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Published ahead of print on April 3, 2003, doi:10.1165/rcmb.2002-0226OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 321-330, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0226OC

Efficacy of CD40 Ligand Gene Therapy in Malignant Mesothelioma

Paul L. Friedlander, Christie L. Delaune, Jennifer M. Abadie, Marisa Toups, Jeffrey LaCour, Luis Marrero, Qiu Zhong and Jay K. Kolls

Department of Otolaryngology and Biocommunication, and the LSUHSC Gene Therapy Program, Department of Medicine, Pulmonary Section at the Louisiana State University Health Sciences Center, New Orleans, Louisiana

Address correspondence to: Jay K. Kolls, M.D., Professor of Pediatrics, Chief of Pediatric Pulmonary, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. E-mail: jay.kolls{at}chp.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene delivery of CD40 Ligand (CD40L) has shown promise in murine models of melanoma and adenocarcinoma; however, its potential for thoracic malignancies such as malignant mesothelioma remains unclear. In this study, we investigated the hypothesis that CD40L gene therapy would be effective in local and distant tumor suppression in mesothelioma using an immunocompetent murine model. Using a recombinant adenovirus encoding murine CD40L (AdCD40L), we demonstrated no suppression of in vitro cell growth for the AC29 (mesothelioma) cell line. However, inoculation of immunocompetent CBA/J mice with AC29 cells treated ex vivo with AdCD40L resulted in significant suppression of tumor formation in vivo when compared with controls (P < 0.001). Intratumoral inoculation of AdCD40L into previously established AC29 tumors yielded similar antitumor results and was associated with increased recruitment of intratumoral CD8+ T cells. Adoptive transfer of CD8+ T cells from AdCD40L-treated tumor bearing mice conferred protection to naive mice given an AC29 tumor challenge. Finally, in mice with two synchronous tumors, treatment of one of the tumors with AdCD40L resulted in a regression of both tumors. These findings demonstrate the development of tumor specific CD8+ T cells by AdCD40L and support the further development of AdCD40L for the treatment of malignant mesothelioma.

Abbreviations: antigen-presenting cells, APC • CD40 Ligand, CD40L • cytotoxic T lymphocytes, CTL • dendritic cells, DC • enhanced green fluorescent protein, EGFP • enzyme-linked immunosorbent assay, ELISA • fluorescent automated cell sorting, FACS • fluorescein isothiocyanate, FITC • interferon, IFN • interleukin, IL • major histocompatability complex, MHC • multiplicity of infection, MOI • phosphate-buffered saline, PBS • plaque-forming units, pfu • Arginine-Glycine-Aspartic acid, RGD


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Advanced solid tumors, such as mesothelioma, are characterized by heterogeneity and an inability to generate an immune response in an immunocompetent host (1–4). These tumors express a variety of antigens; however, they do not stimulate a host immune response. The failure of the host immune system to detect and destroy tumors is attributed to the inability of these tumor antigens to generate a tumor-specific cytotoxic T lymphocyte (CTL) response (5). A variety of strategies have been developed to augment the immune system and to improve the host response to tumors: introduction of cytokine genes into tumors, overexpression of tumor antigens, modification of antigen-presenting cells (APCs) to express tumor antigens, and the transfer of costimulatory molecules to tumor cells (6–17).

Recent studies suggest that the expression of the gene encoding CD40 Ligand (CD40L) may be an effective strategy to enhance the host immune response (18, 19). CD4+ T cells are essential in the generation of an antitumor response (20). Patients with malignant mesothelioma and other solid tumors are also known to have suppression of CD4+ T lymphocyte function (21–23). CD40L has emerged as an important costimulatory molecule that may be used to bypass CD4+ T cells in initiating a CD8+ cytotoxic response. This 33-kD protein is a member of the tumor necrosis factor family, and is expressed on the CD4+ T cells (24, 25). When CD4+ cells are stimulated, CD40L and CD40 (expressed on the APC) interact and the APC becomes activated. CD40 signaling results in upregulation of the major histocompatability complex (MHC) and costimulatory molecule expression is increased in the APC (1). Furthermore, there is an increased production of cytokines, such as interleukin (IL)-12, that facilitates CD8+ T cell activation and result in lysis of tumor cells (1, 26, 27).

Previous studies have demonstrated that ex vivo modification of melanoma and colon cancer cell lines with the CD40L gene results in inhibition of tumor growth (28–30). The effect of CD40L appears to be T cell–mediated through a CD8+ T cell–dependent mechanism (19). The effectiveness of CD40L immunotherapy appears to be tumor-specific, with B16 melanoma and CT26 colon cancer cell lines demonstrating an excellent response, whereas Lewis lung carcinoma cell lines demonstrated less of a response (18, 19).

