules that are important in cognate cell-cell interactions [14 15 16 ]. We have shown that gene transfer of the CD40L causes CD40+ leukemia B cells to become professional APC that are capable of inducing cytotoxic T cells specific for autologous leukemia cells in vitro and in vivo [17 , 18 ]. Furthermore, CD40 ligation on DC results in secretion of cytokines, especially IL-12, that is important in the initial induction of innate and adaptive immunity [19 ]. Thus, CD40-CD40L interactions play a more general role in immune regulation.

Conversely, soluble proteins released from activated T cells and monocytes may contribute to immune activation [15 , 20 ]. TNF-{alpha}, for example, is a protein that can exist as a soluble molecule or as a membrane-associated glycoprotein. Either form of the protein can augment B-cell expression of CD80 and other immune co-stimulatory molecules and cause polyclonal B-cell activation [21 ]. Similar to TNF-{alpha}, we and others [16 , 22 23 24 ] recently found that activated CD4+ T cells not only express the membrane form of CD40L but also produce a soluble form, sCD40L, in vitro and in vivo. Importantly, cell-free supernatants from activated T cells contain sCD40L that can induce CD40-bearing cells to express enhanced levels of the important costimulatory molecules, CD80 and CD86, indicating that sCD40L has biologic activity [23 ]. For these reasons, in this study we examined whether cell-free supernatants from anti-CD3-activated T cells [designated T-cell conditioned medium (TCCM)] that might contain various soluble factors, such as sCD40L, TNF-{alpha}, and IFN-{gamma}, could induce the terminal differentiation of immature DC to professional APC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Human peripheral blood mononuclear cells (PBMCs) were purified from heparin-treated blood from normal healthy donors via density-gradient centrifugation in Histopaque 1077 (Sigma Chemical Co., St. Louis, MO). Monocytes were purified from PBMCs using MACS CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The bead-bound cells were analyzed by flow cytometry and found to be composed of >90% CD14+ monocytes. To isolate T cells, negative cells passed through magnetic column were subsequently incubated with MACS CD19 Microbeads to deplete B cells. Magnetic depletion of bead-unbound cells resulted in >90% pure CD3+ T cells, as determined by flow cytometry.

Preparation of TCCM
TCCM was prepared as follows: Purified and sterilized monoclonal antibodies (mAbs) specific for human CD3 (5 µg/ml), OKT3 (Ortho Biotech, Raritan, NJ), Leu-4 (BD Biosciences, San Jose, CA), UCHT1 (BD PharMingen, San Diego, CA), and NU-T3 (Nichirei Co., Tokyo, Japan) were distributed onto six-well plates and incubated overnight at 4°C. After washing three times, freshly isolated peripheral T cells were added at 1 x 106 cells/well to anti-CD3-coated plates and cultured in RPMI 1640 culture medium. After 2 d, cell-free supernatants were collected and stored at -80°C before use as TCCM. Alternatively, T cells were cultured without stimulation with anti-CD3, and supernatants were collected as negative controls for TCCM. In addition, cell-free supernatants were collected at various times and tested for cytokine concentrations.

Preparation of MCM
MCM was prepared as previously described [12 ]. Briefly, human {gamma}-globulin (Venilon, Kaketsuken, Kumamoto, Japan) at 10 mg/ml was distributed onto six-well plates for 4 h at 4°C. After washing three times with phosphate-buffered saline (PBS), plastic adherent monocytes were added at 1 x 106 cells/well to {gamma}-globulin-coated plates and were cultured in RPMI 1640 culture medium. After 1 d, cell-free supernatants were collected and stored at -80°C before use as MCM.

