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* Laboratory of Experimental Immunology and
Intramural Research Support Program, SAIC-Frederick, Nactional Cancer Institute Center For Cancer Research, Frederick MD 21702-1201;
National Genome Research Institute, National Institutes of Health, 5 Nokbun-dong, Eunpyung-ku, Seoul 122-701, Korea; and
Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV 89557
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Abstract |
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CD40 to both wild-type and CD40–/– mice bearing Renca tumors resulted in substantial amounts of TNF- and MCP-1 in the serum, increased the number of total splenocytes and MHC class II+ CD11c+ leukocytes, and when combined with IFN-
, inhibited the progression of established Renca tumors in vivo in both wild-type and CD40–/– mice. Similarly, treatment of CD40+ A704 and ACHN human RCC lines with mouse anti-human CD40 Ab induced strong expression of genes and proteins for MCP-1, IL-8, and GM-CSF in vitro and in vivo. Finally, in SCID mice, the numbers of ACHN pulmonary metastases were dramatically reduced by treatment with species-specific human CD40 Ab. These results show that CD40 stimulation of CD40+ tumor cells can enhance immune responses and result in antitumor activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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CD40) Ab induces the production of various cytokines (e.g., GM-CSF, IL-1, IL-4, IL-6, IL-8, IL-10, IL-12, RANTES, and TNF-), up-regulation of adhesion and costimulatory molecules (ICAM-1, VCAM-1, CD80, and CD86), and induction of Fas expression (1, 2). The biological importance of CD40–CD40L interactions also has been well documented in diseases such as X-linked hyper IgM syndrome, atherosclerosis, Hodgkin’s disease, and Alzheimer’s disease (1, 2).
Although it has been reported that some solid tumor cells, including bladder, breast, and ovarian carcinoma cells, as well as melanoma cells, express CD40 (3, 4, 5, 6), the biological implications of CD40 expression on most solid tumor cells remain unclear. In this study, we demonstrate that the Renca mouse renal cell carcinoma (RCCs) constitutively expresses CD40, and stimulation of CD40 resulted in expression of GM-CSF and MCP-1 genes and proteins. Stimulation of Renca with agonist CD40 in vitro also caused the secretion of proteins capable of enhancing migration of HUVEC cells. Similarly, stimulation of CD40 on two human RCC lines, ACHN and A704, induced gene and protein expression of IL-8, GM-CSF, and MCP-1, as well as antitumor responses in SCID mice (that lack responsiveness to human CD40).
This study demonstrates that stimulation of CD40 expressed on RCCs induces expression of biologically active genes and proteins that can contribute to antitumor host responses. The results further suggest that therapeutic strategies that include stimulating CD40 on tumors may be useful for cancer treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BALB/c and CB17 SCID mice were obtained from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center (NCI-Frederick), and BALB/c CD40–/– mice were purchased from The Jackson Laboratory. All mice were used between 8 and 10 wk of age. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985).
Tumor cells and cell culture
The Renca renal adenocarcinoma (7) and streptozotocin-induced RCCs (8) of BALB/c origin were maintained in RPMI 1640 medium supplemented with 10% FBS (FBS), 2 mM L-glutamine, 1x nonessential amino acids, and 1 mM sodium pyruvate. Other mouse cell lines (A20, P815, Yac-1, B16, CT26, DA-3, Hepa 1-6, M109, 3LL, RAW-264, and EOMA) and human cell lines (ACHN and A704) also were cultured in RPMI 1640 medium with the same supplements. HUVEC cells were obtained from BioWhittaker and cultured in Endothelial Cells Growth Medium BulletKit (BioWhittaker).
Cytokines and reagents
Recombinant mouse IFN- (specific activity, 1 x 107 U/mg) was purchased from PeproTech. Agonist rat anti-mouse CD40 (clone FGK115) (9) was obtained from B. Blazer (University of Minnesota Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, MN). Briefly, the Ab was produced in pristane-primed mice using the hybridoma FGK115-B3. The ascites fluid was tapped, filtered (0.45 µm), and frozen until purification. The purification was done by salt cut, which consisted of an initial caprylic acid purification, followed by ammonium sulfate precipitation. Protein/IgG quantification was performed by spectrophotometry, and Ab content was determined by rat IgG ELISA. The endotoxin level of the purified Ab was 1.74 EU/mg Ab (BioWhittaker). Purified rat IgG was purchased from Jackson ImmunoResearch Laboratories. Anti-human CD40 (S2C6) was obtained from Seattle Genetics. All other Abs used in these studies were purchased from BD Pharmingen.
