In vivo gene transfer of CD40 ligand into colon cancer cells induces local production of cytokines and chemokines, tumor eradication and protective antitumor immunity

Abstract

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 on antigen-presenting cells, is essential for the initiation of cell-mediated and humoral immune responses. In this study, we investigated the antitumor effect of in vivo gene transfer of CD40L to tumor cells using an adenoviral vector (AdCMVmCD40L) in a murine CT-26 colon cancer model. We found that injection of AdCMVmCD40L caused tumor regression in a dose-dependent manner. A complete regression of tumor was observed in 81% of mice treated with 109 p.f.u. of AdCMVmCD40L. The antitumor effect induced by CD40L was mediated by CD8+ T cells and was associated with the generation of tumor-specific cytolytic T lymphocytes (CTL). Animals that eradicated the tumor were protected against tumor cell rechallenge, and both CD4+ and CD8+ T cells were involved in specific protective immunity. Treatment with AdCMVmCD40L in one tumor nodule also caused complete regression of established tumors at distant sites. The antitumor effect elicited by AdCMVmCD40L was associated with the intratumoral production of IL-12 and IFN-γ and with an increased intratumoral expression of chemokines such as MIP-1α, MIP-1β, MIP-2, RANTES, and eotaxin. These data demonstrate that intratumoral injection of AdCMVmCD40L induces a powerful cascade of chemokines and cytokines in the tumor mass and stimulates an efficient antitumor immunity leading to regression of established colon cancer and protection against tumor cell rechallenge.

Introduction

CD40 is a member of the tumor necrosis factor (TNF) receptor family of molecules and is expressed by a range of cells, including various antigen presenting cells (APCs), B cells, fibroblasts, epithelial cells and endothelial cells.12 CD40 ligand (CD40L, CD154) is a 33-kDa type II membrane glycoprotein belonging to the TNF family and predominantly expressed on CD4+ T cells upon TCR stimulation.23 CD40L–CD40 interaction was originally shown to play a key role in thymus-dependent humoral immune responses, mediating cognate interactions between CD4+ T cells and B cells that are essential for B cell activation and differentiation, class switching, germinal center formation, and the generation of B cell memory.145 Recent evidence suggests that CD40L–CD40 interaction is also important for the activation of antigen presenting cells (APCs), which is a critical step in T cell stimulation.2678 The importance of APC activation through CD40 engagement has been recently highlighted by the demonstration that the requirement of T helper cell function for priming MHC class I-restricted CTLs is mediated through the engagement of CD40 on APCs by its ligand on CD4+ T cells. The resulting activation of APCs is sufficient to drive naive CD8+ T cells to become fully activated effector cells.78 Monoclonal antibody against CD40 and soluble fusion proteins of CD40L are very effective substitutes for T helper cells in ‘conditioning’ APCs both in vitro and in vivo.678 The changes brought about in the APCs during such activation are not fully understood, but probably involve a combination of improved antigen processing, increased expression of co-stimulatory and adhesion molecules, and up-regulation of cytokine and chemokine production.

Cross-linking of CD40 on dendritic cells by cell-bound CD40L results in maturation, activation,9101112 and prolongation of survival of dendritic cells13 together with up-regulation of accessory molecules such as CD58, CD80, and CD86 and production of certain cytokines such as IL-8, MIP-1α, TNF-α and IL-12.9101112 The generation of proinflammatory cytokines and chemokines by APCs is important for the induction of inflammation and attraction of effector cells. In addition, secretion of IL-12 by dendritic cells in particular, is critical for activation of T cells and development of Th1-type of response. The ability to induce this chain of events into the tumor mass may have important implications for the immunotherapy of cancer.

In most low-grade lymphoid malignancies and Hodgkin's disease, triggering of neoplastic cells via CD40 stimulation may protect against apoptosis.141516 By contrast, in vitro CD40 stimulation of high-grade non-HIV-related lymphoma inhibits cell proliferation, up-regu- lates Fas-encoding gene and increases sensitivity to apoptosis.1415 Soluble recombinant human CD40L exerts a cytotoxic effect on breast cancer cells expressing CD40 in vitro and inhibits tumor growth in vivo.17 In addition to the direct effect on tumor cells, recent reports have shown that CD40 activation in vivo can overcome tumor-specific CD4+ and CD8+ tolerance and augment antitumor immunity.1819 Treatment of lymphoma with CD40 antibody induces a rapid cytotoxic T cell response independent of T helper cells, leading to eradication of the lymphoma and protection against tumor cell rechallenge.20 Ex vivo modification of tumor cells with the CD40L gene could elicit antitumor immunity in animal models with different tumors.2122232425 Recently, it has also been demonstrated that in vivo modification of established tumor cells with CD40L by recombinant adenovirus induces tumor-specific cellular immunity and inhibits the growth of pre-existing tumors. However, the detailed mechanism implicated in the generation of antitumor immunity and tumor rejection has not been elucidated in that study.26