Generation of monocyte-derived dendritic cells
Monocytes (approximately 5x105 cells/ml) were cultured in six-well tissue-culture plates (Corning, Corning, NY) in RPMI 1640 culture medium [containing 1 mM sodium pyruvate, antibiotics, and 10% fetal calf serum (FCS) or 2% autologous human serum] supplemented with recombinant human GM-CSF (10 ng/ml; donated by Kirin Brewery Co., Gunma, Japan) and IL-4 (10 ng/ml; PeproTech, Rocky Hill, NJ). Endotoxin levels in all reagents were quite low (<1.0 EU/ml). On day 3, the cultures were fed with fresh RPMI 1640 medium and cytokines. On day 7, nonadherent cells were harvested by gentle pipetting and transferred to new plates. The cultures were supplemented with TCCM (5–50% vol/vol), MCM (5–50% vol/vol), TNF-{alpha} (200 pg/ml, PeproTech), sCD40L (200 pg/ml), and IFN-{gamma} (5 ng/ml, PeproTech). On day 9, monocyte-derived DC were collected, and cell differentiation was monitored by phase-contrast light microscopy, flow cytometry, and mixed lymphocyte reaction (MLR). Biologically active sCD40L was prepared from cell-free supernatants of HeLa cells transfected with the full-length human CD40L (kindly provided by Dr. Thomas J. Kipps, University of California, San Diego, CA) as described previously [24 ]. Neutralization antibodies specific for human CD40L (TRAP-1; eBioscience, San Diego, CA), TNF{alpha}- (MP9-20A4; Caltag Laboratories, Burlingame, CA), or IFN-{gamma} (B27; Caltag) were used at a concentration of 5 µg/ml.

Enzyme-linked immunosorbent assay (ELISA) for cytokines
Human cytokines such as IFN-{gamma}, TNF-{alpha}, IL-4, IL-10, and IL-12 were measured by ELISA (Quantikine®, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The levels of sCD40L in TCCM were measured by an ELISA, which uses two noncross-blocking CD40L mAbs (clones TRAP-1 and M90) as described previously [24 ]. For this, polystyrene microtiter plates (Corning) were coated with TRAP-1 at 3 µg/ml in 10 mM phosphate buffer (pH=9.0). The plates were washed with PBS and treated for 2 h at room temperature with blocking buffer (10% FCS and 0.01% sodium azide in PBS). The plates were washed four times with wash buffer (0.05% Tween-20 in PBS) and then incubated overnight at 4°C with human plasma diluted in blocking buffer. For a standard curve, we used a recombinant sCD154 obtained from Bender MedSystems (Vienna, Austria). Plates were washed four times with wash buffer and then incubated for 1 h with biotinylated M90 mAb at 3 µg/ml in blocking buffer. Washed plates were treated with avidin and then biotinylated horseradish peroxidase (biotin-HRP; Elite VectastainTM, Vector Laboratories, Burlingame, CA). After washing with blocking buffer, plates were allowed to react with substrate 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The optical density at 450 nm was determined using an ELISA microplate reader (Molecular Devices, Menlo Park, CA).

Flow cytometry
DC were washed and then suspended in staining media (SM), consisting of RPMI-1640, 3% FCS, 0.05% NaN3, and 1 µg/ml propidium iodide (PI) with saturating amounts of fluorochrome-conjugated mAbs specific for human HLA-ABC, CD16, CD54, CD80, CD86 (BD Pharmingen), HLA-DR, CD1a, CD14, CD25, or CD83 (Immunotech, Marseille, France). After 30 min at 4°C, the cells were washed with SM and analyzed via flow cytometry using a FACSCalibur® (Becton Dickinson, San Jose, CA). Dead cells staining with PI was excluded from the analyses. The relative expression of surface antigen is described as the mean fluorescence intensity ratio (MFIR) or as overlaid histograms. MFIR equals the MFI of cells stained with a fluorochrome-conjugated antigen-specific mAb divided by the MFI of cells stained with a fluorochrome-conjugated isotype control mAb (Immunotech).

