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. 2003 Sep 10;53(1):53–61. doi: 10.1007/s00262-003-0419-2

Combination of monocyte-derived dendritic cells and activated T cells which express CD40 ligand: a new approach to cancer immunotherapy

Takami Sato 1,✉, Mizue Terai 1, Rie Yasuda 2, Ryuko Watanabe 2, David Berd 1, Michael J Mastrangelo 1, Kenichiro Hasumi 2
PMCID: PMC11032856  PMID: 13680192

Abstract

Interactions between dendritic cells (DCs) and activated T cells are critically important for the establishment of an effective immune response. To develop the basis for a new DC-based cancer vaccine, we investigated cell-to-cell interactions between human monocyte-derived DCs and autologous T cells that are activated to express the CD40 ligand (CD40L). Peripheral blood monocytes were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) to induce differentiation of DCs. Activated T cells (ATs) consisted of autologous peripheral blood lymphocytes that had been activated with phytohemagglutinin (PHA) and then stimulated with calcium ionophore to up-regulate expression of CD40L. Coculture of these DCs and ATs induced significant production of interleukin 12 (IL-12) and also enhanced the production of interferon γ (IFN-γ). The production of IL-12 was blocked by an anti-CD40L antibody or by separation of the DC and AT fractions by a permeable membrane. Furthermore, coculture of DCs and ATs induced DCs to upregulate CD83 expression and stimulated migration of DCs toward the macrophage inflammatory protein 3-β (MIP-3β). ATs also migrated toward the MIP-3β. These results suggest a combination of DCs and ATs as a potentially effective therapeutic strategy.

Keywords: Dendritic cells, Activated T cells, CD40 ligand, Interleukin 12, Macrophage inflammatory protein 3-β

Introduction

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) with the ability to acquire, process, and present antigens to the immune system in the context of major histocompatibility complex (MHC) molecules. DCs also display an array of costimulatory and cell adhesion molecules. They are critically important in initiation of primary immune responses against external pathogens, self-antigens, allografts, and tumors [2, 33]. In their so-called immature state, DCs reside in peripheral tissues, where they search for incoming pathogens or internal antigens recognized as non-self. On encounter with these antigens, DCs are activated and migrate into the regional lymphoid organs to trigger a specific T-cell response. The cumulative evidence that substantiates the critical role of DCs in mounting immune responses and recent advances in cell culture techniques have prompted development of DC-based immunotherapies for the treatment of cancer [5, 10, 24].

Immature DCs, similar to those found in peripheral tissues, can be generated by culturing bone marrow cells or peripheral blood mononuclear cells with cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) [26, 27, 28]. DCs cultured with both GM-CSF and IL-4 are highly endocytotic, but are not fully capable of stimulating antigen-specific T cells or migrating into the regional lymph nodes. In contrast, mature DCs are able to migrate to the regional lymph nodes [9, 32] and to efficiently present antigens to specific T cells [8, 23]. Various stimuli that induce maturation of DCs include inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), lipopolysaccharides (LPS), and CD40 ligand (CD40L) [6, 25, 29]. Maturation of DCs results in up-regulation of adhesion and costimulatory molecules such as CD80 and CD86 and down-regulation of endocytosis [8, 23]. Maturation of DCs results also in up-regulation of CC chemokine receptor 7 (CCR7) and facilitates migration into lymph nodes where the ligands for CCR7, secondary lymphoid tissue chemokine (SLC), and macrophage inflammatory protein 3β (MIP-3β) are present [9, 32, 36].

CD40L, one of the most potent inducers of DC maturation, is mainly expressed on activated CD4+ T lymphocytes and provides a maturation signal to DCs as a part of "T-cell help." CD40L stimulates DCs to produce interleukin 12 (IL-12) and up-regulates expression of the costimulatory molecules CD80 and CD86 [6, 7, 15, 20]. Binding of CD40L to CD40 up-regulates expression of Bcl-2 and renders DCs resistant to apoptosis induced by Fas [4]. Interaction between CD40 and CD40L is considered essential to induction of an immune response and establishment of a Th1-type T-cell response [16].