In the present report, we have used a murine model of syngeneic colon cancer to analyze the anti-oncogenic potential and antitumoral immunity elicited by the in vivo transduction of established tumors with an adenoviral vector expressing CD40L (AdCMVmCD40L). Our results show that intratumoral injection of AdCMVmCD40L was very efficient in causing regression of both treated and distant untreated tumors. This effect was completely dependent on CD8+ T cells and was associated with the generation of CTLs, whereas both CD4+ and CD8+ T cells were involved in specific protective immunity against rechallenge with tumor cells. Treatment with AdCMVmCD40L-induced local production of cytokines such as IL-12 and IFN-γ, as well as several chemokines which might be involved in activation of immune responses and rejection of tumors.

Results

Structure and identification of AdCMVmCD40L

We have constructed recombinant adenovirus carrying mouse CD40L driven by the cytomegalovirus (CMV) immediate–early promoter (AdCMVmCD40L) and the structure of AdCMVmCD40L is shown in Figure 1a. To confirm whether CD40L could be expressed on the membrane of cells after infection with AdCMVmCD40L, we infected murine colon cancer cell line CT-26 with AdCMVmCD40L at different MOI (100, 1000, 2000 and 10000) or with control vector carrying lacZ reporter gene (AdCMVLacZ) at MOI of 10000. Two days after infection, expression of CD40L was measured by cytometry using specific antibody against CD40L. As shown in Figure 1b, no CD40L expression was detected in AdCMVLacZ-infected CT-26 cells. In contrast, a dose-dependent expression of CD40L was found in cells infected with AdCMVmCD40L. When CT-26 cells were infected at an MOI of 100, almost no cells expressed CD40L. When the MOI reached 1000 and 2000, about 35% and 90% of tumor cells expressed CD40L, respectively. If the MOI was up to 10000, all tumor cells expressed high level of CD40L. CT-26 tumor cells did not express CD40 measured by FACS analysis using an antibody against CD40 (data not shown).

Figure 1
figure1

Structure and identification of adenovirus expressing mouse CD40L (AdCMVmCD40). (a) Structure of AdCMVmCD40L. The expression cassette for CD40L is driven by the murine cytomegalovirus (CMV) immediate–early promoter in the E1 region of the Ad5 genome. (b) CD40L expression by CT-26 cells infected with AdCMVmCD40L. CT-26 cells were infected with AdCMVlacZ or AdCMVmCD40L at different multiplicity of infection (MOI). Two days later, cultured cells were labeled with R-PE conjugated anti-murine CD154 antibody and analyzed by FACS. The solid area represents uninfected CT-26 cells and open area represents CT-26 cells infected with AdCMVlacZ or AdCMVmCD40L labeled with R-PE conjugated anti-murine CD154 antibody.

Antitumoral activity of AdCMVmCD40L by intratumoral injection

To evaluate the antitumor activity of AdCMVmCD40L, a colon cancer model was established by subcutaneous implantation of murine CT-26 tumor cells into syngeneic Balb/c mice. When the tumor nodule reached 4 to 6 mm in diameter, tumor-bearing animals were treated by intratumoral injection of AdCMVmCD40L at 108 p.f.u. (n = 12) or 109 p.f.u. (n = 16), or control adenovirus AdCMV lacZ at 109 p.f.u. (n = 10) or PBS (n = 10). Figure 2a shows that the size of tumor in animals treated with PBS or control vector AdCMVlacZ increased progressively. However, treatment with two doses of AdCMVmCD40L (108 or 109 p.f.u.) significantly inhibited the growth of established tumor in a dose-dependent manner (Figure 2a). The tumors disappeared between the 9th and 17th day after vector administration. Treatment with a high dose of AdCMVmCD40L (109 p.f.u.) resulted in complete tumor regression in 13 out of 16 mice (81%) with long-term survival (Figure 2b). Treatment with a low dose of AdCMVmCD40L (108 p.f.u.) resulted in complete tumor regression and long-term survival in four out of 12 mice (33%) (Figure 2b). By contrast all mice treated with AdCMVLacZ or PBS died within 25 days after treatment (Figure 2b).

Figure 2
figure2

Therapeutic effect of AdCMVmCD40L on established tumor. CT-26 tumor cells were injected subcutaneously into syngenic Balb/c mice. When the tumor size reached 4 to 6 mm in diameter, tumor-bearing animals were treated by intratumoral injection of AdCMVmCD40L at 108 p.f.u. (n = 12) or 109 p.f.u. (n = 16), or control adenovirus AdCMVlacZ at 109 p.f.u. (n = 10) or PBS (n = 10). Size of tumor was measured and death of animals was monitored. (a) Size of tumor after treatment with AdCMVmCD40L. (b) Long-term survival of animals after treatment with AdCMVmCD40L.