MLR
An allogeneic MLR (allo-MLR) was performed using a protocol modified from one previously described [17 ]. After cultivation with or without TCCM or MCM, DC were harvested and incubated with mitomycin-C (Sigma Chemical Co.) at a concentration of 40 µg/ml for 1 h at 37°C to inhibit proliferation. These cells were then washed three times and plated into 96-well U-bottom culture plates (Corning) at various cell concentrations for use as stimulators cells. Purified normal T cells were added at 2 x 105 cells/well in a final total volume of 200 µl, and plates were incubated at 37°C in 5% CO2. After 2 d, cell-free supernatants were collected and tested for human IFN-{gamma} by ELISA as described above. After 4 d, the proliferative response was measured by alamarBlueTM (BioSource International, Canarillo, CA) assay according to the manufacturer’s instructions.

For the autologous antigen-specific MLR, DC and T cells were isolated from cytomegalovirus (CMV)-seropositive individuals. CD14+ monocytes were cultured with GM-CSF and IL-4 for 6 d and pulsed for 1 d with complement fixation antigens of CMV (CF-CMV) or control antigens (CF-control) purchased from Denka Seiken Co. (Tokyo, Japan). After washing three times, antigen-pulsed DC were incubated with (20% vol/vol) or without TCCM for 2 d and then cultured with autologous T cells at various responder/stimulator (R/S) ratios. After 2 d, cell-free supernatants were collected and tested for human IFN-{gamma} by ELISA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytokines in TCCM
Activation through the T-cell receptor/CD3 complex with anti-CD3 mAbs has been known to mimic the stimulation of T cells by mitogens and antigens. To investigate the agonistic effect of mAbs specific for CD3 on the induction of T-cell activation, we first examined cytokine production by human peripheral blood T cells stimulated with four available anti-CD3 mAbs: OKT3, Leu-4, UCTH1, and NU-T3. As shown in Figure 1 A , T cells were induced to produce significantly larger amounts of sCD40L (>1000 pg/ml) and IFN-{gamma} (>15,000 pg/ml) when cultured on OKT3- or NU-T3-immobilized plates (P<0.01; Bonferroni t-test) than when cultured on Leu-4- or UCTH1-immobilized plates. By contrast, significant levels of sCD40L and IFN-{gamma} were not detected in the supernatant of unstimulated T cells (unpublished results).



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  		Figure 1. Production of soluble factors by human T cells activated with anti-CD3 mAbs. (A) Human peripheral T cells (1x106 cells/ml) were cultured with various immobilized anti-CD3 mAbs, as indicated. Cell-free supernatants were collected after the stimulation of each cell culture for 48 h and tested for concentrations of human sCD40L and IFN-{gamma} by ELISA. Data represent the mean ± SE of triplicate wells. (B) Time course of cytokine production in TCCM. T cells were stimulated with OKT3 for the times indicated on the abscissa. The calculated concentrations of sCD40L, IFN-{gamma}, and IL-10 in TCCM are listed on the ordinate, and each point represents the mean of triplicate wells.

 
We next assessed the kinetics of production of sCD40L, IFN-{gamma}, and IL-10 during activation of T cells with immobilized OKT3. As shown in Figure 1B , the production of sCD40L and IFN-{gamma} was significantly detected in TCCM at 24 h after anti-CD3 stimulation and then maximally expressed after 48 h, and IL-10 reached maximal expression by 72 h.

We then quantified the levels of soluble factors in TCCM that were prepared from T cells of three individuals with or without anti-CD3 stimulation for 48 h. Consistent with data shown in Figure 1 , approximately 1000 pg/ml of sCD40L was detected in the TCCM from anti-CD3-activated T cells (Table 1 ). Furthermore, high levels of IFN-{gamma} (20–60 ng/ml) and TNF-{alpha} (650–1360 pg/ml) were present in TCCM, whereas control TCCM from nonstimulated T cells did not contain detectable levels of sCD40L, IFN-{gamma}, or TNF-{alpha} (<100, <80, and <40 pg/ml, respectively). In addition, TCCM had low or undetectable levels of IL-4 or IL-10, which are known to be immunosuppressive cytokines. Accordingly, TCCM was prepared from the cell-free supernatants of OKT3-activated peripheral T cells at 48 h.