In the current study we investigated a new approach to a DC-based cancer vaccine. To facilitate DC maturation, we used activated autologous T cells that expressed CD40L (AT) to serve not only as stimulators but also as providers of "T-cell help." Coculture of DCs and ATs resulted in significant production of IL-12 and interferon γ (IFN-γ). This mutual interaction also induced DCs to mature and facilitated migration of DCs and ATs toward MIP3-β. This is the first demonstration of bidirectional stimulation between human DCs and autologous ATs. Such data support the concept of combination immunotherapy with DCs and ATs as a viable approach to the treatment of cancer.

Materials and Methods

Isolation of peripheral blood mononuclear cells (PBMCs)

Leukocytes obtained from healthy donors by leukapheresis were separated by density centrifugation through Histopaque (SIGMA, St. Louis, MO) to yield PBMC. Isolated PBMCs were then washed with PBS with 0.1% EDTA, resuspended in RPMI medium supplemented with 4-mM l-glutamine, 2.5% human serum albumin, and 10% DMSO, and cryopreserved in liquid nitrogen until use.

Generation of cultured dendritic cells (DCs)

Monocyte-derived DCs were generated from frozen PBMCs according to published methods [26, 27, 28]. Briefly, PBMCs were thawed, washed, and resuspended in AIM-V medium (GIBCO, Grand Island, NY) to yield 10×106 cells/ml. Five milliliters of cell suspension was incubated in a T-25 culture flask (Corning-Coster, Cambridge, MA) at 37°C for 2 h. Nonadherent cells were removed gently, and adherent cells were cultured in AIM-V medium supplemented with 800 IU/ml of GM-CSF (Berlex Laboratories, Seattle, WA) and 500 U/ml of IL-4 (BD PharMingen, San Diego, CA) for 7–8 days.

Generation of activated T cells which express CD40L (AT)

PBMCs(2×106/ml AIM-V medium) supplemented with 10 μg/ml phytohemagglutinin (PHA) (SIGMA, St. Louis, MO) were cultured for 3 days in a T-75 culture flask (Corning-Coster, Cambridge, MA). The cells were then stimulated for 3 h with 1 μg/ml of ionomycin (SIGMA, St Louis, MO) [21]. These treatments produce activated T cells expressing CD40L, which were then harvested and washed extensively before use in various experiments.

Determination of optimal ratio of DC to AT

ATs (2×105 in AIM-V medium) were placed in each well of a 96-well flat-bottom plate. DCs were then added as duplicate series of graded concentrations. In another experiment, 5×103 DCs were added to each well first and duplicate series of graded concentrations of ATs were added to the 96-well plates which contained DCs. In control cultures, DCs were incubated either alone or with monocyte-depleted, autologous unstimulated peripheral blood lymphocytes (PBLs). Neither GM-CSF nor IL-4 was added during coculture experiments. The plates were incubated for 18 h at 37°C in 5% CO2. The medium was collected from each individual well and assayed for content of IL-12 and IFN-γ. Potential changes in the morphology of DCs or ATs during the course of the experiments were monitored by phase contrast microscopy. Also, each experimental condition was reproduced in chamber slides (BD PharMingen, San Diego, CA). At the end of the experiment, the cells in chamber slides were fixed and stained with hematoxylin and eosin prior to examination by light microscopy.

Blocking of CD40-CD40L interaction

ATs (2×105 in 50 μl of AIM-V medium) were added to each well of a 96-well flat-bottom plate. The cells were then incubated at 37°C for 30 min with mouse antihuman CD40L (CD154) neutralizing antibody (Clone TRAP1, BD PharMingen, San Diego, CA) at a final concentration of 10 μg/ml, or with the same concentrations of isotype-matched mouse IgG (SIGMA, St. Louis, MO). DCs (5×103 in 100 μl of AIM-V medium) were then added to each well to achieve a DC/AT ratio of 1:40. The cells were incubated for 24 h at 37°C in a humidified incubator with 5% CO2. The medium from each well was then collected and assayed for cytokines.