RT-PCR
Total cellular RNA was isolated using Trizol (Invitrogen Life Technologies) and analyzed via RT-PCR using the SuperScript Preamplification System for first-strand cDNA and priming with oligonucleotide in a 20-µl reaction. The converted cDNA (3 µl) was used as a template for PCR amplification with gene-specific primers. PCR amplification was performed with AmpliTaqGold Polymerase (PerkinElmer) for 10 min of initial denaturation at 94°C for 40 s, 55°C for 40 s, and 72°C for 1 min. PCR products were resolved by 1.2% agarose gel electrophoresis with ethidium bromide staining. The gel picture was inverted by the NIH Image software (
http://rsb.info.nih.gov/nih-image
).
RNase protection assay (RPA)
RNA was isolated from cell lines or whole tumor homogenate using Trizol reagent. RPAs were performed using [-33P]UTP and BD RiboQuant In Vitro Transcription (BD Biosciences) to synthesize riboprobes. The BD RPA kit was used to perform the protection assay, and both procedures were performed according to the manufacturer’s protocols. The protected RNA was separated on a 6% acrylamide gel, and the gel was dried for 1 h at 80°C. The dried gel was exposed to a phosporimager screen overnight, and visualized using the Typhoon Scanner (Molecular Dynamics) and ImageQuant (Amersham Biosciences) software (Molecular Dynamics, Amersham Pharmacia).
Flow cytometric analysis
Cells (0.5–1 x 106) were stained with fluorescence, either FITC- or PE-labeled anti-mouse Abs at 4°C for 20 min. After washing, cells were analyzed on a FACScan flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences).
Migration assay
Renca cells were treated with mouse rIFN- (100 U/ml), CD40 (FGK115; 100 µg/ml), or IFN- plus CD40 for 18 h, and 30 µl of culture medium under these conditions was plated in triplicate in a 48-well Boyden chemotaxis chamber. The chamber was covered with an 8-mm collagen-coated membrane, and 50 µl of HUVEC cells (1 million per ml) was added to the top portion of the chamber. The chemotaxis chamber was incubated for 2 h at 37°C, and the membrane was fixed and stained with HemaQuick (Richard Allen Scientific) in accordance with the manufacturer’s protocol. Migrated cells were counted using a microscope (Olymous BX40).
In vivo tumor models
Renca cells (100,000) were injected into the kidney of BALB/c CD40+/+ or CD40–/– mice. Eleven days after tumor injection, mice were treated with HBSS, CD40 (100 µg i.p.), CD40 (FGK115; 100 µg i.p.) plus mouse rIFN- (50,000 IU i.p.), or IFN- alone (50,000 IU) daily for 4 days. This cycle was repeated one time starting 18 days after tumor injection. Mice were euthanized 15 and 22 days after injection, and tumors were collected and weighed. Spleens and serum also were collected at days 15 and 22. For the human RCC experiments, 1 x 106 ACHN cells were injected i.v. in SCID mice, and established pulmonary metastases in the mice were treated beginning on day 7 with HBSS, CD40 (S2C6; 10 µg), or control IgG Ab (10 µg) daily for two cycles, with 5 days per cycle. Mice were monitored for tumor growth and progression or euthanized at day 26, and lung metastases were counted.
Isolation of spleen cells
Spleens were homogenized using a stomacher, and RBC were removed with ACK lysing buffer (Quality Biologicals) before use for flow cytometry.
Serum cytokine levels
Cytokine levels were measured on pooled mouse serum using the BD Cytometric Bead Array (CBA assay; BD Biosciences) as described by the manufacturer’s instructions.
Immunohistochemistry
Primary tumors were removed 15 days post Ab treatment, fixed in 4% paraformaldehyde, and sectioned in paraffin-embedded sections. The slides were deparaffinized in xylenes, followed by sequential alcohol series, and blocked with 2% BSA/PBS. Slides were then incubated with one of the following primary Abs: F4/80, CD3, CD31, or isotype-matched negative control Abs, followed by incubation with HRP-conjugated secondary Abs and diaminobenzidine chromogen system for visualization. As a positive control, each Ab was first tested on spleen sections (data not shown).