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  		Table 1. Cytokine Concentration in T-Cell-Conditioned Medium

 
TCCM induces immune accessory molecules on DC
Mature DC are typically characterized by the expression of high levels of MHC molecules, costimulatory molecules, and maturation markers. To determine the effect of TCCM on the induction of mature DC, we cultured monocyte-derived immature DC with or without TCCM for an additional 2 d. In contrast to immature DC in the absence of TCCM (Fig. 2 , top histograms), DC cultured with TCCM (20% vol/vol) expressed high levels of MHC molecules (i.e., HLA-ABC), adhesion/costimulatory molecules (i.e., CD54 and CD86), and activation/maturation markers (i.e., CD25 and CD83; Fig. 2 , bottom histograms). Conversely, control TCCM that was prepared from nonstimulated T cells did not up-regulate the expression of these immune accessory molecules on DC (Fig. 2 , middle histograms). Furthermore, we found that the addition of TCCM to immature DC resulted in the down-regulation of CD16 and receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (F-DEX), indicating that TCCM could convert immature DC into mature DC. We also observed significant differences in the morphology and homotypic aggregation of TCCM-treated DC (unpublished results). Cell yield at day 9 was not significantly different in DC with or without TCCM (unpublished results). These results were consistent for DC of each of three individuals tested.



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  		Figure 2. Changes in surface-antigen phenotype of DC treated with TCCM. Human peripheral blood monocytes were cultured with GM-CSF and IL-4 for 7 d and further with medium alone (top), control TCCM (20%, middle), or the TCCM of activated T cells (20%, bottom) for 2 d. DC were examined for surface expression of HLA-ABC, CD54, CD86, CD25, CD83, CD16, and CD14 by flow cytometry. Incorporation of FITC-conjugated dextran (F-DEX) was also assessed by flow cytometry. Open histograms represent staining of DC with fluorochrome-conjugated isotype-control mAb, and shaded histograms represent staining of DC with FITC-conjugated specific mAb.

 
We next examined the dose-response of TCCM on the induction of the immune accessory molecules CD25, CD83, and CD86. As shown in Figure 3 , maximal induction of these molecules occurred with high concentrations of TCCM. However, cell viability and yield decreased in DC treated with 50% TCCM compared with that with 25% TCCM (52% vs. 74%). Given these results, we decided to use a maximal 25% of TCCM for the induction of maturation in DC.



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  		Figure 3. Dose-dependent phenotypic changes in DC treated with TCCM. Immature DC were cultured for 2 d with TCCM at various concentrations as indicated on the abscissa. DC were examined for the expression of surface markers by flow cytometry. The MFIR, comparing the fluorescence intensity of DC stained with fluorochrome-conjugated CD25, CD83, and CD86 with the same stained with a fluorochrome-conjugated isotype-control mAb at each point, is represented by {circ}, {square}, and •, respectively. The scales are depicted on the ordinate, both sides. Viability of DC after cultivation with various concentrations of TCCM was determined by staining with PI and indicated in the figure.

 
Molecules in the TNF family including TNF-{alpha} and CD40L contribute to the maturation of DC. In addition, it has been shown that MCM was more effective than defined cytokines in mediating the maturation of DC [12 ]. We next examined the induction of immune accessory molecules by individual cytokines, MCM and TCCM. Compared with immature DC cultured with GM-CSF plus IL-4 only, the addition of cytokine mixtures [including sCD40L (200 pg/ml), TNF-{alpha} (200 pg/ml), and IFN-{gamma} (5 ng/ml)] or MCM (20% vol/vol) could up-regulate the expression of immune accessory molecules on DC (MFI CD80: 9 vs. 34 and 69; CD83: 5 vs. 13 and 23; CD86: 11 vs. 36 and 54; HLA-DR: 52 vs. 187 and 284; CD1a: 9 vs. 11 and 14; Fig. 4 , middle histograms). It is important that the addition of 20% TCCM markedly increased the percentage of mature DC expressing high levels of costimulatory molecules and maturation markers (MFIR CD80: 83; CD83: 46; CD86: 114; HLA-DR: 387; CD1a: 23; Fig. 4 , bottom histograms). Remarkably, these phenotypic changes in DC were induced even by the addition of only 5% of TCCM but not by the addition of equal volume of MCM (unpublished results). Similar phenotypic changes were observed in TCCM-treated DC cultured in medium supplemented with autologous human serum.