Long-term antitumor immunity induced by intratumoral injection of AdCMVmCD40L

To test whether AdCMVmCD40L treatment could induce a long-term specific protective antitumor immunity, mice with complete tumor regression were rechallenged with 5 × 105 CT-26 cells s.c. on the opposite flank 30 or 100 days after adenovirus treatment. Figure 3a and b shows that all mice which have been cured by AdCMVmCD40L treatment were resistant to CT-26 tumor cell rechallenge, whereas all control mice developed tumors at the expected time. However, if the cured mice were rechallenged with irrelevant but syngeneic BNL cells (a mouse hepatocellular carcinoma cell line), all animals developed tumor at a similar time as the control mice (data not shown). These results suggest that intratumoral treatment with AdCMVmCD40L not only caused regression of established tumors, but also induced long-term specific protective immunity against tumor cell rechallenge.

Figure 3
figure3

Rejection of rechallenged CT-26 tumor cells after treatment with AdCMVmCD40L. Mice that have been cured by AdCMVmCD40L (109 p.f.u.) treatment were rechallenged with 5 × 105 CT-26 cells subcutaneously on the opposite flank 30 days (a) or 100 days (b) after AdCMVmCD40L treatment. The day of tumor appearance in mice was recorded. Control group, n = 6; AdCMVmCD40L-treated group, (a) n = 10 and (b) n = 8.

Involvement of immunocytes in the regression of established tumors and production of protective immunity by AdCMVmCD40L

In order to determine the involvement of immunocytes in regression of established tumors and in the generation of protective immunity induced by AdCMVmCD40L, CD4+ or/and CD8+ cells or NK cells were depleted by intraperitoneal administration of the corresponding antibody 3, 2 and 1 days before treatment with the adenovirus or rechallenge with tumor cells and thereafter once weekly to maintain depletion until completion of the experiment. The efficiency of cell depletion was greater than 99% (data not shown), as measured by flow cytometry. As represented in Figure 4, all AdCMVmCD40L-treated mice which were depleted of CD4+ T cells experienced complete tumor regression as it occurred in similarly treated normal mice, whereas mice with depletion of CD8+ alone or of both CD4 and CD8+ T lymphocytes, showed progressive tumor growth which was comparable to that observed in control groups receiving AdCMVLacZ or PBS. Figure 5 shows that all mice depleted of CD4+ T cells, CD8+ T cells or NK cells alone rejected tumor cells after rechallenge while depletion of both CD4+ and CD8+ T lymphocytes caused 60% of mice to develop tumoral growth after tumor cell rechallenge. These results suggest that tumor regression induced by intratumoral injection of AdCMVmCD40L is strictly dependent on CD8+ T cells and that both CD4+ and CD8+ T cells are involved in induction of long-term protective immunity in animals subjected to this therapy.

Figure 4
figure4

Involvement of T cell subsets in the regression of tumor induced by treatment with AdCMVmCD40L. Depletion of CD4+ and/or CD8+ T cells was carried out in animals with subcutaneous tumors (4 to 6 mm in diameter) by intraperitoneal injection of corresponding antibodies 3, 2 and 1 days before intratumoral injection of AdCMVmCD40L or AdCMVLacZ (109 p.f.u.) and once weekly thereafter to maintain depletion until completion of the experiment. The tumor size was monitored after treatment. Five animals per group.

Figure 5
figure5

Involvement of immunocytes in long-term protection induced by treatment with AdCMVmCD40L. Mice that have been cured by AdCMVmCD40L (109 p.f.u.) treatment were rechallenged with 5 × 105 CT-26 cells subcutaneously on the opposite flank 100 days after treatment. Depletion of CD4+ and/or CD8+ T cells, or NK cells was carried out by intraperitoneal injection of corresponding antibody 3, 2 and 1 days before tumor cell rechallenge and once weekly thereafter to maintain depletion until completion of the experiment. Tumor appearance was monitored after injection of tumor cells. Five animals per group.

