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Clinical Cancer Research Vol. 11, 8763-8772, December 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Natural Killer Cells License Dendritic Cell Cross-Presentation of B Lymphoma Cell–Associated Antigens

Tao Dao, Marta Gomez-Nunez, Christophe Antczak, Barry Kappel, Jaspreet Singh Jaggi, Tatyana Korontsvit, Victoriya Zakhaleva and David A. Scheinberg

Authors' Affiliation: Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New York

Requests for reprints: David A. Scheinberg, Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-8635; Fax: 212-717-3068; E-mail: d-scheinberg{at}ski.mskcc.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Presentation of exogenous antigen by MHC class I molecules, or cross-presentation, is a property of dendritic cells, which is considered crucial for the priming of cytotoxic T-cell response to tumor antigens. However, the precise mechanisms of this process are not fully understood.

Experimental Design and Results: We show here in a human in vitro system, using B lymphoma cells as a tumor model, that the cross-presentation of cell-associated antigens to T cells by dendritic cells requires "help" from natural killer cells. When autologous dendritic cells that had taken up apoptotic B lymphoma cells and induced to a fully mature state were used to stimulate nonadherent cells of peripheral blood mononuclear cells from healthy donors, they induced strong cytotoxicity against B lymphoma cells in a HLA-A0201-restricted manner. The cells failed to induce cytotoxicity, however, when purified T cells were used as effector cells. Depletion of CD56+ cells, but not CD14+ or CD19+ cells, abrogated the cytotoxicity of nonadherent cells, showing that the help was provided by natural killer cells. Further, when natural killer cells were present in the cultures, a strong and persistent production of interleukin-18, but not interleukin-12 and interleukin-15, was observed. Blocking interleukin-18 significantly reduced the cytotoxicity of nonadherent cells against B lymphoma cells.

Conclusions: These results suggest that capture of tumor cells and a full maturation status of dendritic cells are not sufficient to cross-prime CD8 T cells. Effective cross-priming requires further activation of dendritic cells by natural killer cells and an abundant production of interleukin-18, which, along with other yet undefined mechanisms, contribute to the generation of CTL response against B-cell lymphoma.

Immunotherapy using monoclonal antibody (mAb) against cell surface antigens, such as CD20 and CD52, has made significant progress in treating B-cell leukemia and lymphoma (1, 2). However, the majority of patients ultimately relapse and new therapeutic strategies are required. One promising approach for targeting B-cell malignancies is to induce CTLs specific for tumor-associated antigens on B cells. To date, vaccination with tumor-specific immunoglobulin idiotype has proven to induce not only humoral but also cellular anti-idiotype response, which correlate with improved relapse-free and overall survival in B-cell lymphoma patients (3–6). In addition, active immunization with peptides or DNA vaccines comprising B cell surface antigens, such as CD19, CD20, and CD22, have been shown to induce antigen-specific CTL responses, which protect mice from B lymphoma cell challenge, and kill human B lymphoma cells in vitro (7–9).

Induction of CTL response requires presentation of target antigens as peptide fragments in the context of the MHC class I. Because the MHC class I–restricted presentation is almost exclusively to components synthesized within the presenting cells itself, this pathway is often termed the "endogenous pathway" (10). In contrast to this direct presentation, extracellular antigens can also gain access to the MHC class I processing pathway to elicit CTL responses, a process called cross-priming (11). Various antigens, including soluble proteins, immune complexes, intracellular pathogens, and, most importantly, cellular antigens, can be cross-presented (12–16). This is particularly important in antitumor immunity, because direct presentation of antigens by tumor cells is inefficient in eliciting CTL response (17). Therefore, the uptake and presentation of tumor cells by specialized immune cells, such as professional antigen-presenting cells (APC), could induce effective antitumor CTL responses. Dendritic cells have been shown to be the principal cell type involved in cross-priming because of their capacity for highly efficient phagocytosis and antigen processing as well as their expression of a high-level MHC molecules and costimulatory molecules, which are required for priming naive T cells (18, 19).

