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Immunology |
Center for Surgery Research-FF50, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 [J. K., J. T., J. A. K., K. R., S. S.], and Providence Portland Medical Center, Earle A. Chiles Research Institute, Portland, Oregon 97213 [A. W.]
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ABSTRACT |
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INTRODUCTION |
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There are several known molecules that can provide costimulation (5 , 9) . The best characterized costimulatory signal is the one delivered to the T-cell CD28 receptor by its ligand B7.1 (CD80) or B7.2 (CD86). In many murine tumors, transduction of tumor cells to express B7.1 or B7.2 resulted in the loss of tumorigenicity (10, 11, 12) . Immunization with such modified tumor cells elicited protective immunity against challenges with wild-type tumors and in some cases, mediated regression of existing tumors (10) . In addition, several other membrane-bound receptor-ligand pairs can also serve to be costimulators for T-cell activation. In particular, members of the tumor necrosis factor receptor superfamily have been shown to share the ability to enhance or costimulate the process of T-cell activation (13, 14, 15, 16, 17) . This family consists of the CD30, CD40, CD27, Fas (CD95), DR3, 4–1BB, and OX-40. The 4–1BB receptor binds to a high-affinity ligand (4–1BBL) expressed on several APCs such as dendritic cells, macrophages, and activated B cells. Expression of 4–1BB is somewhat restricted to primed CD4, CD8 T cells, and natural killer cells (18) . Of particular significance is the observation that administration of 4–1BB mAb as a single agent could eradicate well-established tumors in mice (19) . Although both CD4 and CD8 participated in the antitumor immune responses, the stimulation of a CD8 CTL response was particularly striking. The CTL activity generated from 4–1BB mAb-treated mice was increased up to approximately 65 times compared with that of spleen cells from control animals. Thus, ligation of costimulation receptors in vivo may augment natural immunity to the growing tumors sufficient to induce their regression.
Until recently, the OX-40 receptor-ligand costimulation system has received relatively little attention for exploitation of it ability to enhance antitumor immunity because due to the fact that the receptor expression was reportedly confined to primed CD4 T cells only (20) . The OX-40R has a very distinct pattern of expression in animals with EAE. At the inflammatory site, it appears that T cells expressing the OX-40R are cells that recognized the autoantigen and were involved in the pathogenesis of EAE (21, 22, 23) . Additional analyses of tumor-infiltrating lymphocytes and tumor-draining lymph nodes from melanoma, breast cancer, and head and neck cancer patients identified the presence of OX-40R+ cells (24 , 25) . These findings along with our recent demonstration that tumor-specific CD4 T cells isolated from L-selectinelow cell population of tumor-draining lymph nodes mediated tumor regression without the participation of CD8 T cells (26) have inspired the inception of the hypothesis that in vivo ligation of the OX-40R on T cells may augment of antitumor immunity.
Recent work has tested the therapeutic efficacy of the OX-40L:immunoglobulin fusion protein as well as specific OX-40R mAb for the treatment of four antigenically and histologically distinct murine tumors (25) . In each tumor, treatment by ligation of OX-40R in vivo resulted in a significant improvement in survival of the tumor-bearing mice. The anti-OX-40R effects were dose-dependent and immunologically mediated. In the current study, we further analyzed the immunomodulatory function of the in vivo administered OX-40R mAb in mice bearing tumors of various immunogenicities as well as tumors inoculated at different anatomical sites. Our results suggest that the therapeutic responses may be predicated to the extent of OX-40R+ T lymphocytes infiltrating the growing tumor.
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MATERIALS AND METHODS |
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Tumors.