In separate experiments, direct interactions between DCs and ATs were interrupted with a permeable membrane. DCs (6×104 in 600 μl of AIM-V) were added to each well of a 24-well plate (Corning-Coster, Cambridge, MA). Transwell culture inserts with 0.4-μm pore size (Becton Dickinson, Franklin Lakes, NJ) were inserted into each well. ATs (1.2×106 in 300 μl of AIM-V) were then added to the upper chamber (DC/AT ratio 1:20). The cells were incubated for 24 h at 37°C in a humidified incubator with 5% CO2. The medium from each chamber of the wells was collected and assayed for cytokines. As controls, DCs alone and ATs alone were cultured in the same conditions.

Kinetics of production of IL-12 and IFN-γ during coculture of DCs and ATs

DCs (5×103) were plated into each well of a 96-well flat-bottom plate. These cells were then cultured with ATs (2×105 cells/well) for either 3, 6, 12, 18, 24, or 37 h. Media collected at the end of each incubation period were assayed for content of IL-12 and IFN-γ. Control cultures included DCs stimulated with 100 ng/ml of LPS (SIGMA, St. Louis, MO), as well as ATs cultured in the absence of DCs.

Flow cytometry

Murine monoclonal antibodies for flow cytometric analysis included anti-CD40 (IgG1), anti-CD11c (IgG1), anti-CD86 (IgG1), anti-CD14 (IgG2a), anti-CD83 (IgG1), anti-CD69 (IgG1), anti-CD154 (CD40L) (IgG1), anti-HLA-DR (IgG2a), anti-CD3 (IgG1), anti-CD4 (IgG1), anti-CD8 (IgG1), conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Mouse IgG1 and IgG2a, conjugated to either FITC or PE, were used as negative controls. All were purchased from BD PharMingen (San Diego, CA).

DCs were analyzed with FITC- or PE-conjugated antibodies to CD11c, CD86, CD40, HLA-DR, CD14, or CD83 with the direct method. ATs were analyzed with FITC- or PE-conjugated antibodies to CD3, CD4, CD8, CD69, or CD154 (CD40L). The stained cells were fixed with 1% paraformaldehyde and analyzed with an EPICS XL-MCL flow cytometer (Beckman-Coulter, Miami, FL).

Assay for cytokines

The content of IL-12 (p40 and p70) and IFN-γ in media were determined with commercially available ELISA kits (BIOSOURCE International, Camarillo, CA). Sensitivity of the IL-12 kit is 2 pg/ml and the effective assay range is 7.8–500 pg/ml. Sensitivity of the IFN-γ kit is 4 pg/ml and the effective assay range is 15–1,000 pg/ml. The reagents in these kits do not cross-react with other known human cytokines.

Measurement of cellular migration to MIP-3β

Migration of cells to the chemotactic factor MIP-3β was measured in Boyden chambers using the previously published methods [9, 14]. MIP-3β was purchased from R&D Systems (Minneapolis, MN). First, DCs were labeled with 100 μg/ml of Dextran conjugated with FITC (Molecular Probe, Eugene, OR). After the 1-h incubation with Dextran-FITC at 37°C, DCs were washed thoroughly to remove uncoupled Dextran-FITC. More than 99% of DCs showed significant fluorescence in the cytoplasm after incubation with Dextran-FITC.

MIP-3β was diluted with AIM-V medium to the final volume of 600 μl and added to the lower chambers of a 24-well culture plate (Corning-Coster, Cambridge, MA). Two different concentrations were tested (100 ng/ml and 1,000 ng/ml) [9]. AIM-V medium without MIP-3β was used as control. Transwell culture inserts (Corning-Coster, Cambridge, MA) with a 6.5-mm diameter and 5.0-μm pore size were inserted into each well. DCs (1×104 per well) mixed with or without ATs (10×104 per well) in AIM-V medium at the final volume of 100 μl were added to the upper chamber of the wells. DCs (1×104 per well) mixed with autologous unstimulated PBLs (10×104 per well) were also tested. After 24 h of incubation, the cells in the lower chamber were spun on the microslides and FITC-positive DCs were counted with a fluorescence microscope. In other experiments, ATs (10×104 per well) or unstimulated PBLs (10×104 per well) were placed in the upper chamber of the wells and migration toward MIP-3β was investigated. After 4 h, 9 h, and 24 h of incubation, total cell number in the lower chamber was counted.

