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[Cancer Research 63, 4128-4135, July 15, 2003]
© 2003 American Association for Cancer Research

Immunology

CpG Oligodeoxynucleotides Enhance the Capacity of Adenovirus-mediated CD154 Gene Transfer to Generate Effective B-Cell Lymphoma Vaccines1

Roman Rieger and Thomas J. Kipps2

Division of Hematology/Oncology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0663


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of CD40 by CD154 induces antigen-presenting cells (APC) to express immune costimulatory molecules, thereby enhancing their APC activity. Oligonucleotides (ODN), containing immunostimulatory DNA sequences (ISS) with nonmethylated CpG dinucleotides in a defined motif, also can induce similar changes in APC. In this study, we examined whether infection with recombinant adenovirus (Ad) encoding CD154 and/or treatment with ISS-ODN could enhance the capacity of A20 murine B lymphoma cells to function as APCs capable of inducing a syngeneic antilymphoma immune response. High-level expression of CD154 after infection with Ad-CD154 induced up-regulation of immune costimulatory molecules on A20 cells, as did incubation with ISS-ODN. Treatment of A20 cells with ISS-ODN also enhanced surface expression of {alpha}v integrins, making them significantly more susceptible to Ad infection than nontreated A20 cells. In syngeneic mixed-lymphocyte reactions with BALB/c splenocytes, A20 cells activated with ISS-ODN and then transduced with Ad-CD154 were significantly more effective APCs than Ad-CD154 transduced cells, which, in turn, were significantly more effective than A20 cells treated with ISS-ODN alone. Also, injection of mice with ISS-activated, Ad-CD154-infected cells induced significantly better A20-specific immune responses against A20 cells, as assessed via enzyme-linked immunospot analysis in vitro and immune prophylaxis against subsequent challenge with A20 lymphoma cells in vivo. These data demonstrate that CpG-containing oligonucleotides can serve as an adjuvant for Ad-mediated gene therapy of B-cell malignancies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD40 and its ligand CD154 are important molecules in immune activation. CD154 is expressed transiently on activated T-helper cells on T-cell receptor ligation (1) . Ligation of its receptor CD40 on APCs3 is essential for the initiation of immune responses, including antitumor immunity (2) . This interaction induces APC, such as B cells, dendritic cells, and macrophages, to express immune costimulatory molecules, such as CD80 (B7–1) and CD86 (B7–2), and adhesion molecules, such as CD54 (ICAM-1), and to up-regulate cytokine production. These phenotypic changes enhance the ability of APCs to activate antigen-specific T cells in response to presented foreign or "altered-self" tumor antigens.

CD154 has been examined for its capacity to enhance antitumor immunity against solid tumors or B-cell malignancies. There are different approaches in animal models using either intratumoral injection of viral vectors encoding CD154 (3) or cell-based vaccines comprised of either dendritic cells or tumor cells that had been modified to express CD154 (4, 5, 6, 7, 8) . Transduction of primary follicle center lymphoma B cells (9) or CLL B cells (10) with Ad encoding CD154 can enhance autologous cellular immune recognition of neoplastic cells. Moreover, Ad-CD154 transduced CLL B cells showed promising results in a Phase I clinical trial study (11) .

ISS, containing nonmethylated CpG dinucleotides within a defined, species-specific motif (5'-pur-pur-CpG-pyr-pyr-3' for mouse and 5'-pur-pyr-CpG-pyr-pyr-3' for human; Refs. 12, 13, 14 ), can induce similar changes in APCs. These ISS were identified as the active immune-stimulatory component in Mycobacterium Bacille Calmette-Guérin (15 , 16) . Treatment with Bacille Calmette-Guérin can enhance development of antitumor immunity in mouse tumor models or in patients with transitional cell bladder cancer (17) . CpG ODNs can stimulate natural killer cells and APC to express immune costimulatory molecules and/or adhesion receptors, and certain proinflammatory cytokines, such as interleukin 6, interleukin 12, or IFN-{gamma} (12 , 18 , 19) .

The use of CpG-ODN as an antitumor agent has been described in several reports (20, 21, 22) . For example, vaccination with CpG-DNA that is covalently linked to a protein antigen induces CD8+ T cell-mediated protection against a lethal tumor challenge (23) . Systemic administration of CpG-DNA in experimental animals can protect against challenge with acute myelogenous leukemia cells (20) or inhibit metastatic spread of primary tumor (21) . Furthermore, CpG-ODN can also serve as an effective adjuvant for immunization against murine B-cell lymphoma and neuroblastoma antigens (22 , 24 , 25) . However, to our knowledge, it has not been fully investigated whether tumor cells that were treated with ISS-ODN could serve as an effective antitumor cell vaccine.

In this study, we examined the phenotypic changes and immune-stimulatory activity of A20 cells or A20 cells that had been infected previously with Ad-CD154, treated with ISS-ODN, or that had been treated with ISS-ODN and then transduced with Ad-CD154. In addition, we examined the capacity of treated and untreated A20 cells to induce protective immunity in BALB/c mice against subsequent lethal challenge with syngeneic A20 lymphoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Cell Culture.
Female BALB/c mice, 6–8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the UCSD animal facility. All of the animal studies were approved by the Animal Subjects Committee and the Biosafety Committee of the UCSD School of Medicine and were performed in accordance with institutional guidelines. The BALB/c pre-B lymphoblastoid cell line A20 was obtained from American Type Culture Collection and maintained in RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (Omega-Scientific, Tarzana, CA), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY). A20 cells were passaged in vivo by injection of 105 tumor cells into the tail vein of syngeneic mice to enhance tumorigenicity on subsequent adoptive transfer to BALB/c mice. The A20 tumor cells were isolated from abdominal lymph nodes 4 weeks after injection.