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  		Figure 4. Induction of surface-antigen phenotype of DC treated with cytokines, MCM and TCCM. Immature DC were cultured for 2 d with cytokine mixtures including sCD40L, TNF-{alpha} plus IFN-{gamma}, MCM (20%), or TCCM (20%). Expression of CD80, CD83, CD86, HLA-DR, and CD1a was assessed by flow cytometry. Open and shaded histograms represent staining of DC with fluorochrome-conjugated isotype-control or -specific mAb, respectively. Calculated, the MFI values of each DC are indicated.

 
To examine whether soluble factors produced by activated T cells contribute to changes in DC surface phenotype, we added neutralizing mAbs specific for these cytokines to DC cultures in the presence of TCCM. The ability of TCCM to induce expression of CD86 could be inhibited by anti-CD40L, anti-TNF-{alpha}, or anti-IFN-{gamma} mAbs (Fig. 5 ). The mixture of these mAbs almost completely abrogated the changes in DC phenotype. Collectively, these results imply that TCCM from activated T cells can efficiently induce the expression of immune accessory molecules on DC and that this activity is mainly mediated by sCD40L, TNF-{alpha}, and IFN-{gamma}.



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  		Figure 5. Inhibition of CD86 induction in TCCM-treated DC by anti-cytokine-neutralizing mAbs. Immature DC were cultured for 2 d with TCCM (12%) in the presence (5 µg/ml) or absence of irrelevant control mAb or mAbs specific for CD40L, TNF-{alpha}, and IFN-{gamma}. Horizontal bars represent MFI of CD86 expression.

 
Allogeneic mixed T-cell reaction by TCCM-treated DC
Although previous studies showed that MCM, which contains several factors including TNF-{alpha}, IL-1ß, IL-6, and IFN-{alpha}, consistently induced the maturation of DC, we were interested in comparing the effects of TCCM and MCM on DC maturation. IL-12 heterodimer p70 (IL-12 p70) is produced in large amounts by mature, not by immature, DC and therefore is a suitable factor to determine DC maturation. As shown in Figure 6 A , DC stimulated with TCCM could produce significantly higher levels of IL-12 p70 (205 pg/ml) than those stimulated without or with MCM (<5 and 47 pg/ml, respectively; P<0.01; Bonferroni t-test). To determine whether these phenotypic changes and cytokine production correlated with APC function, we next examined the function of MCM- or TCCM-treated DC as stimulator cells in allogeneic MLR. Responder T cells from a nonrelated donor were co-cultured with DC pretreated without or with MCM and TCCM. Four days after the initiation of the MLR, we assessed the proliferatve response by a colorimetric growth indicator based on detection of metabolic activity. As shown in Figure 6B , TCCM-treated DC were able to stimulate allogeneic T cells to proliferate to a much greater extent than control cultures of nontreated DC, MCM-treated DC, or responder T cells alone. Based on the findings that TCCM-treated DC produce high levels of IL-12 p70, we next examined the concentration of IFN-{gamma} in the culture supernatants of MLR by ELISA. The supernatants of the MLR stimulated with TCCM-treated DC contained significantly higher levels of IFN-{gamma} (80 ng/ml) than that of MLR cultures with non- or MCM-treated DC (0.5 and 5 ng/ml, respectively; Fig. 6C ). These significantly higher productions of IFN-{gamma} by responder T cells stimulated with TCCM-treated DC were observed in all R/S ratios (P<0.01; Bonferroni t-test; Fig. 6D ). The supernatants of responder T cells alone or of stimulator DC alone did not contain detectable amounts of IFN-{gamma} (<0.1 pg/ml).



