| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, Portland, OR 97213;
Department of Surgery, Section of Surgical Oncology, Oregon Health Science University, Portland, OR 97201;
Cantab Pharmaceuticals, Cambridge, United Kingdom; and
§
Computer and Statistical Services, National Cancer Institute-Frederick Research and Development Center, Frederick, MD 21702
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The OX-40R (CD134) is a lymphocyte-specific member of a growing family
of receptors for membrane-bound and soluble cytokines that has been
termed the TNFR superfamily (13). In addition to the TNFR,
this family also contains the CD30 Ag, CD40, CD27, FAS (CD95), DR3, and
4-1BB, all of which are expressed predominantly on cells of
hematopoietic lineage (13). A common function of the TNFR
superfamily seems to be in the regulation of activation/proliferation
or apoptosis of lymphocytes. For example, a signal transmitted through
CD40 is pivotal for Ig switching and prevents programmed cell death of
germinal center B cells (14). In contrast, signaling
through FAS induces apoptosis; the absence of a functional FAS protein
leads to a generalized autoimmune syndrome that may result from
impaired deletion of autoreactive lymphocytes (15, 16).
The OX-40R is a membrane-associated glycoprotein with an apparent m.w.
of 47,000–51,000, which is found primarily on activated
CD4+ T cells and not on normal resting peripheral
blood lymphocytes (17). The OX-40 ligand (OX-40L) is a
type II membrane protein of 
34,000 m.w. (18), which is
expressed on activated B cells (19), activated endothelial
cells, dendritic cells, and activated macrophages (20). It
is not expressed on normal resting cells. The OX-40L delivers a potent
costimulatory signal to OX-40R+ T cells and an
OX-40R/OX-40L interaction appears to be directly involved in an
adhesion event between endothelial cells and T cells
(20).
The presentation of a tumor Ag to T cells by MHC class I or II molecules is insufficient to prime an immune response in vivo. At least two signals are necessary to activate a CD8 or CD4 T cell response (21). The first signal is delivered through the T cell Ag receptor by Ag (peptide) bound to MHC class I or II. If only the first signal occurs, the T cell becomes tolerant or can undergoes apoptosis. A second signal involving the ligation of a costimulatory molecule appears to be required for optimal T cell activation. The best-characterized second signal is the one delivered to the T cell CD28 receptor by its ligand B7.1 or B7.2 (now known as CD80 and CD86, respectively). Both B7.1 and B7.2 bind to two determinants on T cells, CD28 and CTLA-4; the former is constitutively expressed on 95% of CD4+ and 50% of CD8+ T cells, and the latter is expressed only on activated T cells (21). The interaction of CD80 or CD86 with CD28 results in increased production of IL-2, which is necessary for the development of a beneficial T cell response. In contrast, the interaction of B7 with CTLA-4 delivers a negative signal (22) to the T cell and could negate the activation signal of CD28. The OX-40R is expressed on activated CD4+ T cells and when engaged causes a potent costimulatory signal to effector T cells that can enhance long-term survival (23, 24).
The OX-40R has a very distinct pattern of expression in autoimmune disease, in that it is expressed on CD4+ T cells within the inflammatory compartment but not on peripheral CD4+ T cells (25, 26). Approximately 15–40% of the T cells that invade the inflammatory tissue during an acute episode of autoimmune encephalomyelitis (an animal model for multiple sclerosis) are OX-40R+, whereas 100% of the invading T cells express the IL-2R (25, 27). Spinal cord T cells isolated from the CNS of rats with experimental autoimmune encephalomyelitis (EAE), sorted based on OX-40R expression, had their TCR CDR3 regions (Ag-binding region) sequenced. The sequence analysis revealed that 16 of 17 clones isolated from the OX-40R+ fraction contained myelin-specific binding motifs, whereas only 5 of 17 in the OX-40R-negative population showed the same motif (28). These data provide strong evidence that the OX-40R+ T cells within the inflammatory site were indeed the cells that recognized autoantigen in vivo. Further support for this hypothesis was derived from experiments that depleted OX-40R+ T cells in animals with EAE. An anti-OX-40 immunotoxin was injected into animals with ongoing signs of EAE, and after treatment the disease did not progress and the autoantigen reactive cells were no longer present within the inflammatory site (26). We propose that the OX-40R+ T cells found within the inflammatory compartments in cancer (29) are the tumor-reactive T cells and if they can be specifically expanded it should lead to an increase in tumor-specific immunity.