Ad Vectors and Ad Infection of A20 Cells.
The construction of Ad vectors encoding ß-galactosidase (Ad-lacZ) and murine CD154 (Ad-CD154) have been described previously (10 , 26) . Ad vectors were propagated in 293 cells. Purified, high-titer Ad preparations were produced by Molecular Medicine, LLC (San Diego, CA) by anion-exchange chromatography as described (27, 28, 29) . The virus titer and purity was determined by plaque assay with 293 cells and HPLC chromatography. Viral titers ranged from 1 x 1010 to 2 x 1010 plaque-forming units per milliliter. For infection, A20 cells (106) were suspended in 0.1 ml of culture medium and then infected with Ad vectors at various MOI ratios (e.g., 1–1000). Cells were incubated for at least 24 h at 37°C before being analyzed for transgene expression.

Nucleic Acid Reagents and Activation of A20 Cells with ISS-ODN.
Phosphorothioate single-stranded ODNs ODN1018 (5'-TGACTGTGAACGTTCGAGATGA-3') and ODN1019 (5'-TGACTGTGAAGGTTAGAGATGA-3') were obtained from Dr. Eyal Raz (UCSD) and Dynavax Technologies (Berkeley, CA). For activation with CpG-DNA, A20 cells (starting concentration: 5 x 105 cells/ml) were cultured for 72 h in the presence of 1 µg/ml ODN1018 or ODN1019, respectively. The cells were then washed and analyzed by flow cytometry for expression of activation surface antigens.

Flow Cytometry.
The cells were washed and then suspended in staining medium, composed of deficient RPMI 1640 (Irvine Scientific), 3% FCS, and 1 µg/ml propidium iodide. The cells were then incubated with saturating amounts of fluorochrome-conjugated mAb for 30 min at 4°C. The cells were washed with staining medium and analyzed using a FACSCaliber flow cytometer (Becton Dickinson, San Jose, CA). Dead cells staining with propidium iodide were excluded from the analyses. The relative expression of given surface antigen was described as the MFIR and/or as the percentage of cells that expressed that antigen. The MFIR equals the mean fluorescence intensity of cells stained with a fluorochrome-conjugated antigen-specific mAb divided by the mean fluorescence intensity of cells stained with a fluorochrome-conjugated isotype control mAb. All of the antibodies were purchased from BD PharMingen (San Diego, CA), which included phycoerythrin-conjugated mAb specific for mouse CD154, CD40, CD51 ({alpha}v-integrin), CD54, CD61 (ß3-integrin), CD80, CD86, CD95, H-2Kd (MHC class I), and fluorescein-conjugated mAb specific for mouse CD29 (ß1-integrin) and I-Ad (MHC class II).

Autologous MLR.
Thirty-six h after the infection with Ad, or after 72 h of treatment with ODN, A20 cells were harvested, washed with culture medium, and then irradiated with 90 Gy to block subsequent cell proliferation. In each well of a 96-well U-bottomed plate we placed 2 x 105 irradiated treated or untreated A20 cells to act as stimulator cells in the MLR. Splenocytes were isolated from 6–8-week-old female BALB/c mice. For this, the spleens were removed, and a single-cell suspension was obtained by teasing the tissue through a wire mesh in RPMI 1640 cell culture medium. After incubation for 5 min at room temperature with ACK lysing buffer (BioWhittaker, Walkersville, MD) to lyse RBCs, splenocytes were washed three times with culture medium and then mixed with A20 stimulator cells at a 1:1 cell ratio in a total culture volume of 200 µl. Three days after initiation of the culture, aliquots of the culture supernatant were collected to measure IFN-{gamma} production by ELISA. To measure cell proliferation, the cultures were supplemented with 1 µCi/well [3H]thymidine 72 h after the initiation of the MLR. After 12–16 h, the cultures were harvested onto a nitrocellulose membrane using a cell harvester (Tomtec, Hamden, CT). [3H]Thymidine incorporation was determined with a liquid scintillation ß-counter (Wallac, Turku, Finland).

Cytokine ELISA.
The rat antimouse IFN-{gamma} capture antibody and the biotinylated rat antimouse IFN-{gamma} detection antibody were purchased from BD PharMingen (San Diego, CA). Half-area 96-well plates (Costar, Cambridge, MA) were coated with capture antibody diluted according to the manufacturer’s recommendations in phosphate buffer [0.1 M Na2HPO4 (pH 9.0)], overnight at 4°C. Plates were washed with PBS containing 0.05% Tween 20 and then blocked for 2 h at 37°C with blocking buffer (10% fetal bovine serum in PBS). Plates were washed and then incubated with tissue culture supernatant from syngeneic MLR overnight at 4°C. Recombinant mouse IFN-{gamma} (BD PharMingen) was used for standard curve (1:2 serial dilutions in blocking buffer). The plates were washed four times in wash buffer and then incubated for 1 h at room temperature with detection antibody diluted according to the manufacturer’s recommendations in blocking buffer. The plates were washed twice and incubated with avidin and biotinylated horseradish peroxidase (Elite Vectastain; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. After plates were washed 5 times, the 3,3',5,5'-tetramethyl-benzidine dihydrochloride (TMB) peroxidase substrate was added (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The reaction was stopped with 1 M phosphoric acid (Sigma Chemical Co., St. Louis, MO), and the absorbance at 450 nm was measured using an ELISA microplate reader (Molecular Devices, Menlo Park, CA). IFN-{gamma} concentrations were extrapolated from the standard curve using linear regression.