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  		Figure 6. Allogeneic and autologous T-cell responses induced by TCCM-treated DC. (A) Production of IL-12 p70 by DC treated with TCCM. Immature DC were cultured for 2 d without or with MCM or TCCM (20% vol/vol). Cell-free supernatants were collected and tested for the concentration of human IL-12 p70 by ELISA. Horizontal bars represent the mean concentration of triplicate wells, and error bars depict the standard error. (B) Allogeneic T-cell response induced by MCM- and TCCM-treated DC. Allogeneic T cells (2x105) were cultured in a 96-well microplate with 2 x 104 various stimulator DC as described. Wells were supplemented with alamarBlue for the final 12 h of a 4-d culture. Proliferative index is presented on the abscissa according to the manufacturer’s instruction. (C) The concentration of IFN-{gamma} in the supernatants of MLR after 2 d is indicated on abscissa. Error bars denote the standard error about the mean concentration of triplicate wells. (D) Allogeneic T cells (2x105 cells/well) as responder cells were cultured with various numbers of DC pretreated with (•) or without ({circ}) TCCM as a stimulator. (E) Antigen-specific autologous T-cell responses induced by TCCM-treated DC. Immature DC were pulsed with CMV antigen (•, {blacksquare}) or control antigen ({circ}, {square}) for 1 d. After washing, DC were treated with TCCM (squares) or control media (circles) for an additional 2 d. Autologous T cells (2x105 cells/well) were cultured with various numbers of DC as indicated on the abscissa. The concentration of IFN-{gamma} in the supernatants after 48 h of culture is indicated on the ordinate, and each point represents the mean ± SE of triplicate wells.

 
Autologous T-cell response to virus antigen presented on TCCM-treated DC
T cells isolated from the same donor were examined for their ability to respond to DC pulsed with recall virus antigen. Immature DC, prepulsed with CMV or control antigens, were grown with or without TCCM for 2 d and then co-cultured with autologous T cells for an additional 2 d. As shown in Figure 6E , the supernatants from autologous MLR cultures stimulated with control DC and pulsed with control antigens ({circ}) contained negligible amounts of IFN-{gamma} (<100 pg/ml). We detected modest production of IFN-{gamma} (<10,000 pg/ml) in cultures with control DC pulsed with CMV (•) or TCCM-treated DC with control antigen ({square}) in an R/S ratio-dependent manner. By contrast, TCCM-stimulated DC pulsed with CMV ({blacksquare})-induced autologous T cells to produce significantly more IFN-{gamma} than any of the other cultures at R/S ratios of 4 and 8 (P<0.05; Bonferroni t-test). Collectively, these results correlated with the findings that TCCM could induce immune accessory molecules on DC, indicating that functionally mature DC as professional APC could be efficiently generated by culturing with TCCM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we developed an efficient approach for the induction of phenotypically and functionally differentiated DC using TCCM, which contains high levels of biological active forms of sCD40L, TNF-{alpha}, and IFN-{gamma}.

CD40L ordinarily exists as a 39-kDa type II membrane glycoprotein [13 ]. It is known that the membrane form of CD40L is expressed transiently by activated T cells and subsequently cleaved from the cell surface, releasing a soluble fragment, sCD40L, of 18–20 kDa [16 , 23 , 24 ]. As with TNF-{alpha} and lymphotoxin-ß (LT-ß), sCD40L retains its ability to form trimers, to bind CD40, and to deliver biological signals, indicating that sCD40L also acts as a cytokine. sCD40L can also induce B-cell activation or differentiation. Moreover, several in vitro studies have revealed that sCD40L or an sCD40L chimeric protein can induce APC to express immune accessory molecules (i.e., CD80, CD83, and CD86) and cytokines (i.e., IL-12, IFN-{alpha}, and TNF-{alpha}) in a manner similar to that of the membrane-bound form of CD40L on activated T cells [16 , 19 , 25 ]. Previously, we found that culture supernatants of anti-CD3-activated T cells or HeLa cells transfected with the full-length human CD40L could induce CD40-positive leukemia B cells to express high levels of CD54, CD86, and CD95 in a CD40-CD40L dependent manner [24 ]. In addition, other cytokines such as TNF-{alpha} and IFN-{gamma}, which are also secreted by activated T cells, are involved in the up-regulation of adhesion molecules and costimulatory molecules on a variety of tumor cells. However, it has not been examined whether supernatants of activated T cells can act as a substitute for T cells during DC maturation. Reddy et al. [12 ] proposed that MCM was more effective than defined cytokines in mediating the maturation of DC. MCM contains several cytokines including TNF-{alpha}, IL-1ß, IL-6, and IFN-{alpha}, which are important for immature DC to become active. Active DC are effective at stimulating the proliferation of naive CD4 T cells but rarely secrete IL-12 as a key cytokine for the differentiation of Th1 cells. From the MCM results, we were struck with the idea that supernatant from activated T cells (designated TCCM) might be a strong candidate for inducing terminal maturation of DC.

