Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-γ or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

Cytokine and/or costimulatory gene transfer have been shown to enhance the immunogenicity of tumor cell-based cellular vaccines (1). Different strategies can be used to manipulate the antitumor immune response by selecting a particular cytokine and/or costimulatory gene. Our approach has encompassed the transfer of genes whose products might favor the interaction of the injected vaccine with host dendritic cells (DC)4 rather than directly with T lymphocytes. This strategy obviates the need for isolation of DC and exploits the continuous availability of precursors undergoing maturation and migration in vivo, which allows their encounter with Ag under physiological conditions. Indeed, we showed that DC can functionally bridge the interaction between T lymphocytes and C26 colon carcinoma cells cotransduced with both GM-CSF and CD40 ligand (CD40L) (2). DC isolated from mice injected with the GM-CSF and CD40L double-transduced tumor were shown to capture and present tumor-associated Ag (TAA) and to prime naive mice for CTL activation (2). Despite their activity in promoting T cell priming, C26/GM/CD40L cells used as a cellular vaccine to treat C26 lung metastases showed a cure rate (40%) comparable to that of our C26/IL-12 vaccine (50%) (our unpublished results and Ref. 3). Therefore, we turned to another costimulatory molecule, OX40 ligand (OX40L), which might enhance the curative effect by providing a sustained T cell boost. OX40L is a member of the TNF ligand superfamily and is expressed on activated professional APCs, including DC. CD40-activated, but not freshly isolated, DCs express OX40L in both mice (4) and humans (5). The receptor for OX40L, OX40, is expressed primarily on activated CD4+ T lymphocytes (reviewed in Ref. 6). The mRNAs for OX40L and OX40 are up-regulated in lymph nodes around the time of CD4+ T cells priming (7), and cross-linking of OX40 with OX40L amplifies T cell activation, proliferation, and cytokine production, including that of IL-2 and IFN-γ. Ligation of OX40 by OX40L also leads to the migration of CD4+ T cells from the T zone to B follicles of the lymph node where the germinative centers form (4). Ag stimulation plus OX40 engagement results in a marked inhibition of peripheral T cell deletion and increases Ag-specific T cell memory development by enhancing primary clonal expansion (8, 9) and, as recently reported, by inhibiting apoptosis through induction of Bcl-xL and Bcl-2 expression (10). Moreover, signaling through OX40 can break an existing state of tolerance in the CD4+ T cell compartment (11). In the pathogenesis of experimental autoimmune encephalomyelitis (EAE), effective OX40L-OX40 interaction requires both intact CD28 and CD40 signals (12), possibly through the ability of these molecules to up-regulate OX40 on T cells (13).

OX40+ T cells infiltrate a variety of human cancers such as melanoma, head and neck cancer, and mammary carcinoma (14, 15). In some murine tumor models, treatment with OX40L-Ig or anti-OX40 Ab early after tumor injection improved tumor-free survivorship rates due to stimulation and expansion of tumor-associated lymphocytes (9, 16). However, only s.c. tumors, not experimental metastases, were cured by treatment with anti-OX40 Ab (17).

To determine whether tumor-specific T cells might be activated by transducing C26 colon carcinoma cells with the OX40L gene (C26/OX40L), and whether cotransduction with granulocyte/monocyte colony-stimulating factor (GM-CSF) might favor priming by APC, which might be needed, since C26 is MHC class II-negative and OX40L acts mainly on CD4+T cells, we tested mice injected with C26/GM/OX40L cells for tumor development. Indeed, GM-CSF, either recombinant or released by tumor cells, exerts a paracrine effect at the injection site that recruits a mixed cellular infiltrate, including APCs, eosinophils, and T and B cells capable of recognizing tumor Ags (18, 19, 20, 21). C26/GM/OX40L tumors regressed in 85% of injected mice by means of CD4+, CD8+ T cells and APC-mediated CD40-CD40L cosignaling. C26/GM/OX40L cells cured 83% of mice with pre-established C26 lung metastases, underlining the potential therapeutic application of this tumor cell vaccine designed to improve APC recruitment and T cell priming as well as to boost the T cell response.

Female BALB/cn AnCr (H-2d) mice, 8–10 wk old, were purchased from Charles River Laboratories (Calco, Italy). BALB/c-Ifg (tm1 129) IFN-γ-deficient (GKO) (22) and IL-12p35-deficient (IL-12p35KO) (23) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained at the Istituto Nazionale Tumori under standard conditions according to institutional guidelines. CD40-deficient mice (CD40KO) (24) on a BALB/c background were provided by L. Adorini (Roche, Milan, Italy).

C26 murine colon adenocarcinoma cells were derived from BALB/c mice treated with N-nitroso-N-methylurethane (25). Cells were cultured in DMEM supplemented with 10% FCS (all from Life Technologies, Paisley, U.K.).