Statistical analysis

Each experiment was performed at least three times using PBMCs from three healthy donors to confirm consistency of data between and among preparations and donors. Each set of conditions in an experiment was tested in either duplicate or triplicate. Average values and standard errors for each set of conditions were calculated and statistical significance was determined using Student's t-test. Differences were considered to be significant if the P value was less than 5% in two-tailed analysis.

Results

Development of monocyte-derived DCs

Monocyte-derived DCs with surface veils or processes were obtained from PBMCs after 7–8 days of culture with GM-CSF and IL-4. After vigorous pipetting with AIM-V, DCs were collected from the culture flask and tested for surface markers. Flow cytometric analysis revealed the cultured DCs to be strongly positive for CD11c (>90%) and HLA-DR (>80%), but negative or only weakly positive for CD14 (<20%). Approximately 40% of the cells expressed CD40 and 60–90% expressed CD86. The representative flow cytometry data are shown in Fig. 1.

Fig. 1a–e.

Fig. 1a–e.

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Surface markers on cultured DCs. PBMCs were cultured with GM-CSF and IL-4 for 7 days. The surface markers on cultured DCs were tested with PE-conjugated CD11c (a), FITC-conjugated CD86 (b), CD40 (c), HLA-DR (d), and CD14 (e) (shaded curve). Dead cells and lymphocytes were excluded from analysis by light scatter properties. Isotype-matched control antibodies were included in each histogram (unshaded curve)

Expression of CD40L on ATs

Incubation of PBMCs with PHA for 3 days resulted in the development of cells that expressed CD3 (>99%) and CD4 (>70%) and that spontaneously produced significant amounts of IFN-γ (>200 pg/ml/105 cells/day). A small fraction of T cells weakly expressed CD154 (CD40L) (<15%) after PHA stimulation. Further incubation of these cells with 1-μg ionomycin/ml enhanced the expression of CD154 (CD40L). Expression of CD154 on T cells dramatically increased (>70%) after 2 h of treatment with ionomycin (data not shown). Approximately 70–90% of T cells that were positive for CD154 (CD40L) were also positive for CD4. The representative flow cytometry data on activated T cells are shown in Fig. 2.

Fig. 2a–d.

Fig. 2a–d.

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Expression of CD40L on ATs. PBMCs were stimulated with PHA for 3 days. The cells were then further incubated with ionomycin for 2 h. The surface markers on activated T cells (ATs) were tested with FITC-conjugated CD4 (c) and PE-conjugated CD154 (CD40L) (d) (shaded curve). Dead cells were excluded from analysis by light scatter properties (a). Isotype-matched control antibodies were included in each histogram (unshaded curve). The histogram of two-color flow cytometry using FITC-CD4 and PE-CD40L is also shown (b)

The ratio of DCs to ATs affects synthesis of IL-12

When different numbers of DCs were incubated with a fixed number of ATs for 18 h, synthesis of IL-12 was maximal at the DC/AT ratios of 1:20 to 1:40 (Fig. 3). DC incubated alone or cultured with unstimulated PBLs did not produced IL-12 (data not shown). The production of IL-12 was lower in the wells in which DCs and ATs were cultured at the ratio of 1:10 despite the presence of twice the number of DCs in these wells. These findings were contrary to our expectation that the amount of IL-12 synthesized would be essentially proportional to the number of DCs in the culture. It was unlikely that the lower production of IL-12 in the wells with the DC/AT ratio of 1:10 was due to overcrowding because total number of cells in the well increased less than 5% from 210,000 to 220,000.

Fig. 3.

Fig. 3.

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Effect of ratio of DCs to ATs on synthesis of IL-12 when the number of ATs added to the culture remains constant. ATs at 2×105/well were placed in a 96-well flat-bottom plate. Various numbers of DCs were then added to each well. The cells were cultured together for 18 h, after which the culture medium was collected and assayed for content of IL-12. Data are representative of at least five separate experiments in three different individuals

To investigate whether DC/AT ratio is critical in IL-12 production, we set up a related experiment in which the same number of DCs (5×103/well) was placed in a 96-well plate, and then different numbers of ATs were added. IL-12 production was maximum at the DC/AT ratio of 1:20 to 1:40 (Fig. 4). ATs alone produced minimum amounts of IL-12.