The MCA 203 and MCA 205 fibrosarcomas are 3-methylcholanthrene-induced
tumors of B6 origin (27)
. The tumors have been routinely
passed in vivo by serial s.c. transplantation in syngeneic
mice and were used within the fifth to the eight transplantation
generation. Single-cell suspensions were prepared from solid tumors by
digestion with a mixture of 0.1% collagenase, 0.01% DNase, and 2.5
units/ml hyaluronidase (Sigma, St. Louis, MO) for 3 h at room
temperature. The cells were filtered through a layer of no. 100 nylon
mesh, washed, and resuspended in HBSS. B16/D5 is a poorly immunogenic
subclone of the spontaneously arising B16/BL6 melanoma
(28)
. The B16/D5 tumor does not exhibit a detectable level
of MHC class I (H-2 Db and
Kb) and class II molecules. These tumor cells
were maintained in culture in CM. CM consisted of RPMI 1640
supplemented with 10% heat-inactivated FCS, 0.1
mM nonessential amino acids, 1
µM sodium pyruvate, 2 mM
fresh L-glutamine, 100 µg/ml streptomycin, 100
units/ml penicillin, 50 µg/ml gentamicin, and 0.5 µg/ml fungizone
(all from Life Technologies, Grand Island, NY), and 5 x 10-5 M
2-mercaptoethanol (Sigma). GL261 glioma, originally induced by
intracranial implantation of methylcholanthrene pellets in a B6 mouse,
was obtained from the Division of Cancer Treatment Repository
(Frederick, MD; 29
). The GL261 tumor cells was maintained
in continuous culture in CM. Cultured B16/D5 or GL261 tumor cells were
harvested after a short incubation at 37°C with a solution containing
0.25% trypsin and 0.02% EDTA (Life Technologies, Inc., Grand Island,
NY). The tumor cells were washed and resuspended in HBSS for animal
inoculation.
Tumor Inoculation.
B6 mice were given s.d. injections with 1 x 105 to 1.5 x 106 MCA 205 tumor cells suspended in 100 µl of
HBSS to initiate tumor growth. The diameters of s.d. tumors were
measured twice weekly with a Vernier caliper, and size was recorded as
an average of perpendicular measurements and presented as individual
tumor growth curves. Mice were killed when the dermal tumor reached a
size greater than 20 mm. To establish intracranial tumors, B6 mice were
anesthetized with 0.8 mg of pentobarbital i.p. and inoculated with 10
µl of tumor cell suspension transcranially using a 27-gauge needle
and glass tuberculin syringe (Perfectum; Popper & Sons, Inc, New Hyde
Park, NY). The needle insertion was perpendicular to the skull and in
line with the anterior margin of the ear and the medial half of the
right eye. The depth of insertion was controlled by placement of
electric wire insulation as a collar over the needle with exposure of
the terminal 4 mm. To establish pulmonary metastases, mice were given
i.v. injections with 1 x 105 MCA
205 tumor cells suspended in 1 ml of HBSS. On day 21, mice were killed,
and metastatic tumor nodules on the surface of the lung were enumerated
after counterstaining with India ink.
mAb to OX-40R and Treatment Protocol.
Hybridoma that produced the antimurine OX-40R-specific mAb (termed
OX-86) was obtained from the European Collection of Animal Cell
Cultures (30)
. The hybridoma was grown in RPMI
medium containing 10% FCS that was depleted of immunoglobulin
by passing it over protein G columns. The cells were grown to high
density, and the supernatant was poured over a protein G column, and
purified antibody was eluted. Antibody concentrations were quantitated
by absorption at 280 nm. In most experiments, tumor-bearing mice were
treated with the mAb on days 3 and 7 after tumor inoculation by i.p.
injections. Doses of mAb are indicated in the figure legends.
Depletion of T-Cell Subsets in Vivo.
Ascites for the rat IgG2b mAb against murine CD4 (GK1.5, L3T4) and CD8
(2.43, Lyt 2.2) was produced in sublethally irradiated (500 R),
cyclophosphamide (2 mg/mouse)-treated DBA/2 mice. Mice with
3-day established intracranial GL261 tumors were depleted of T-cell
subsets by i.v. injection of 0.2 ml of ascites fluid diluted to 1.0 ml
with HBSS prior to OX-40R mAb treatment. T cell depletion was confirmed
by flow cytometric analyses of spleen cells from treated animals.
Isolation and Characterization of T Cells from Intracranial
Tumors and Lymphoid Organs.