C26 cells expressing murine GM-CSF (C26/GM) were obtained in our laboratory as previously described (2). The cDNA for OX40L was cloned by RT-PCR from mouse splenocytes cultured in the presence of irradiated C26/GM/CD40L cells (2), which induce proliferation and activation of B220+ B lymphocytes (our unpublished observations). OX40L cDNA was amplified using specific primers containing 5′ and 3′ HpaI and BamHI sites, respectively, and the 614-bp insert was cloned into HpaI and BamHI of LXSH (26) to obtain vector mOX40LSH. Retroviral vectors were transfected into the amphotropic Am12 packaging cell line by standard calcium phosphate coprecipitation, and the 48-h culture supernatant was used to infect the ecotropic gp+E86 packaging cell line. Infected gp+E86 were selected with hygromycin and used to generate helper-free virus-containing supernatants. C26 and C26/GM target cells were infected by four cycles of exposure to undiluted supernatant for 2 h in the presence of polybrene (8 μg/ml). At 48 h after infection, cells were diluted and selected in hygromycin. Bulk cultures and single resistant colonies were expanded and screened by FACS analysis for OX40L expression (see below).

Expression of OX40L on transduced cell lines was assayed by flow cytometry after staining with biotin-conjugated anti-OX40L mAb (clone RM134L; BD PharMingen, San Diego, CA) followed by STREP-PE. Expression of OX40 on purified anti-CD3 activated CD4+ and CD8+ T cells was assayed with anti-OX40 rat IgG1 Ab (RDI-MCD134; BD PharMingen), followed by anti-rat FITC (BD PharMingen). Analysis was performed on a FACScan (BD Biosciences, Mountain View, CA). Data were collected on 5,000–10,000 viable cells and analyzed using Winmdi software (available at http://facs.scripps.edu/software.html).

T cells were purified from spleen after RBC lysis and nylon wool purification using CD4+ and CD8+ microbeads (Minimacs; Miltenyi Biotec, Calderara di Reno, Italy). Proliferation assays were performed in 200-μl reaction mixtures in 96-well, flat-bottom microplates (Corning, Corning, NY). CD4+ and CD8+ T cells (1 × 105 cells/well) were stimulated in wells coated with anti-CD3 (0.5 μg/well) and cultured in the presence of irradiated (15,000 rad) control or transduced C26 cells (104/well). Where indicated, anti-OX40L Ab or control IgG (50 μg/ml) were added to the culture. Cultures were incubated at 37°C in a humidified 5% CO2 atmosphere for 5 days. [3H]TdR (1 mCi/well: NEN, Boston, MA) was added to each well 16 h before harvest, and [3H]TdR incorporation was measured in a microplate scintillation counter (Tomtec; Wallac, Turku, Finland).

Tumor growth and size were recorded twice each week in mice injected s.c. in the left flank with 5 × 104 control or transduced C26 cells in 0.2 ml or, as indicated, with 105 or 2 × 105 cells. Tumor growth was expressed as percentage of tumor-free mice among total injected mice at the indicated time points, while tumor size was measured with a caliper and calculated as longest diameter × shortest diameter (2) (in cubic millimeters).

Mice whose C26/GM/OX40L tumor had regressed were challenged s.c. with wild-type tumor cells at 105 cells/0.2 ml/mice. For in vivo depletion of T lymphocytes, 0.2 mg/mouse of anti-CD4 (clone GK1.5; American Type Culture Collection, Rockville, MD) or anti-CD8 (clone 3.155; American Type Culture Collection) was injected i.p. at 48 h before tumor inoculation and once weekly thereafter, while 0.05 mg/mouse of anti-GR1 (clone RB6-8C5; American Type Culture Collection) was injected i.p. at 48 and 24 h before tumor inoculation and then twice weekly. These conditions achieved at least 95% depletion of the specific splenocytes subsets. Mice immunized with irradiated tumor cells were treated i.v. with rat IgG2a isotype-matched control or with an agonist mAb to CD40 (clone FGK45, a gift from V. Bronte, University of Padua, Italy; 0.1 mg/mouse) starting the day of immunization.

Five-day supernatants from proliferation experiments were harvested, and levels of IL-2 and IFN-γ were measured by standard ELISA (BD PharMingen). GM-CSF production by transduced cells was measured by ELISA (BD PharMingen) after 48-h incubation of 106 cells/ml.

Naive BALB/c or CD40KO mice were inoculated into the footpad with gamma-irradiated (15,000 rad) parental or transduced tumor cells at a dose of 1 × 106 cells/mouse. After 5 days popliteal lymph nodes were removed, and lymphoid cells were suspended to 5 × 105 cells/ml in RPMI 1640 medium supplemented with 10% FCS and restimulated in vitro for 5 days at 37°C in 5% CO2 in the presence of irradiated C26 tumor cells (5 × 104 cell/ml) in mixed lymphocyte tumor culture. Effector cells were used in cell-mediated cytotoxicity assays with C26 cells as specific targets.