Vaccination with dendritic cells loaded with tumor-associated antigens has been increasingly applied for cancer immunotherapy (20–22). Immature dendritic cells that are mainly involved in antigen capture are not equipped to induce immunity but rather to promote tolerance. Mature dendritic cells that have up-regulated MHC class II and costimulatory molecules CD80, CD86, and CD40 are capable of effective T-cell priming. Therefore, phenotypic maturity of dendritic cells has been a gold standard for T-cell priming. To date, the most common dendritic cell vaccine approach for clinical trials in various cancers requires the maturation of dendritic cells by inflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor-{alpha} (TNF-{alpha}), and prostaglandin E2, or CD40 ligation before injection (23–28). However, recent progress in dendritic cell biology has revealed the existence of a dendritic cell maturational state devoid of immunologic potential. In vitro studies have shown that dendritic cells matured by inflammatory cytokines induce CD8 T-cell proliferation, but these T cells are rendered tolerant unless dendritic cells are further stimulated by helper immune cells (29). In certain circumstances, mature dendritic cells bearing antigens in draining lymph nodes induce CD4+ T-cell proliferation but not effector responses (30, 31). These series of studies thus provide important evidence that maturity, based on the "classic" phenotypic markers (abundant expression of MHC class II and costimulatory molecules) and the ability to stimulate naive T-cell proliferation, is not synonymous with the capacity for priming T cells.

The mechanisms underlying cross-presentation and its regulation are beginning to be elucidated. Earlier studies have shown that certain maturation stimuli that target dendritic cell surface receptors, such as DEC-205 (32), stimulating dendritic cells with Toll-like receptor agonists (33) or targeting antigens to Fc{gamma} receptors on dendritic cells using antigen-antibody complexes (34), can enhance cross-presentation. Increasing evidence has also shown that dendritic cells that have captured exogenous antigens do not necessarily cross-prime effectively. Dendritic cells also need to receive appropriate activation signals to become competent to induce cross-priming, a process called licensing. In viral infection, dsRNA produced during virus replication and type 1 IFN license dendritic cells to cross-prime virus-specific CD8+ T cells (35–37).

The licensing process for promoting dendritic cell cross-presentation with tumor-associated antigens is poorly understood. A recent in vitro study using human peripheral blood mononuclear cells has shown that coating melanoma cells with anti-syndecan-1 mAb promoted a HLA-restricted CTL response against melanoma cells. The enhancement of this cross-priming was partially mediated by activation of dendritic cells via Fc{gamma} receptors. However, both dendritic cell maturation and phagocytosis were not enhanced compared with the controls without antibody opsonization (38). This raises the question of whether maturation phenotype of dendritic cells reflects the functional ability for T-cell priming and suggests that cross-priming may be a far more complex process than a simple dendritic cell event. This and some other similar studies use nonadherent cells from peripheral blood mononuclear cells as effector cells for CTL induction. Therefore, the potential role of other cell types, particularly natural killer (NK) cells, is unclear.

NK cells are effectors of innate resistance that are able to lyse a variety of tumor cells without prior sensitization (39). Growing recognition of immunoregulatory function of NK cells has brought much attention to the interaction between NK cells and dendritic cells. Whereas dendritic cells are able to stimulate proliferation and cytotoxic function of fresh NK cells (40, 41), activated NK cells can license or provide "help" for dendritic cells to promote CTL response against pathogens (42) or tumor (43). In the present study, using in vitro human model, we show that dendritic cells that have taken up apoptotic B lymphoma cells cannot by themselves induce cytotoxic T cells. Effective cross-priming requires dendritic cell licensing by NK cells. This process was characterized by abundant IL-18 production. This cytokine, along with other potentially undefined mechanisms, contributes to the induction of cytotoxic T-cell activity against B lymphoma cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. Human Burkitt's lymphoma cell lines Ramos (HLA-A3, B51, B44, C16), B-Jab (HLA-A2, A1, B13, B35, C6, C4), Daudi (HLA-A1, A10, B17, B16; Cw3, Cw6), and SKLY-16 (HLA-A2, A32, B 35, C4) were obtained from American Type Culture Collection (Manassas, VA). Classic human NK cell target LCL.721 cell line was provided by Dr. Christian Munz (Rockefeller University, New York, NY). These cell lines were cultured in RPMI 1640 fortified with 5% FCS, penicillin/streptomycin, and glutamine. HLA type of the cell lines was assessed by genotyping.

Cytokines and antibodies. Human granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-4, IL-6, IL-15, TNF-{alpha}, and prostaglandin E2 were purchased from R&D Systems (Minneapolis, MN). Neutralizing mAb to human IL-18 and an isotype control mouse IgG were purchased from MBL (Watertown, MA). The antibodies used for immunofluorescence assays, including mAbs to human CD3, CD4, CD8, CD56, CD14, CD19, CD25, CD16, CD45, CD83, CD86, CD11C, CD14, CD40, HLA-DR, and isotype controls, were obtained from BD Biosciences (San Diego, CA). Antibodies against HLA-A0201 (clone BB7.1) and HLA-DR (clone L-243; ref. 44) were obtained from the Monoclonal Antibody Facility at Memorial Sloan Kettering Cancer Center (New York, NY). ELISA kits for human IFN-{gamma} was purchased from BD Biosciences (San Diego, CA), IL-12 p70 active form was from Pierce Biotechnology, Inc. (Rockford, IL), and IL-15 and IL-18 were from Cell Sciences, Inc. (Canton, MA).