In this study, we chose to investigate the effectiveness of CD40L gene therapy on malignant mesothelioma using a syngeneic mouse model. To date, there are no data to indicate that CD40L gene therapy may be effective in pulmonary neoplasms. The aim of this study was to determine if the expression of the CD40L gene by these tumors would induce tumor-specific cellular immunity, and investigate the effect of this therapy on multifocal or distant disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Male CBA/J mice and athymic nude female mice (6–8 wk) were obtained from the Charles River Laboratory (Wilmington, MA). The Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee approved all procedures prior to performing the study. The mice were housed in the animal care facility and were cared for according to institutional standards. The mice remained there until they were killed following the completion of their respective study.

In all the animal experiments, the mice were anesthetized with an intraperitoneal injection of 45.5/4.5 mg/kg body weight of ketamine/xylazine (Schering-Plough Animal Health Corporation, Union, NJ) for cell injections, intratumoral injections, and resection of spleens.

Cell Culture
AC29 mesothelioma cells of CBA/J origin were obtained from Dr. Bruce Robinson of the Queen Elizabeth II Medical Center, Perth, Australia, and were maintained in Dulbecco's modified Eagle's medium/F12 supplemented with 10–15% certified fetal bovine serum (Life Technologies, Rockville, MD), 50 IU/ml penicillin/50 µl/ml streptomycin (Life Technologies, Rockville, MD), and 2 mM L-glutamine (BioWhittaker, Inc., Walkersville, MD) (31, 32).

Adenoviral Vectors
AdEGFP, AdLuc, and AdCD40L were obtained from the Vector Core Laboratory of the LSU Health Sciences Center Gene Therapy Program in New Orleans, LA. AdEGFP is an E1/partial E3–deleted adenovirus, which encodes the enhanced green fluorescent protein (EGFP). AdLuc is a recombinant adenovirus on the same backbone, which encodes the firefly luciferase gene. AdCD40L is a recombinant E1/E3-deleted adenovirus encoding the murine CD40L gene. All transgenes were driven by the CMV promoter. The adenoviral vectors were propagated on the 911 cell line, purified on CsCl density gradients, assayed for replication competency, and plaque titered as previously described (33).

Transfection of Cell Lines with Adenoviral Vectors
To assess transduction efficiency, AC29 cells were transduced with AdEGFP at a multiplicity of infection (MOI) of 0, 5, 10, 50, and 100. Fluorescence was measured via Fluorescent Automated Cell Sorting (FACS) Analysis (FACSCalibur; Becton Dickinson Immunocytometry Systems, San Jose, CA). Based on these results, AC29 cells were transfected at an MOI of 10, 50, or 100 using AdCD40L. Forty-eight hours later, CD40L expression was measured by FACS using biotin–anti-mouse CD40L as the primary antibody (Pharmingen, San Diego, CA), followed by StreptAvidin–fluorescein isothiocyanate (FITC) (Pharmingen).

Cell Proliferation Assay
To examine the effect of the adenoviral vectors on cell growth, the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit, using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI) was used. Five thousand cells were plated in wells of a 96-well plate. AC29 cells were subsequently treated with phosphate-buffered saline (PBS) or transfected with AdLuc or AdCD40L at an MOI of 100. Each treatment was performed in replicates of five. Cell proliferation using the manufacturers protocol was measured at 24, 48, and 72 h using an enzyme-linked immunosorbent assay (ELISA) reader (µQuant; Bio-Tek Instruments, Inc., Winooski, VT) at 490 nm.

Ex Vivo Therapy Model
AC29 tumor cells were transduced ex vivo with AdCD40L or AdLuc at an MOI of 100 or mock transduced with PBS. Forty-eight hours later, 2 x 106 AC29 cells from the three different transduction groups were injected subcutaneously in the right flank of CBA/J mice (4–6/group). Tumor growth was observed by measuring the two perpendicular diameters of the mass with a vernier caliper. Tumor volume was calculated using this formula: tumor volume = 0.4(ab2), where "a" is the length of the longer diameter and "b" is the length of the shorter diameter. Mice were killed when the tumor volume measured 1,500 mm3 or greater or when food and water intake fell below 75% of baseline. Experiments were performed in at least duplicate. In addition, ex vivo therapy was repeated using athymic nude mice.