Groups of three mice were euthanized at 3, 7, and 14 days after tumor inoculation. Acetone-fixed cryostat sections were immunostained with mAb against DEC205 (clone NLDC 145; Cedarlane, Hornby, Canada) or anti-CD11b/CD18 (clone M1/70.15), anti-OX40 (clone RDI-MCD134), anti-OX40L (clone RM134L) and anti-VCAM-1 (CD106, clone 429 MVCAM.A; all from BD PharMingen), or anti-CD8 (Ly/T2, clone YT5 169.4) and anti-CD4 (LT34, clone YT5.191.1.2; from Sera-lab, Crawley Down, U.K.) or anti-GR1 (clone RB6-8C5; American Type Culture Collection), anti-IL-1β (clone B122; Genzyme, Cambridge, MA), anti-TNF-α (clone MP6-XT22; Immuno Kontact, Frankfurt, Germany), anti-IFN-γ (clone XMG1.2; provided by Dr. S. Landolfo, University of Turin, Turin, Italy), anti-ICAM-1 (CD54, clone 3E2; Santa Cruz Biotechnology, Santa Cruz, CA), anti-ELAM-1 (CD62E, clone 10E9.6; provided by Dr. A. Vecchi, Negri Nord, Milan, Italy). After washing, sections were overlaid with biotinylated goat anti-rat, anti-hamster, and anti-rabbit and horse anti-goat Ig (Vector Laboratories, Burlingame, CA) for 30 min. Unbound Ig was removed by washing, and slides were incubated with ABC complex/AP (DAKO, Glostrup, Denmark). Quantitative studies of stained sections were performed independently by three pathologists in a blinded fashion. Expression of cytokines and adhesion molecules was scored as absent (−), low (±), moderate (+), or frequent (++). Cell counts were obtained in 10 randomly chosen fields under a microscope (×400 field; 0.180 mm2/field).

To induce lung metastases, mice were injected i.v. with 104 cells of the C26 tumor, clone 5A6, on day 0. Immunotherapy was started on day 1 and repeated on days 3, 8, and 11 by injecting s.c. 2 × 106 irradiated (15,000 rad) transduced C26 tumor cells. For survival experiments, mice were euthanized when they displayed respiratory symptoms; surviving mice were considered cured 2 mo after the end of treatment.

For counting lung metastases, mice were sacrificed 10 days after the last immunization (20 days after tumor injection). Lungs were insufflated with 15% India ink and bleached in Fekete solution, and metastases were counted after dissection of lung nodules as previously described (3).

Statistical analysis was performed using Student’s t test (Microsoft Office).

Parental C26 colon carcinoma and C26 cells transduced to express GM-CSF (C26/GM; 10–12 ng/ml from 106 cells in 48 h) were transduced with the retroviral vector containing the OX40L cDNA together with the selectable marker hygromycin. Colonies obtained after transduction were screened for expression of OX40L by FACS analysis (Fig. 1, A and B). Two colonies (C26/OX40L and C26/GM/OX40L) were selected to express similar amounts of OX40L and used for subsequent experiments.

FIGURE 1.

Expression of OX40L on transduced C26 tumor cells and of OX40 on activated CD4+ and CD8+ purified T cells. Cell surface expression of murine OX40L on selected C26/OX40L (A) and C26/GM/OX40L (B) cell colonies was determined by FACS analysis. The presence of OX40 was tested on 3-day anti-CD3-activated CD4+ (C) and CD8+ (D) purified T cells.

FIGURE 1.

Expression of OX40L on transduced C26 tumor cells and of OX40 on activated CD4+ and CD8+ purified T cells. Cell surface expression of murine OX40L on selected C26/OX40L (A) and C26/GM/OX40L (B) cell colonies was determined by FACS analysis. The presence of OX40 was tested on 3-day anti-CD3-activated CD4+ (C) and CD8+ (D) purified T cells.

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To test OX40L biological activity in transduced C26 cells, T cell proliferation, and cytokine production were measured as function of OX40 engagement on T cells (27). Purified naive CD4+ or CD8+ T cells were incubated with irradiated C26 or C26/OX40L tumor cells in the presence of anti-CD3, which is required to up-regulate OX40 on T cells (Fig. 1, C and D). C26/OX40L cells significantly increased the in vitro proliferation and IL-2 production of CD4+ T cells (Fig. 2, A and B; p < 0.05), while they had little effect on CD8+ T cell proliferation or on IFN-γ (Fig. 2, C and D) or IL-2 (data not shown) production. Addition of a neutralizing anti-OX40L Ab prevented CD4+ T cell proliferation and cytokine production, indicating the specificity of transduced OX40L-mediated effects.

FIGURE 2.

CD4+ and CD8+ T cell proliferation and cytokine production in the presence of C26 or C26/OX40L tumor cells. CD4+ and CD8+ T cells were stimulated with coated anti-CD3 in the presence of irradiated C26 or C26/OX40L cells, with or without anti-OX40L Ab or control IgG. CD4+ T cell proliferation (A) and IL-2 production (B) or CD8+ T cell proliferation (C) and IFN-γ production (D) were measured on day 5. Data are given as the mean (±SE) of four independent experiments. Basal proliferation of CD4+ T or CD8+ T cells incubated with C26 cells is 6320 or 3824 cpm, respectively. ∗, Significant difference between C26 and C26/OX40L tumors (p < 0.05).

FIGURE 2.

