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Experimental Therapeutics, Preclinical Pharmacology |
H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Mice bearing mammary adenocarcinoma expressing a model tumor antigen, influenza virus HA (DA3-HA), and parental tumor (DA3) were treated with different doses of paclitaxel with or without DCs. Paclitaxel was injected three times weekly, DCs were injected either i.v. or into tumor site (t.s.) 36 h after each injection of paclitaxel. Apoptosis was measured using Annexin V binding or terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays. CD8-mediated response of T cells to HA-derived peptide epitope was measured in an enzyme-linked immunospot (ELISPOT) assay. CD4-mediated response of T cells to HA-derived peptide was measured in proliferation assay. Nonspecific T-cell proliferation was measured in response to ConA and immobilized anti-CD3 and anti-CD28 antibodies.
Results: We have selected the dose of paclitaxel that induced a substantial level of apoptosis and moderate inhibition of T-cell function. Combined treatment resulted in the induction of HA-specific CD8-mediated response in all nine of the tested mice, and CD4-mediated responses in four of six treated mice. These effects were observed only if DCs were injected into tumor site, but not when injected i.v. No specific responses were found in mice treated with either chemotherapy or DCs alone. Injection of dexamethasone together with paclitaxel did not affect the induction of immune responses. Significant antitumor effect of combined treatment was observed in DA3-HA tumor-bearing mice as well as in mice bearing parental DA3 tumor.
Conclusion: The combination of DC administration with repeated cycles of chemotherapy and dexamethasone (conditions similar to real clinical practice) resulted in the induction of antitumor response despite the immunosuppression induced by such treatment.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor cells themselves are the richest TAA source. However, the mere presence of DCs in the vicinity of tumor has been insufficient to induce antitumor responses. DCs were recently identified to have unique pathways for phagocytosis, processing, and presentation of antigens from apoptotic cells (3 , 4) , and are capable of inducing potent immune responses (5 , 6) . It appears that the optimal presentation of antigens from tumor cells would require not only phagocytosis of apoptotic cells by DCs that have not matured yet, but also activation and maturation of DCs via exposure to the necrotic tumor cells or their products (7 , 8) . These new findings suggest a therapeutic possibility: exposure of DCs to tumor apoptotic bodies to provide TAAs together with an activation/maturation environment. The combination of apoptosis-inducing treatment with DC administration also eliminates the requirement for patient selection based on MHC class I type, for future clinical development. Similarly, it eliminates subject selection criteria based on the expression of defined TAAs and reduces the problem of TAA loss during tumor progression. Recently, several groups, including ours, described induction of antitumor response after DC administration into tumors with high spontaneous rates of apoptosis (9) or after the combination of DC administration with apoptosis-inducing therapy (10 , 11) . These experiments established proof-of-concept that the combination of the intratumoral placement of ex vivo generated autologous DCs in concert with apoptosis-inducing therapy (chemotherapy or radiation) might lead to a generation of systemic antitumor immunity. In cancer patients conventional chemotherapy is given in several cycles over relatively long periods of time. It is known that chemotherapy is associated with systemic immune suppression. The obvious question is whether this environment will be permissible for the induction of effective immune response. This study, for the first time, has tried to address this question. We used a tumor model of mammary adenocarcinoma DA3. DC administration was combined with three cycles of paclitaxel at the dose that induces apoptosis and moderate immune suppression. We have found that such combined treatment induces significant antitumor immune response. This response was associated with marked antitumor effect.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß T-cell receptor specific for MHC class II-restricted SFERFEIFPKE peptide, derived from influenza HA, were originally obtained from Harold von Boehmer (Basel Institute for Immunology, Basel, Switzerland) and then were crossed to a BALB/c background for more than 10 generations.
Description of the Experimental Model.