In Vivo Therapy Model
Naive CBA/J or athymic nude mice were injected with 2 x 106 AC29 cells suspended in 100–200 µl of cell media. When tumor volume reached 100 mm3 ± 30 mm3 (~ 7 x 6 mm), the mice were inoculated with PBS, AdLuc, or AdCD40L. CBA/J mice received a single intratumoral injection of 5 x 109 plaque-forming units (pfu) of virus or PBS at Day 1 (5/group). Mice were anesthetized and tumors were immobilized with calipers for injections to ensure delivery of the adenovirus to the tumor and not to surrounding tissue. Tumor volume was measured daily. Mice were killed using the same criteria as above. Experiments were performed in at least duplicate. In a synchronous tumor model, naive CBA/J mice were injected with 2 x 106 AC29 cells suspended in 100–200 µl of cell media in both the right and left flank. When tumor volumes reached 100 mm3 ± 30 mm3, one flank tumor was inoculated with AdLuc or AdCD40L (4–5/group). No injection was performed in the contralateral flank. Tumor volume was measured as previously described. Mice were killed when the tumor volume measured 2,000 mm3 or greater because it was necessary to see the effect on the treated tumor growth versus the untreated tumor.

IL-12 and Interferon-{gamma} ELISAs
Serum was obtained from mice 2 and 6 d after intratumoral injections with PBS, AdLuc, or AdCD40L. Serum was assayed for IL-12 and interferon (IFN)-{gamma} using the OptEIA Mouse IL-12 (p70) kit and the OptEIA Mouse IFN-{gamma}, respectively (PharMingen, San Diego, CA). The protocol from the manufacturer was followed. Plates were read at 450 nm.

Immunofluorescence
Tumors were resected 72 h after intratumoral injections and placed in 4% formaldehyde (methanol-free), then transferred to 30% sucrose (Bio-Rad Laboratories, Hercules, CA). Tissue was then transferred to OCT Compound (Sakura Finetek USA, Inc., Torrance, CA). Five-micron frozen sections were cut using a Microm HM505E Cryostat (Richard Allen Scientific Co., Kalamazoo, MI). Sections were blocked with 5% rat serum and 1% bovine serum albumin with 0.1% Tween-20 (Sigma, St. Louis, MO). Biotin–anti-mouse CD40L antibody was added to the sections followed by NeutrAvidin Oregon Green 488 conjugate (Molecular Probes, Eugene, OR). Alexa Fluor 568 Phalloidin (Molecular Probes) and DAPI (Molecular Probes) were also added to the sections to visualize actin filaments and cell nuclei, respectively. ProLong Antifade Kit (Molecular Probes) was used to mount and preserve the cells for microscopy. Sections were visualized using the x40 and x60 objectives of a Leica DMRXA epifluorescence microscope (Meyer Instruments, Houston, TX) with ultraviolet, FITC, and Texas Red filter cubes. Images were captured with a CCD camera operated using SlideBook deconvolution software (Intelligent Imaging Innovations, Denver, CO).

To assess CD4+ and CD8+ T cell infiltration, tumors were resected between 4 and 13 d and fixed in 4% formaldehyde, then transferred to 30% sucrose followed by embedding in OCT. Frozen sections (5 and 50 µm) were cut using a cryostat. The 5-µm sections were blocked with goat serum (Sigma) and were double labeled. Either anti-CD4 (clone H129.19, rat-{alpha}-mouse) or anti-CD8a (clone 53–6.7) (Research Diagnostics, Inc., Flanders, NJ) was added to the sections followed by Alexa Fluor 488 goat-{alpha}-mouse IgG (Molecular Probes). A Biotin Blocking System Kit from DAKO Corp. (Carpinteria, CA) was used to block endogenous biotin. Subsequently, CD3{epsilon} (CD3 and chain) biotin–anti-mouse (PharMingen), NeutrAvidin Texas Red (Molecular Probes), and DAPI were added to the sections. ProLong Antifade Kit was used to mount and preserve the cells for microscopy. Sections were visualized using deconvolution microscopy as previously described. For the 50-µm sections, CD4 or CD8 (primary antibody) was added to tissue samples, followed by Alexa Fluor 488 goat-{alpha}-mouse IgG (secondary antibody). SYTOX Orange (Molecular Probes) was added to identify the nuclei. ProLong Antifade kit was used to preserve tissue sections for microscopy. Confocal microscopy was utilized to visualize 50-µm sections. A Nikon TE300 microscope (Melville, NY) equipped with a BioRadiance laser scanning system with a krypton/argon laser and dual PMT detectors was used. Imaging was achieved using Laser Sharp 2000 confocal software (Bio-Rad).