CD4+ and CD8+ T cell proliferation and cytokine production in the presence of C26 or C26/OX40L tumor cells. CD4+ and CD8+ T cells were stimulated with coated anti-CD3 in the presence of irradiated C26 or C26/OX40L cells, with or without anti-OX40L Ab or control IgG. CD4+ T cell proliferation (A) and IL-2 production (B) or CD8+ T cell proliferation (C) and IFN-γ production (D) were measured on day 5. Data are given as the mean (±SE) of four independent experiments. Basal proliferation of CD4+ T or CD8+ T cells incubated with C26 cells is 6320 or 3824 cpm, respectively. ∗, Significant difference between C26 and C26/OX40L tumors (p < 0.05).

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Syngeneic BALB/c mice injected s.c. with 5 × 104 C26/OX40L cells displayed a delayed tumor onset (Fig. 3,A) and a reduced tumor volume (Fig. 3,B) compared with mice injected with parental C26 cells (p < 0.01). In contrast, only 30% of mice injected with C26/GM/OX40L cells developed tumors, and half of the mice showed subsequent tumor regression (Fig. 3,A). Thus, the overall survival of mice injected with C26/GM/OX40L was 85%; all the mice rejected a subsequent challenge with live C26 cells, indicating the development of immunological memory (Fig. 3,A). When the dose of injected C26/GM/OX40L cells was increased to 105, the tumor grew in all mice and was still subsequently rejected in 85% of mice, whereas at 2 × 105 cells, the tumor regressed only in 45% of mice (Fig. 3 C). Thus, tumor burden is a major obstacle to immune-mediated rejection.

FIGURE 3.

In vivo tumorigenicity of C26 and transduced cell variants. A, Tumor cells (5 × 104) were injected s.c. into the left flank of female BALB/c mice. C26 (○), C26/OX40L (□), and C26/GM (•) tumors grew in all mice tested, whereas the C26/GM/OX40L tumor (▪) was rejected in 85% of mice. Cured mice remained tumor-free after challenge with 105 parental C26 cells injected in the right flank. The pooled results of five experiments (n = 35) are shown. B, Tumor volumes of C26/OX40L (□) were significantly smaller than parental C26 (○) tumors (∗, p < 0.01). C, C26/GM/OX40L cells injected at 5 × 104 (▪) and 105 (▿) were rejected in 85% of mice, while at 2 × 105 (▴) they were rejected only in 42% of animals. The pooled results of two experiments are shown (n = 14).

FIGURE 3.

In vivo tumorigenicity of C26 and transduced cell variants. A, Tumor cells (5 × 104) were injected s.c. into the left flank of female BALB/c mice. C26 (○), C26/OX40L (□), and C26/GM (•) tumors grew in all mice tested, whereas the C26/GM/OX40L tumor (▪) was rejected in 85% of mice. Cured mice remained tumor-free after challenge with 105 parental C26 cells injected in the right flank. The pooled results of five experiments (n = 35) are shown. B, Tumor volumes of C26/OX40L (□) were significantly smaller than parental C26 (○) tumors (∗, p < 0.01). C, C26/GM/OX40L cells injected at 5 × 104 (▪) and 105 (▿) were rejected in 85% of mice, while at 2 × 105 (▴) they were rejected only in 42% of animals. The pooled results of two experiments are shown (n = 14).

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To dissect the role of cells of the innate and acquired immune system and the effector mechanisms responsible for tumor rejection, leukocyte subset-depleted and gene-targeted mice were analyzed. C26/GM/OX40L cells injected into mice depleted of GR-1+, CD4+ or CD8+ cells formed tumors in all animals, with no late regression (Fig. 4,A). To test whether the requirement for the CD4+ T cell subset reflects the need for interaction between APC and CD4+ T cells (since C26 is MHC class II-negative), we evaluated C26/GM/OX40L tumor take in CD40KO mice, whose APC lack CD40; 100% of mice developed tumors, and no tumor rejection was detected (Fig. 4,B). Since CD40 engagement on APC induces IL-12 production (28) and, in turn, IFN-γ production, which is critical for tumor rejection (29), tumor take was evaluated in IL-12p35KO and GKO mice; both mouse strains rejected the C26/GM/OX40L tumor (Fig. 4 B). Thus, failure of CD40KO mice to reject the tumor was not due to defective IL-12 or IFN-γ production.

FIGURE 4.

C26/GM/OX40L tumorigenicity in leukocyte subset-depleted and gene-targeted mice. Groups of undepleted BALB/c control mice (▪) or CD4 (□), CD8 (○), GR-1-depleted (▵) mice (A; n = 7), and groups of CD40KO (⋄), IL-12p35KO (▴), and GKO (•) mice (B) were injected s.c. into the left flank with 5 × 104 C26/GM/OX40L cells (n = 14). Subset-depleted animals and CD40KO, but not IL-12p35KO or GKO, mice were unable to reject C26/GM/OX40L tumors.

FIGURE 4.

C26/GM/OX40L tumorigenicity in leukocyte subset-depleted and gene-targeted mice. Groups of undepleted BALB/c control mice (▪) or CD4 (□), CD8 (○), GR-1-depleted (▵) mice (A; n = 7), and groups of CD40KO (⋄), IL-12p35KO (▴), and GKO (•) mice (B) were injected s.c. into the left flank with 5 × 104 C26/GM/OX40L cells (n = 14). Subset-depleted animals and CD40KO, but not IL-12p35KO or GKO, mice were unable to reject C26/GM/OX40L tumors.