To TCCM, we stimulated monocyte-depleted peripheral T cells with immobilized anti-CD3 mAb under various conditions. It is interesting that stimulation with two mAbs, OKT3 and NU-T3, which are both mouse immunoglobulin (Ig)G2a isotype, could induce T cells to produce significantly more sCD40L and IFN-{gamma} than treatment with the other two mAbs, Leu-4 and UCTH1, which are mouse IgG1 isotype. The differences in levels of cytokine production are certainly a result of differences in affinity, epitope, and isotype of these mAbs. Because contamination with endotoxin or micoplasma can trigger the maturation of DC [11 , 26 ], we used the OKT3 mAb, which is available as a clinical grade, endotoxin-free reagent for the production of TCCM. As represented in Figure 1B , extremely high levels of sCD40L (1500 pg/ml) and IFN-{gamma} (>10,000 pg/ml) were detected in TCCM at 48 h of stimulation. By contrast, IL-10 was maximally secreted at 72 h of stimulation. Previous studies have revealed that culture supernatants of some tumor cells or regulatory T cells containing IL-10 are able to down-regulate the expression of costimulatory molecules and secretion of IL-12, indicating that IL-10 is significantly involved in the inhibition of DC maturation and APC function [27 , 28 ]. Furthermore, we found increased levels of lactate dehydrogenase derived from dead cells and acidification in TCCM after 72 h (unpublished results). We harvested supernatants of OKT3-activated T cells at 48 h for use as TCCM.

We found several potential advantages to using TCCM for the induction of mature DC compared with the use of individual cytokines or MCM. First, it is possible to collect a large volume of TCCM (10 ml from 15 ml whole blood), greater than the volume of MCM derived from same volume of peripheral blood (1 ml). Furthermore, the ability of TCCM to induce the expression of costimulatory molecules on DC is superior to that of MCM (shown in Figs. 4 and 6 ). As a substitution for MCM, Jonuleit and colleagues [29 ] previously demonstrated that pro-inflammatory cytokines (i.e., TNF-{alpha}, IL-1, and IL-6) and prostaglandins induce maturation of immunostimulatory DC under FCS-free conditions. We also found similar phenotypic changes in TCCM-treated DC under FCS-free medium containing autologous human serum. Second, TCCM reproducibly contains high levels of sCD40L, which has biologic activity. In our previous findings, TCCM could induce CD40-positive B cells to express immune accessory molecules in a CD40/CD40L-dependent manner [17 ], indicating that sCD40L in TCCM is biologically active. Similar to the processing of TNF-{alpha} or Fas ligand, the release of sCD40L requires a matrix metalloproteinase(s) that is induced on activated lymphocytes [24 ]. Although sCD40L released from activated T cells apparently forms homotrimers, lyophilized recombinant human CD40L protein derived from Escherichia coli forms primarily monomers, exhibiting only faint biological activity to stimulate CD40-positive cells. Third, TCCM, but not MCM, contains high levels of IFN-{gamma}. Recently, Ito et al. [30 ] demonstrated that IFN-{gamma} was the most potent cytokine for enhancing the maturation of CD11c+ myeloid DC, supporting our result that TCCM is effective on the maturation of DC. Forth, conditioned medium that is generated by stimulating T cells with anti-CD3 mAb may contain other candidate factors for modulating DC activity. Recently, it was shown that new members of the TNF family TRANCE/RANKL (TNF-related activation-induced cytokine/receptor activator of nuclear factor kappa B ligand) could augment the ability of DC to stimulate naive T cells and promote their survival [31 , 32 ]. Notably, this factor is selectively expressed in activated and memory T cells, raising the possibility that TCCM might contain soluble versions of this molecule. Further studies are required to determine whether the unknown factors in TCCM contribute to the phenotypic and functional maturation of DC.