Tumors were resected 2 or 6 d after intratumoral injection with PBS, AdLuc, or AdCD40L to further subjectively characterize MHC class I and II presence and quantitate possible dendritic and CD8+ T cell infiltration. Tumors were snap-frozen in isopentane submerged in liquid nitrogen. Five-micron sections were cut using a Leica CM3050S cryostat (Meyer Instruments). All slides were fixed with -20°C acetone for 10 min, thoroughly washed with PBS, and subsequently blocked with 5% goat serum in 1% bovine serum albumin for 10 min. Next, sections were divided for double labeling into CD11c+/MHC II+ and CD8a+/MHC I+ groups. The first primary antibody incubation included biotinylated rat-{alpha}-mouse CD11c and hamster-{alpha}-mouse H- (Pharmingen) added to their corresponding group of slides at 10 µg/ml for 60 min at room temperature. After several washes in PBS, the sections were incubated with secondary detection reagent NeutrAvidin conjugated to Texas Red fluorophore. Next, the sections were washed and the second primary antibody incubation was performed on both anti-CD11c and anti-MHC I slides with anti-MHC II-FITC and unconjugated anti-CD8a (clone 53–6.7), respectively. The second primaries were also diluted to10 µg/ml and incubated for 60 min at room temperature. Finally, all slides were washed and the anti-CD8a was indirectly conjugated to a secondary goat anti-rat IgG Alexa Fluor 488 antibody. All slides were counterstained with blue DAPI nucleic acid dye. Cells were mounted in ProLong and visualized using a Leica DMRXA epifluorescence microscope (Meyer Instruments). Quantitation was performed for a total of 15 fields using x400 magnification and mask sub-sampling with Slidebook software (Intelligent Imaging Innovations). A deconvolution algorithm was applied to selected fields using the same software and for presentation purposes only.

Adoptive Transfer
Thirty-five days after receiving indirect therapy, or 17 d after receiving direct therapy, CBA/J mice (control tumor volumes were < 1,000 mm3) were killed and spleens were processed through a 70-µm cell strainer to obtain cell suspensions. Cells were enumerated visually using a hemocytometer and trypan blue stain was used for viability determination. The splenocytes were separated into CD4+ and CD8+ cells via Magnetic Automated Cell Sorting (AutoMACS) using the protocol for magnetic labeling of cells from lymphoid tissue. The cells were first exposed to the CD8+ (Ly-2) microbeads and then the CD4+ L3T4 microbeads (Miltenyi Biotec, Auburn, CA). To determine purity of the separation, splenocytes were stained for FACS analysis. The cells were double-labeled; FITC–{alpha}-mouse CD4 (RM4–5) (Pharmingen) and PE-{alpha}-mouse CD8a (Pharmingen) were used. The purity of positive selected cells was >= 97%. Mice were adoptively transferred by injecting 1 x 107 CD4+ cells or 0.8 x 108 CD8+ cells intraperitoneally. Two weeks after receiving the splenocytes, the animals were injected subcutaneously with 2 x 106 AC29 cells in the right flank (4/group). Tumor size was measured using the previously described method. Experiments were not repeated.

Statistical Analyses
All statistical analyses were performed using StatView for Windows, Version 4.57 (Abacus Concepts, Inc., Berkeley, CA). A Mann-Whitney unpaired two-group test was used to determine significance. ELISA data was analyzed using an ANOVA. P values of < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AC29 Cells Can Be Transduced with CD40L with Minimal Effect Upon Cell Proliferation
To investigate whether AdCD40L could be an effective in vivo antitumor therapy, pilot studies were undertaken to examine efficiency of transduction and expression of CD40L in AC29 cells. Initial transduction experiments were conducted with AdEGFP followed by FACS analysis with an MOI of 0, 5, 10, 50, and 100. These experiments revealed that an MOI of 100 resulted in over 95% of AC29 cells staining positive for EGFP (data not shown).

Similar MOIs were used to investigate expression of surface CD40L by FACS. Again, AC29 cells showed over 95% positivity with an MOI of 100 (Figure 1). Untransfected or mock-transfected cells showed no detectable surface CD40L expression (data not shown). We next determined if the expression of CD40L altered in vitro cell proliferation. An MTS assay was used to evaluate the effect of transfection of CD40L upon cell growth. AC29 cells were mock-transduced in vitro with PBS, or transduced with AdLuc or AdCD40L at an MOI of 100. The MTS assay demonstrated no significant decrease in mitochondrial activity for all three groups at 24, 48, and 72 h (data not shown).



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  		Figure 1. Expression of CD40L on transduced AC29 cells by FACS analysis. Cells were transduced with AdCD40L at an MOI of 0 (unshaded) or 100 (shaded), followed by FACS analysis to determine cell surface expression of CD40L.