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To determine whether C26/GM/OX40L tumor growth observed in CD40KO mice might rest in defective T cell activation, the ability of transduced C26 cells to induce CTL activation was compared in BALB/c and CD40KO mice (Fig. 5). In BALB/c mice, C26/GM and C26/GM/OX40L cells each primed CTL more efficiently than C26 or C26/OX40L cells (Fig. 5,A), indicating that GM-CSF, but not OX40L, is important in the early phases of CTL induction. In CD40KO mice, C26/GM/OX40L cells induced weak or undetectable CTL activity (Fig. 5,B), possibly due to a defective CD4+ T cell-APC interaction (30, 31). Indeed, CD4+ T cell-depleted BALB/c mice immunized with irradiated C26/GM/OX40L cells and treated with agonistic mAb to CD40 showed CTL induction, whereas CD4+-depleted mice injected with an isotype-matched unrelated Ab did not (Fig. 5 C). These results indicate that CD40-dependent T cell help is crucial in priming CTL and in rejecting the C26/GM/OX40L tumor.

FIGURE 5.

Impaired CTL induction in the absence of the CD40 signaling pathway. A, Cytotoxicity of lymphocytes from popliteal lymph nodes of BALB/c mice primed with C26 (○), C26/GM (•), C26/OX40L (□), or C26/GM/OX40L (▪) against C26 target cells. B, CTL induction by C26/GM/OX40L cells in BALB/c (▪) and CD40KO (⋄) mice. C, CTL induction by C26/GM/OX40L cells in BALB/c mice (▪) or CD4-depleted BALB/c treated with the agonistic mAb to CD40 (▵) or with an isotype-matched irrelevant Ab (▴). Data are from one of two experiments with similar results.

FIGURE 5.

Impaired CTL induction in the absence of the CD40 signaling pathway. A, Cytotoxicity of lymphocytes from popliteal lymph nodes of BALB/c mice primed with C26 (○), C26/GM (•), C26/OX40L (□), or C26/GM/OX40L (▪) against C26 target cells. B, CTL induction by C26/GM/OX40L cells in BALB/c (▪) and CD40KO (⋄) mice. C, CTL induction by C26/GM/OX40L cells in BALB/c mice (▪) or CD4-depleted BALB/c treated with the agonistic mAb to CD40 (▵) or with an isotype-matched irrelevant Ab (▴). Data are from one of two experiments with similar results.

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Initial time-course analysis of C26 and its transduced variants indicated that the optimal time point for a wider comparative analysis among the tumors was day 7. At that time the number of infiltrating macrophages, PMNs, CD8+, CD4+, and DC (DEC205+) was significantly higher (p < 0.005) in mice injected with C26/GM/OX40L as compared with those injected with C26, C26/GM, or C26/OX40L tumor cells (Table I). The increased leukocyte infiltration was paralleled by increased expression of adhesion molecules and pro-inflammatory cytokines at the tumor site (Table I). More importantly, most of the CD4+ and CD8+ T cells infiltrating the C26/GM/OX40L tumors from BALB/c, but not CD40KO mice were OX40-positive (Fig. 6). This observation may explain the inability of the OX40L-expressing tumor to provide any costimulatory signal to infiltrating T cells (Table I and Fig. 6). Precise quantitation of OX40L+ tumor-infiltrating reactive cells is difficult, since the OX40L transduced into C26 cells accounts for the diffuse and homogeneous pattern of OX40L staining. Nevertheless, particularly at the edges of the C26/GM/OX40L and C26/OX40L tumors from BALB/c mice, frequent and moderate infiltration, respectively, by OX40L+ reactive cells was observed (Fig. 6,k; data not shown). By contrast, only few reactive cells infiltrated C26/GM/OX40L tumors from CD40KO mice and OX40L expression was weak (Fig. 6 l).

Table I.

Immunohistochemical analysis of tumor growth/rejection area 7 days after tumor cell challenge of mice

C26C26/OX40LC26/GMC26/GM/OX40L (in BALB/c)C26/GM/OX40L (in BALB/c CD40KO)
Reactive cellsa      
DEC 205 16 ± 3 23 ± 5b 13 ± 3b 34 ± 6b 16 ± 3c 
Mφ 15 ± 3 56 ± 9b 85 ± 11b 103 ± 15b 72 ± 9c 
PMN 7 ± 2 57 ± 10b 88 ± 12b 113 ± 17b 58 ± 7c 
CD8 3 ± 1 5 ± 2 11 ± 3b 19 ± 4b 4 ± 1c 
CD4 4 ± 1 4 ± 1 30 ± 5b 37 ± 6b 22 ± 4c 
Costimulatory molecules      
OX40 2 ± 1 5 ± 2b 11 ± 3b 49 ± 3b 0c 
Adhesion molecules      
VCAM − +d ± ± 
ELAM − − ± 
ICAM ± ± ± 
Cytokines      
IL-1β − ± ± − 
TNF-α − − ± ± ± 
IFN-γ − ± ± ++ − 
IL-4 − ± − ± 
C26C26/OX40LC26/GMC26/GM/OX40L (in BALB/c)C26/GM/OX40L (in BALB/c CD40KO)
Reactive cellsa      
DEC 205 16 ± 3 23 ± 5b 13 ± 3b 34 ± 6b 16 ± 3c 
Mφ 15 ± 3 56 ± 9b 85 ± 11b 103 ± 15b 72 ± 9c 
PMN 7 ± 2 57 ± 10b 88 ± 12b 113 ± 17b 58 ± 7c 
CD8 3 ± 1 5 ± 2 11 ± 3b 19 ± 4b 4 ± 1c 
CD4 4 ± 1 4 ± 1 30 ± 5b 37 ± 6b 22 ± 4c 
Costimulatory molecules      
OX40 2 ± 1 5 ± 2b 11 ± 3b 49 ± 3b 0c 
Adhesion molecules      
VCAM − +d ± ± 
ELAM − − ± 
ICAM ± ± ± 
Cytokines      
IL-1β − ± ± − 
TNF-α − − ± ± ± 
IFN-γ − ± ± ++ − 
IL-4 − ± − ± 
a