ELISPOT Assays.
Ninety-six-well nitrocellulose plates (Multiscreen HA; Millipore, Bedford, MA) were coated overnight at 4°C with an antimouse IFN-{gamma} antibody (clone R4–6A2; BD PharMingen) at 4 µg/ml in sterile PBS. The plates were washed four times with PBS and then blocked with RPMI 1640 containing 10% FCS for at least 1 h at 37°C. Splenocytes from mice that had been vaccinated with Ad-infected A20 cells or ODN-treated A20 cells were isolated, as described for the MLR reaction. Then 2 x 105 splenocytes were plated out into separate wells of an ELISPOT plate and either cultured alone or cocultured with mitomycin C (Sigma Chemical Co.) treated A20 stimulator cells in a 1:1 cell ratio at a total culture volume of 200 µl. The plates were incubated for 48 h at 37°C in a humidified atmosphere of 5% CO2 and then washed three times with PBS, followed by three washes with PBS containing 0.05% Tween 20. To each well, 50 µl of biotinylated anti-IFN-{gamma} mAb (clone XMG1.2; PharMingen) were added to achieve a final IFN-{gamma} concentration of 2 µg/ml in PBS/0.05% Tween 20. The plates were incubated overnight at 4°C. After four washes with PBS/0.05% Tween 20, 100 µl of a 1:500 dilution of horseradish peroxidase-conjugated streptavidin in PBS/0.05% Tween 20 were added to each well. The plates were incubated for 60 min at room temperature and then washed three times with PBS/0.05% Tween 20 followed by three washes with PBS. Fresh 3-amino-9-ethylcarbazole substrate was prepared by dissolving 4 mg of 3-amino-9-ethylcarbazol (Sigma Chemical Co.) in 1 ml of dimethylformamide and then diluting this into 9 ml of sodium acetate buffer (pH 5.0) that contained 5 µl of freshly added 30% hydrogen peroxide. One-hundred µl of the sterile-filtered 3-amino-9-ethylcarbazole substrate was added to each well, and the plates were incubated for >=5 min at room temperature. The substrate solution was discarded, and the plates were rinsed extensively with water and air-dried. After the plates were completely dry, spots were counted using the ImmunoSpot image analyzer and software (Cellular Technology Ltd., Cleveland, OH), and triplicate wells were used to calculate the average number of spots ± SE.

Animal Studies.
Preliminary studies determined that 105 A20 cells could induce palpable s.c. tumors in BALB/c mice after ~1 week in 100% of the injected animals. For splenocyte activation studies, mice received s.c. 100 µl injections in the left hind flank on day 1 and day 7 with 3 x 105 A20 cells that either had been infected with Ad or treated with ODN. On day 14, the mice were sacrificed and spleen cells isolated for use in the MLR. For tumor challenge studies, mice received two s.c. injections in the left flank with A20 cells or A20 cells, which have been incubated with ODN1018 or ODN1019, or infected with Ad-lacZ or Ad-CD154, respectively, at the times indicated in the text. The A20 cells were irradiated with 90 Gy in the presence of culture medium and washed four times with PBS before injection. Seven days after the last injection, the mice were injected s.c. into the right flank with 105 A20 cells in 100 µl PBS. Tumor progression was monitored biweekly over the course of the experiment by measuring the length and width of the developing tumor using calipers starting 1 week after the challenge with wild-type A20 cells. Tumor volumes were calculated by the following formula: volume = 0.5 x length x (width)2. Mice were sacrificed when length exceeded 20 mm. All of the animal experiments have been conducted at least twice.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Murine CD154 on A20 B Lymphoma Cells after Infection with Ad Encoding CD154.
We infected A20 cells with Ad-CD154 at increasing MOI and then examined for cell surface expression of CD154 by flow cytometry. Infection of A20 at an MOI of >=300 resulted in high-level surface expression of CD154 48 h postinfection (Fig. 1)Citation . Analysis of the infected cells over time revealed that CD154 expression reached maximum levels at ~3 days after initial infection (data not shown). CD154 expression on A20 cells was analyzed by flow cytometry subsequently 48 h postinfection, if not noted otherwise.



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  		Fig. 1. Expression of CD154 on Ad-CD154-infected A20 B lymphoma cells. A20 cells were infected with Ad-CD154 at the indicated MOI and then analyzed for expression of CD154 by flow cytometry 48 h later. The shaded histograms depict the fluorescence of cells stained with anti-CD154 phycoerythrin mAb, whereas the open histograms depict the fluorescence of cells stained with an isotype control mAb of irrelevant specificity. The percentage of CD154-positive cells is indicated in each histogram.

 
Induction of Immune Accessory Molecules on Ad-CD154-infected or ISS-ODN-treated A20 Mouse Lymphoma Cells.
A20 B lymphoma cells were infected with Ad-CD154 or Ad-lacZ and then examined for changes in surface antigen expression by flow cytometry. In contrast to noninfected or Ad-lacZ-infected cells, A20 cells infected with Ad-CD154 at an MOI of 1000 were induced to express significantly higher levels of immune accessory molecules, such as CD54, CD80, CD95, and MHC class II. However, infection with Ad-CD154 did not alter the expression levels of CD86 or MHC class I (H-2Kd; data not shown) molecules, and actually significantly reduced expression of CD40 (Fig. 2)Citation . Incubation of A20 cells with heat-inactivated Ad-CD154 did not result in detectable expression of CD154 or change in surface antigen expression, indicating that expression of CD154 was dependent on infection with Ad-CD154 (data not shown).



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  		Fig. 2. Phenotypic changes of A20 cells after infection with Ad or treatment with ODN. A20 cells were infected with Ad-lacZ or Ad-CD154 at an MOI of 1000 and then analyzed 48 h later by flow cytometry. Alternatively, A20 cells were treated with the control-ODN (ODN1019) or the ISS-ODN (ODN1018) at a concentration of 1 µg/ml for 72 h. The cells were analyzed by flow cytometry for the expression of various surface antigens. The bar charts show the mean MFIR value (bars, ±SD) of three representative experiments. * indicate a statistically significant difference (P < 0.05, Bonferroni t test) in the expression of the listed antigen compared with that observed on nontreated A20 cells.

 
On the other hand, A20 cells were treated with either an ODN that contained a CpG-motif (ODN1018) or a control mutated sequence (ODN1019) at 1 µg/ml for 72 h. This concentration was found optimal for activating murine B cells and macrophages in preliminary studies (data not shown) and prior studies (18) . Significantly increased levels of CD40, CD54, CD80, CD86, CD95, and MHC class II were observed on A20 cells treated with ODN1018 compared with nontreated A20 cells or A20 cells treated with the control oligonucleotide, ODN1019. In contrast to infection with Ad-CD154, ODN1018 enhanced the expression levels of CD40 on A20 cells (Fig. 2)Citation . Moreover, ODN1018-activated A20 cells maintained high-level expression of CD80 for at least 5 days after treatment (data not shown).