Flow cytometry and phenotypic analysis. For cell surface staining, cells were incubated with appropriate mAbs for 30 minutes on ice, washed, and incubated with secondary antibody reagents when necessary. Analysis was done on a BD Biosciences FACScan (San Diego, CA) or BD Biosciences LSR (Mountain View, CA).

Generation of apoptotic tumor cells. Apoptotic tumor cells were generated by serum deprivation. Tumor cells in culture were collected by centrifugation and serum-free RPMI 1640 was used to culture the cells. The percentage of apoptosis was measured by Annexin V staining (BD Biosciences, San Diego, CA). Cell viability was measured by trypan blue exclusion. The cells were washed, irradiated with 3Gy, and immediately added to immature dendritic cells or used to stimulate effector cells.

Generation of monocyte-derived dendritic cells and loading with tumor cells. Peripheral blood mononuclear cells were obtained from healthy donors or from leukocyte concentrate (Memorial Sloan Kettering Cancer Center) by Ficoll-Hypaque centrifugation (Amersham Biosciences, Piscataway, NJ). Monocyte-derived dendritic cells were differentiated from adherent cells of peripheral blood mononuclear cells using RPMI 1640 supplemented with 1% autologous plasma, GM-CSF, and IL-4. On day 5, immature dendritic cells were cocultured overnight with apoptotic tumor cells at a ratio of 1:1. The maturation of these dendritic cells was then induced by adding granulocyte-macrophage colony-stimulating factor, IL-1β, IL-4, IL-6, TNF-{alpha}, and prostaglandin E2 to the cultures for 1 to 2 days as described previously (45).

Evaluation of tumor cell uptake by dendritic cells. To detect tumor cell uptake by dendritic cells, the tumor cells were labeled with PKH67 green (Sigma-Aldrich, St. Louis, MO) before coculturing with dendritic cells. Before or after dendritic cell maturation, the cells were stained with phycoerythrin-conjugated antibodies against dendritic cell markers CD83 or CD86. Phagocytosis was measured by flow cytometry on double-positive cells. Alternatively, trypan blue was used to stain the dying tumor cells before adding them to the dendritic cell culture. By serum deprivation, we could obtain 98% to 100% dead or dying cells. Tumor cell uptake was measured by the disappea-rance of trypan blue–positive cells after coculturing with dendritic cells.

Stimulation of effector cells. T cells were purified from nonadherent peripheral blood mononuclear cells using a Pan T-Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) by negative immunomagnetic cell separation. The CD3– fraction was used for generation of NK/lymphokine-activated killer cells by culturing the cells with IL-15 (10 ng/mL) for 7 to 8 days. The depletion of CD56+, CD19+, or CD14+ cells was done by standard methods using magnetic beads (Miltenyi Biotec). Purity of the resulting cells was determined by flow cytometry after staining the cells with mAbs against various cell surface markers for T cells, B cells, and monocytes/macrophages. The purity of the cells was always >98%. T cells, CD56+, CD19+, or CD14+ depleted or nonadherent cells (2 x 106/mL) were stimulated with autologous mature dendritic cells that had taken up tumor cells at a ratio of 10:1 in RPMI 1640 with 10% autologous plasma. On day 7, cultures were restimulated with identical dendritic cells at a ratio of 30:1. Five or 6 days after secondary stimulation, the effector cells were harvested and their cytotoxicity was tested by 51Cr-release assay.

Evaluation of CTL response. MHC class I–restricted CTL response was measured via 51Cr-release assay using HLA-A locus-matched or unmatched B lymphoma or leukemia cells. LCL.721 cells were used as targets for measuring NK cell cytotoxicity. Briefly, target cells (1 x 106) were incubated with 50 µCi 51Cr for 1 hour at 37°C and then extensively washed of unincorporated 51Cr. Target cells (100 µL/well) were added to effector cells (100 µL/mL) in 96-well round-bottomed plates at different E:Ts as indicated. The cells were incubated at 37°C for 4 to 5 hours. 51Cr in supernatants were collected and measured in a gamma counter (Beckman, Milan, Italy). Specific percentage of 51Cr release was calculated using a standard formula: [(Sample release – Spontaneous release) / (Maximum release – Spontaneous release)] x 100. For some experiments, mAbs against HLA-A0201, HLA-DR, or isotype control were added to the target cells before coculturing with effectors.