Treatment with AdCMVmCD40L elicited cytotoxic T lymphocyte (CTL) response

As mentioned, our results indicate that CD8+ T cells play a key role in tumor regression after intratumoral administration of AdCMVmCD40L. We further investigated whether this treatment was able to stimulate a CTL response against tumor cells. Spleens from tumor-bearing animals were obtained 2 weeks after treatment with 109 p.f.u. of AdCMVmCD40L, control adenovirus AdCMVlacZ or PBS. Splenocytes were used to analyze specific CTL activity against CT-26 or irrelevant BNL cells. As shown in Figure 6, there was no significant CTL activity against CT-26 cells in animals treated with PBS or AdCMVlacZ. In contrast, a manifest CTL response against CT-26 cells was detected in splenocytes from animals treated with AdCMVmCD40L. These cells however, did not exhibit cytotoxicity against BNL cells. These results indicate that local expression of CD40 ligand on tumor cells induces a potent, systemic and specific CTL response.

Figure 6
figure6

Induction of tumor-specific cytotoxic T cells by AdCMVmCD40L treatment. Effector cells were generated from splenocytes obtained 14 days after treatment with AdCMVmCD40L or AdCMVLacZ (109 p.f.u.) or PBS by culture with mitomycin C-treated (150 μg/ml for 30 min) CT-26 cells for 5 days. Effector cells and target cells (CT-26 or BNL) were cultured at different ratios (50:1, 25:1, 12.5:1 and 6.25:1) at 37°C for 5 h as described in Materials and methods. (a) CT-26 cells as target cells; (b) BNL cells as target cells. Representative results of two independent experiments are shown.

Local AdCMVmCD40L treatment induced distant antitumoral effects

Since a systemic antitumoral CTL activity was elicited by local administration of AdCMVmCD40L, we evaluated whether the injection of AdCMVmCD40L into a tumor nodule had any effect on a distant tumoral lesion. For this purpose we used an animal model with two tumor nodules (one in each hind flank) and we treated only one of the two lesions. Mice were injected subcutaneously at the two sites with 5 × 105 CT-26 cells and, when tumor size reached 4 to 6 mm in diameter on both sides, right side tumors were treated with intratumoral injection of 109 p.f.u. AdCMVmCD40L or AdCMVlacZ. All mice which received AdCMVlacZ showed progressive tumor growth of both treated (Figure 7a) and distant tumors (Figure 7b), while those mice which were given AdCMVmCD40L exhibited complete regression of treated tumors in five out of six animals (Figure 7c) and, most importantly, complete elimination of distant tumors was also observed in four out of six mice (Figure 7d). These data suggest that local treatment with AdCMVmCD40L not only induces regression of treated tumors, but also of distant non-treated neoplasms.

Figure 7
figure7

Local AdCMVmCD40L gene therapy of one tumor (right side) induces distant antitumoral effect on the contralateral tumor (left side) in animals with bilateral established tumors. Half a million CT-26 tumor cells were injected subcutaneously into both left and right hind flanks of mice. When tumor size reached 4 to 6 mm in diameter in both sites, animals were treated by intratumoral injection of 109 p.f.u. AdCMVmCD40L or AdCMVLacZ at one side of the tumor (right side). Growth of treated and non-treated tumor was monitored after treatment. (a) Size of AdCMVLacZ-treated tumor; (b) Size of left-side tumor (non-treated tumor) in animals in which the right-side tumor was treated with the control vector AdCMVlacZ. (c) Size of AdCMVmCD40L treated tumor; (d) Size of left-side tumor (non-treated tumor) in animals in which the right-side tumor was treated with AdCMVmCD40L.

Local production of cytokines and chemokines following intratumoral injection of AdCMVmCD40L

Activation of dendritic cells by CD40L–CD40 interaction results in increased expression of IL-12, TNF-α, IL-8 and macrophage inflammatory protein (MIP)-1α.1112 To examine if AdCMVmCD40L treatment induced production of cytokines within the tumor mass in our model, a time course of expression of IL-12, IFN-γ and chemokines following intratumoral injection of 109 p.f.u. of either AdCMVmCD40L or AdCMVLacZ was performed along a period of 8 days. We found that in tumors treated with AdCMVmCD40L the absolute and relative (per milligram of tumor weight) values of IL-12 were much higher than in tumors injected with AdCMVLacZ (Figure 8a and b). IL-12 production reached maximal values at day 3 after the administration of AdCMVmCD40L (Figure 8a). Since IL-12 is a strong inducer of IFN-γ2728 we also measured the production of IFN-γ within tumors treated with AdCMVmCD40L or AdCMVLacZ. As shown in Figure 8c and d, tumors which received AdCMVmCD40L showed higher local levels of IFN-γ than those treated with AdCMVLacZ treatment.

Figure 8
figure8

Local expression of IL-12 and IFN-γ in tumor following treatment with AdCMVmCD40L. Tumor-bearing mice were treated by intratumoral injection of 109 p.f.u. of either AdCMVmCD40L or AdCMVLacZ and two mice from each group were killed on days 1, 3, 5 and 8, and the production of IL-12 (a and b) and IFN-γ (c and d) in tumor homogenates was measured using an ELISA as described in Materials and methods. (a and c) Total cytokine production within tumor following adenovirus treatment; (b and d) cytokine production per milligram tumor following adenovirus treatment. Representative results of two independent experiments are shown.