Increased Susceptibility to Ad Infection of CpG-ODN-treated A20 Cells.
To examine whether treatment with ODN1018 could enhance signaling via the CD40-CD40L pathway, we infected ODN-treated A20 cells with Ad-CD154 at increasing MOI and analyzed for the expression of CD154 and CD80 48 h after infection by flow cytometry. A20 cells that were pretreated with ODN1018 and then infected with Ad-CD154 (MOI 300) expressed significantly higher levels of CD80 (MFIR 142 ± 17) than A20 cells that were treated with ODN1018 alone (MFIR 10.5 ± 2.2) or that only were infected with Ad-CD154, even at an MOI of 1000 (MFIR 8.6 ± 2.8; Fig. 3BCitation ). Furthermore, we found that Ad-CD154-infected A20 cells that had been pretreated with ODN1018 expressed significantly higher levels of CD154 than Ad-CD154-infected cells that had not been treated or that had been pretreated with ODN1019, at any given MOI. Moreover, the expression levels of CD154 on infected A20 cells that had been pretreated with ODN1018 were higher than that of nontreated or control-treated A20 cells that were infected with Ad-CD154 at more than one-half log higher MOI (Fig. 3A)Citation . As such, it appears that treatment with ODN1018 enhanced the susceptibility of A20 cells to Ad infection. Consistent with this, we observed that treatment with ODN1018 also enhanced the susceptibility of A20 cells to infection with Ad-lacZ (data not shown).



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  		Fig. 3. A, infection of ODN-treated A20 cells with Ad-CD154. A20 cells were incubated with ODN for 72 h at 1 µg/ml. After washing, cells were infected with Ad-CD154 at increasing MOI, as indicted each histogram ({Delta} = heat-inactivated virus, 65°C, 20 min). Cells were analyzed 48 h later by flow-cytometry for CD154. Mean CD154 MFIR values of triplicate experiments were plotted on a logarithmic scale; bars, ±SD. * indicate statistically significant difference (P < 0.05, Bonferroni t test) in expression of CD154 compared with that observed on control-treated or nontreated A20 cells infected at the same MOI. B, up-regulation of CD80 expression on ODN-treated and/or Ad-CD154-infected A20 cells. Cells either were treated for 72 h with ODN1018 or ODN1019 (1 µg/ml), or infected with Ad-CD154 at an MOI ratio of 300 or 1000. In parallel ODN1018-treated or ODN1019-treated A20 cells were infected with Ad-CD154 at an MOI of 300. The cells were analyzed 48 h after infection by flow cytometry for expression of CD80. Shown is the mean CD80 MFIR of two independent experiments; bars, ±SD. The * indicates a statistically significant increase (P < 0.05, Bonferroni t test) in expression of CD80 compared with that of all other treatment groups.

 
Treatment of A20 Cells with ODN1018 Up-Regulates Expression of {alpha}v, ß1/3 Integrins.
To investigate why pretreatment with ODN1018 increased the susceptibility of A20 cells to Ad infection, we examined for ODN-induced changes in the expression levels of surface antigens that facilitate virus attachment and entry. We found that A20 cells expressed negligible levels of the CAR. However, the expression level of CAR did not change after treatment with ODN1018 (data not shown). Also, treatment with A20 cells with ODN1018 did not induce increased expression of {alpha}M (data not shown), an integrin that can facilitate Ad internalization (30) . However, we found that treatment of A20 cells with ODN1018 significantly increased surface expression of other integrins that facilitate Ad entry, namely {alpha}v, ß1, and ß3 (Fig. 4A)Citation . Treatment of A20 cells with ODN1018 increased expression levels of these integrins ~3-fold over that of nontreated A20 cells or A20 cells treated with ODN1019.



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  		Fig. 4. A, increased surface expression of integrins on ISS-ODN-treated A20 cells. A20 cells were treated for 72 h with ODN and then analyzed by flow cytometry for expression of {alpha}v, ß1, and ß3 integrins. Histograms depict the fluorescence intensity of untreated (———), ODN1019-treated (····), or ODN1018-treated () A20 cells that were labeled with fluorochrome-conjugated anti-integrin mAb. These are depicted together with A20 cells stained with a fluorochrome-conjugated isotype-control antibody (filled black histogram). B, blocking of Ad-CD154 infection of ISS-ODN activated A20 cells with an anti-{alpha}v antibody. ODN-treated A20 cells (as above) were incubated for 90 min at 4°C with increasing concentrations of anti-{alpha}v antibody (H9.2B8). After this, the cells were infected with Ad-CD154 at a low MOI of 30. Surface expression of CD154 was determined 24 h after infection by flow cytometry. The surface expression of CD154 on noninfected, Ad-CD154-infected, or ODN1018-treated A20 cells examined in parallel is shown for comparison. The data represent the mean percentage of CD154-positive cells at each condition in two experiments; bars, ±SD. The * indicate a statistically significant difference (P < 0.05, Bonferroni t test) in the expression of CD154 of the anti-integrin-treated A20 cells compared with that of control-treated A20 cells after infection with Ad-CD154.

 
To determine whether the increased susceptibility of ODN1018-treated A20 cells to Ad-CD154 infection is because of increased expression of {alpha}v integrins, we incubated ODN1018-activated A20 cells with increasing concentrations of anti-{alpha}v integrin antibody (H9.2B8) before infection with Ad-CD154 at an MOI of 30 and then measured Ad-mediated gene delivery 24 h after infection by flow cytometry. We found that pretreatment of ODN1018-activated A20 cells with anti-{alpha}v integrin antibody could significantly block Ad-CD154 infection in a dose-dependent manner, reducing transgene expression to levels observed after infection of nonactivated cells (Fig. 4B)Citation .