Statistical analysis
The comparison of mean values between groups was analyzed by ANOVA and Dunnett’s comparison test. All p values <0.05 were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Expression of CD40 was detected by RT-PCR using a panel of mouse tumor cell lines. Most mouse tumor cell lines of different histotypes were negative for CD40 gene expression. However, strong expression of the CD40 gene was found on both the mouse Renca RCC and the recently derived SIRCC-1 streptozotocin-induced RCC, as well as the A20 B cell line (Fig. 1A). High constitutive cell surface expression of CD40 protein was confirmed by flow cytometry on the same cell lines (Fig. 1B). The constitutive expression of CD40 on A20, which was used as a positive control, was not surprising because it is a B cell line and was known to be CD40+ (10). Although some other normal and neoplastic cells have been previously reported to express CD40 (10), the strong expression by both mouse renal cell lines had been previously unreported.
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Stimulation of CD40 on some carcinoma cells has been reported to induce production of IL-1, TNF, and GM-CSF (3). This result suggested that CD40 expressed on RCCs also might be functional. Renca cells were stimulated with agonist CD40 (FGK115; 10 µg/ml) for 24 h in vitro, and expression of cytokines was then screened by RPA and/or ELISA. Ligation of CD40 induced strong expression of both MCP-1 and GMCSF genes (Fig. 2A), and substantial levels of both GMCSF and MCP-1 proteins also were induced by CD40 in a dose-dependent manner (Fig. 2, B and C). IFN- is known to up-regulate CD40 expression, and our studies showed that IFN- plus CD40 enhanced the level of MCP-1 protein expression over that observed with either agent alone (Fig. 2D). This enhancement also was seen for both Fas and ICAM-1 proteins (data not shown). The demonstration of strong increases in cytokine production and cell surface expression of other proteins after CD40 stimulation clearly demonstrated that ligation of CD40 expressed on several mouse renal cancer cells can activate a variety of functions that could be associated with increased immunogenicity (GMCSF) and/or recruitment of leukocytes (MCP-1). In either case, these biological activities might thus contribute to, or amplify, early host responses against cancer. To rule out the potential for contaminating TLR agonists in the Ab preparation, we generated DC from CD40–/– mice and exposed them to the CD40 Ab for 48 h, because it has been reported that cytokine production by DC via CD40 triggering requires microbial/TLR signaling (11, 12). We did not detect any cytokines (e.g., IL-12, TNF-, MCP-1, and IL-6) in the cell culture supernatants from these Ab-treated DC cultures (data not shown). However, we could confirm that these DC secreted significant levels of cytokines, such as IL-6, upon LPS treatment (data not shown).
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CD40 enhances HUVEC migration
To assess whether the increase in gene expression of chemotactic factors would result in functional changes in vitro, Renca cells were pretreated with 100 µg of mouse CD40 for 18 h to up-regulate chemotactic proteins. Supernatant from CD40-treated cells caused increased migration of HUVEC cells, compared with untreated Renca (Fig. 3). This effect was further enhanced with CD40 plus IFN- treatment. These results demonstrate a functional effect of CD40-induced signaling, in this case, an ability to attract human endothelial cells.
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CD40 enhances chemokine expression in vivo
The data presented above demonstrate that the increased expression of chemotactic factors upon stimulation of CD40 is accompanied by the appropriate functional activities in vitro. The studies presented in Fig. 4 demonstrate similar effects in vivo. Administration of agonist CD40 to tumor-bearing wild-type (WT) and CD40–/– mice results in substantial levels of both TNF- and MCP-1 (Fig. 4a), proteins shown to be released after exposure of Renca to CD40 in vitro. Interestingly, even in CD40–/– mice, CD40 induced up to 200 pg/ml MCP-1 (Fig. 4b), demonstrating that the CD40 expressed on the tumor can in of itself be a functional target for CD40-induced biological effects in vivo. As a specific control, we implanted a CD40– tumor, DA3, into both WT and CD40–/– mice. The DA3 tumor was selected because it was determined to be negative for CD40 expression (see Fig. 1), and it is syngeneic for the same BALB/c background as the Renca tumor. As shown in Fig. 4, treatment of WT mice bearing the DA3 tumors led to the up-regulation of TNF- as well as MCP-1, albeit to levels lower than that for the CD40+ Renca tumors. Furthermore, no up-regulation of either cytokine was observed when DA3 tumors were injected into CD40–/– mice (Fig. 4b).