 
Antitumor Effects of Ex Vivo Transfection with AdCD40L
AC29 cells were mock-transfected with PBS, or transduced with AdLuc or AdCD40L at an MOI of 100. Forty-eight hours later, CBA/J mice were injected subcutaneously with 2 x 106 AC29 cells from each transduction group. Mock-transduced cells (PBS) or cells transduced with AdLuc resulted in in vivo tumor formation (Figure 2A). However, AC29 cells transduced with AdCD40L did not result in tumor formation. These differences in tumor formation were highly statistically significantly different (P < 0.01). Because Figure 2A demonstrates mean tumor growth, it appears as that tumor volume in the PBS group spontaneously regressed; however, this was due to survival bias. Two animals in the PBS group had to be killed due to large tumor volumes in addition to ulcerations on the flank; thus, the loss of these mice in the protocol lowered the mean of the cohort. As demonstrated above using the MTS assay, ex vivo modulation with AdCD40L did not decrease cellular growth. This is further supported by tumor growth in all three treatment regimens in a nude mouse model (Figure 2B). There was no significant difference in tumor growth among the three groups. We hypothesized that the decreased ability to form tumors was secondary to an antitumor immune response. Adoptive transfer experiments were performed to test this hypothesis.



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  		Figure 2. AdCD40L-dependent suppression of tumor growth after ex vivo transduction. Significant tumor suppression was observed after ex vivo transduction of AC29 cells by AdCD40L compared with AdLuc and PBS (A). An asterisk denotes a significant difference between PBS and AdCD40L. A number sign (#) denotes a significant difference between AdLuc and AdCD40L (n = 4–6, P < 0.05). Lack of antitumor effect in athymic nude mice. Tumor growth was not suppressed in athymic nude mice (B, n = 5). Gray line, PBS; dashed line, AdLuc; solid black line, AdCD40L.

 
Adoptive Transfer of Splenocytes of Mice Exposed to Tumors Expressing CD40L Imparts Antitumor Immunity
Immunocompetent mice were injected with AC29 cells that had been treated ex vivo with either AdLuc or AdCD40L (MOI 100). After 4 wk, the animals were killed and the splenocytes were harvested. The splenocytes were separated into either CD4+ or CD8+ cells. The purity (> 97%) of the separation was confirmed using FACS analysis with anti-CD4 and anti-CD8 antibodies (data not shown).

Tumor-naive CBA/J mice were inoculated with either 1 x 107 CD4+ or 0.8 x 107 CD8+ cells. Eight days later, they were challenged with 2 x 106 AC29 cells. There was a significant decrease in tumor formation and size in mice that had received either AdCD40L CD4+ or AdCD40L CD8+ cells when compared with the AdLuc groups (P < 0.05) (Figure 3). Inoculation with the AdCD40L CD8+ T cells also demonstrated a statistically significant decrease in tumor growth when compared with the AdCD40L CD4+ T cells (P < 0.05).



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  		Figure 3. Antitumor effect of adoptively transferred CD4+ or CD8+ cells obtained from mice inoculated with ex vivo–transduced AC29 cells. The figure depicts the mean tumor volume of mice inoculated with AC29 cells after adoptive transfer of CD4+ or CD8+ cells obtained from mice inoculated with AC29 cells transduced ex vivo. There was a marked antitumor effect by the transfer of either CD4+ or CD8+ T cells from animals that received AdCD40L-transduced AC29 cells. A tilde denotes a significant difference between AdLuc CD4+ and AdCD40L CD4+; a number sign denotes a significant difference between AdLuc CD8+ and AdCD40L CD8+ (n = 4, P < 0.05).

 
Efficacy of AdCD40L in Established Mesothelioma Tumors
We next examined the efficacy of gene therapy using AdCD40L to impart antitumor immunity in established tumors. Tumors were established in the flanks of CBA/J using 2 x 106 AC29 cells. After tumor volume reached 100 ± 30 mm3, mice were treated with a low dose (1 x 109 pfu) or high dose (5 x 109 pfu) of AdLuc, AdCD40L, or PBS was injected directly into the tumors. Low dose AdCD40L resulted in a decrease in tumor size; however, there was still significant tumor growth (data not shown). Using a dose of 5 x 109 pfu of AdCD40L, tumor growth significantly regressed in the AdCD40L group (Figure 4A). Both the control and the AdLuc group demonstrated marked tumor growth at 2 wk. Thus, the efficacy of AdCD40L appears to be dose-dependent and correlated with the number of cells that had been transduced, because intratumoral injection of AC29 tumors to 1 x 109 pfu in vivo resulted in transfection efficacy of ~ 10%, whereas exposure to 5 x 109 pfu resulted in a transfection efficacy of 30–35% (data not shown). Again, we failed to observe in vivo antitumor effect of AdCD40L in athymic nude mice (Figure 4B).



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  		Figure 4. Antitumor effect of AdCD40L in established AC29 tumors in vivo. 5 x 109 pfu of AdCD40L was injected intratumorally in CBA/J mice containing previously established AC29 tumors. This resulted in a significant antitumor effect compared with PBS or AdLuc intratumoral injections (A). An asterisk denotes a significant difference between PBS and AdCD40L (n = 5, P < 0.05). Lack of antitumor effect in athymic nude mice. Tumor growth was not suppressed in athymic nude mice (B, n = 5). A: gray line, PBS; dashed line, AdLuc; solid black line, AdCD40L. B: dashed line, PBS; gray line, AdLuc; solid black line, AdCD40L.