ODN1018-activated and CD154-expressing A20 Cells Are Highly Effective Stimulator Cells in Syngeneic MLRs.
We evaluated whether A20 cells treated with ODN1018 and/or infected with Ad-CD154 could stimulate BALB/c splenocytes to produce IFN-{gamma} and to proliferate in a MLR. Splenocytes from syngeneic BALB/c mice were isolated and cocultured at a stimulator:effector ratio of 1:1 with irradiated A20 cells or irradiated A20 cells that had been treated previously with ODNs, infected with Ad-CD154 or Ad-lacZ at an MOI of 1000, or that had been pretreated with ODN and then infected with Ad-CD154 at an MOI of 300. After a 3-day MLR, we assessed the concentration of IFN-{gamma} in the culture supernatants by ELISA. The MLR with ODN1018-treated A20 cells generated significantly higher amounts of IFN-{gamma} (590 ± 40 pg/ml, mean ± SE; n = 3) than did MLR cultures with control-ODN-treated A20 cells (100 ± 20 pg/ml), Ad-lacZ-infected A20 cells (160 ± 20 pg/ml), or A20 cells alone (90 ± 10 pg/ml; P < 0.05, Bonferroni t test; Fig. 5ACitation ). Also, the effector splenocytes or A20 stimulator cell preparations did not produce significant amounts of IFN-{gamma} when cultured alone (Fig. 5A)Citation . A20 cells infected with Ad-CD154 at an MOI of 1000 stimulated syngeneic BALB/c splenocytes to produce significantly more IFN-{gamma} (5330 ± 200 pg/ml) than did A20 cells treated with ODN1018 (P < 0.01, paired t test). However, supernatants from cultures with stimulator cells that had been pretreated with ODN1018 and then infected with Ad-CD154 at an MOI of 300 produced amounts of IFN-{gamma} (7400 ± 20 pg/ml) that were significantly higher than that produced by cultures stimulated with A20 cells that only had been infected with Ad-CD154 or that had been treated with ODN1019 and then infected with Ad-CD154 (6,000 ± 250 pg/ml, P < 0.05, Bonferroni t test). On the other hand, culture supernatants with stimulator cells that had been pretreated with ODN1018 and then infected with Ad-lacZ (Fig. 5ACitation ; ODN1018 + lacZ) produced amounts of IFN-{gamma} (640 ± 40 pg/ml) that were similar to that of cultures stimulated with A20 cells that had been treated with ODN1018 alone (590 ± 40 pg/ml), demonstrating that infection with Ad per se did not enhance the stimulating capacity of A20 cells in a syngeneic MLR.



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  		Fig. 5. Syngeneic MLR. A20 cells either were treated with ODN at 1 µg/ml for 72 h or infected with Ad-lacZ (A20-lacZ) or Ad-CD154 (A20-CD154) at an MOI of 1000. Also, ODN1018-treated or ODN1019-treated A20 cells were infected with Ad-CD154 or Ad-lacZ at an MOI of 300 (ODN1018+Ad-CD154, ODN1019+Ad-CD154, or ODN1018+Ad-lacZ). Thirty-six h later these cells were irradiated (9000 rad) and then used as stimulator cells in a syngeneic MLR. For this, 2 x 105 stimulator cells were cultured alone ({square}) or with 2 x 105 BALB/c splenocytes () in 96-well U-bottomed plates. The type of stimulator cell used is indicated on the left side of each figure. A, after 3 days of culture, the supernatants were assessed for IFN-{gamma} by ELISA. B, after 3 days of culture, the cells were pulsed with 1 µCi/well [3H]thymidine. We monitored for incorporation into acid-precipitable DNA 14 h later using a ß-scintillation counter. The bars represent the mean ± SE of triplicate wells.

 
After 4 days, T-cell proliferation was analyzed by measuring the incorporation of [3H]thymidine into DNA. ODN1018-treated A20 stimulator cells induced significantly more [3H]thymidine incorporation (44,500 ± 6,600 cpm, mean ± SE; n = 3) than did control ODN1019-treated A20 cells (26,600 ± 1,600 cpm), Ad-lacZ-infected A20 cells (23,900 ± 1,200 cpm), or mock-treated A20 cells (20,900 ± 2,500 cpm, P < 0.05, Bonferroni t test; Fig. 5BCitation ). Ad-CD154-transduced A20 stimulator cells induced significantly more [3H]thymidine incorporation (119,900 ± 9,500 cpm) than did A20 stimulator cells that had been treated with ODN1018 or had been pretreated with ODN1018 and then infected with Ad-lacZ (41,500 ± 2,400 cpm, P < 0.05, Bonferroni t test). However, cultures with stimulator cells that had been pretreated with ODN1018 and then infected with Ad-CD154 at MOI 300 incorporated significantly higher amounts of [3H]thymidine (177,700 ± 9,500 cpm) than did cultures stimulated with Ad-CD154-infected A20 cells or ODN1019-treated and then Ad-CD154-infected A20 cells (140,100 ± 4,600 cpm, P < 0.05, Bonferroni t test). Isolated splenocytes or irradiated A20 cells incorporated negligible amounts of [3H]thymidine when cultured alone (Fig. 5BCitation , empty bars).