Anesthetized B6 mice with 10-day established GL261 or B16/D5
intracranial tumors were perfused through the left ventricle of the
heart with 3 ml of HBSS before removal of the brain. Single-cell
suspensions were prepared by digesting minced brain tissue in 40 ml of
HBSS containing 10 mg collagenase (type IV, Sigma) for 60 min at room
temperature. The cell suspensions were washed in HBSS, resuspended in
10 ml of 50% Percoll (Pharmacia, Uppsala, Sweden), and then overlaid
with 4 ml of 30% Percoll to form a discontinuous gradient in a 15-ml
centrifuge tube. The gradient was centrifuged at 4°C for 40 min at
1000 x g. Cells recovered from the
interphase were washed in HBSS before flow cytometric analysis.
Single-cell suspensions from lymph nodes or spleens were
prepared mechanically by teasing organs with needles followed by
pressing tissue fragments with the blunt end of a plastic syringe.
Cells were stained by indirect immunofluorescence for the expression of
OX-40R using OX40L:immunoglobulin fusion protein and FITC-conjugated
antihuman IgG (Caltag, South San Francisco, CA). Cells were also
stained with PE-conjugated anti-CD4 or CD8. The membrane
fluorescence was analyzed using FACSCalibur (Becton Dickinson,
Sunnyvale, CA).
Statistical Analysis.
The significance of differences in numbers of pulmonary metastases
between groups was analyzed by the Wilcoxon rank-sum test. Differences
of numbers of cells infiltrating tumor tissues were analyzed by the
Student’s t test. A two-tailed P of 
0.05 was considered significant.
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depicts the results in which mice inoculated with 1 x 105 (Fig. 1
, Expt. 1) or
3 x 105 (Fig. 1
, Expt.
2) MCA 205 tumor cells responded to the treatment resulting
in complete tumor eradication in 3 of 5 animals. In mice inoculated
with 1.5 x 106 tumor cells (Fig. 1
, Expt. 3), treatment with OX-40R mAb resulted in some
retardation and delay of tumor growth, but all eventually succumbed to
the progressive tumors. In all of the tumor doses, inoculation led to
progressive s.d. tumor growth in control mice. These results. thus,
indicate that tumor burden may be a limiting factor to the therapeutic
response of the OX-40R mAb treatment.
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. Treatment with higher antibody doses, either by four
administrations of 150 µg on days 3, 7, 11, and 15 or by two
administrations of 300 µg on days 3 and 7, demonstrated a higher
therapeutic efficacy resulting in complete eradication of tumors in
50% of the treated mice. Similarly, for the treatment of MCA 203
sarcoma and GL261 glioma, i.p. injection of mice with 300 µg of
OX-40R mAb on days 3 and 7 prolonged survival, and 3 and 4 of 6 treated
animals were cured of tumors, respectively (Fig. 2, B and C)
. Increasing the dose of GL261 tumor cells in the tumor
inoculum resulted in decreased therapeutic effects of the antibody
treatment (Fig. 2D)
. In an additional experiment, mice
inoculated intracerebrally with 105 MCA 205 tumor
cells were treated with two injections of 300 µg of OX-40R mAb 4 days
apart commencing on day 3, 5, or 7. Whereas 2 of 5 mice treated
beginning on day 3 were cured, all of the mice succumbed to the
progressive growing tumors when treatment was delayed (data not shown).
These results thus confirm that the tumor burden is a limiting factor
as seen in previously experiments (Fig. 1)
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A similar strategy was used to treat pulmonary metastases derived from
the MCA 205 sarcoma. Mice were injected i.v. with 1 x 105 tumor cells suspended in 1.0 ml of HBSS to
establish multiple metastases in the lung. Such a model system has been
used extensively in the past for evaluating therapeutic efficacy of
both active and adoptive immunotherapies. Mice bearing pulmonary
metastases were treated exactly as described above with two i.p.
injections of 300 µg of OX-40R mAb on days 3 and 7. On day 21, all of
the mice were killed and metastatic nodules on the surface of the lung
were estimated. Despite its effectiveness for the treatment of s.d. and
intracranial MCA 205 tumors, the antibody failed to effect tumor growth
as judged by the numbers of metastatic nodules in treated mice as
compared with controls (Fig. 2F)
.