Cytokine ELISAs. Supernatants from cell cultures at different time points were collected and stored at –80°C. IFN-{gamma}, IL-12 p70 active form, and IL-18 secretion was directly measured from the supernatants. For IL-15 detection, supernatants were concentrated 10-fold by ultrafiltration (Vivaspin, 10-kDa exclusion size; Vivaspin, Goettingen, Germany). All the cytokines were quantified with commercial ELISA kits according to the manufacturer's instruction.

Proliferation assay. A week after first stimulation with tumor-captured dendritic cells, nonadherent or T cells (1 x 105/well) were incubated with 3 x 104 dendritic cells loaded with B lymphoma cells for 5 days in RPMI 1640 supplemented with 10% pooled human serum in 96-well round-bottomed microtiter plates, 1 µCi [3H]thymidine was added to each well for the last 6 hours of the 5-day culture. Cells were harvested with a Harvester Mach IIIM (Tomtec, Hamden, CT) and counted in a 1450 MicroBeta TriLux (Wallac, Turku, Finland). The measured counts/min represented mean values of triplicate microwell cultures.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Dendritic cells phagocytose apoptotic B lymphoma cells. B lymphoma cells (Ramos) were induced to undergo apoptosis by serum deprivation. More than 98% of the cells become trypan blue positive after culture in serum-free medium for 7 to 10 days. These cells were washed, irradiated (3Gy) to eliminate possible proliferation, and immediately fed to immature dendritic cells at a ratio of dendritic cell to lymphoma cells of 1:1. One day after the coculture, dendritic cells were matured by adding the maturation cytokine cocktail for another 1 or 2 days. The phagocytosis of tumor cells was measured by labeling tumor cells with dye PKH67 green (Fig. 1A) before the coculture with dendritic cells and staining the dendritic cells with mAb to CD83 (Fig. 1B) before or after maturation. More than 93% of the Ramos cells were taken up by the dendritic cells (Fig. 1C). This result was confirmed by assessment of residual Ramos cells by microscopy. Although ~98% of cells were trypan blue positive before they were added to the dendritic cell culture, after overnight coculture, fewer than 7% of the cells remained trypan blue positive (Fig. 1D). This was not due to breakdown and disappearance of dead cells because the control culture with dying Ramos cells alone still showed almost all trypan blue positive (data not shown).


Figure 1
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  		Fig. 1. Phagocytosis of Ramos cells by dendritic cells determined by flow cytometry. Apoptotic Ramos cells were labeled with PKH67 green (A). Dendritic cells alone (B) or mature dendritic cells that had captured PKH67 green–labeled Ramos (C) were stained with phycoerythrin-conjugated mAb to CD83. The double-positive cells show the percentage of phagocytosis. Uptake of Ramos cells by dendritic cells was also assessed by trypan blue staining of dying Ramos cells before or after coculture with dendritic cells. Reduction of trypan blue–positive cells indicates the ingestion of Ramos cells by dendritic cells (DCs; D). Representative data from three similar experiments. Points, average of triplicate microwell cultures; bars, SD.

 
The maturation status of dendritic cells was assessed by expression of HLA-DR, CD11C, CD83, CD40, and CD86 after maturation induction. The dendritic cells expressed high levels of HLA-DR and CD83, CD11C, CD40, and CD86 (Fig. 2) but not CD14 (data not shown). The expression level of these markers were similar between dendritic cells that had taken up B lymphoma cells (right) and control dendritic cells that had been generated in parallel without tumor cell uptake (left). These results confirm that mature dendritic cells that had taken up apoptotic Ramos cells were the population used for T-cell priming.


Figure 2
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  		Fig. 2. Assessment of dendritic cell maturation states. Dendritic cells alone (left) or dendritic cells that have captured apoptotic Ramos cells (right) were induced to maturation by cytokine cocktails for 1 day, and the cells were stained with mAbs conjugated with different fluorescence to dendritic cell markers, including HLA-DR versus CD11C, CD83, CD86, and CD40, and analyzed by flow cytometry. Representative data from three similar experiments.