Activation of Splenocytes in Mice Vaccinated with ODN1018-treated and Ad-CD154-infected A20 Cells.
We examined whether A20 cells treated with Ad-CD154 or Ad-lacZ, and/or ODN1018 or ODN1019 could induce a cellular immune response against parental A20 cells in syngeneic BALB/c mice. We injected BALB/c mice on days 0 and 7 s.c. with 3 x 105 live A20 cells that had been infected with Ad, or treated with ODN or a combination of both. After 14 days, splenocytes were isolated for the ELISPOT assay to determine the proportion of cells that could produce IFN-{gamma} when cocultured with A20 cells. Splenocytes from mice that had been vaccinated with the various A20 cell populations were either cultured alone or cocultured with mitomycin C-treated A20 cells in a stimulator:effector cell ratio of 1:1 for 48 h. When cocultured with mitomycin C-treated A20 cells, the splenocytes from mice injected with Ad-CD154-transduced A20 cells had a significantly higher proportion of IFN-{gamma}-producing cells per 106 splenocytes (280 ± 22, mean ± SE; n = 3; Fig. 6Citation , shaded bars) than did splenocytes from mice immunized with A20 cells (25 ± 12.5), ODN1018-treated A20 cells (145 ± 17), ODN1019-treated A20 cells (125 ± 14.5), or Ad-lacZ-infected A20 cells (115 ± 28, P < 0.05, Bonferroni t test). However, when mice were vaccinated with A20 cells that had been activated with ODN1018 and then infected with Ad-CD154 at an MOI of 300, a significantly higher number of IFN-{gamma} secreting splenocytes (730 ± 60.5) was detected after coculture with A20 cells compared with vaccination with Ad-CD154-infected cells or ODN1019-treated, Ad-CD154-transduced cells (410 ± 19, P < 0.05, Bonferroni t test). Also, splenocytes from mice immunized with ODN1018-activated and Ad-CD154-transduced cells also had significantly higher proportions of IFN-{gamma}-producing cells (230 ± 31.5) than did the splenocytes from mice immunized with Ad-CD154-transduced (80 ± 18.5), ODN1018-treated (30 ± 6.5), or Ad-lacZ-infected cells (50 ± 10, P < 0.05, Bonferroni t test; Fig. 6Citation ). Negligible numbers of splenocytes from nonimmunized BALB/c mice produced IFN-{gamma} when cultured alone (Fig. 6Citation , empty bars).



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  		Fig. 6. ELISPOT assay for IFN-{gamma} producing spleen cells. Six to 8-week-old female BALB/c mice were injected s.c. in the hind flank on days 0 and 7 with 3 x 105 A20 cells (A20) or A20 cells that first were treated for 72 h with 1 µg/ml of ODN1019 (A20 ODN1019) or ODN1018 (A20 ODN1018), or infected with Ad-lacZ (A20-lacZ) or Ad-CD154 (A20-CD154) at an MOI ratio of 1000 36 h before injection. Alternatively, the mice were injected with ODN1018-treated or ODN1019-treated A20 cells (as above) that subsequently were infected with Ad-CD154 at an MOI ratio of 300 36 h before injection. On day 14, the spleen cells were harvested for ELISPOT assay. For this, 2 x 105 spleen cells were cultured alone ({square}) or with 2 x 105 mitomycin C-treated A20 cells () for 48 h in a 96-well nitrocellulose plate. The numbers of IFN-{gamma} producing spleen cells were determined in an ELISPOT assay. The bars depict the mean number of IFN-{gamma}-producing spleen cells per 106 splenocytes of triplicate wells; bars, ±SE.

 
Antitumor Immunity of Mice Immunized with A20 Cells.
We evaluated whether any of the A20 cell preparations could induce an immune response against adoptive transfer of A20 into BALB/c mice. On day -14 and day -7, BALB/c mice (8 animals/group) were injected s.c. in the left flank with 3 x 105 irradiated (90 Gy) A20 cells or A20 cells that had been treated with ODN1018 or ODN1019, or had been infected with Ad-CD154 or Ad-lacZ at an MOI ratio of 1000, or that had been activated with ODN1018 and then infected with Ad-CD154 at an MOI ratio of 300. On day 0, the animals were challenged with a lethal dose of A20 cells (1 x 105 cells) injected into a contralateral site. All of the mice immunized with A20 cells (Fig. 7Citation , {triangleup}) formed tumors within 1 week of challenge, similar to what was observed for nonimmunized mice (Fig. 7Citation , {blacksquare}). In the groups that were injected with A20 cells that had been treated with ODN1018 (Fig. 7Citation , {blacktriangledown}), ODN1019 (Fig. 7Citation , x), or infected with Ad-lacZ (Fig. 7Citation , {circ}) 1 of 8 mice (12.5%) resisted tumor challenge. However, in the group of mice immunized with A20 cells infected with Ad-CD154 at an MOI ratio of 1000 (Fig. 7Citation , {square}), 5 of 8 (62.5%) resisted tumor challenge. Furthermore, vaccination with A20 cells that were first activated with ODN1018 and then infected with Ad-CD154 at an MOI of 300 resulted in a tumor incidence of 62.5% (5 of 8 animals; Fig. 7Citation , {blacktriangleup}) providing significantly better protection than injections of ODN1018-treated or Ad-lacZ-infected A20 cells alone (P < 0.03, log-rank test). A repeat experiment with 8 animals per group yielded similar results (data not shown).



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  		Fig. 7. A20 tumor growth in mice that had been vaccinated previously with ODN-treated or Ad-infected A20 cells. Six to 8-week-old female BALB/c mice were injected on days 0 and 7 s.c. into the left hind flank with 3 x 105 irradiated A20 cells ({triangleup}), or A20 cells that either were treated for 72 h with ODN1019 (x) or ODN1018 ({blacktriangledown}) at 1 µg/ml, or infected with Ad-lacZ ({circ}) or Ad-CD154 ({square}) for 36 h at an MOI ratio of 1000, or that first were treated with ODN1018, as above, and then infected with Ad-CD154 for 36 h at an MOI ratio of 300 ({blacktriangleup}). As a control, one group of mice did not receive A20 cells ({blacksquare}). On day 14, the mice were challenged by s.c. injection of 105 parental A20 cells. The graph shows the percentage of mice per group (8 animals/group) that remained tumor-free over time after the challenge (in weeks).

 
We examined whether increasing the number of cells given as a vaccine would have a beneficial effect on tumor protection. Therefore, we injected BALB/c mice (8 animals/group) twice in a 2-week interval s.c. with 106 irradiated A20 cells that were treated with ODN1018 at 1 µg/ml for 72 h, infected with Ad-CD154 at an MOI ratio of 1000, or first treated with ODN1018 and then infected with Ad-CD154 at an MOI ratio of 300. One week after the last vaccination, the mice received a s.c. injection of 1 x 105 live A20 cells into the contralateral flank. As shown in Fig. 8ACitation , mice injected with ODN1018-treated A20 cells were significantly better protected against a tumor challenge than nonvaccinated mice (P < 0.02, log-rank test) with 25% of mice remaining tumor-free >=18 weeks after tumor challenge (Fig. 8A)Citation . Injection of ODN1018-treated cells also slowed tumor growth. Compared with nontreated mice, vaccinated mice had significantly smaller tumors 5 weeks after challenge (P < 0.05, paired t test), and the tumors were not significantly different from tumors in mice that were vaccinated with any of the other A20 cell populations (Fig. 8B)Citation . Seven of 8 (87.5%) mice injected with Ad-CD154 (MOI 1000) transduced A20 cells remained tumor-free after 19 weeks, whereas 100% of the mice were protected when they were injected with A20 cells that were treated sequentially with ODN1018 and then Ad-CD154 (Fig. 8A)Citation .