Cells were counted in 10 randomly chosen fields per sample at ×400 in a 0.180-mm2 field. Results are the mean ± SD number of positive cells per field evaluated on cryostat sections by immunohistochemistry.

b

Values significantly different (p ≤ 0.005) from corresponding values in C26, (b) C26/OX40L (c) and C26/GM (d) tumors.

c

Values significantly different (p ≤ 0.005) from corresponding values in C26/GM/OX40L tumors growing in BALB/c mice.

d

Expression of adhesion molecules and cytokines was defined as absent (−), low (+/−), moderate (+), or frequent (++).

FIGURE 6.

Immunohistochemical features of tumor growth/rejection area 7 days after tumor cell challenge in BALB/c or CD40KO mice. Few CD4+ (a), CD8+ (b), OX40+ T lymphocytes (g), and PMN (m) were detectable in C26 tumors growing in BALB/c mice. By contrast, CD4+ (b), CD8+ T lymphocytes (e), and PMN (n) heavily infiltrated the C26/GM/OX40L tumor rejection area in these mice. Furthermore, distinct expression of OX40 was observed in the majority of infiltrating T lymphocytes (h). In CD40KO mice, C26/GM/OX40L tumors were characterized by a less pronounced T lymphocyte (c and f) and PMN (o) infiltration associated with lack of OX40 (i) expression. Unlike the C26 parental tumor (j) in which OX40L was not expressed, a homogeneous and diffuse pattern of OX40L expression was clearly evident in C26/GM/OX40L tumors (k and l). However, in tumors from BALB/c (k), but not CD40KO (l) mice, OX40L expression was detected in several tumor-infiltrating reactive cells.

FIGURE 6.

Immunohistochemical features of tumor growth/rejection area 7 days after tumor cell challenge in BALB/c or CD40KO mice. Few CD4+ (a), CD8+ (b), OX40+ T lymphocytes (g), and PMN (m) were detectable in C26 tumors growing in BALB/c mice. By contrast, CD4+ (b), CD8+ T lymphocytes (e), and PMN (n) heavily infiltrated the C26/GM/OX40L tumor rejection area in these mice. Furthermore, distinct expression of OX40 was observed in the majority of infiltrating T lymphocytes (h). In CD40KO mice, C26/GM/OX40L tumors were characterized by a less pronounced T lymphocyte (c and f) and PMN (o) infiltration associated with lack of OX40 (i) expression. Unlike the C26 parental tumor (j) in which OX40L was not expressed, a homogeneous and diffuse pattern of OX40L expression was clearly evident in C26/GM/OX40L tumors (k and l). However, in tumors from BALB/c (k), but not CD40KO (l) mice, OX40L expression was detected in several tumor-infiltrating reactive cells.

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Mice were treated s.c. with irradiated C26/GM, C26/OX40L, C26/GM/OX40L, or nontransduced C26 cells on days 1, 3, 8, and 10 after i.v. injection of C26 cells and evaluated for survival. Vaccination with C26/GM/OX40L cells cured 83% of treated mice, while vaccination with parental C26, C26/GM, or C26/OX40L cured 0, 16, and 28% of mice, respectively (Fig. 7). The results clearly indicate the synergistic activity of GM-CSF and OX40L when cotransduced into a tumor cell vaccine.

FIGURE 7.

Survival of mice bearing C26 lung metastases after vaccination with transduced C26 tumor cells. Mice were injected i.v. with C26 cells on day 0 and vaccinated s.c. on days 1, 3, 8, and 10 with irradiated parental C26 cells (○) or C26 cells transduced with GM (•), OX40L (□), and GM and OX40L (▪) or were left untreated (▵). The pooled results of two experiments are shown (n = 14). The best therapeutic effect was obtained by vaccination with C26/GM/OX40L cells, which cured 83% of treated mice.

FIGURE 7.

Survival of mice bearing C26 lung metastases after vaccination with transduced C26 tumor cells. Mice were injected i.v. with C26 cells on day 0 and vaccinated s.c. on days 1, 3, 8, and 10 with irradiated parental C26 cells (○) or C26 cells transduced with GM (•), OX40L (□), and GM and OX40L (▪) or were left untreated (▵). The pooled results of two experiments are shown (n = 14). The best therapeutic effect was obtained by vaccination with C26/GM/OX40L cells, which cured 83% of treated mice.