 
NK cells provide help for cross-priming of B lymphoma cell–associated antigens by dendritic cells. To evaluate if the mature dendritic cells loaded with tumor cells stimulate specific CTL activity in autologous T cells, we used different B lymphoma cells with distinct HLA as a readout system. Autologous mature dendritic cells that had taken up apoptotic Ramos cells (A0201–) were used as APCs to stimulate effector cells from HLA-A0201+ donors. When nonadherent cells were stimulated twice with dendritic cells loaded with Ramos cells, a strong cytotoxicity against B-Jab (A0201+; Fig. 3A) but not Daudi (A0201–; Fig. 3B) cells was induced, indicating that B lymphoma cell–associated antigens can be cross-presented to autologous T cells to elicit an A0201-restricted killing. To monitor NK-mediated killing, LCL.721 cells were used as targets in parallel. Weak killing against LCL.721 was induced (Fig. 3C), suggesting that this killing might be mediated by NK cells remaining in nonadherent cell population. Surprisingly, when purified T cells were used in parallel as effectors, the overall cytotoxicity against B-Jab was greatly reduced (Fig. 3A). However, cell proliferation was similar between nonadherent cells and T cells as measured by [3H]thymidine incorporation (Fig. 3D). We also monitored the cell counts by trypan blue exclusion and never found the reduction of the cell number in the culture of T cells alone compared with nonadherent cells (data not shown). This suggests that both nonadherent and T cells are activated by dendritic cells loaded with Ramos cells, but purified T cells alone failed to differentiated into cytotoxic effector cells. To confirm that the cytotoxicity of nonadherent cells is truly induced by dendritic cell cross-presentation of B lymphoma cell–associated antigens rather than by alloresponse against Ramos cells, we stimulated nonadherent or T cells with dendritic cells that had taken up apoptotic Ramos cells and with apoptotic Ramos cells alone in parallel. The cytotoxicity was tested against a panel of B lymphoma cell lines that have a different HLA-A locus. As summarized in Table 1, nonadherent cells stimulated with either dendritic cells that had captured Ramos or Ramos alone showed a similar expansion (Table 2) but a very different target specificity. The most striking difference is that nonadherent cells stimulated with dendritic cells that had captured Ramos cells kill the target B-Jab (HLA-A2) and SKLY-16 (HLA-A2) cells but not Daudi cells (HLA-A2–). This confirmed that autologous dendritic cells captured and cross-presented cellular antigens of B lymphoma and elicited the cytotoxicity against B lymphoma target cells in a HLA-A0201-restricted manner. However, when apoptotic Ramos cells alone were used as stimulators, there was no cytotoxicity against B-Jab and SKLY-16 cells. Both stimulators induced cytotoxicity of nonadherent cells against Ramos. This is not surprising, because both conditions could induce allogenic antigen-specific CTL against Ramos. It has already been shown that the generation of allo-CTL requires help from NK cells (46). Interestingly, T cells expanded as well as nonadherent cells when they were stimulated with dendritic cells that had captured Ramos but had very poor expansion when stimulated with only Ramos. This shows that B lymphoma itself is a poor stimulator/APC for T cells (Table 2). In contrast, dendritic cells that had taken up the tumor cells are strong stimulators for T cells; however, without the presence of NK cells, they failed to induce differentiation of CD8 T cells to cytotoxic effectors cells. In addition, the NK/lymphokine-activated killer cells generated from the same donors killed all targets, including Daudi and LCL.721 cells (Table 1). This further supports the results that killing of B-Jab and SKLY-16 targets is restricted to HLA-A0201 presentation. These results showed that dendritic cells are able to cross-present B lymphoma cell–associated antigens to elicit CTL response and that this process requires the presence of NK cells.


Figure 3
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  		Fig. 3. Induction of HLA-A2-restricted cytotoxicity by dendritic cells loaded with Ramos. Nonadherent cells (diamonds) or purified T cells (squares) from HLA-A0201+ donor were stimulated with autologous dendritic cells that have captured Ramos cells for two rounds. The cytolytic activity against B lymphoma cells B-Jab (A), Daudi (B), or NK target LCL.721 (C) was measured by 51Cr-release assay. Representative data from five similar experiments. Proliferation of nonadherent cells (NA) or T cells was assessed by [3H]thymidine incorporation after coculture with dendritic cells that have taken up apoptotic Ramos cells at the E:T of 30:1 for 5 days (D). To determine the cytotoxicity via MHC class I or II pathway, target B-Jab cells were pretreated with antibodies against HLA-A0201, HLA-DR, or isotype control immunoglobulin before coculturing with effector nonadherent cells (at E:T of 30:1) in 51Cr-release assay (E). Points, average of triplicate microwell cultures; bars, SD.