Cumulatively, this series of experiments confirms the therapeutic effectiveness of OX-40R ligation in the induction of tumor eradication as claimed previously (25) . The successful treatment is mAb dose-dependent and effected by the intrinsic immunogenicity of tumors. It is also evident that the response of a particular tumor to the treatment varies and is dependent on the histological location of tumor growth.
Specificity and Long-Term Immunity after Successful Treatment.
To determine whether a long-lasting immunity was induced in mice that
were cured by the OX-40R mAb treatment, we challenged survival animals,
60 days after initial tumor inoculation, with either the same tumor or
an antigenically distinct tumor. All of the mice cured of MCA 205
primary s.d. tumors (see Fig. 1
) resisted a s.d. rechallenge with
3 x 105 MCA 205 tumor cells,
whereas control, naive mice showed progressive tumor growth after tumor
challenge (Fig. 3A)
. Similarly, mice that survived the intracranial MCA 205
tumors were resistant to a second intracranial challenge (1 x 105) with the same tumor (Fig. 3B)
. We also tested the specificity of this long-term
immunity by rechallenging the mice that survived the intracranial GL261
tumor as a consequence of OX-40R mAb treatment with the antigenically
distinct MCA 205 tumor. As can be seen in Fig. 3C
, the
growth of MCA 205 intracranial tumors has not been affected by the
initial rejection of the GL261 tumor. These results indicate that the
tumor eradication triggered by OX-40R mAb established a long-lasting
tumor-specific immunological memory.
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20% in untreated mice to <2% of CD4 or
CD8 T cells in the treated mice. In the experiment depicted in Fig. 4
, mice treated with OX-40R mAb without T-cell depletion showed
prolongation of survival and three of six mice were cured of the tumor.
In animals depleted of either CD4 or CD8 T cells, the antitumor effects
of the OX-40R mAb were abrogated. Although OX-40R has been primarily
expressed on activated CD4 T cells, and the GL261 glioma does not
express MHC class II molecules, our results suggest that tumor
regression induced by the OX-40R mAb treatment required the
participation of both CD4 and CD8 host T cells.
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). One possible mechanism responsible for the tumor
susceptibility is the abundance of tumor-sensitized precursor T cells
during the growth of immunogenic tumors. We, therefore, attempted to
define the OX-40R expression on tumor-associated T cells isolated from
10-day-old tumors of both the susceptible immunogenic GL261 glioma and
the resistant poorly immunogenic B16/D5 melanoma. Because successful
OX-40R mAb treatment resulted in tumor-specific systemic immunity, we
also examined the expression of OX-40R on splenic T cells from
tumor-bearing mice. Quantitatively, in the intracranial GL261 tumor,
there were 25% tumor-associated T cells, which is 6-fold more T
cells than could be detected in the B16/D5 tumor (4.5%, Table 1
). It is noteworthy that among tumor-infiltrating T cells, there were
26 ± 3% of the GL261 tumor-associated CD4 cells
expressing OX-40R, whereas only 11 ± 3% of
OX-40R+ CD4 cells were detected in the B16/D5
tumor (Fig. 5)
. Unexpectedly, 11 ± 3% and 4 ± 1% of
CD8 cells isolated from GL261 and B16/D5 tumors were
OX-40R+, respectively. Analysis of OX-40R
expression on splenic T cells of GL261-bearing mice revealed that
15% of CD4 cells expressed OX-40R, whereas only 7% of CD4 T
cells from B16/D5-bearing mice were OX-40R+. In
both tumor systems, no OX-40R+ CD8 T cells were
detected in the spleen (Fig. 5)
or lymph nodes (data not shown). It is
possible that OX-40R+ T cells in the tumors are
the candidate for targeting by the mAb. The observations that the
therapeutic efficacy is dependent on both the CD4 and CD8 T cells and
the immunogenicity of the treated tumor, support this possibility.