DA3 cells (provided by Dr. Diana M. Lopez, University of Miami School of Medicine, Miami, FL) is a well-characterized tumor cell line that was derived from the D1-DMBA-3 mammary tumor syngeneic to BALB/c mice (12)
. The DA3 cell line produces tumors in BALB/c mice with the same growth kinetics as the parent tumor. s.c. injection of 1 x 106 DA3 tumor cells results in a solid tumor in the area of injection after about 5–7 days. Approximately 10 days after s.c. implantation, micrometastases are evident in the draining lymph nodes of tumor-bearing animals, and, as tumor progresses, additional metastases are found in the lungs but not in the liver or spleen. DA3-HA cells were generated by the transfection of DA3 cells with the construct pIHA, which encodes the HA molecule of the influenza virus A/PR/8/34 (H1N1). A stable transfectant was selected and expanded, and expression of the HA gene was confirmed by the presence of HA protein on the cell surface, as determined by FACS analysis using the anti-HA antibody H-18 as described elsewhere (13)
. HA contains two defined antigenic epitopes. One is MHC class I (Kd) restricted (IYSTVASSL). A CD8+ T-cell clone specific for a Kd-restricted peptide epitope of HA (14)
proliferated vigorously and produced significant levels of IL-2 when incubated with DA3-HA cells in vitro, whereas no IL-2 was detected in the supernatants of the same T cells cultured with DA3 wild type (data not shown). The other one is MHC class II (IEd) restricted (SFERFEIFPKE). It induces a strong CD4+ T-cell response (15
, 16)
. It should be pointed out that the expression of HA did not alter the immunogenicity of DA3 cells, because s.c. injection into BALB/c mice with either DA3-WT or DA3-HA resulted in tumor growth with similar kinetics (data not shown).
Reagents.
The following antibody-producing hybridomas were obtained from American Type Culture Collection (Manassas, VA) and used as culture supernatants: anti-CD4 (L3T4, TIB-207), anti-CD8 (Lyt-2.2, TIB-210), anti-MHC class II (I-Ad, TIB-120). Mouse GM-CSF, and IL-4 were obtained from RDI (Flanders, NJ), ConA, purified anti-CD3 and anti-CD28 antibodies, FITC- or phycoerythrin-conjugated anti-CD11c, CD11b, CD86 (B7-2), and IAd antibodies were purchased from PharMingen (San Diego, CA). Anti-clonotypic TCR (clone 6.5) were obtained from Caltag (Burlingame, CA). Isotype matched FITC- and phycoerythrin-conjugated IgG was used as a control of nonspecific binding. Complete culture medium included RPMI 1640, supplemented with 10% FCS, antibiotics, and 5 x 10-5 2-mercaptoethanol.
Cell Preparation and Analysis of Cell Surface Receptors.
DCs were generated from bone marrow of naïve syngeneic mice in complete culture medium supplemented with 20 ng/ml murine GM-CSF, 10 ng/ml IL-4 (RDI), and 50 µm 2-mercaptoethanol. The cells were maintained at 37°C in a 5% CO2 humidified atmosphere. One-half of the medium was replaced on day 3. After 5–6 days in culture, cells were collected and enriched by centrifugation over a 13.5% metrizamide gradient (Accurate Chemical, Westbury, NY). The purity of the DC fraction was higher than 80% as determined by FACS analysis of surface molecules expression.
For evaluation of the specific and nonspecific T-cell responses a single-cell suspension was prepared from spleens, and red cells were removed by hypotonic shock using ACK lysis buffer. An enriched population of T cells were obtained from spleens using T-cell enrichment columns (R&D Systems).
Antigen-specific T-Cell Proliferation.
Splenocytes were placed in triplicates into U-bottomed 96-well plates (105/well) in the presence of 10 µg/ml HA-derived peptide (amino acids 110–120, SFERFEIFPKE) or control OVA-derived peptide (amino acids 323–339 ISQAVHAAHAEINEACR) and were cultured for 4 days. Eighteen h before harvesting, cells were pulsed with [3H]thymidine (1 µCi/well; Amersham, Arlington Heights, IL). [3H]thymidine uptake was counted using a liquid scintillation counter and expressed as cpm.
Nonspecific T-Cell Proliferation.