 

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  		Table 1. Cytotoxicity of nonadherent cells, T cells, or NK/lymphokine-activated killer cells against a panel of B lymphoma cell lines

 

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  		Table 2. Effector cell numbers

 
To confirm that the cytotoxicity of nonadherent cells against B-Jab cells was indeed mediated by HLA-A0201 pathway, we added mAbs to HLA-A0201 or HLA-DR to the target cells B-Jab before coculturing with nonadherent cells in 51Cr-release assays. The mAb to A0201 significantly blocked the killing of nonadherent cells against B-Jab, but control antibody and mAb to HLA-DR did not show any significant effect on the killing (Fig. 3E). Similarly, we also did experiments across different HLA-A haplotypes. We used Ramos to feed dendritic cells from HLA-A1 donor and generated CTL against HLA-A1 targets but not HLA-A2 target cells (data not shown).

Purification of T cells was done by depleting B cells (CD19+), monocytes/macrophages (CD14+), and NK cells (CD56+) from nonadherent cells. It was important to determine which cell populations provided help for the CTL induction. We therefore also separately depleted CD56+, CD14+, or CD19+ cells from nonadherent cells. The purity of the cells was determined by flow cytometry after staining the cells with respective antibodies. The purity of the cells was always >98% (data not shown). Because no CD14+ cells remained after the adherent procedure (Fig. 4A), a possible role for CD14+ cells among the nonadherent cells could be excluded. When nonadherent, T cells, CD56-depleted or CD19-depleted cells were used as effectors, cytolytic activity against B-Jab was reduced only in the samples with CD56-depleted cells or T cells alone but not CD19-depleted cell populations (Fig. 4B). Cytotoxicity of nonadherent and CD19-depleted cells against Daudi and LCL.721 was <10% (data not shown). Our results showed that, although the dendritic cells loaded with apoptotic B lymphoma cells appear phenotypically to be fully mature, they are not capable of promoting cross-priming by themselves. The induction of a CTL response requires the presence of NK cells in the cultures.


Figure 4
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  		Fig. 4. Induction of cytotoxicity against B-Jab by different effector cell populations. Nonadherent cells were stained with mAbs to CD14 and CD3 and analyzed by flow cytometry. Data show the depletion of CD14+ cells after adherent procedure of peripheral blood mononuclear cells (A). Nonadherent cells (filled circles), CD56-depleted cells (squares), CD19-depeleted cells (triangles), or T cells (open circles) from HLA-A0201+ donor were stimulated with dendritic cells loaded with Ramos cells for two rounds (B). The cytotoxicity against B-Jab was measured by 51Cr-release assay. Representative data from three similar experiments. Points, mean of triplicate microwell cultures; bars, SD.

 
Induction of CTL by licensed dendritic cells was partially mediated via IL-18. The helper function of NK cells for dendritic cells is mediated by certain cytokines, such as IFN-{gamma} (42, 43). To determine the mechanisms by which NK cells help dendritic cell cross-priming in our experimental model, we examined the soluble factors produced in the effector cell cultures. IFN-{gamma} secretion was tested first, because this cytokine can be produced by activated NK cells or T cells, which in turn activate APCs, such as dendritic cells and macrophages. As shown in Fig. 5A, a strong IFN-{gamma} secretion was induced in the cultures regardless of the presence of NK cells. Because T cells are the predominant population in these cultures, it is reasonable that the production of IFN-{gamma} by T cells may mask the amount of IFN-{gamma} produced by NK cells. Although IFN-{gamma} would positively regulate the function of dendritic cells, our results show that CTL development depends on whether NK cells are present in the culture.


Figure 5
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  		Fig. 5. Cytokine secretion. The supernatants from the cultures of nonadherent cells, CD56-depleted cells, or purified T cells were collected on days 2 and 7 after first stimulation and days 2 (day 9) and 7 (day 14) after second stimulation. The IFN-{gamma} (A) or IL-18 (B) secretion was measured by ELISA kits. Representative data from two similar experiments. Points, mean of triplicate microwell cultures; bars, SD.

 
It is well described that cytokines, such as IL-12, IL-15, and IL-18, produced by activated dendritic cells play an important role in the expansion, survival, and induction of cytotoxicity in T cells. We therefore tested if any of these cytokines were involved in downstream CTL induction in our experimental model. Although there was a significant and persistent increase of IL-18 secretion in the culture supernatant of nonadherent cells compared with the T cells or CD56-depleted cells, we could not detect any significant secretion of IL-12 p70 active form or IL-15 (data not shown). IL-18 secretion was significantly higher in the cultures of nonadherent cells compared with the cultures of CD56-depleted or T cells (Fig. 5B). The increase of IL-18 became even more striking with increasing culture time, when the effectors cells were harvested for measuring their cytotoxicity (day 7 after the second stimulation). This result indicates that the persistent secretion of IL-18 may activate cytotoxic T cells.