Fig. 4.

Fig. 4.

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Effect of ratio of DCs to ATs on synthesis of IL-12 when the number of DCs added to the culture remains constant. DCs (5×103) were placed into each well of a 96-well flat-bottom plate. Various numbers of ATs were then added. The cultures were continued for 18 h, at which time the medium was removed and assayed for content of IL-12. Data are representative of at least five separate experiments in three different individuals

Morphologically, DCs and ATs were firmly adhered to each other in those wells in which there was significant synthesis of IL-12 (Fig. 5a). ATs were attached to the long processes of DCs which had developed in the coculture. In contrast, significant cellular interaction was lacking in DCs cocultured with autologous, unstimulated PBLs (Fig. 5b). Furthermore, DCs became rounded and showed morphological characteristics of macrophages if they were incubated in AIM-V medium alone or with autologous, unstimulated PBLs. These data indicate that direct interaction between DCs and ATs is critically important in synthesis of IL-12 and stabilization of the morphology of DCs.

Fig. 5a, b.

Fig. 5a, b.

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Effects of ATs on the morphology of DCs. DCs were cultured with either ATs (a) or unstimulated PBLs (b) for 24 h in chamber slides. After 24 h of culture, the medium was removed and the slides were stained with hematoxilin and eosin (x200)

Blocking of CD40-CD40L interaction

To determine whether binding of ligand to CD40 is critical to synthesis of IL-12 production, CD40L on the surface of ATs was blocked with anti-CD40L (CD154) neutralizing antibody prior to coculture with DCs. Isotype-matched mouse IgG was used as a control. As shown in Fig. 6, IL-12 production by DCs was almost completely blocked by anti-CD40L antibody (P<0.01). In contrast, the anti-CD40L antibody only slightly decreased synthesis of IFN-γ (Fig. 6). The importance of direct interactions between DCs and ATs in production of IL-12 was investigated by separating DCs from ATs with a permeable membrane. When direct interaction between DCs and ATs was prevented with a permeable membrane, the production of IL-12 decreased from 500 pg/ml to 100 pg/ml (data not shown).

Fig. 6.

Fig. 6.

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Anti-CD40L (CD154) antibody suppresses synthesis of IL-12 but only slightly suppresses IFN-γ production. ATs were incubated with an anti-CD40L (CD154) antibody or isotype-matched mouse IgG antibody for 30 min prior to coculture with DCs. The content of IL-12 and IFN-γ were measured in medium obtained after 24 h of coculture of DCs and ATs. *P<0.05, **P<0.01; no antibody vs anti-CD40L (CD154) antibody

Kinetics of IL-12 and IFN-γ productions in coculture of DCs and ATs

Alterations in the morphology of the DCs during coculture prompted the speculation that interaction between DCs and ATs would result in bidirectional stimulation of these two cell fractions. To confirm this hypothesis, DCs were cultured with ATs at a 1:40 ratio and media were collected and analyzed at various times thereafter. IL-12 accumulated in a linear fashion throughout the 37-h incubation period (Fig. 7). In contrast, accumulation of IL-12 reached a plateau in 18 h when DCs were treated only with LPS.

Fig. 7.

Fig. 7.

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Kinetics of accumulation of IL-12 in cultures of DCs and ATs. DCs were cocultured with ATs at the DC/AT ratio of 1:40. Culture media were collected at various times and assayed for content of IL-12. As controls, DCs were either stimulated with LPS (100 ng/ml), or cultured alone. Solid triangles DCs cocultured with ATs (DC&AT), open squares DCs cultured without ATs or LPS (DC alone), open circles DCs stimulated with LPS (DC&LPS)

IFN-γ also accumulated in a linear fashion in the medium when antigen-pulsed DCs were cultured with ATs at a 1:40 ratio (Fig. 8). No significant increase was observed when ATs were cultured without DCs.

Fig. 8.

Fig. 8.