In this manuscript, we report the antitumor effects of OX-40R engagement with Ab or OX-40L:Ig in vivo during tumor priming. Injection of OX-40L:Ig or anti-OX-40R on days 3 and 7 after s.c. tumor inoculation enhanced tumor-free survival of mice compared with untreated controls. The effect, which was dose dependent, was observed in four separate tumor models derived from four different tissues. Anti-OX-40R-treated mice that survived the initial tumor challenge exhibited enhanced tumor-specific CD4+ T cell memory. Examination of surgically removed human breast cancers revealed that OX-40R+ T cells were found in close proximity to tumor cells. Therefore, the OX-40R-specific antitumor effects observed in the mouse tumor models may also be applicable to the immunotherapy of human cancer.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Both the B16/F10 and MCA 303 were produced on the C57BL/6 (B6) background, whereas the CT26, Renca, and SM1 tumors were produced on the BALB/c background. Therefore, the mice used in this study were either female B6 or BALB/c purchased from The Jackson Laboratory (Bar Harbor, ME) at 4 wk of age. All mice used for injection of tumor were 6–12 wk old, and each individual experiment used mice of the same age. All mice were housed at the Earle A. Chiles Research Institute animal care facility (Providence Portland Medical Center) and were cared for according to National Institutes of Health guidelines, with the exception of the CT26 experiments, which were performed at Cantab Pharmaceuticals (Cambridge, U.K.) according to British animal facility guidelines.
Tumor models
All of the tumor lines used for this study were MHC class II negative and did not express the OX-40L, as assessed by flow cytometry (data not shown). The B16/F10 (F10) melanoma line was originally described by Hart (30) and was selected as an invasive variant of the original B16 line. The F10 variant is considered poorly immunogenic because it does not protect against subsequent challenge to parental tumor when irradiated and used as a vaccine. In all experiments, the F10 tumor was inoculated with in vitro passaged cells s.c. in the flank at a dose of 1 x 105 cells, a dose which usually necessitates the mice to be sacrificed within 25–30 days after inoculation. The D5G6 melanoma is a variant of B16 that was transfected with the GMCSF cDNA (31). The transfection vector was constructed so that the only gene product produced is GMCSF. This line produces 450 ng/106/24 h of GMCSF and can be cultured and maintained in vitro.
The MCA 303 tumor model was originally described by Huntzicker and Fox
(32) at the Earle A. Chiles Research Institute and is one
of a series of methylcholanthrene-induced sarcomas. It has been shown
to be moderately immunogenic because it protects 50% of mice
against subsequent challenge to parental tumor when used as an
irradiated vaccine (32). MCA 303 grows slowly in vitro and
for this reason we have been using in vivo passaged tumor for the
inoculations, except for in Fig. 3
A where in vitro passaged
tumor was used. We have found with each in vivo passage, the MCA 303
tumor becomes more tumorigenic (data not shown) and the in vitro
passaged tumor appeared to be more immunogenic and easier to treat with
the anti-OX-40R reagents (see Fig. 3A).
|
CT26 is an N-nitroso-N-methylurethane-induced
undifferentiated colon carcinoma (34). All of the mice
inoculated with the CT26 cell line in this study were given 1 x
105 cells s.c. (see Fig. 6). The Renca tumor is a
spontaneous arising murine renal cell carcinoma and was isolated and
maintained as described previously (35). The mice in Fig. 6B were inoculated s.c. with 1 x
105 Renca cells. All mice were sacrificed when
the tumor reached 200 mm2 in the flank, except
for the CT26- and Renca-injected mice which were sacrificed when the
tumor size reached 100 mm2.
|
Both the murine and human OX-40L:Ig fusion proteins were
produced at Cantab Pharmaceuticals by constructing a chimeric cDNA that
contained the C-terminal region of the OX-40L (extracellular domain
without the membrane spanning region) fused to the constant region of
human IgG1 (36). The human DR3 receptor:Ig fusion protein
was produced at SmithKline Beecham Pharmaceuticals (Philadelphia, PA)
and the extracellular domain (excluding the membrane spanning region)
was fused to the constant region of human IgG1. All of these proteins
were purified by protein G chromatography and were subjected to
SDS-PAGE analysis under reducing conditions and showed a single band of
the appropriate m.w. The hybridoma that produces the anti-murine
OX-40R-specific mAb was obtained from the European Cell Culture
Collection (37) and is referred to as OX-86. The hybridoma
was grown in RPMI 1640 with FCS that was stripped of Ig by passing it
over a protein G column. The cells were grown to a high density and the
supernatant was then poured over a protein G column and purified Ab was
eluted. Ab concentrations were quantitated by absorption at 280 nm. All
of the injections of fusion proteins or Abs were given i.p. on days 3
and 6 or 7 (except for Fig. 6
) after tumor inoculation at amounts
ranging from 25 to 250 µg per injection. The dose for each individual
experiment is indicated in the figure legends.