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DISCUSSION |
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The results of current studies have extended previous findings that in vivo engagement of OX-40R by administration of OX-40L:immunoglobulin fusion proteins or OX-40R mAb resulted in significant therapeutic benefits in four histologically and immunologically distinct murine tumors (25) . Whereas our results emphasized the critical role of the immunogenicity of tumors to the effects of OX-40R mAb treatment, the histological location of tumor growth was also a determining factor affecting the outcome of treatment. It is clear that s.d. and intracranial tumors were susceptible to the antibody treatment in a dose-dependent manner. Established pulmonary metastases from the same tumor seemed to be refractory to the effects of mAb. To explain the difficulty in treating metastases in the lung, it is possible that micrometastases may not facilitate sufficient intratumoral infiltration of activated T cells for mAb targeting as compared with solitary s.d. and intracranial tumors. This is supported by our recent observation, in which therapy with the adoptive transfer of tumor-specific T cells in conjunction with OX-40R mAb administration significantly accelerated the antitumor immunity against 10-day established pulmonary metastases.4 It is, therefore, possible that advanced pulmonary metastases might be more susceptible to the treatment effects of mAb. This hypothesis is being tested experimentally.
The responsiveness of intracranial tumors to the systemic OX-40R mAb
treatment is intriguing because the brain is considered to be an
immunologically privileged site because of the existence of the BBB. It
is also however, possible that the growing tumor in the brain disrupts
the integrity of the BBB, which allows the entry of antibodies. Because
the OX-40R mAb does not directly interact with tumor cells, and its
function is targeting the activated T cells by ligation of OX-40R on
their surface (23)
, an initial immune response must have
occurred to generate activated T cells that express the OX-40R.
Although inflammatory cells are rare in the normal brain, increased
leukocyte entry occurs in pathological conditions. The physical BBB
using tight endothelial junctions and glial end-feed, blocks passive
entry of large molecules but does not stop the active entry of living
cells (37
, 38)
. Furthermore, in the normal brain, MHC
expression is minimal. However, both MHC class I and II expression can
be up-regulated on microglia and endothelial cells under the influence
of IFN-
and other cytokines (39
, 40)
. These cells may
serve the function of APCs; thus, the growing brain tumors may have
created a microenvironment conducive to initiating an immune response.
Indeed, not only have we observed the expression of OX-40R on the
tumor-associated or tumor-infiltrating lymphocytes but the total number
of cells and percentages of OX-40R positive cells seems to predict
their responses to the OX-40R mAb treatment.
We are aware that the intracerebral route of injection may have damaged the BBB, perhaps permitting local entry of lymphocytes and the mAb. However, this is unlikely to occur solely after a local injury, because there is a lack of inflammatory response in the brain after intracerebral injection of sterile PBS (41) . The progressive growth of a tumor, on the other hand, may disrupt the integrity of the BBB even in naturally occurring tumors. It has been reported that the primary human gliomas are often infiltrated with lymphocytes, and in the peripheral circulation, there exists lymphocytes specifically reactive to glioma cells (42 , 43) . In fact, survival of the glioma patients correlates with the degree of lymphocytic infiltration in the tumor. Nevertheless, we cannot entirely exclude minor injury as a contributing factor in the success of antibody-mediated tumor regression.
The finding that the poorly immunogenic B16/D5 melanoma was not
responsive to the therapeutic effects of OX-40R mAb has several
explanations. It is possible that B16/D5 tumor cells either lack
molecules that can serve sufficiently as tumor antigens recognized by T
cells or are deficient in the processing, transportation or
presentation of such molecules by APCs. It is, relevant therefore, to
note that MHC molecules (both class I and II) are not detectable on
B16/D5 cells. In many poorly immunogenic tumors, increasing MHC class I
expression by transfection with MHC class I genes or IFN- cDNA
resulted in an enhanced sensitivity to CTL lysis in vitro
and an increased infiltration of tumor by CD8 lymphocytes in
vivo (10
, 44)
. Furthermore, B16 melanoma cells
transduced with a gene encoding granulocyte-macrophage
colony-stimulating factor could elicit a protective immunity against
challenges with wild-type tumor cells (45)
. This finding
may be explained by the production of cytokines and/or enhanced
expression of MHC and costimulatory molecules induced by
granulocyte-macrophage colony-stimulating factor. Taken together, the
failure to treat the B16/D5 melanoma may be primarily attributable to
insufficient triggering of the initial immune response. If this is the
case, transduction of tumor cells to express MHC molecules or IFN-
may provide a means of increasing responsiveness to the OX-40R mAb
treatment against poorly immunogenic tumors.