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The ability of CD40 to stimulate cytokine and chemokine production even in mice where only the tumor itself is positive for CD40, suggested that such tumor-dependent CD40 mediated effects might alter leukocyte subset number and incidence. Spleens isolated from Renca-bearing BALB/c treated with CD40 or CD40 plus IFN- contained an increased number of total leukocytes, compared with untreated or control Ab-treated mice (Fig. 5a; p < 0.05). Similar increases also were seen in the liver (data not shown). Interestingly, although the increases were most pronounced in WT mice, there also was a 70% increase in total splenocytes in tumor-bearing CD40–/– mice (Fig. 5b). This effect was lost in CD40–/– tumor-bearing mice treated with control Ab, or in CD40–/– mice that did not have a tumor showing that the biological effects obtained occurred by direct stimulation of CD40 on the tumor itself. In addition, the MHC class II (MHC-II)+, CD11c DC subset was as effectively increased in tumor-bearing CD40–/– mice as in WT mice after CD40 treatment (Fig. 5c), showing that selective triggering of CD40 expressed by tumor cells can result in increased numbers of DCs, a critical element in the initiation of adaptive immune responses. Again, CD40 expressed on tumor cells are shown to be sufficient for this effect, because no increases in DC number were observed in WT or CD40–/– mice treated with control Ab, or in non-tumor-bearing CD40–/– mice treated with active CD40.
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To directly assess the effects of CD40 ligation upon leukocyte recruitment and blood vessel distribution, we performed immunohistochemistry on fixed Renca tumor sections. Renca tumors were injected into either WT or CD40–/– mice as before, and mice were treated with either CD40 or a rat isotype control Ab. Tumor sections were immunostained with Abs for F4/80, CD3, and CD31, as described in Materials and Methods. As shown in Fig. 6, F4/80 staining revealed intense reactivity that was indistinguishable between WT and CD40–/– mice. We also noted strong reactivity in tumor vessels for CD31 immunoreactivity that also was indistinguishable between WT and CD40–/– mice (Fig. 6). Finally, CD3 immunostaining was elevated over isotype control staining in both the WT and CD40–/– mice. For all three Ags, immunoreactivities appeared indistinguishable between the WT and CD40–/– mice, and the corresponding negative control Ab staining was always negative. Taken together, these data support the hypothesis that CD40 expression by the tumors leads to significant leukocyte recruitment, which appears to be consistent with the particularly high levels of MCP-1 that we could detect in vivo (Fig. 4).
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The ability of CD40 to increase DCs in both WT and CD40–/– mice, and the previous finding that GM-CSF gene expression was enhanced by treatment of CD40+ tumors in vitro (Fig. 2b), suggested the ligation of tumor-associated CD40 would result in increased GM-CSF expression in vivo. Analysis of RNA involved from the tumor site revealed an elevation of GM-CSF gene expression in WT tumor-bearing mice after treatment with CD40 and the combination of CD40 plus IFN- (Fig. 7, a and b). Interestingly, impressive increases in GM-CSF gene expression also were seen in tumor-bearing CD40–/– mice, and this effect was further increased by the coadministration of IFN- with the CD40. These results confirm that strong biological responses in the tumor microenvironment can be achieved solely through stimulation of CD40 expressed on tumor cells.
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CD40 plus IFN- leads to a reduction in tumor size that can occur in the absence of CD40 on normal host cells
Because ligation of CD40 expressed on tumors can result in strong biological effects in vitro and in vivo, even in the absence of CD40 expression on host cells (Figs. 4–7), studies were designed to determine whether ligation of CD40 on tumor cells could contribute to tumor regression. In these studies, treatment of tumor-bearing WT mice with CD40 or CD40 plus IFN- resulted in a significantly smaller tumor burden, compared with all other groups (Fig. 8a). Interestingly, although treatment with CD40 or IFN- alone failed to induce substantial tumor responses in CD40–/– mice, the combination of CD40 plus IFN- reduced mean tumor size by 
50%, compared with untreated controls, IFN- or CD40 alone, or mice treated with a control Ab (Fig. 8b). These results suggested that the presence of IFN-, which potentates the ability of CD40 to increase DC numbers and GM-CSF levels in tumor-bearing CD40–/– mice, also complements the ability of CD40 ligation to negatively impact tumor growth (Fig. 8b). Overall, these results suggest that the most pronounced effects of CD40 on tumor progression do depend predominantly on stimulation of CD40 expressed on host cells; however, the results also reveal a component of the response that may be enhanced by ligation of CD40 expressed on the tumor itself.