Close modal

Additional experiments were performed to evaluate the number of lung metastases in mice vaccinated or not with C26/GM/OX40L cells. According to survival studies, vaccination eradicated metastases formation. Moreover, vaccination of CD8-depleted or CD40KO mice showed impaired vaccine efficacy, confirming that both CD8+ T cells and intact CD40 signaling are required for therapeutic effect (Fig. 8).

FIGURE 8.

Eradication of lung metastases by vaccination with C26/GM/OX40L cells requires CD8+ T cells and intact CD40 signaling. BALB/c mice depleted or not of CD8+ T cells and CD40KO mice were injected i.v. with C26 cells and vaccinates as indicated in Fig. 7. Mice were sacrificed 10 days after the last vaccination, and surface lung metastases were scored. •, A single mouse or mice with identical number of metastases. The solid lines represent the median of seven mice per experimental group.

FIGURE 8.

Eradication of lung metastases by vaccination with C26/GM/OX40L cells requires CD8+ T cells and intact CD40 signaling. BALB/c mice depleted or not of CD8+ T cells and CD40KO mice were injected i.v. with C26 cells and vaccinates as indicated in Fig. 7. Mice were sacrificed 10 days after the last vaccination, and surface lung metastases were scored. •, A single mouse or mice with identical number of metastases. The solid lines represent the median of seven mice per experimental group.

Close modal

Our functional and immunohistochemical data indicate that GM-CSF and OX40L coexpressed in C26 colon carcinoma cells induce a strong antitumor immune response, most likely through the activation of APC and interaction with CD4+ T cells, as summarized in Fig. 9. Both OX40+/CD4+ and OX40+/CD8+ T cells are generated for possible further interaction with the OX40L on transduced tumor cells. Moreover, our data provide evidence for a synergistic effect of these two molecules in the therapy of experimental lung metastases.

FIGURE 9.

Schematic representation of APC bridging the interaction between tumor cells, transduced with GM-CSF and OX40L, and T cells. GM-CSF is required to favor APC cross-priming of TAA, since tumor is MHC class II negative. A loaded APC can then interact with CD4+ T cells via both MHC/TCR and CD40/CD40L (and probably other costimulators not investigated here) for full CD4 activation, including up-regulation of OX40 and further APC licensing to constrict CD8+ T cells for CTL activity. Our data show that both CD4+ T cells and CD40-CD40L interaction are needed to find CTL activity and tumor rejection. Also they show that OX40L on tumor cells cannot bypass OX40L signaling by APC at least during the priming phase, while it probably boosts the effector phase.

FIGURE 9.

Schematic representation of APC bridging the interaction between tumor cells, transduced with GM-CSF and OX40L, and T cells. GM-CSF is required to favor APC cross-priming of TAA, since tumor is MHC class II negative. A loaded APC can then interact with CD4+ T cells via both MHC/TCR and CD40/CD40L (and probably other costimulators not investigated here) for full CD4 activation, including up-regulation of OX40 and further APC licensing to constrict CD8+ T cells for CTL activity. Our data show that both CD4+ T cells and CD40-CD40L interaction are needed to find CTL activity and tumor rejection. Also they show that OX40L on tumor cells cannot bypass OX40L signaling by APC at least during the priming phase, while it probably boosts the effector phase.

Close modal

In addition to CD8+ T and GR-1+ cells, which are the common effector mechanisms in this and other tumor models (32, 33), CD4+ T cells are required in the rejection of C26/GM/OX40L cells. In GM-CSF-secreting tumors, the priming phase has been shown to involve the recruitment of bone marrow-derived APC to present tumor Ags to both CD4+ and CD8+ T cells (34). Our extensive analysis of the C26 colon carcinoma model, including its transduction with several cytokine genes, points to a very restricted role for CD4+ T cells in tumor rejection, i.e., in GKO mice injected with IL-12-transduced C26 cells, the role of CD4+ T cells was linked to GM-CSF production, which, in turn, sustained the PMN and CD8+ T cell effector phase. Indeed, CD4+ T cell depletion led to a decline in PMN and CD8+ T cells number at the tumor site and abrogated in situ detection of GM-CSF (35). The presence of GM-CSF in combination with OX40L facilitates the priming via APC, a necessary step in activating CD4+ T cells, since C26 cells are MHC class II negative. OX40 is induced in both activated CD4+ and CD8+ T cells (Fig. 1) (36), but CD4+ T cells appear to be the most responsive to receptor engagement (Fig. 2) (37). In addition to providing regulatory signals required for CTL priming, CD4+ T cells might be required at the effector phase (34). We did not specifically investigate the role of CD4+ T at the effector phase, but it is possible that activated CD4+ T cells infiltrating the C26/GM/OX40L tumors are restimulated via OX40 by OX40L-transduced cells. As reported by others, this event appears to occur when agonist anti-OX40 mAb are used (15).