 
CD4+ and CD8+ T Cells, Dendritic Cells, and Cells Expressing MHC I Are Recruited to the Tumor Bed after Injection with AdCD40L
The tumor beds of CBA/J mice that had undergone in vivo therapy with intratumoral inoculation of AdCD40L were examined after either 2 or 6 d to determine the presence of CD11c+ cells, a marker of dendritic cells (DCs), CD4+ or CD8+ T cells, and the presence of MHC Class I. After either 2 or 6 d following intratumoral inoculation with AdCD40L, there was an increased recruitment of CD4+ and CD8+ T cells in the tumor bed when compared with cells transduced with AdLuc (Figures 5A and 5B). This was particularly true of CD8+ T cells (Figure 5B). Immunohistochemical stains for CD11c were also performed on the resected tumors. A significant increase of CD11c cells was also seen in the tumor bed for mice treated with AdCD40L versus those treated with AdLuc or PBS (P < 0.05) (Figure 6A). This effect was seen both 2 and 6 d after intratumoral inoculation with AdCD40L (Figure 6A). Immunohistochemical analysis for the expression of MHC Class I revealed positive staining within the tumor, but no significant change at Days 2 or 6 for tumors treated with AdCD40L versus AdLuc or PBS (Figure 6B). These data suggest that the antitumor effect of AdCD40L is related to a stimulation of the CD8+ T cell response. This is supported by a lack of effect of intratumoral inoculation with AdCD40L in the athymic nude mouse (Figure 4B).




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  		Figure 5. Immunofluorescent detection of intratumoral CD4+ and CD8+ T cells. Immunofluorescent examination of representative frozen sections from the tumors revealed an increased number of CD8+ cells in the tumor bed 6 d after intratumoral injection with AdCD40L. Sections were taken from the tumor, and DAPI staining was performed as well as stains for either CD4+ or CD8+ cells. The scale bar in the lower right corner represents 10.1 µm. (A) Quantification of intratumoral CD8+ T cells after receiving intratumoral AdCD40L therapy. Immunofluorescent detection of CD8+ cells was performed as described in MATERIALS AND METHODS. CD8+ cells were quantified using a total of 15 fields at x400 magnification. There were significantly more CD8+ cells in the AdCD40L-treated animals compared with control animals at Days 2 (filled bars) and 6 (open bars) (B). An asterisk denotes a significant difference between PBS and AdCD40L, and a number sign denotes a significant difference between AdLuc and AdCD40L (n = 3, P < 0.05).

 



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  		Figure 6. Detection of intratumoral CD11c+ cells after direct therapy with AdCD40L. CD11c+ cells were quantified using a total of 15 fields at x400 magnification. There were significantly more CD11c+ cells in the AdCD40L-treated animals compared with control animals at Days 2 (filled bars) and 6 (open bars) (A). An asterisk denotes a significant difference between PBS and AdCD40L, and a number sign denotes a significant difference between AdLuc and AdCD40L (n = 3, P < 0.05). Immunofluorescent detection of class I MHC and CD8+ T cells after intratumoral AdCD40L therapy (B). Representative frozen sections were obtained from mice at Days 2 and 6, and were stained for CD8+ T cells (green), class I MHC (red), and nuclei were stained with DAPI (blue).

 
The enhanced CD8+ T cell effect at Days 2 and 6 was also associated with increased serum levels of IL-12 and IFN-{gamma}. At Day 2, IL-12 levels were significantly increased in AdCD40L-treated mice versus AdLuc or PBS mice (310 ± 71 versus 148 ± 47 versus 50 ± 35 pg/ml, respectively, P < 0.05). However IL-12 levels fell to near control levels at Day 6 in AdCD40L-treated mice (45.7 + 30 pg/ml, P = NS). In contrast, IFN-{gamma} levels were significantly increased in AdCD40L-treated mice versus AdLuc and PBS mice at both Day 2 (649 ± 118 versus 136 ± 78 versus 110 ± 95 pg/ml, respectively, P < 0.05) and Day 6 (218 ± 113 versus 40 ± 23 versus 0 + 0 pg/ml, respectively, P < 0.05).