T cells isolated from spleens were seeded in triplicates at a concentration 105/well into U-bottomed 96-well plates. Cells were cultured for 4 days with various concentrations (1–10 µg/ml) of ConA. Cell proliferation was evaluated as described above. In parallel, wells were coated either with 0.25 µg/ml anti-CD3 antibody or with anti-CD3 antibody and 0.5 µg/ml anti-CD28 antibody. After the wells were washed, T-cells (105/well) were incubated in triplicates for 4 days. Cell proliferation was evaluated as described above.
ELISPOT Assay.
The number of IFN-
-producing cells was measured using ELISPOT assay. Briefly, Millipore MultiScreen-HA plates were coated with antimouse IFN- antibody (PharMingen). Splenocytes (2 x 105 cells/well) were cultured for 24 h at 37°C in a 5% CO2 incubator in the complete medium alone or in the presence of the specific or control peptides at a concentration of 10 µM. After that time, wells were washed and then incubated overnight at 4°C with a different clone of biotinylated anti-IFN- antibody (PharMingen). Reactions were visualized using avidin-alkaline phosphatase and BCIP/NBT substrate. The number of spots per 106 splenocytes, which represented the number of IFN- producing cells, was calculated blindly by two investigators (S.K. and D.G.).
IL-2 and IFN- ELISA.
ELISA was performed using antibodies and protocol developed by PharMingen. The sensitivity of the assays was 6 pg/ml.
Apoptosis Assay.
To evaluate induction of apoptosis of tumor cells by Taxol, tumors were excised and minced on ice. Connective tissue was digested by 30-min incubation with 400 units/ml collagenase type V (Sigma) at 37°C, and single-cell suspension was prepared. Apoptosis of tumor cells was measured by staining with FITC-conjugated Annexin-V and PI (PharMingen) and by TUNEL assay using Apo-Direct kit (PharMingen) according to the manufacturer’s instructions.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Chemotherapy on Function of T Cells.
An adequate function of T cells is critically important for the success of immunotherapy. The main perceived limitation of the combination of chemotherapy and DC administration is immunosuppression induced by chemotherapy during the prolonged exposure to the drugs. Here, we tested the effect of different doses of paclitaxel on the function of T cells in DA3-HA-bearing mice. T cell proliferation and IL-2 production in response to ConA, to immobilized anti-CD3 antibody, or to a combination of anti-CD3 and anti-CD28 antibodies were evaluated. Paclitaxel at the highest dose (15 mg/kg) induced a profound defect in T-cell function by day 7 after injection (Fig. 1)
. No defects in T-cell function were detected after the injection of 10 mg/kg or 7.5 mg/kg of the drug. Chemotherapy of cancer patients includes several cycles of treatment separated by 3–4 weeks. Repeated administration of drugs may cause much more serious defects of the immune system than does the single dose. To clarify this issue in our experimental system, paclitaxel was injected into mice three times with a 7-day interval. Mice were sacrificed 7 days after the second and third injections. Two injections of paclitaxel at a dose of 10 mg/kg caused profound decrease in T-cell response to all stimuli (Fig. 1)
. The dose of 7.5 mg/kg slightly reduced T-cell response to the combination of anti-CD3 and anti-CD28 antibodies. Three injections of the drug at the dose of 7.5 mg/kg resulted in a statistically significant, although moderate, decrease in T-cell response to all stimuli (Fig. 1)
. Thus, these data demonstrated that paclitaxel at the dose 7.5 mg/kg induced a substantial level of apoptosis in DA3-HA tumor with a moderate inhibition of T-cell function. This dosage was used in subsequent experiments.
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production (Fig. 2)
. This effect was less pronounced at the 7.5-mg/kg dose of paclitaxel. This dose did not affect peptide-specific T-cell proliferation (Fig. 2A)
. However, it substantially decreased peptide-specific IL-2 and IFN- production (Fig. 2, B and C)
. These data are consistent with the results of a study of nonspecific T-cell function and indicate that even a low dose of paclitaxel is capable of inducing a moderate decrease in antigen-specific T-cell function.