To confirm that IL-18 is a key mediator for the development of the cytotoxic T cells, we added a neutralizing mAb for IL-18 to the cultures of nonadherent cells on days 0 and 4 after the first and second stimulations. The presence of this antibody significantly inhibited the cytotoxicity of nonadherent cells against B-Jab cells (Fig. 6). The cytotoxicity of nonadherent cells against Daudi and LCL.721 was <10% with or without antibodies (data not shown). These results show that production of IL-18, possible along with other undefined mechanisms, plays an important role in the induction of CTL response against B lymphoma cells.


Figure 6
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  		Fig. 6. Blockage of anti-IL-18 antibody on cytotoxicity against B-Jab. Nonadherent cells from HLA-A0201+ donor were stimulated with autologous dendritic cells loaded with apoptotic Ramos cells as described in Fig. 1. The control mouse IgG or mAbs against human IL-18 were added to the cultures at a concentration of 1 ng/mL on days 0 and 4 for first and second stimulations. The cytotoxicity was measured by 51Cr-release assay. Diamonds, nonadherent cells; squares, nonadherent cells treated with control mouse immunoglobulin; triangles, nonadherent cells treated with mAb to IL-18. Representative data from two similar experiments. Points, mean of triplicate microwell cultures; bars, SD.

 
To test the possibility of if CD4+CD25+ T regulatory cells are induced in the culture or if T cells are not fully activated without the presence of NK cells, we compared phenotype between nonadherent cells and T cells before or after stimulations. No significant changes were found. Therefore, these factors are unlikely to be involved.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Vaccine-based immunotherapy with dendritic cells has been a promising approach for the treatment of cancer. The majority of studies done to date use tumor-associated antigenic peptides (47, 48). However, because of the high rate of mutation in tumor cells allowing for loss of expression of a single antigen, it is likely that use of multiple antigens will induce a broader, longer-lasting, and more effective tumor-specific immune response. Therefore, apoptotic tumor cells can be an excellent source of tumor antigens for dendritic cell loading, because potential uncharacterized antigens could be presented to T cells without particular requirement for specific HLA typing. This approach may also overcome the limitation of using single antigenic peptides (49–51). Other than immunoglobulin idiotype, there have been no highly effective vaccine candidate antigens defined for B-cell lymphoma. Therefore, dendritic cells loaded with apoptotic lymphoma cells may be a strategy that warrants investigation for immunotherapy for B lymphoma.

The signals that lead to dendritic cell activation and priming of an adaptive immune response are not fully understood. The ability of dendritic cells to prime effective immune response has been evaluated by phenotypic maturity of dendritic cells that can be induced by inflammatory cytokines, microbial products, and CD40L stimulation. However, a series of recent studies have challenged this traditional view, showing that phenotypically "mature" dendritic cells are not necessarily functional dendritic cells capable of T-cell priming. Studies by Mailliard et al. have shown that the induction of effective antitumor CTL requires NK cells, along with other factors, to provide help for dendritic cells. In their studies, this help was mainly mediated by IFN-{gamma} and TNF-{alpha}, which polarize dendritic cells into type 1 dendritic cells, characterized by superior production of IL-12 p70 in response to subsequent interaction with T cells (43). Similarly, an in vivo study by Smith et al. showed that licensing of dendritic cells by cognate CD4+ T cells is required to generate CD8+ T-cell immunity (35).

In the present study, we describe the important helper role of NK cells in the cross-priming of B lymphoma cell–associated antigens by dendritic cells. Autologous immature dendritic cells that have captured apoptotic B lymphoma cells were induced to maturation by inflammatory cytokines before stimulating T cells. These dendritic cells showed a fully mature phenotype, such as a high level of expression of MHC class II, costimulatory molecules CD83, CD86, and CD40. They stimulated autologous T cells to proliferate as well as nonadherent cells did. However, they failed to induce functional cytotoxic T cells. A CTL response was induced only when NK cells were present in the culture, thereby showing a crucial "helper" role for NK cells in cross-priming.

The dendritic cell licensing function of NK cells and the subsequent T-cell responses can be mediated by both soluble factors and cell-to-cell contact. IFN-{gamma} and TNF-{alpha} have been shown to be major factors that mediate NK help for dendritic cells (52). However, a cell-to-cell contact mechanism has also been shown to play an important role in the interaction between NK and dendritic cells. Piccioli et al. reported that maturation of dendritic cell by activated NK cells was completely dependent on cell-to-cell contact (53). Similarly, Gerosa et al. showed that coculture of fresh human NK cells with monocyte-derived immature dendritic cells induced mutual activation of both NK cells and dendritic cells. In their system, the activation of dendritic cells by NK cells was mostly cell contact dependent (54).