We further investigated the production of chemokines within tumors treated with AdCMVmCD40L, AdCMVLacZ or PBS using a ribonuclease protection assay (RPA). As shown in Figure 9, 2 days after administration of AdCMVmCD40L the intratumoral mRNA values of RANTES, Eotaxin, MIP-1β, MIP-1α and especially of MIP-2 were higher than those found in tumors treated with AdCMVLacZ or PBS. Five and 7 days after vector administration, mRNA levels of all these chemokines in tumors treated with AdCMVmCD40L fell to values similar to those found in neoplasms which received AdCMVLacZ. AdCMVmCD40L treatment seemed to have no apparent effect on the expression of Ltn, IP-10, MCP-1 and TCA-3. To confirm whether the observed elevation in chemokine transcripts corresponded with increased production of chemokine protein, we measured MIP-1α and MIP-2 by specific ELISA. As shown in Figure 10, AdCMVmCD40L treatment induced a much higher level of MIP-1α and MIP-2 within tumors than control animals receiving AdCMVLacZ or PBS.

Figure 9
figure9

Gene expression of chemokines in tumor following treatment with AdCMVmCD40L. (a) Total RNA was extracted from tumors 2, 5 and 7 days after treatment with 109 p.f.u. of AdCMVmCD40L or AdCMVLacZ or PBS and subjected to ribonuclease protection assays as described in Materials and methods to measure mRNA level of Ltn, RANTES, Eotaxin, MIP-1β, MIP-1α, MIP-2, IP-10, MCP-1, TCA-3 mRNA. L32 and GAPDH are housekeeping genes. Representative results of two independent experiments are shown. (b) The L32 signal was used as a control for the amount of input RNA and to quantify the relative expression of chemokine mRNA by measuring density of images.

Figure 10
figure10

Local expression of MIP-1α and MIP-2 in tumor following treatment with AdCMVmCD40L. Tumor-bearing mice were treated by intratumoral injection of 109 p.f.u. of either AdCMVmCD40L or AdCMVLacZ and three mice from each group were killed on days 2 and 7. Production of MIP-1α (a) and MIP-2 (b) in tumor homogenates was measured using an ELISA as described in Materials and methods and chemokine production is expressed per milligram of total protein from tumor homogenates following adenovirus treatment.

Discussion

The data presented here show that local injection of adenovirus carrying CD40L into colon cancer lesions in mice generates an efficient antitumor immunity leading to eradication of established tumors. In addition, this treatment not only causes regression of the tumor injected with AdCMVmCD40L, but also eliminates non-treated established tumors located at a distant site and induces specific protective immunity against tumor cell rechallenge. Recently, Kikuchi and Crystal26 reported that treatment of established tumors by intratumoral injection of adenovirus expressing CD40L had a strong antitumor effect in the animal models with CT-26 cells and B16 cells. In contrast, a study from Chiodoni et al29 showed that only 22% of animals receiving C-26 engineered ex vivo to express CD40L using retroviruses remained tumor-free. Other studies for induction of antitumor immunity by ex vivo transduction of tumor cells with vectors containing CD40L demonstrated that poor immunogenic tumor cells, murine neuroblastoma neuro-2a and fibrosarcoma cells, expressing CD40L resulted in a slower growth of the tumor.2223

The antitumor effect derived from the inoculation of the tumors with adenovirus containing CD40L cannot be attributed in our model to a direct interaction of the transgene with CD40 on the tumor cell membrane since CT-26 cells do not express this molecule and infection of CT-26 cells with AdCMVmCD40L did not interfere with their rate of growth as compared with non-infected cells or with cell infected in vitro with the control vector (data not shown). Our observations clearly indicate that the antitumor effect of AdCMVmCD40L is due to an indirect effect of CD40L via activation of host immunocytes. The in vivo depletion studies demonstrate that CD8+ T cells, but not CD4+ T cells are necessary for regression of treated primary tumors. Specific CTL activity against CT-26 cells could be generated only in animals treated with AdCMVmCD40L, but not in control animals. The data from Kikuchi and Crystal26 further support our observation by showing infiltration CD8+ T cells and cells expressing B7-2 and IL-2Ra in the tumor treated by adenovirus expressing CD40L.