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  		Fig. 8. Development of palpable tumors in mice injected with A20 cells after vaccination with ISS-ODN-treated, Ad-CD154-infected A20 cells. Six to 8-week-old female BALB/c mice were injected twice over 2 weeks in the s.c. tissue of the left hind flank with 106 irradiated A20 cells that either were treated for 72 h with ODN1018 ({blacksquare}) at 1 µg/ml, infected with Ad-CD154 ({blacktriangleup}) 36 h earlier at an MOI of 1000, or treated with ODN1018 and then infected with Ad-CD154 at an MOI of 300 ({square}). One control group of mice did not receive A20 cells ({diamondsuit}). One week after the last vaccination, the mice were challenged by s.c. injection of 105 parental A20 cells. A, graphs depict the percentage of mice in each treatment group (n = 8) that remained tumor-free over time after the challenge. B, graphs depict the mean size of s.c. tumors of the mice that developed tumors in each of treatment group (n = 8) over time after tumor challenge; bars, ±SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor cells generally do not present antigens efficiently to T lymphocytes and are ineffective in inducing an antitumor immune response (31) . A promising approach to overcome this problem is to modify tumor cells to express immune costimulatory molecules or to secrete cytokines and thereby enhance their capability to function as effective APCs. Gene transfer of CD154 into tumor cells, for example, can induce immune responses in various tumor models (32) . For immunotherapy of B-cell malignancies, gene transfer of CD154 can be accomplished using an Ad vector encoding CD154. Successful transduction of malignant B cells, such as CLL B cells (10) , follicular lymphoma B cells (9) , or murine A20 lymphoma cells (8) , has resulted in improved tumor APC activity. This is confirmed in the present study, demonstrating that infection of A20 lymphoma cells with Ad-CD154 at high MOI ratios resulted in high-level expression of CD154. Expression of CD154 on A20 cells induced significant up-regulation of CD80, CD54, CD95, and MHC class II (I-Ad), immune accessory molecules that play a vital role in T-cell costimulation by APCs (2) . However, we also observed that expression of CD40 on A20 cells decreased after infection with Ad-CD154, most likely secondary to ligand-receptor down-modulation. Consistent with this notion, we observed that the level of CD40 expressed on such cells decreased after coculture with cells expressing CD154 (data not shown).

Other candidates that are currently under investigation for cancer immunotherapy are immunostimulatory (ISS) or CpG ODNs (33) . ISS-ODNs contain unmethylated CpG-dinucleotides within a defined motif and can stimulate expression of immune accessory molecules and cytokines by B cells, dendritic cells, or macrophages, and can induce proliferation of murine and human normal or leukemia B cells (12 , 18 , 34, 35, 36) . We examined in this study the effects of immunostimulatory DNA on A20 lymphoma cells. We observed that incubation of A20 lymphoma cells with an ISS-ODN, ODN1018, but not with a mutated control oligonucleotide, induced expression of immune accessory molecules CD40, CD54, CD80, CD86, CD95, and MHC class II. However, in contrast with A20 cells that were transduced with Ad-CD154, we found the A20 cells treated with ODN1018, but not the control ODN, were induced to express significantly higher levels of CD40. The phenotypic changes that were induced after treatment of A20 cells with ISS-ODN were apparent for at least 5 days after treatment and enhanced the ability of treated A20 cells to act as APCs in cognate cellular immune interactions.

Besides the induction of costimulatory molecules after incubation of A20 cells with ISS-ODN, we found that ISS-ODN-treated cells exhibited an increased susceptibility to Ad infection. Infection of cells with type 5 Ad involves at least two different types of cellular receptors/surface molecules. First, attachment of the virus is mediated by interaction of the virus fiber protein with a 46-kDa cellular receptor, CAR (37) . Internalization of Ad into the cell is then mediated by binding of the penton-base proteins of Ad to integrins of the {alpha}v family, such as {alpha}vß1, {alpha}vß3, and {alpha}vß5 (38, 39, 40) . Because of limited or deficient expression of CAR on hematopoietic cells, Ad transduction efficiency is reduced and virus entry into those cells is mainly mediated by {alpha}v integrins (30 , 39) . In our study, we observed that successful Ad transduction of A20 lymphoma cells required a very high MOI. However, susceptibility to Ad infection could be increased by treatment of A20 cells with ODN1018 but not a control ODN. When analyzed for the expression of cell surface receptors that have been reported to mediate Ad entry into target cells, we found that ODN1018 treatment induced expression of {alpha}v, ß1, and ß3 integrins. However, treatment of A20 cells did not induce expression of CAR or the {alpha}M integrin. The involvement of {alpha}v integrins in Ad entry in ISS-ODN-activated A20 cells could be demonstrated additionally by specifically blocking Ad infection with an anti-{alpha}v antibody. Preincubation of ODN1018-treated A20 cells with an anti-{alpha}v antibody significantly decreased the percentage of infected cells to levels seen after Ad-CD154 infection of untreated A20 cells.