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Kinetics of accumulation of IFN-γ in cultures of DCs and ATs. ATs were cocultured with DCs (DC to AT 1:40), or cultured alone. Medium was collected at various times after initiation of the culture and assayed for content of IFN-γ. Solid triangles DCs cocultured with ATs (DC&AT), open squares ATs without coculture with DCs (AT alone)

Up-regulation of surface markers on DCs

Expression of CD83, as measured by flow cytometry, was taken as evidence of maturation of DCs after coculture with ATs. DCs were cocultured with ATs at the DC/AT ratio of 1:10 for 24 h, at which time the expression of CD83 on DCs was measured. CD83 expression by DCs increased from 30% to 63% after coculture with ATs for 24 h (Fig. 9). No significant up-regulation of CD83 was observed when DCs were cocultured with unstimulated PBLs (data not shown).

Fig. 9a–c.

Fig. 9a–c.

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Effects of ATs on up-regulation of CD83 by DCs. DCs were cocultured with ATs at the DC/AT ratio of 1:10 for 24 h, at which time the expression of CD83 on DCs was measured. The data were collected from the DC fraction on the bitmap. a Isotype matched mouse IgG, b CD83 DCs without coculture with ATs, c CD83 DCs after coculture with ATs

In vitro migration assay

One of the key questions in using DCs as cancer immunotherapy is whether DCs migrate to regional lymph nodes after administration to patients. It has been reported that mature DCs up-regulate CCR7 and down-regulate CC chemokine receptor 1 (CCR1) [9, 32, 36]. Since coculture of DCs with ATs apparently induces maturation of DCs, as measured by expression of CD83, such mature DCs would be expected to migrate towards the chemokine MIP-3β or SLC which are abundant in lymph nodes This hypothesis was tested by an in vitro chemotaxis assay performed in Boyden chambers. As shown in Fig. 10, it was found that DCs that had been cocultured with ATs migrated toward MIP-3β in a dose-dependent manner. In contrast, DCs cocultured with unstimulated PBLs did not show significant chemotaxis toward MIP-3β (data not shown). It is of note that ATs alone also migrated toward MIP-3β (Fig. 11). The chemotaxis of ATs occurred as early as after 4 h of incubation in the Boyden chambers. Unstimulated PBLs did not show the dose-dependent chemotaxis toward MIP-3β (data not shown)

Fig. 10.

Fig. 10.

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Migration of DCs towards MIP-3β. DCs were labeled with FITC as described in "Materials and methods." MIP-3β was added to AIM-V medium at various concentrations (0 ng/ml, 100 ng/ml, or 1,000 ng/ml) and placed in the lower chamber of a 24-well plate. Transwells were then inserted and 104 of DCs mixed with ATs (105) in AIM-V medium were placed in the upper chambers. Incubations were continued for 24 h when the cells that migrated to the lower chamber were collected on cytospin slides. The number of FITC-positive DCs was counted under fluorescent microscopy. *P<0.05, **P<0.01

Fig. 11.

Fig. 11.

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Kinetics of migration of ATs toward MIP-3β. MIP-3β was added at various concentrations (0 ng/ml, 100 ng/ml or 1,000 ng/ml) to the wells of a 24-well plate. Transwells were then inserted and 105 ATs in AIM-V medium were placed in the upper chamber. The number of cells that migrated to the lower chamber in 4, 9, or 24 h after the initiation of the cultures was counted with a hemocytometer. *P<0.05, **P<0.01

Discussion

Our goal in this study was to establish a basis for a new DC-based cancer vaccine. To this end, we chose autologous T cells that were activated to express CD40L so that they could induce maturation of DCs. These activated T cells mimic the in vivo environment to which the T cells likely contribute "helper factors" as well as stimulation through CD40-CD40L interaction. In this setting, usage of CD40L-expressing ATs might be more effective than free CD40L molecules since ATs could migrate into the regional lymph nodes to directly interact with other types of immune cells such as B cells or CD8+ cytotoxic T cells.

The current study demonstrates that interactions between DCs and ATs are not only useful for the maturation of DCs but also important for the migration of DCs and synthesis of IL-12. Direct contact between DCs and ATs and interaction through CD40 and CD40L are required for IL-12 production since IL-12 was not synthesized when DCs and ATs were separated by a permeable membrane, or when interactions between CD40 and CD40L were blocked by anti-CD40L antibody. The CD40L molecule is known to induce significant production of IL-12 by DCs and up-regulation of adhesion and costimulatory molecules on DCs [1, 7, 12, 16]. IFN-γ augments the effect of CD40L on IL-12 synthesis by DCs [7, 31]. Therefore, using ATs that express CD40L and also produce IFN-γ would be more effective than the CD40L molecule itself in inducing a Th1 type immune response.