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To more definitively assess the potential of CD40 expressed on tumors to serve as a molecular target for CD40-directed therapy, several human RCC lines were tested for CD40 expression. Both ACHN and A704 exhibited constitutive expression of CD40, which was up-regulated by IFN- pretreatment (Fig. 9). Like the mouse Renca RCC, CD40 stimulation increased cytokine message levels for MCP-1 and IL-8 (Fig. 10a), and levels of MCP-1, GMCSF, and IL-8 proteins (Fig. 10b) in vitro. ACHN cells also were injected intrarenally into SCID mice and then treated with HBSS, human CD40, or a control IgG Ab. Analysis of serum from the mice showed an increased level of human MCP-1 protein after one cycle (day 22) or two cycles (day 29) of treatment (Fig. 11). The protein detected was produced solely from ACHN, because the assay detected only human MCP-1, and the human D40 does not react with expression of mouse CD40 on host tissue.
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CD40 (S2C6) induces cancer regression of established lung metastases
To definitively determine whether CD40 expressed on tumor cells could serve as a target for inducing antitumor response, mice were injected i.v. with ACHN cells to selectively establish lung metastases and treated with HBSS, human CD40 (S2C6), or control Ab. Mice receiving CD40 therapy had significantly fewer detectable lung metastases, compared with HBSS- or control Ab-treated mice (Fig. 12). Thus, in this human tumor xenograft model, where the human CD40 recognizes only the CD40 expressed on the tumor, significant suppression of tumor growth was achieved.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Some recent studies have suggested that CD40 expressed in the tumor microenvironment may contribute to tumor growth. For example, CD40 up-regulation in the tumor vessels of renal carcinomas and Kaposi’s sarcoma suggests a possible role for this molecule in tumor angiogenesis (15), whereas sarcoma patients with tumors expressing CD40 in >50% of cells have an unfavorable prognosis, compared with patients with CD40– tumors (16). In addition, investigations using CD40L gene therapy in a bladder cancer model have shown that CD40 activation in the tumor milieu can cause regression of smaller tumors and inhibit progression of larger tumors while inducing IL-12 and reducing IL-10 message (17).
In the present study, we report that both mouse and human RCCs also express cell surface CD40. Our studies demonstrate strong constitutive expression of CD40 on mouse and human RCC cell lines, and expression of CD40 also has been observed on human renal proximal tubule cells (18). In addition, most primary cultures from RCCs also express CD40 (19), suggesting that fresh RCCs also can express CD40. These results may suggest that CD40 expression may be up-regulated during neoplastic development or perhaps during development of metastatic potential. CD40 activation has been shown to up-regulate the expression of various molecules necessary for effective Ag presentation, such as B7.1, B7.2, ICAM-1, and VCAM-1, and induces the production of cytokines such as IL-1, IL-6, IL-8, IL-10, IL-12, and TNF- (1, 2), which are strongly associated with the induction of both Th1 and Th2 immune responses. Strong induction of GM-CSF, MCP-1, and IL-8 protein and/or message was observed in both mouse and human RCC cells after CD40 stimulation. Consistently, we observed that CD40 ligation, even in CD40–/– mice, elicited significant monocyte as well as T cell recruitment into the tumors. The pattern of induction of chemokines and cytokines varied between different CD40+ cells. The expression of CD40 also was observed on mouse endothelial cells (EOMA) (data not shown) as well as A20 (B lymphoma) and RAW-264 (monocytic tumor cells). However, the induction of cytokines or chemokines in EOMA and A20 cells was not observed after CD40 stimulation in vitro, whereas the expression of IL-18 and metastasis-reducing protein NM23 genes was up-regulated in RAW-264 cells after CD40 engagement (data not shown). This cell type-specific induction of cytokines and chemokines suggests that CD40 ligation could play differing biological roles in various CD40+ tumors by inducing unique profiles of genes.