Adoptive Transfer of Splenocytes of Mice with Previously Established Tumors Transduced with AdCD40L Imparts Antitumor Immunity
Immunocompetent mice with previously established AC29 tumors were transduced with either AdLuc or AdCD40L. Adoptive transfer experiments with purified CD4+ or CD8+ T splenocytes harvested from these treated animals demonstrated an anti-tumor effect that was transferable. Naive mice that received splenocytes harvested from animals transduced with AdCD40L had significantly decreased amount of tumor growth when compared with animals that had received splenocytes from animals transfected with AdLuc. In addition, transfer of AdCD40L CD8+ T cells demonstrated a statistically significant decrease in tumor growth when compared with the AdCD40L CD4+ T cells (P < 0.05) (Figure 7A).



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  		Figure 7. Antitumor effect of adoptively transferred CD4+ or CD8+ cells obtained from mice after intratumoral AdCD40L therapy. The figure depicts the mean tumor volume of CBA/J mice inoculated with AC29 cells after adoptive transfer of CD4+ and CD8+ cells derived from mice with previously established AC29 tumors who had undergone intratumoral therapy with either AdLuc or AdCD40L (A). The figure demonstrates a marked antitumor effect for transfer of CD8+ cells from animals exposed to AdCD40L versus AdLuc. There also is a significant decrease in tumor size for animals exposed to AdCD40L CD8+ cells versus AdCD40L CD4+ cells. A tilde denotes a significant difference between AdCD40L CD4+ and AdCD40L CD8+; a number sign denotes a significant difference between AdLuc CD8+ and AdCD40L CD8+ (n = 4, P < 0.05). Local and distant tumor regression after solitary intratumoral injection of AdCD40L. AC29 tumors were established in both rear flanks of CBA/J mice. After tumor volume was 100 ± 30 mm3, one tumor was inoculated with either AdLuc or AdCD40L. Tumor volume was recorded over time for the treated or untreated tumor (B). An asterisk denotes a significant difference between AdCD40L-treated tumors and AdLuc-treated tumors. A number sign denotes a significant difference between AdCD40L-treated tumors and AdLuc-untreated tumors. A tilde denotes a significant difference between AdCD40L-untreated tumors and AdLuc-treated tumors (n = 4–5, P < 0.05). A: dotted line with diamonds, AdLuc CD4+; dashed line with squares, AdLuc CD8+; gray line, AdCD40L CD4+; solid black line, AdCD40L CD8+. B: solid black line, AdCD40L-treated; solid gray line, AdCD40L-untreated; dashed gray line, AdLuc-treated; dashed black line, AdLuc-untreated.

 
Injection of AdCD40L into Tumors Imparts a Systemic Effect in a Synchronous Tumor Model
CBA/J mice were inoculated with AC29 cells in both posterior flanks. After tumor volume reached 100 ± 30 mm3, either 5 x 109 pfu of AdCD40L or AdLuc was injected into the larger tumor. Inoculation with AdCD40L resulted in a significant decrease in tumor size for the injected and contralateral tumor when compared with AdLuc (Figure 7B). Thus, AdCD40L imparts a systemic response that inhibits tumor growth at sites distant from direct exposure.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented in this study demonstrate that delivery of the CD40L gene by an adenoviral vector can impart antitumor immunity to one class of thoracic malignancies: mesothelioma. This treatment does not affect cell growth in vitro. In the immunocompetent mouse model, either ex vivo delivery or direct intratumoral injection of CD40L resulted in the failure of tumor growth or tumor regression. Moreover, direct therapy of tumors with CD40L resulted in tumor regression at local and distant sites. This antitumor response was demonstrated to be a CD8+-dependent cellular response.

Previous studies have demonstrated a variable effect of the CD40L protein on in vitro cell growth. Some tumor cells express the CD40 receptor, and a variable response has been demonstrated when tumor cell lines are exposed to AdCD40L. Apoptosis and growth inhibition have been demonstrated in certain cell lines, whereas other cell lines have demonstrated stimulation of cell growth (34, 35). However, we do not believe this effect to be operational, as we observed no effect of AdCD40L transduction in in vitro cell growth or tumor formation in nude mice. Moreover, AC29 cells do not stain for CD40 by FACS (data not shown).

In contrast, exposure to either AdLuc or high dose AdCD40L resulted in significant suppression or regression of tumor growth in the immunocompetent murine model. A nonspecific adenoviral effect was seen in both ex vivo and intratumoral gene therapy with AdLuc and likely represents an immune response to genetically modified cells in the immunocompetent mouse model. However, these effects were less significant than those induced by AdCD40L, and furthermore, they were not tumor-specific, as evidenced by the fact that the effects were not transferable through adoptive transfer of splenocytes. Similar effects of wild-type adenovirus with reporter genes have been seen through a variety of animal models (36–38).