Although infiltrated by some OX40+ T cells, the C26/GM tumor was not rejected, probably due to the impaired expression of OX40L or other costimulatory molecules on APC as a consequence of tumor-immunosuppressive mechanisms (38). Indeed, immunohistochemical findings suggest that APC near the tumor site express little OX40L (data not shown). OX40L is physiologically up-regulated on APC only in highly inflammatory situations, such as infection with mouse mammary tumor virus or in EAE (39, 40), which might result from OX40-OX40L cosignaling as occurs in C26/GM/OX40L tumors. Indeed, we found a 5-fold increase in the number of CD4+ OX40+ T cells infiltrating the C26/GM/OX40L tumors compared with the C26/GM tumors (Table I). The pronounced expression of proinflammatory cytokines and endothelial adhesion molecules detected in the tumor rejection area (Table I) is consistent with this view.

OX40L activity is secondary to a successful priming phase as shown by the requirement for intact CD40-CD40L cosignaling in C26/GM/OX40L tumor rejection. Accordingly, progressive tumor growth (Fig. 4), undetectable CTL activity (Fig. 5), and lack of OX40 expression on T cells (Fig. 6) were observed in CD40KO mice injected with C26/GM/OX40L cells, probably due to defective APC-T cell cross-talk. Treatment with agonistic mAb to CD40 restored CTL activity and tumor rejection in CD4+ T cell-depleted BALB/c mice (Fig. 5,C and data not shown). The data indicate that APC and CD4+ T lymphocytes must functionally interact to activate CD40L expression on CD4+ T cells for CD40 cognate triggering on APC. T cell help for CTL induction is mediated by CD40-CD40L interaction (30, 31, 41). Mackey et al. (42) reported that CD40 expression on APC is required for their maturation and for generation of protective antitumor immunity and suggested that impaired antitumor response in the absence of CD40/CD40L interaction might result from a lesion in APC function, i.e., IL-12 production. However, rejection of the tumor in IL-12p35KO and GKO mice (Fig. 4) ruled out the possibility that C26/GM/OX40L tumor rejection depends on IL-12 or IL-12-induced IFN-γ. Consistent with this conclusion is the demonstration that APC-derived IL-12 is not required for CTL generation in vivo (43). Pardoll and collaborators (44) documented the existence of CD40-independent pathways of T cell help in CTL priming based on both CD40-independent DC sensitization and on direct lymphokine-dependent CD4+ and CD8+ T cell communication. In our system these pathways may be insufficient to activate T cells to a stage suitable for OX40 expression, considering the low immunogenicity of the dominant C26 TAA and the possible limiting dose of Ag in vivo (45).

In conclusion, the function of the tumor-associated OX40L molecules appears to depend on whether efficient priming activates T cells to express OX40 on their surface. The data obtained in CD40KO mice also suggest that OX40L expressed on tumor cells cannot substitute for OX40L on APC, whose expression is strictly CD40 dependent (4, 5, 46).

The synergistic activity of OX40L and GM-CSF was evidenced in a therapeutic setting where C26/GM/OX40L cured 83% of mice, compared with only 28 and 16% cured by C26/OX40L and C26/GM vaccination, respectively (Fig. 7). We previously showed that tumor cells transduced with GM-CSF and CD40L induce proliferation and maturation of hemopoietic cells, stimulate DC accessory properties, and enhance the antitumor immune response (2). However, those cells cured no >40% of mice with C26 lung metastases (data not shown). Thus, the approach used in the present study, which exploited a T cell costimulatory molecule other than B7 to boost the induced immune response, had a better therapeutic effect.

Recent adoptive immunotherapy experiments have shown that the coadministration of anti-OX40 Ab reduces the number of transferred T cells required to obtain remission of pulmonary metastasis and intracranial tumors (17). While adoptive immunotherapy might be useful in a prospective clinical setting as a therapy for advanced tumors, vaccination with GM-CSF- and OX40L-transduced tumors might be effective against minimal residual disease and in the control of tumor recurrences. Moreover, genetically modified tumor cell vaccines provide the advantage of containing the entire repertoire of potential tumor Ags, optimizing the probability of inducing an endogenous immune response against relevant TAA. In fact, although several tumor Ags have been identified, it is still not clear which of them are appropriate helper and target epitopes for inducing therapeutic antitumor immunity (47).

We are indebted to Dr. Andrew Weinberg for critically reading the manuscript and for his suggestions and encouragement. We also thank Mariella Parenza, Ivano Arioli, and Barbara Cappetti for technical help and care of the mice.

1

This work was supported by Associazione Italiana per la Ricerca sul Cancro, Instituto Superiore di Sanità-Italy-USA (T00.A15), and Consiglio Nazionale delle Ricerche Finalized Project on Biotechnology (PF49). G.G. was supported by an Italian Foundation for Cancer Research Fellowship.

4

Abbreviations used in this paper: DC, dendritic cell; CD40KO, CD40 knockout; CD40L, CD40 ligand; EAE, experimental autoimmune encephalomyelitis; TAA, tumor-associated Ag; GKO, IFN-γ knockout; IL-12p35KO, IL-12 p35 subunit knockout; OX40L, OX40 ligand; PMN, polymorphonuclear cells; TAA, tumor-associated Ag.

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