We detected high-level secretion of IFN-{gamma} in the cultures with or without NK cells. Thus, we did not exclude a possible role for NK-produced IFN-{gamma} in the initial activation of dendritic cells. Coculturing NK cells with antigen-captured dendritic cells may rapidly activate NK cells by producing IFN-{gamma} and/or other factors, which in turn activate dendritic cells. T cells primed by these "licensed" dendritic cells then generate abundant IFN-{gamma} in the cultures. Because T cells are the predominant population in the cultures, it is difficult to distinguish the IFN-{gamma} production between T and NK cells in this system. Although we do not challenge the possible role of IFN-{gamma} in activation of dendritic cells, we suggest that dendritic cell licensing by NK cells may mainly depend on direct contact between dendritic cells and NK cells and/or other factors that result in downstream development of CTL in our system. According to Mailliard et al., although IFN-{gamma} and TNF-{alpha} seem to be the key mediators for NK cells to polarize dendritic cells to become DC1, characterized by abundant production of IL-12 and ability to stimulate Th1 and CTL responses, the presence of NK cells always leads to a better dendritic cell function in inducing Th1 and CTL responses (43). This suggests that interaction between NK cells and dendritic cells is a complex process, which may involve both cell membrane molecules and soluble factors. NK cells express several activating and inhibitory receptors. NK cells exert their function through the interactions between these receptors with their ligands on target cells (39). Activating receptors NKp30 and other NKp30-independent pathways have been shown to be responsible for NK activation by dendritic cells in a cell-to-cell contact basis (40). However, there has been limited information concerning the molecules directly involved in dendritic cell licensing by NK cells.

IL-12, IL-15, and IL-18, produced by activated APCs, have been shown to act as signals for dendritic cell activation and T-cell priming (42, 43). In both murine and human systems, ability of dendritic cells to promote Th1 and CTL responses was characterized by enhanced IL-12 production (43). Surprisingly, IL-12 secretion was undetectable in the supernatants of the cultures and therefore is unlikely involved in the CTL induction in our experimental model. Instead, we detected secretion of IL-18, which correlates with enhanced cytotoxicity of T cells against B lymphoma cells. Indeed, when neutralizing antibody to IL-18 was added to the culture of nonadherent cells, the cytotoxic activity of the cells was reduced. IL-18 is a potent cytokine that enhances cytotoxicity of T and NK cells as well as their cytokine production (55). Systemic administration of IL-18 has shown considerable therapeutic activity in several murine tumor models (56, 57). IL-18 secretion may well be a signal of dendritic cell licensing by NK cells and a mediator for enhancing CD8+ T-cell response. Furthermore, IL-18 may also further activate NK cells, forming a positive feedback loop in the cultures, which would provide a means for the amplification of NK cell action. Because anti-IL-18 antibody did not completely abrogate the CTL response, additional yet undefined mechanisms are likely to be involved. NK cells may also directly instruct dendritic cells to switch on the cross-priming machinery. It is interesting to note that both IL-12 and IL-18 are produced by activated APC, such as dendritic cells and monocytes/macrophage, and the two cytokines share many functional similarities. How the differential signals for these cytokines was switched on in different situations is unknown. The experiment system by Mailliard et al. (58) is very different from ours and therefore cannot be compared directly. It may depend on antigen types and origins as well as on the access routes of the antigens to the cells. It has been shown recently that soluble and particle antigens, including cells, use differential pathways for the cross-priming (59).

The ability of NK cells to license dendritic cells for cross-priming of cytotoxic T cells against lymphoma has important implications for improving dendritic cell–based cancer immunotherapy. For example, effective strategies for the immunotherapy of cancer patients could include (a) licensing of vaccine-carrying dendritic cells by use of activated NK cells ex vivo, (b) use of NK-derived cytokines to license dendritic cells ex vivo before their use as a vaccine, or (c) inclusion of ex vivo expanded NK cells mixed in dendritic cell–based vaccines.


   Acknowledgments
 
We thank Drs. James W. Young, Christian Munz, and Adam Boruchov for helpful discussions.


   Footnotes
 
Grant support: NIH grants PO1 (CA23766) and RO1 (CA55349), Tudor Fund, and The Lymphoma Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: D.A. Scheinberg is a Doris Duke distinguished scientist.

Received 5/ 4/05; revised 9/ 9/05; accepted 9/27/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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