Transferable tumor suppression was seen after both ex vivo AdCD40L transduction of AC29 cells or with intratumoral administration of AdCD40L. Adoptive transfer studies demonstrated that primarily CD8+ cells mediate this effect. A similar effect of CD40L has been demonstrated in both colon adenocarcinoma and melanoma cell lines. Kikuchi and coworkers demonstrated in vivo tumor suppression of colon carcinoma cell lines using gene therapy with AdCD40L. This effect was enhanced in the presence of dendritic cells, and could be negated with the use of a monoclonal antibody directed against CD40L (18, 19). Sun and colleagues also delivered CD40L to colon cancer cells using an adenoviral vector. They demonstrated similar antitumor immunity and found increased amounts of IL-12 as well as IFN-{gamma}. They also observed tumor regression at sites distant from the initial injection (39). Thus, in these models, the antitumor effect of CD40L appears systemic and transferable.

We postulate that the mechanism of protection may be due to either direct transduction of tumor cells in vivo or the result of transduction of intratumoral dendritic cells, which express CD40. Solid tumors are poorly immunogenic (17, 40). These cancer cells have a variety of genetic alterations and produce abnormal proteins. These proteins are shuttled to the cell surface and expressed on MHC class I molecules.

Tumor expression of MHC antigens relies upon APCs found in the tumor bed. The immature dendritic cells are able to process and capture antigens, but cannot activate T cells. Maturation of APCs is dependent upon interaction with CD4+ T lymphocytes. When activated, these cells express CD40L, which can bind to the CD40 receptor found on the APCs, resulting in maturation of the APCs resulting in the production of IL-12 (1, 26, 27). After maturation occurs, the APCs are able to effectively present cell surface antigens to CD8+ cells, inducing a CTL response (21). Problems with induction of a robust immune response against tumors include a paucity or dysfunction of CD4+ T lymphocytes (21).

Expression of CD40L protein within the tumor resulted in stimulation of a CD8+ immune response against the tumor. Whether CD4+ T cells are required for the induction of this response has not been tested in this model, but studies with depleting anti-CD4 antibodies are in progress. In our experiment, we demonstrated protection of tumor formation in naive mice through the transfer of CD8+ T cells derived from mice that had been exposed to AC29 cells transfected with AdCD40L. This suggests that the CD40L protein expressed on the tumor cells themselves or in intratumoral dendritic cells was able to interact with CD40 found on APCs, which resulted in a systemic IL-12 response and a better CD8+ T cell response compared with control mice. In support of this systemic immune response, we also observed significant effects on synchronous uninjected tumors that may be due to recirculation of antigen-specific CD8+ T cells. Moreover, we also observed an increase in CD11c+ cells, presumably dendritic cells after AdCD40L therapy.

In this study, we also demonstrated a dose dependent response for the efficacy of AdCD40L. Adenoviral-based gene therapy is limited by the amount of dose that can be delivered without inducing local or systemic toxicity. A dose of 5 x 109 pfu was required to induce antitumor immunity using direct therapy. A lower dose of 109 pfu was not able to effectively produce this response. Increasing the viral dose by 1/2 log caused an increased response in the AdCD40L animals. The ability to induce antitumor immunity may be related to the number of cells that express the immunostimulatory CD40L protein, as the lower dose of 109 pfu only resulted in ~ 10% transduction efficiency, whereas the higher dose of 5 x 109 pfu resulted in 30–40% transduction (data not shown).

Other strategies employed to increase the efficacy of AdCD40L have been to amplify the response to CD40L through the use of intratumoral injection of dendritic cells. Kikuchi and coworkers have shown an increase in the efficacy of AdCD40L when simultaneously injecting tumors with dendritic cells (19). The disadvantage of this approach in the clinical setting is the fact that two reagents per patient would have to be produced.

Another potential strategy is to increase transfection efficacy by tumor targeting through the use of modified adenoviruses. Curiel and colleagues have developed a capsid-modified adenovirus containing a RGD-motif in the H-I loop of the knob protein, which binds to cells via a coxsackie adenovirus receptor–independent pathway. They have demonstrated increased transfection efficacy in ovarian and squamous cell carcinoma cell lines (41, 42). The use of capsid-modified adenoviruses may be an important strategy to increase gene delivery to tumors in vivo and the induction of antitumor immunity. Clearly, more work is required to determine both the number and types of cells expressing CD40L required to induce effective antitumor immunity in vivo; however, our data suggest that AdCD40L may be an attractive candidate for mesothelioma gene therapy.


   Acknowledgments
 
The authors acknowledge Dr. Oliver Sartor and the Stanley S. Scott Cancer Center and the Louisiana Gene Therapy Research Consortium for their continuing support. This work was supported by the Stanley S. Scott Cancer Center in New Orleans, Louisiana and the Louisiana Gene Therapy Research Consortium.

Received in original form October 25, 2002

Received in final form March 19, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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