Because of the central role of CD40 in the genesis and amplification of immune responses, the in vivo effects of CD40 stimulation are actively being studied in a variety of murine cancer models. In some settings, absence of some critical level of CD40 stimulation may result in an ineffective antitumor response. Therefore strategies that specifically target CD40 may enhance the quality or strength of a cellular immune response against cancer. This approach has proven successful in targeting and killing of CD40+ leukemia cells (20) and studies targeting CD40– solid tumors suggest that CD40 does not always need to be expressed on the tumors themselves. For example, growth of established solid tumors (B16-F10 and CT26) that are CD40– was inhibited (21) by treatment with an agonistic CD40 mAb that mimics the stimulation of CD154 and this response was shown to require CD8+ cells. Murphy et al. (22) proved that CD40 engagement combined with IL-2 administration induced tumor regression in mice bearing metastatic Renca tumors, and showed that this response involved both CD8+ T cells as well as DCs. Our present studies have used this model to investigate directly whether CD40 expressed on RCCs can influence the ability of CD40-specific reagents to modify tumor growth and progression. The results suggest that ligation of CD40 in WT mice can result in expression of numerous cytokines and chemokines, and increase the number of DCs. This is consistent with previous reports showing stimulation of CD40 on APC leads to the secretion of cytokines (including GM-CSF), an increase in costimulatory molecules such as ICAM-1, CD80, and CD86 and subsequent priming of T cells (3). However, our studies in CD40 deficient mice, show that CD40 plus IFN- is capable of eliciting a modest yet consistent reduction in tumor size in the absence of CD40 expression on normal tissue.
Previous studies have proven that CD40 need not be expressed by the tumor itself for the therapy to be effective (21), and clinical data imply that the degree of CD40 expression on some human cancer cells correlates with metastatic spread (23). Our data suggest the possibility that a tumor, which is capable of signaling through CD40 and producing some cytokines and costimulatory molecules, such as GM-CSF, MCP-1, and ICAM-1, might be more effectively targeted through a strategy that assures delivery of CD40-specific reagents to the tumor microenvironment. In addition to showing that CD40 plus IFN- treatment causes an increase in CD8+ T cells, our results also show that other vital leukocyte compartments can be affected because there is an increase in CD4+ T cells and macrophages. However, these effects are primarily host mediated, because the increases in CD4+ and CD8+ T cells and activated macrophages are not seen in CD40–/– mice under the same treatment conditions. For example, we found that the CD40/IFN--mediated increase in total splenocytes was lost in CD40–/– mice. Some recent studies have demonstrated that IFN- treatment up-regulates CD40 expression on several cell types, including DCs (24). Thus, it is conceivable that the IFN--responding cells from the CD40–/– mice fail to up-regulate CD40 expression and respond to the simultaneous ligation via CD40. Taken together, these data suggest that the biological contributions of targeting CD40 on the tumor itself are qualitatively different from the effects that are achieved when stimulation of CD40 on host immune cells is exclusively achieved.
Immunotherapy has great potential for cancer prevention and treatment and may provide considerable advantage for therapy of those cancers, including RCCs, which are most resistant to conventional chemotherapy. By analyzing the role of a CD40+ tumor in both CD40 competent and CD40–/– hosts, we have been able to demonstrate that if stimulated properly, the tumor itself may be induced to play an active role in its own destruction. This also leads to the possibility that an even more potent antitumor response would be obtained by optimally targeting CD40 on both normal host immune cells, as well as the tumor itself.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported in whole or in part by with federal funds from the National Cancer Institute (NCI) and National Institutes of Health (NIH) Contracts N01-CO-12400 and R01-CA-95572 (to W.J.M.). This research also was supported in part by the Intramural Research Program of the NIH/NCI. 
2 The publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
3 Address correspondence and reprint requests to Dr. Robert H. Wiltrout, Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute at Frederick, Building 560, Room 31-93, Frederick, MD, 21702-1201. E-mail address: wiltroutr{at}ncifcrf.gov.
4 Abbreviations used in this paper: DC, dendritic cell; RCC, renal cell carcinoma; CD40L, CD40 ligand; MHC-II, MHC class II; WT, wild type.
Received for publication March 17, 2005. Accepted for publication March 22, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy. J. Clin. Invest. 108: 51-62. [Medline] production by human cord blood monocyte-derived dendritic cells. Ann. Hematol. 84: 423-428. [Medline]
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