There is evidence showing that activation of host APCs by CD40–CD40L interaction may protect dendritic cells from tumor-induced apoptosis, enhance presentation of tumor-associated antigens, generate cytotoxic T lymphocytes and induce production of cytokines.2022242630313233 In dissecting the immune mechanisms responsible for antitumor effect in our system we found that although CD4+ T cells are not involved in the regression of primary tumor, they are involved in the generation of protective immunity against tumor cell rechallenge, since depletion of either CD4+ T cells or CD8+ T cells alone did not abrogate this effect and only depletion of both cell types eliminates protection against tumor growth. These observations can be interpreted according to data showing that anti-CD40 monoclonal antibodies with agonistic functions, or soluble CD40L, are effective substitutes for T-helper cells in activation of APCs both in vitro and in vivo.678 These data together with evidence showing that activated APC can substitute CD4+ cells for induction of cytotoxic CD8+ cells78 suggest that, in our system, activated APC by tumoral cells expressing CD40L can bypass helper T cells for stimulation of CD8+ cytotoxic T cells which are the efficient effectors in eradicating the established neoplasm in our model. In constrast, both CD4+ and CD8+ appear to be sufficient for rejection of challenging tumoral cells in protection experiments.

Data from the present paper show that intratumoral injection of AdMCVmCD40L results in enhanced production of IL-12 and IFN-γ within the tumor mass. IL-12 is synthesized by B cells, dendritic cells and macrophages and it acts on T cells and NK cells stimulating proliferation and production of cytokines, especially IFN-γ, and activating cytotoxic lymphocytes.27 Previous work by our group has shown that intratumoral injection of adenovirus containing IL-12 resulted in regression of colon cancer and induction of antitumor immunity.34 In the present work, the local production of IL-12 within the tumor nodule might be derived from activation of dendritic cells or macrophages after interaction with tumor cells expressing CD40L following transduction of the tumor with AdCMVmCD40L. In fact, it has been shown that CD40L can stimulate macrophages and dendritic cells in vitro to up-regulate the expression of B7 and to secrete IL-12, TNF-α, IL-8 and MIP-1α.1112 Also enhanced expression of MHC class I and II, as well as of B7-1 and B7-2, by dendritic cells has been observed in vivo in animal tumor models treated by gene transfer of CD40L.29

Our results show that intratumoral injection of AdCMVmCD40L induced high transcriptional expression of various chemokines, including MIP-1α, MIP-β, MIP-2 and RANTES. The increased level of MIP-1α and MIP-2 was also confirmed at the protein level by ELISA tests. MIP-1α is a chemoattractant for immature dendritic cells, macrophages, activated T cells, NK cells and neutrophils3536373839 and MIP-2 is one of the IL-8 homologues in the mouse and is a chemoattractant mainly for neutrophils.4041 These novel observations demonstrating up-regulation of chemokines MIP-1α and MIP-2 inside the tumor nodule after in vivo gene transfer of CD40L provides information on the mechanisms underlying the potent antitumoral effect which results from the in vivo transduction of the neoplasm with CD40L. Future studies will be focused on the role of chemokines and attraction of dendritic cells, T cells and neutrophils to the tumoral lesion on the induction of antitumor immunity by CD40L gene transfer.

The data presented here show that CT-26 colon cancer cells are relatively resistant for adenovirus infection, since infection of cells with MOI of 1000 only induces transgene expression in 35% of cells and MOI of 2000 can reach 90% of cells with transgene expression. This could be related to low or no expression of Coxsackie-adenovirus receptor (CAR) and alpha v integrins that are important for adenovirus infection.4243 However, a number of tumor cells (about 10% in injection site) could be transduced in vivo by intratumoral injection of 108 p.f.u. AdCMVlacZ, as reported in our previous study.34 This may suggest that tumor cells permissive to adenovirus infection could have a better antitumor effect by such treatment.

In summary, our data demonstrate that in vivo gene transfer of CD40L into tumor cells mediated by an adenoviral vector induces regression of established tumors and that this antitumor effect is dependent on CD8+ T cells and is associated with the generation of specific cytotoxic T lymphocytes. This procedure also causes regression of established tumors implanted at a distant site and generates protective immunity against rechallenge with neoplastic cells. Local production of cytokines and chemokines may be involved in the initiation and promotion of immune responses against tumors. These data suggest that local expression of CD40L can be used as a potent tool for immunogene therapy of cancer.

Materials and methods

Mice and cell lines

Female BALB/c (H-2d) mice, 6 to 8 weeks old, were purchased from Charles River (Barcelona, Spain). During the experimental period, animals were housed in standard conditions and all animal procedures were performed according to approved protocols and in accordance with recommendations for proper care and use of laboratory animals.