In an attempt to elicit anti-tumor immunity, numerous tumor cell-, protein-, and gene-based strategies have been devised for priming DC to present tumor-associated antigens (TAA) [5 , 33 , 34 ]. It is known that mature DC are the only cells that efficiently present virus antigen and TAA [35 , 36 ]. Several murine studies have shown the successful elimination of established tumors using DC pulsed with TAA [36 , 37 ]. In humans, vaccinations using DC pulsed with TAA were shown to be effective for patients with B-cell lymphoma and melanoma [38 , 39 ]. Nestle et al. [39 ] have shown that vaccination with TAA-loaded DC activated with keyhole limpet hemocyanin as a helper antigen resulted in the induction of TAA-specific cytolytic T lymphocyte (CTL) responses in some melanoma patients. Furthermore, induction of a CTL response after vaccination with HER-2/neu-pulsed mature DC was observed in patients with breast and ovarian cancer [40 ]. Accordingly, TAA-pulsed DC stimulated to mature by TCCM appear capable of inducing a host anti-tumor immune response that may be therapeutic. We are undergoing studies to examine whether an autologous T-cell response can be generated by TCCM-treated DC pulsed with TAA, such as MART-1 and SART-1. Furthermore, we found that murine-derived TCCM could induce murine bone marrow-derived DC to express high levels of MHC and costimulatory molecules (data to be presented elsewhere). To examine the anti-tumor effect elicited by TCCM-treated DC, an in vivo study of tumor-bearing mice is also in progress.

Alternatively, engineering human myeloid and lymphoid leukemia cells to provide APC function could potentially result in polyvalent immunization to multiple tumor antigens [17 , 41 42 43 ]. These leukemic DC expressing costimulatory molecules and producing IL-12 were potent stimulators of CTL specific for autologous leukemia cells. To extend these findings into an efficient strategy for the induction of anti-leukemia immune response, we examined whether the exposure of leukemia cells to TCCM could induce the morphological and phenotypic changes that were observed in DC. Culturing the leukemia cell lines Ramos (B-cell lymphoma) and THP-1 (monocytic leukemia) with TCCM or mononuclear cells of chronic myelogenous leukemia patients with GM-CSF, IL-4, and TCCM significantly induced the expression of a variety of immune accessory molecules such as MHC class II, CD80, CD83, and CD86 (unpublished results). We are now investigating allogeneic and autologous T-cell responses induced using TCCM-treated leukemia cells such as APC. In conclusion, TCCM may be more efficient at inducing professional APC from monocyte-derived DC and leukemia cells, allowing us to consider this approach for the immunotherapy of patients with cancer.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grants-in-Aid for Scientific Research on Priority Areas "Cancer" from the Ministry of Education, Science, Culture, Sports and Technology of Japan, by a Grants-in-Aid from ONO Medical Research Foundation (to K. K.), by a Grants-in-Aid for Second Term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare, by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, and by the Foundation for Promotion of Cancer Research in Japan (to Y. T. and H. W.). We thank Drs. Thomas J. Kipps (UCSD, CA), Masao Takei (Kirin Brewery Co.), and Shigehiro Sato (Iwate Medical University, Morioka, Japan) for their helpful discussions. We also thank Drs. Rumiko Asada-Mikami, Yoshiko Inoue, Yukie Harada, Atsushi Yasumoto, Nathalie Allain, and Sachiyo Hatada for their technical assistance.

Received April 23, 2001; revised August 18, 2001; accepted August 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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