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. LPS markedly activated DCs. The proportion of CD11c+IAd+ cells was increased 1.5-fold, CD11c+B7-2+ cells almost 2-fold, and CD11c+CD40+ more than 20-fold (Fig. 3)
. These data indicate that the population of DCs generated in our experiments was indeed immature, and could be activated on exposure to one of the DC activation stimuli LPS.
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production by splenocytes in response to HA-derived MHC class I-restricted peptide IYSTVASSL using ELISPOT assay. Irrelevant p53-derived peptide was used as a control peptide. Practically no IFN--producing cells were detected in all of the six nontreated tumor-bearing mice as well as in all five of the mice treated with paclitaxel alone (Fig. 4A)
. i.v. injections of DC alone or in combination with paclitaxel resulted in slightly higher background levels of IFN--producing cells. However, no response to the specific peptide has been observed. Injection of DCs into tumor site induced significantly higher background, but again, no specific response was found in any of six tested mice. In contrast, the combination of DCs and paclitaxel led to a specific response in all nine of the tested mice (Fig. 4A)
. The magnitude of the response was different. In four mice, the difference between the response to specific and control peptides was higher than 3 SD, and, in the remaining five mice, it was lower than 3 SD but higher than 2 SD (Fig. 4A)
. Overall, antigen-specific response was observed only in the group of mice treated with a combination of chemotherapy and DC administration into the tumor site (Fig. 4B)
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. A statistically significant specific response was detected in one of six mice treated with injections of DCs into the tumor site. At the same time, such a specific response was determined in four of six mice treated with a combination of paclitaxel and DCs (Fig. 5A)
. The differences between groups were highly statistically significant (Fig. 5B)
. The background levels of IL-2 production were significantly increased in mice treated with DCs with or without paclitaxel. No statistically significant differences between responses to control and specific peptides were found in any of the tested mice (data not shown). Thus, the combination of chemotherapy and DC administration resulted in the induction of TAA-specific CD8+ and CD4+ T-cell responses. Because no signs of immune reactivity against specific epitopes were detected in any other groups, we did not use i.v. administration of DCs in additional experiments.
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. However, combined treatment increased median survival from 29 days in control group to 45 days (P = 0.012; Fig. 6
). In clinical practice, chemotherapy is combined with the administration of glucocorticoids to alleviate the toxic effect of chemotherapy. The standard dose of treatment includes the administration of 24–60 mg dexamethasone over 12–24 h, together with taxane infusion. This treatment is repeated with each cycle of chemotherapy. Glucocorticoids are known to suppress the immune system. We investigated the effect of glucocorticoids by comparing the effect of combined treatment with and without comparable doses of dexamethasone (3 µg/mouse). Inclusion of dexamethasone in three cycles of the treatment with paclitaxel and DC administration did not affect immune response (Fig. 7)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the last year, three groups have reported the successful results of combining apoptosis-inducing treatment of tumors with intratumoral administration of DCs. Candido et al. (9)
demonstrated that injections of DCs into the tumor with a high rate of spontaneous apoptosis resulted in antitumor effect. We have demonstrated (10)
that the combination of -radiation with local DC administration into the tumor site resulted in the induction of tumor-specific immune response and had significant antitumor effect. Tong et al. (11)
have shown that the combination of chemotherapy and DC administration led to a potent antitumor effect. These studies have established proof-of-concept and have demonstrated the therapeutic potential of this approach.