As discussed earlier, the administration of 4–1BB mAb eradicates established tumors in mice (19) . Analysis of mechanisms of tumor rejection revealed the participation of both CD4 and CD8 in tumor-bearing mice. Because the 4–1BB glycoprotein is expressed on both of the activated CD4 and CD8 T cells (18) , it is logical to hypothesize that the antibody amplifies both CD4 and CD8 immune responses. In contrast to the 4–1BB, the OX-40R has been repeatedly demonstrated to be preferentially expressed by activated CD4 T cells (20 , 23 , 24) . Our recent studies have demonstrated that appropriately sensitized and activated CD4 T cells alone could mediate potent antitumor responses when adoptively transferred to tumor-bearing mice (26) . Because of these findings, we hypothesized a restricted role of OX-40R mAb to be amplifying CD4 T cell-mediated antitumor reactivity. However, T cell phenotype analysis indicated that both CD4 and CD8 T cells were required for effective treatment with the OX-40R mAb. It was first interpreted that OX-40R signaling on CD4 T cells provided an increased T-helper function, which in turn facilitated a CD8 CTL response. To support this hypothesis is the observation that the adoptive transfer of CD8-depleted spleen cells from OX-40L:immunoglobulin cured mice conferred resistance to tumor challenge in naive mice (25) . Because of the demonstration of OX-40R+ CD8 T cells in tumor-infiltrating lymphocytes, it is also possible that the mAb directly binds to CD8 T cells and activates them. CD8 T cells have the potential to express the OX-40R after stimulation with potent mitogens such as ConA and PHA (46) .
The mechanism by which administered OX-40R mAb may assist the
generation of antitumor responses or enhance existing antitumor
immunity is poorly understood. It has previously been shown that
sensitized CD4 T cells exposed to Ag and cocultured with B7.1/OX-40L
expressing fibroblasts, demonstrate enhanced proliferation, IL-2
secretion, and prolonged survival (47)
. Thus, one
possibility is that the tumor-infiltrating T lymphocytes, sensitized
in vivo to tumor antigens, were at a stage of
differentiation that permitted proliferation and activation by the
OX-40R mAb ligation. Because most antigen-sensitized T cells become
susceptible to activation-induced cell death, and only a minority
differentiate to become memory T cells (48)
, ligation of
the OX-40R on antigen-activated T cells may abrogate or delay the
impact of activation-induced cell death leading to increased
Ag-specific memory (49)
. In addition, OX-40R mAb treatment
may shift the balance between Th1 and Th2 immune responses. Blocking of
OX-40R signaling has resulted in reducing in vivo transcript
for Th1 cytokines such as tumor necrosis factor-
, IFN-, IL-2 and
IL-12 in animals with EAE and bowel disease (34
, 35)
.
However, in studies with naive CD4 T cells, in vitro
activation by the OX-40L resulted in promoting Th2 cell development
(50
, 51)
. Shifting of type 1/type 2 T cell functions as a
possible mechanism of OX-40R mAb-mediated tumor regression remains to
be determined.