Besides lowering the amount of Ad required to infect A20 cells, treatment of A20 with ISS-ODN also enhanced the expression of immune costimulatory molecules noted after infection with Ad-CD154. When ISS-activated cells were infected with Ad-CD154, we observed high-level expression of costimulatory and adhesion molecules on infected cells, as shown for CD80. Moreover, cells that first were treated with ISS-ODN and then infected with Ad-CD154 were significantly more activated, expressing at least 10 times more CD80 than cells incubated with ISS-ODN alone or that were infected with Ad-CD154 at an MOI of 1000. The enhanced expression of these costimulatory molecules probably is because of the capacity of ISS-ODN to enhance A20 expression of CD40. This apparently enables ODN1018-activated A20 cells to be better stimulated through the CD40-CD154 signaling pathway, allowing for the induction of high levels of immune costimulatory molecules (41) . This assumption is backed by the observation that coculture with cells transfected to express CD154 also induced significantly higher expression of CD80 on ODN1018-treated A20 cells than on untreated or ODN1019-treated A20 cells (data not shown).

The in vivo use of ISS-DNA in tumor immunotherapy has been described in several reports (33) , where ISS-ODN was given either systemically (20 , 21) or locally along with tumor antigen, mAbs, or irradiated tumor cells (22 , 24 , 25) . To our knowledge, the use of ISS-ODN-treated APCs as tumor vaccines has not been investigated in detail. In this study, we demonstrated an enhanced APC activity in vitro of ISS-ODN-treated, Ad-CD154-transduced, or both ISS-ODN-treated and Ad-CD154-transduced A20 cells. This in vitro activity corresponded to the observed enhanced capacity of ISS-activated and Ad-CD154-infected A20 cells to serve as cellular vaccines in vivo. We observed that all of the mice vaccinated with 106 ISS-activated and Ad-CD154-infected A20 cells were protected against a subsequent tumor challenge, and that 87.5% of mice receiving only Ad-CD154-transduced A20 cells remained tumor-free. Mice that received ISS-ODN-treated cells also resisted tumor challenge more than nonvaccinated animals, but only 25% of such animals remained tumor-free over a period of 19 weeks after tumor challenge. Nevertheless, tumor growth in such mice that developed tumors was significantly slower than that of tumors that developed in control mice, indicating that ISS-activated neoplastic B cells also could serve as effective tumor vaccines.

The antitumor protection observed with Ad-CD154-infected A20 cells cannot be attributed only to the induction of immune accessory molecules on A20 cells. Both Ad-CD154-infected or ISS-ODN-stimulated A20 cells had similar expression levels of immune costimulatory molecules such as CD80 and CD86. However, Ad-CD154-infected A20 cells were significantly more effective in stimulating a cellular immune response than ISS-ODN-treated cells. The difference between these two cell preparations apparently could be attributed to an "adjuvant effect" caused by concomitant immune stimulation against coexpressed Ad antigens by Ad-infected A20 cells. A20 cells treated with ISS-ODN and then infected with Ad-lacZ were not significantly more effective APCs in the MLR than A20 cells treated with ISS-ODN alone. Moreover, vaccination with Ad-lacZ-transduced A20 cells did not induce immune responses against A20 cells that were significantly greater than that observed after injection of noninfected A20 cells, as assessed via tumor challenge experiments and ELISPOT assays of vaccinated animals.

Conceivably, the down-regulation of CD40 in Ad-CD154-infected A20 cells and the up-regulation of CD40 in ISS-ODN-treated cells accounts in part for the differences noted in the relative immunogenicity of these two cell preparations. The relatively high levels of CD40 expressed by ISS-ODN-activated A20 cells could serve as a decoy receptor for CD154 expressed by activated helper T cells (42 , 43) , thereby competing with the CD40 expressed by other, more proficient APCs (e.g., dendritic cells) that might be present within the tumor cell microenvironment. Ad-CD154-infected A20 cells, on the other hand, can function like activated helper T cells because they express a functional ligand for CD40. This allows Ad-CD154-infected A20 cells instead to activate bystander A20 cells and neighboring CD40-expressing APCs, such as dendritic cells or monocytes. Neighboring activated dendritic cells could acquire tumor antigens from apoptotic tumor cells and induce an immediate CTL response by presenting these antigens directly to CD8+ T cells (1 , 44, 45, 46, 47) . This hypothesis is supported by data from Kikuchi and Crystal (3) , who demonstrated dendritic-cell activation by CD154-expressing tumor cells and CD8+ T-cell infiltration into tumors that had been injected with Ad-CD154. It also is supported by our finding that mice vaccinated with Ad-CD154-infected A20 cells have significantly higher proportions of IFN-{gamma} producing splenocytes, a key cytokine in a cytotoxic T-cell response that generates protective immunity (48) .

In conclusion, we demonstrate that treatment of A20 lymphoma cells with ISS-ODN not only enhances APC activity of A20 cells, but also increases their susceptibility to Ad infection. Transduction of such ISS-ODN-treated A20 cells with Ad-CD154 generated highly proficient APCs that could induce immunity to A20 tumors when administered as a tumor-cell vaccine. As such, ISS-ODN potentially could serve as an effective adjuvant for Ad-mediated CD154 gene therapy of hematologic malignancies.


   ACKNOWLEDGMENTS
 
We thank Dr. William G. Wierda for helpful discussions and suggestions and Dr. Eyal Raz for generously providing immunostimulatory oligonucleotides.


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

1 Supported in part by PO1-CA81534 for the Chronic Lymphocytic Leukemia Research Consortium (CRC), and by a grant from the Dana Foundation. Back

2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663. Phone: (858) 534-5417; Fax: (858) 534-5620; E-mail: tkipps{at}ucsd.edu Back

3 The abbreviations used are: APC, antigen-presenting cell; ISS, immunostimulatory DNA sequence; ODN, oligodeoxynucleotide; Ad, adenovirus; CLL, chronic lymphocytic leukemia; CAR, coxsackievirus-adenovirus-receptor; pur, purine; pyr, pyrimidine; MLR, mixed-lymphocyte reaction; ELISPOT, enzyme-linked immunospot assay; MFIR, mean fluorescence intensity ratio; mAb, monoclonal antibody; MOI, multiplicity of infection; UCSD, University of California, San Diego. Back

Received 3/12/03. Accepted 5/ 9/03.


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 RESULTS
 DISCUSSION
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