The 293 cell line (adenoviral E1 transformed human embryonic kidney cells) and murine BNL 1MEA.7R.1 (BNL) (methylcholanthrene transformed liver cell line) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). CT-26, an undifferentiated murine colon adenocarcinoma cell line, was derived by intrarectal injection of N-nitroso-N-methylurethane in a female BALB/c mouse.44 The 293 and BNL cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. CT-26 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Construction of recombinant adenoviral vectors

Adenovirus carrying the LacZ reporter gene under the control of CMV promoter (AdCMVlacZ) was produced as reported previously.45 For construction of AdCMVmCD40L, mouse CD40L cDNA was removed from pBS/mCD40L (Immunex, Seattle, WA, USA) by XhoI and XbaI, and inserted into XbaI site of pMV100 which carries CMV promoter and polyA signal45 by blunt-end ligation. The resultant plasmid was digested with HindIII to release the CD40L expression cassette (CMV-mCD40L-polyA) which was subcloned into HindIII site of adenovirus plasmid pMV60 to get pMV60/ mCD40L. pMV60/mCD40L and pJM17 were co-transfected into 293 cells by calcium phosphate precipitation. Recombinant adenoviruses were isolated from a single plaque, expended in 293 cells, and purified by double cesium chloride ultracentrifugation.45 Purified virus was extensively dialyzed against 10 mM Tris/1 mM MgCl2 and stored in aliquots at −80°C. Titration was made by plaque assay.

CD40L expression on AdCMVmCD40L transfected CT-26 cells

CT-26 cells were infected with AdCMVLacZ or AdCMVmCD40L at different multiplicity of infection (MOI). Two days after infection, tumor cells were detected for CD40L expression. CT-26 cells infected with vectors were allowed to react with R-PE-conjugated anti-mouse CD154 antibody (CD40L, PharMingen, San Diego, CA, USA) for 30 min at 4°C. For detection of CD40 expression on CT-26 tumor cells, tumor cells were incubated with FITC-conjugated anti-mouse CD40 (PharMingen). Irrelevant isotype-matched antibodies were used as controls. Samples were washed twice in FACS buffer (PBS with 3% FBS and 0.02% sodium azide). Samples were analyzed for fluorescence using a FACScan (Becton Dickinson, San Jose, CA, USA).

Animal studies

For colon carcinoma establishment, 5 × 105 of CT-26 tumor cells were injected subcutaneously in the right hind flanks of mice. When the tumor size reached 4 to 6 mm in diameter, they were treated by intratumoral injection of adenovirus vectors (109 p.f.u. for AdCMVLacZ, 108 and 109 p.f.u. for AdCMVmCD40L) in PBS or with PBS alone. Tumor growth was monitored two to three times a week by measuring two perpendicular tumor diameters using calipers, the tumor volume was calculated from the longest diameter and average width, assuming a prolate spheroid. The animals were killed when the longest diameter was greater than 20 mm or when any two measurements were greater than 10 mm, and this was recorded as the date of death for survival studies.

For evaluation of the induction of protective immunity by treatment with AdCMVmCD40L, mice with complete regression of tumor were rechallenged with 5 × 105 CT-26 cells or 5 × 106 BNL cells subcutaneously into the left hind flank regions at day 30 or 100 days after treatment. Age-matched naive mice received the same amount of CT-26 cells or BNL cells as a control. Animals were examined two to three times weekly for the emergence of palpable tumors.

Distant therapeutic effect of AdCMVmCD40L

Animals with bilateral established tumors were established by injection of 5 × 105 tumor cells in both left and right hind flanks of mice. When tumor size reached 4 to 6 mm in diameter in both sides, adenovirus vector (109 p.f.u. AdCMVLacZ or AdCMVmCD40L) in PBS was injected into tumors on the right side. Tumor growth in both sides was monitored after treatment.

In vivo depletion of T cell subsets

CD4+ T cells and/or CD8+ T cells were depleted in BALB/c mice by intraperitoneal injection of anti-CD4 antibody (hybridoma GK1.5, from ATCC) or anti-CD8 antibody (hybridoma 53-6.72, from ATCC). Sixty microliters of ascitic fluid containing the corresponding antibody were administered in a final volume of 200 μl with saline on days −3, −2 and −1 before AdCMVmCD40L (109 p.f.u.) treatment or tumor cell rechallenge and once weekly thereafter to maintain depletion until completion of the experiment. One mouse per group was killed at the time of treatment or tumor cell rechallenge to confirm the in vivo depletion. Splenocytes taken from dead mice were analyzed for CD4+ and CD8+ T cell subpopulations by dual-immunofluorescence staining with R-PE-conjugated mAb CD4 (clone H129.19, from Sigma, St Louis, MO, USA) and FITC-conjugated mAb CD8a (clone 53–6.7, from Sigma) using FACScan. Results showed more than 99% depletion of T cell subtypes.