In summary, ligation of OX-40R in vivo with antibodies can lead to delaying of tumor progression and, in some cases, eradicating tumors. The demonstration of OX-40R+ T cells in tumor-infiltrating lymphocytes suggests that they are the targets of antibody binding and activation. Therapeutic efficacy of the antibody, however, seemed to be limited by the tumor burden. Recently, it has been demonstrated that immune responses are regulated, at least in part, by naturally occurring CD4+, CD25+ T cells in normal animal (52) . In some animal tumor model systems, depletion of this population of regulatory cells resulted in enhanced immune responses and tumor eradication (53 , 54) . Because the therapeutic effect of the OX-40R mAb is indirect through the induction of antitumor immune responses, it may be possible to improve the therapeutic efficacy by the removal of CD4+, CD25+ T cells. This hypothesis is being tested in our laboratory. Finally, the significance of this approach is underscored by the fact that similar OX-40R+ T cells have been demonstrated in several human malignancies including melanoma, head and neck carcinoma, and carcinoma of the breast (24 , 25) . If these cells prove to be tumor-sensitized T lymphocytes, ligation of OX-40R mAb may provide tumor Ag-specific therapeutic benefits in cancer patients.
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FOOTNOTES |
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1 Supported in part by USPHS Grants RO1-CA81383,
RO1-CA78263, and R37-CA67324 from the National Cancer Institute, and by
National Cancer Institute Grant R01-CA81383–01 and Cantab
Pharmaceuticals grant (to A. W.). 
2 To whom requests for reprints should be
addressed, at Center for Surgery Research-FF50, The Cleveland Clinic
Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216)
445-3800; Fax: (216) 445-3805; E-mail: shus{at}ccf.org
3 The abbreviations used are: APC,
antigen-presenting cell; mAb, mononuclear antibody; EAE, experimental
autoimmune encephalomyelitis; OX-40R, OX-40 receptor; OX-40L, OX-40
ligand; CM, complete medium; s.d., subdermal/subdermally; BBB,
blood-brain barrier; IL, interleukin.
Received 3/24/00. Accepted 8/ 3/00.
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) monoclonal antibody. Cancer Res., 59: 3128-3133, 1999.This article has been cited by other articles:
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 D. Hirschhorn-Cymerman, G. A. Rizzuto, T. Merghoub, A. D. Cohen, F. Avogadri, A. M. Lesokhin, A. D. Weinberg, J. D. Wolchok, and A. N. Houghton OX40 engagement and chemotherapy combination provides potent antitumor immunity with concomitant regulatory T cell apoptosis J. Exp. Med., May 11, 2009; 206(5): 1103 - 1116. [Abstract] [Full Text] [PDF]
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W. L. Redmond, M. J. Gough, B. Charbonneau, T. L. Ratliff, and A. D. Weinberg Defects in the Acquisition of CD8 T Cell Effector Function after Priming with Tumor or Soluble Antigen Can Be Overcome by the Addition of an OX40 Agonist J. Immunol., December 1, 2007; 179(11): 7244 - 7253. [Abstract] [Full Text] [PDF]
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S.-W. Lee, Y. Park, A. Song, H. Cheroutre, B. S. Kwon, and M. Croft Functional Dichotomy between OX40 and 4-1BB in Modulating Effector CD8 T Cell Responses J. Immunol., October 1, 2006; 177(7): 4464 - 4472. [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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M. Gerloni, P. Castiglioni, and M. Zanetti The Cooperation between Two CD4 T Cells Induces Tumor Protective Immunity in MUC.1 Transgenic Mice J. Immunol., November 15, 2005; 175(10): 6551 - 6559. [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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J. Dannull, S. Nair, Z. Su, D. Boczkowski, C. DeBeck, B. Yang, E. Gilboa, and J. Vieweg Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand Blood, April 15, 2005; 105(8): 3206 - 3213. [Abstract] [Full Text] [PDF]