Chemotherapy is the most actively used modality in the treatment of advanced-stage cancer. It is well established that it is associated with significant immunosuppression (20, 21, 22)
. Because the success of combined treatment heavily depends on the adequate function of the immune system, this effect of chemotherapy may be critical for the success of combined treatment. Previous studies did not address this problem. In experiments with spontaneous apoptosis, no additional treatment was used (9)
. Local -irradiation of the tumor site used in our previous work has very little systemic immunosuppressive effect (10)
. In most of their experiments, Tong et al. (11)
used two or three injections of the drugs every day or every other day with completion of the treatment within 3 or 4 days. In clinical practice, chemotherapy is a relatively long-term therapy that usually includes multiple cycles of the treatment separated by several weeks. This type of treatment may affect the immune system more profoundly than short-term administration of the same drug. Our experiments in the present study indeed confirmed this hypothesis. Paclitaxel at the dose of 10 mg/kg did not affect T-cell function after a single cycle of treatment. However, two cycles resulted in a profound decrease in T-cell response to stimulation. The same effect was observed at the dose of 7.5 mg/kg. Two cycles of treatments with the drug practically did not affect T-cell function. Three cycles, however, resulted in a significant decrease in T-cell function. These data indicate that repeated cycles of chemotherapy have a negative cumulative effect on the function of the immune system. To evaluate the effect of combined treatment, we have selected the dose of paclitaxel that induces a substantial level of apoptosis and moderate immunosuppression.
To accurately measure antitumor immune response, we used mammary adenocarcinomas that express HA. HA contains at least two known epitopes: matching MHC class I (Kd) and MHC class II (IEd). We hypothesized that, if combined treatment was able to induce tumor-specific immune response, it might manifest in peptide-specific responses. And, indeed, we detected specific CD8-mediated response in all nine of the mice treated with the combination of chemotherapy and DC administration, and we detected CD4-mediated response in four of six treated mice. Such a response was not observed if mice were treated either with chemotherapy alone or with DC administration alone. However, administration of DCs into the tumor site without chemotherapy resulted in increased background in the ELISPOT assay. Although the precise mechanism of this phenomenon is not clear, we may suggest that DCs became activated after administration into the tumor site and subsequent migration to the draining lymph nodes. Such activated DCs are able to present a variety of tumor-nonspecific antigens and stimulate T cells. It is important to note that i.v. injections of DCs were also ineffective. We have previously demonstrated that only a small proportion of DCs injected i.v. could reach the tumor site (10) . Apparently, the number of DCs after i.v. injection was not sufficient to induce immune response. In clinical practice, chemotherapy is combined with the administration of glucocorticoids to alleviate the toxic effects of chemotherapy. We tested the effect of dexamethasone on the induction of antitumor immune response. In our experimental model, short-term administration of dexamethasone did not have a negative effect on the induction of antitumor immune response.
Combined treatment had a clear antitumor effect, which manifested in the prolonged survival of the mice. An antitumor effect was observed not only in mice bearing tumor expressing HA (DA3-HA) but also in mice bearing parental tumor (DA3). The antitumor effect observed in our study was less dramatic than in the previously reported work (11) and did not result in tumor eradication. Evidently, repeated cycles of chemotherapy, which resulted in moderate immunosuppression, might decrease the efficacy of combined treatment.
Thus, in this study, we have demonstrated, for the first time, that the combination of DC administration with repeated cycles of chemotherapy and dexamethasone (conditions similar to real clinical practice) resulted in the induction of antitumor immune response despite the immunosuppression induced by such a treatment. This combined treatment led to a significant antitumor effect. Our results indicate that the clinical success of this method may depend on careful selection of the dosage of the drugs. The effect of the treatment may also be increased if we find the optimal ways to decrease chemotherapy-induced immunosuppression.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at H. Lee Moffitt Cancer Center, University of South Florida, MRC-2E, Room 2067, 12902 Magnolia Drive, Tampa, FL, 33612. Phone: (813) 903-6863; Fax: (813) 632-1328; E-mail: dgabril{at}moffitt.usf.edu 
2 The abbreviations used are: TAA, tumor-associated antigen; DC, dendritic cell; HA, hemagglutinin; FACS, fluorescence-activated cell sorting; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor; ELISPOT, enzyme-linked immunospot (assay); TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; TCR, T cell receptor.
Received 5/ 6/02; revised 9/ 3/02; accepted 9/ 3/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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irradiation and dendritic cell administration in treatment of advanced cancer. Intern. J. Cancer, 94: 825-833, 2001.[CrossRef][Medline]
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