The importance of CD40 and CD40L interaction in T-cell response was demonstrated in an allogeneic mixed lymphocyte response (MLR) [20]. When the interaction was inhibited, there was a significant decrease in proliferation of T cells during the MLR. Since these studies did not use autologous T cells, the interaction between human DCs and autologous T cells was not studied. In contrast, the data reported herein demonstrate that interactions between DCs and CD40L-expressing, autologous ATs result in significant production of IL-12 and IFN-γ, and facilitate migration of DCs and ATs toward MIP-3β. As a form of cancer immunotherapy, autologous T cells offer an advantage over allogeneic T cells in that they are not subject to immune rejection and will not induce a graft-versus-host reaction.

It is of note that the CD40-CD40L interaction is essential to IL-12 production, while it is not required for production of IFN-γ. Since T cells were activated by PHA and ionomycin in our study, a CD40-CD40L independent pathway could have triggered synthesis of IFN-γ. In fact, conjugation of PHA to N-acetylgalactosamine on the surface of T cells increases cytosolic Ca2+ and triggers expression of IL-2 receptors, and stimulates excretion of IL-2 [17, 22, 34]. Therefore, PHA stimulation itself can cause production of IFN-γ, although coculture with DCs further increases the amount produced. In this regard, PHA could be replaced with other stimuli to T cells such as anti-CD3 monoclonal antibody. Production of IFN-γ by T cells also would be enhanced by IL-12 and by the interactions of costimulatory molecules such as CD80/CD86 with CD28 and intercellular adhesion molecule 1 (ICAM-1) with leukocyte function-associated antigen 2 (LFA-2) [19].

It has been reported that CD40-CD40L interaction is critically important in the development of cytotoxic T-cell (CTL) responses against cancer cells [3, 18, 30]. Signaling through CD40 primes helper-dependent CD8+ CTL responses as part of the antitumor immune response [30]. Ligation of CD40 and/or the presence of IL-12 facilitate DCs in presenting an otherwise poorly immunogenic tumor peptide [3]. Absence of interaction between CD40 and CD40L results in a failure to generate systemic antitumor immunity [18]. This lack of interaction results also in impairment of production of IL-12 by APCs and shifts the immune response toward a Th-2 type response [13].

The results of this study impact on the development of a specific DC-based anticancer immunotherapy. For example, DCs pulsed with tumor antigens could be administered intradermally with autologous ATs which express CD40L. It is expected that the in vivo interaction of DCs and ATs at the injection sites would result in maturation of DCs. Mature DCs expressing specific chemokine receptors, such as CCR7, would migrate into the regional lymph nodes where they would produce IL-12. A fraction of ATs might also migrate into the same regional lymph nodes and produce IFN-γ and provide "T-cell help" to tumor antigen–specific CD8+ CTL. It is also possible that the small fraction of antigen-specific T cells in the autologous PBLs would be stimulated by DCs at the injection sites.

Finally, this concept could be brought to practice in patients whose tumor cells are not available for ex vivo vaccine preparation. In these patients, DCs and ATs could be directly injected into a tumor site. Production of IL-12 and IFN-γ in the tumor site would potentially break the tolerance evoked by tumor cells [11, 35] and allow injected DCs to carry tumor antigens to the regional lymph node, where they could stimulate naïve T cells in collaboration with ATs. As a consequence, a tumor-specific immune response could be developed and long-term memory would also be established. Thus, an immunotherapy based on the interactions between DCs and ATs has rationales and experimental support. Clinical and preclinical studies to test this concept are warranted.

Acknowledgements

We thank Dr Kenneth P. Chepenik for his assistance in editing this manuscript. The results of the current study were presented at the 91st and 92nd annual meetings of American Association for Cancer Research and 16th annual meeting of Society of Biological Treatment. This research work was supported by the Hasumi Research Fund provided by Shukokai.

References

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