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J. Lustgarten, A. L. Dominguez, and M. Thoman Aged Mice Develop Protective Antitumor Immune Responses with Appropriate Costimulation J. Immunol., October 1, 2004; 173(7): 4510 - 4515. [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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 A. Ladanyi, B. Somlai, K. Gilde, Z. Fejos, I. Gaudi, and J. Timar T-Cell Activation Marker Expression on Tumor-Infiltrating Lymphocytes As Prognostic Factor in Cutaneous Malignant Melanoma Clin. Cancer Res., January 15, 2004; 10(2): 521 - 530. [Abstract] [Full Text] [PDF]
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X. Yuan, A. D. Salama, V. Dong, I. Schmitt, N. Najafian, A. Chandraker, H. Akiba, H. Yagita, and M. H. Sayegh The Role of the CD134-CD134 Ligand Costimulatory Pathway in Alloimmune Responses In Vivo J. Immunol., March 15, 2003; 170(6): 2949 - 2955. [Abstract] [Full Text] [PDF]
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G. Gri, E. Gallo, E. Di Carlo, P. Musiani, and M. P. Colombo OX40 Ligand-Transduced Tumor Cell Vaccine Synergizes with GM-CSF and Requires CD40-Apc Signaling to Boost the Host T Cell Antitumor Response J. Immunol., January 1, 2003; 170(1): 99 - 106. [Abstract] [Full Text] [PDF]
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T. De Smedt, J. Smith, P. Baum, W. Fanslow, E. Butz, and C. Maliszewski Ox40 Costimulation Enhances the Development of T Cell Responses Induced by Dendritic Cells In Vivo J. Immunol., January 15, 2002; 168(2): 661 - 670. [Abstract] [Full Text] [PDF]
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D. E. Evans, R. A. Prell, C. J. Thalhofer, A. A. Hurwitz, and A. D. Weinberg Engagement of OX40 Enhances Antigen-Specific CD4+ T Cell Mobilization/Memory Development and Humoral Immunity: Comparison of {alpha}OX-40 with {alpha}CTLA-4 J. Immunol., December 15, 2001; 167(12): 6804 - 6811. [Abstract] [Full Text] [PDF]
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J. Kjaergaard, L. Peng, P. A. Cohen, J. A. Drazba, A. D. Weinberg, and S. Shu Augmentation Versus Inhibition: Effects of Conjunctional OX-40 Receptor Monoclonal Antibody and IL-2 Treatment on Adoptive Immunotherapy of Advanced Tumor J. Immunol., December 1, 2001; 167(11): 6669 - 6677. [Abstract] [Full Text] [PDF]
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A. Kotani, T. Ishikawa, Y. Matsumura, T. Ichinohe, H. Ohno, T. Hori, and T. Uchiyama Correlation of peripheral blood OX40+(CD134+) T cells with chronic graft-versus-host disease in patients who underwent allogeneic hematopoietic stem cell transplantation Blood, November 15, 2001; 98(10): 3162 - 3164. [Abstract] [Full Text] [PDF]
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J. A. Kim, B. J. Averbook, K. Chambers, K. Rothchild, J. Kjaergaard, R. Papay, and S. Shu Divergent Effects of 4-1BB Antibodies on Antitumor Immunity and on Tumor-reactive T-Cell Generation Cancer Res., March 1, 2001; 61(5): 2031 - 2037. [Abstract] [Full Text]
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), rat IgG (
, 300
µg/injection on days 3 and 7), or OX-40R mAb (
, 150 µg/injection
on days 3, 7, and 11; 
, 150 µg/injection on days 3, 7, 11, and 15;
, 300 µg/injection on days 3 and 7). In B, B6 mice
in groups of six were inoculated i.c. with 1 x 105 MCA 203 tumor cells. On days 3 and 7, mice were treated
by i.p. injections of HBSS, rat IgG (300 µg/injection), or OX-40R mAb
(300 µg/injection). In C and D, B6 mice
in groups of six were inoculated i.c. with 1 x 105 (C) or 2 x 105 (D) GL261 glioma cells. On days 3 and 7,
mice received i.p. injections of HBSS, rat IgG (300 µg/injection), or
OX-40R mAb (300 µg/injection). In E, B6 mice in groups
of 10 were inoculated i.c. with 1 x 103
B16/D5 melanoma cells. On days 3 and 7, mice were treated by i.p.
injections of HBSS, rat IgG (300 µg/injection), or OX-40R mAb (300
µg/injection). In F, B6 mice in groups of five were
inoculated i.v. with 1 x 105 MCA 205 tumor
cells to establish pulmonary metastases. On days 3 and 7, mice were
treated by i.p. injections of HBSS, rat IgG (300 µg/injection), or
OX-40R mAb (300 µg/injection). Compilations of two independent
experiments because of similar results.
