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* Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy; and
Department of Oncology and Neuroscience, G. D’Annunzio University of Chieti, Chieti, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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--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.).
Vector construction and retroviral infection
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).
Flow cytometry and mAb
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).
Proliferation assay
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 x 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).
In vivo tumorigenesis assays
Tumor growth and size were recorded twice each week in mice injected s.c. in the left flank with 5 x 104 control or transduced C26 cells in 0.2 ml or, as indicated, with 105 or 2 x 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 x 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.
Assay of cytokine production
Five-day supernatants from proliferation experiments were
harvested, and levels of IL-2 and IFN- 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.
Cell-mediated cytotoxicity assay
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 x 106 cells/mouse. After 5 days popliteal lymph nodes were removed, and lymphoid cells were suspended to 5 x 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 x 104 cell/ml) in mixed lymphocyte tumor culture. Effector cells were used in cell-mediated cytotoxicity assays with C26 cells as specific targets.
Immunohistochemical analysis
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-1
(clone B122; Genzyme,
Cambridge, MA), anti-TNF-
(clone MP6-XT22; Immuno Kontact,
Frankfurt, Germany), anti-IFN- (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
(x400 field; 0.180 mm2/field).
Vaccination with transduced C26 tumor cells
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 x 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
Statistical analysis was performed using Student’s t test (Microsoft Office).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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, 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- (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.
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Syngeneic BALB/c mice injected s.c. with 5 x
104 C26/OX40L cells displayed a delayed tumor
onset (Fig. 3A) and a reduced
tumor volume (Fig. 3B) 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. 3A). 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. 3A). 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 x
105 cells, the tumor regressed only in 45% of
mice (Fig. 3C). Thus, tumor burden is a major obstacle to
immune-mediated rejection.
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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. 4A). 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. 4B). Since CD40 engagement on
APC induces IL-12 production (28) and, in turn, IFN-
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. 4B). Thus, failure of
CD40KO mice to reject the tumor was not due to defective IL-12 or
IFN- production.
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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. 5A), 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. 5B), 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. 5C). These
results indicate that CD40-dependent T cell help is crucial in priming
CTL and in rejecting the C26/GM/OX40L tumor.
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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. 6k; data not shown). By contrast, only few
reactive cells infiltrated C26/GM/OX40L tumors from CD40KO mice and
OX40L expression was weak (Fig. 6l).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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.
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) (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. 5C 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-. 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).
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Acknowledgments |
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Footnotes |
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2 G.G. and E.G. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Mario P. Colombo, Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. E-mail address: mcolombo{at}istitutotumori.mi.it
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- knockout; IL-12p35KO, IL-12 p35 subunit knockout; OX40L, OX40 ligand; PMN, polymorphonuclear cells; TAA, tumor-associated Ag.
Received for publication May 1, 2002. Accepted for publication October 25, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes. Science 259:1739. in mediating the antitumor efficacy of interleukin-12. J. Immunother. Emphasis Tumor Immunol. 17:71.[Medline]
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  S. Piconese, B. Valzasina, and M. P. Colombo OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection J. Exp. Med., April 14, 2008; 205(4): 825 - 839. [Abstract] [Full Text] [PDF]
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 R. S. Kornbluth and G. W. Stone Immunostimulatory combinations: designing the next generation of vaccine adjuvants J. Leukoc. Biol., November 1, 2006; 80(5): 1084 - 1102. [Abstract] [Full Text] [PDF]
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 S. Murata, B. H. Ladle, P. S. Kim, E. R. Lutz, M. E. Wolpoe, S. E. Ivie, H. M. Smith, T. D. Armstrong, L. A. Emens, E. M. Jaffee, et al. OX40 Costimulation Synergizes with GM-CSF Whole-Cell Vaccination to Overcome Established CD8+ T Cell Tolerance to an Endogenous Tumor Antigen J. Immunol., January 15, 2006; 176(2): 974 - 983. [Abstract] [Full Text] [PDF]
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A. Song, X. Tang, K. M. Harms, and M. Croft OX40 and Bcl-xL Promote the Persistence of CD8 T Cells to Recall Tumor-Associated Antigen J. Immunol., September 15, 2005; 175(6): 3534 - 3541. [Abstract] [Full Text] [PDF]
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E. Biagi, G. Dotti, E. Yvon, E. Lee, M. Pule, S. Vigouroux, S. Gottschalk, U. Popat, R. Rousseau, and M. Brenner Molecular transfer of CD40 and OX40 ligands to leukemic human B cells induces expansion of autologous tumor-reactive cytotoxic T lymphocytes Blood, March 15, 2005; 105(6): 2436 - 2442. [Abstract] [Full Text] [PDF]
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A. D. Weinberg, D. E. Evans, C. Thalhofer, T. Shi, and R. A. Prell The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement J. Leukoc. Biol., June 1, 2004; 75(6): 962 - 972. [Abstract] [Full Text] [PDF]
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 S. Andarini, T. Kikuchi, M. Nukiwa, P. Pradono, T. Suzuki, S. Ohkouchi, A. Inoue, M. Maemondo, N. Ishii, Y. Saijo, et al. Adenovirus Vector-Mediated in Vivo Gene Transfer of OX40 Ligand to Tumor Cells Enhances Antitumor Immunity of Tumor-Bearing Hosts Cancer Res., May 1, 2004; 64(9): 3281 - 3287. [Abstract] [Full Text] [PDF]
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C. Massa, C. Guiducci, I. Arioli, M. Parenza, M. P. Colombo, and C. Melani Enhanced Efficacy of Tumor Cell Vaccines Transfected with Secretable hsp70 Cancer Res., February 15, 2004; 64(4): 1502 - 1508. [Abstract] [Full Text] [PDF]
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), 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 rejected in 85%
of mice, while at 2 x 105 (
) they were rejected
only in 42% of animals. The pooled results of two experiments are
shown (n = 14).
)
mice (A; n = 7), and groups of
CD40KO (
), IL-12p35KO (