Cytotoxicity assays

Cytotoxicity assay was performed according to a standard protocol.3446 Two weeks after treatment of established tumor with 109 p.f.u. of AdCMVmCD40L, AdCMVLacZ or PBS, splenocytes were isolated and the red blood cells were lysed by ACK lysing buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2–7.4) at room temperature for 5 min. In vitro stimulation was performed for 5 days in 24-well plates, each well containing 8 × 106 splenocytes and 8 × 105 CT-26 cells previously treated with 150 μg/ml of mitomycin C (Sigma) for 30 min at 37°C. In vitro-restimulated lymphocytes were tested for their cytolytic reactivity against CT-26 tumor cells and syngeneic HCC tumor cell line BNL in a standard 5-h 51Cr release assay. A total of 1 × 106 target cells (CT-26 or BNL) were radiolabeled with 50 μCi of Na 2 51 Cr O 4 for 90 min at 37°C. The labeled tumor cells were washed three times and adjusted to 5 × 104/ml, and 100 μl of this cell suspension was added to 96-well round-bottom plate and incubated with effector cells at 37°C for 5 h in different E/T cell ratios. The percentage of specific lysis was calculated as follows: % lysis = 100 × ((experimental release − spontaneous release)/ (maximum release − spontaneous release)). Target cells incubated in medium alone or in medium containing 5% Triton X-100 were used to determine spontaneous and maximum 51Cr release, respectively.

ELISA for cytokines

One, 3, 5 and 8 days after adenovirus treatment, tumors were removed from mice and immediately frozen in liquid nitrogen. Frozen tumors were homogenized in PBS (0.5–1 ml, depending on tumor size) containing 100 μM PMSF (Sigma) and 10 μM/ml aprotinin (ICN Biomedicals, Costa Mesa, CA, USA). The homogenate was then cleared by centrifugation in a microfuge for 5 min at room temperature. Tumor homogenates were stored at −20°C.

ELISA for mouse IL-12 and IFN-γ was performed according to the manufacturer's protocol (PharMingen). Ninety-six-well microtiter plates were coated overnight at 4°C with 50 μl purified anti-cytokine capture antibody (4 μg/ml anti-IL-12 or anti-IFN-γ, PharMingen) per well. Then plates were washed four times with PBS/Tween solution and non-specific binding was blocked by adding 200 μl PBS containing 10% FCS per well and incubated at room temperature for 30 min. The blocking buffer was removed and the wells were washed three times. Standards and samples diluted in blocking buffer/Tween were added at 100 μl per well and incubated overnight at 4°C. The wells were washed four times and 100 μl bitinylated anti-cytokine detecting antibody (2 μg/ml in blocking buffer/Tween) was added followed by 1 h incubation at room temperature. The wells were washed six times and then 1:1000 diluted streptavidin-POD (Boehringer Mannheim Biochemica, Penzberg, Germany) conjugated enzyme was added at 100 μl per well, the plates protected from light and incubated at room temperature for 30 min. The plates were washed eight times and 100 μl ABTS (Boehringer Mannheim Biochemica) substrate solution was added per well, the plates protected from light and incubated at room temperature for 30 to 80 min. The plates were read at OD 405 nm. A standard IL-12 and IFN-γ curve containing known concentration of each cytokine was performed. ELISA for mouse MIP-1α and MIP-2 was performed according to the manufacturer's protocol (R&D System, Minneapolis, MN, USA).

Ribonuclease protection assays

Total RNA was obtained from tumors 2, 5 and 7 days after treatment with adenoviruses using ULTRASPEC RNA isolation system (BIOTECX, Houston, TX, USA). Ten micrograms of RNA from each sample was hybridized to a 32P-labeled antisense RNA probe set (mCK-5, PharMingen) and digested with RNase and T1 nuclease, and the protected probe fragments were resolved on 5% polyacrylamide gels according to the manufacturer's protocols. Band intensity of chemokines was quantified by ID demo2 quantitative analysis with calibration curve in ExcelGel SDS using ImageMaster from Pharmacia Biotech (UK), and normalized to the intensity of the L32 probe.

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Acknowledgements

This work was supported in part by SAF 98-0146 from CICYT, Inés Bemberg Grant and also by J Vidal, Dr Cervera and M Mendez grants for Gene Therapy.

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Correspondence to C Qian.

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Sun, Y., Peng, D., Lecanda, J. et al. In vivo gene transfer of CD40 ligand into colon cancer cells induces local production of cytokines and chemokines, tumor eradication and protective antitumor immunity. Gene Ther 7, 1467–1476 (2000). https://doi.org/10.1038/sj.gt.3301264

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Keywords

  • gene therapy
  • adenovirus
  • CD40 ligand

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