CD8 depletion and adoptive transfer
The OX-40L:Ig-treated mice in Fig. 3 all survived the initial
MCA 303 tumor challenge and were rechallenged 60 days later with MCA
303. Fifty-three days after the second challenge, the mice were
inoculated with MCA 303 and 10 days later were injected with
anti-CD8 (Lyt-2) i.p. to delete CD8+ T cells.
These mice were sacrificed 3 days later, and the spleens were analyzed
by flow cytometry for both CD4+ and
CD8+ cells. Typically, the spleens were <2%
CD8+ and the majority of T cells were
CD4+. One spleen equivalent of the CD8-depleted
cells (1.45 x 107 cells/transfer) were
transferred i.p. into one naive recipient for three mice. The recipient
mice were then rechallenged with MCA 303 15 days after adoptive
transfer (Fig. 3B). All of the mice in this experiment were
given tumor doses of 1 x 105
cells/challenge. The control mice in Fig. 3B were injected
with the same volume of saline i.p. that was transferred with the
CD8-depleted cells.
Analysis of OX-40R+T cells isolated from tumor-draining lymph nodes
Blck/6 mice were immunized with the D5G6 tumor (31)
described above (1 x 106 in four flanks).
The cells from the lymph nodes (inguinal, axillary, brachial) were
harvested 9–11 days after inoculation. The lymph nodes were processed
and put into single-cell suspension as described (31).
From five mice our yields were typically 250–300 x
106 total cells, of which 2–4% were
OX-40R+. The cells were then incubated with
OX-40L:Ig, washed, and incubated with anti-huIg beads (Miltenyi
Biotech, Auburn, CA). The cells were then passed over a
VS+ column, and the positive and negative
fractions were saved for FACS analysis and cytokine assays. The cells
were put in culture for 7 days with 50 U/ml of IL-2 and then stimulated
with irradiated tumor-draining lymph nodes. The cells were set up in
96-well round-bottom plates with 20,000 lymph node cells stimulated
with 1 x 106 irradiated APC. The APC were
isolated from D5G6-draining lymph nodes or MCA 205-draining lymph nodes
(12), irradiated with 5000 rad, washed, and used as APC in
the assay. The cells were stimulated for 72 h, and the
supernatants were collected from triplicate wells and analyzed in a
standard ELISA assay for IFN-
. The anti-IFN- Abs were
obtained from PharMingen (La Jolla, CA) and a standard quantitative
assay was performed (12). The data represented in Fig. 4, A and B, respectively, were performed as separate
experiments, and the MHC class II knockout (ko) mice in Fig. 4B were on the Blck/6 background and were able to elicit
tumor-specific CD8+ T cells that caused
regression of lung (B. Fox, unpublished data).
|
We obtained paraffin-embedded blocks from two breast cancer patients whom had primary tumor and adjacent lymph nodes removed surgically. The patients signed a standard surgical consent form, and the tissue was removed for the sole purpose of treating and diagnosing the disease. The blocks were sectioned and placed on a histostaining slide for analysis. The sections were hydrated and transferred into containers containing 0.5 M Tris (pH 10). The sections were then subjected to a microwave Ag-retrieval technique as described previously (38). Serial sections were then stained with the primary Abs in a humidity chamber overnight. The primary Abs used were anti-human CD4 (Vector Laboratories, Burlingame, CA) used at a dilution of 1/40, anti-human OX-40 (PharMingen) used at a dilution of 1/100, and anti-rat CD45 (PharMingen) used at a dilution of 1/100. All of the Abs used were mouse IgG1 isotype and the anti-rat CD45 Ab (ox-1) was used as a negative control for all samples. The tissue was then washed and incubated with a 1/400 dilution of a biotinylated anti-mouse Ig Ab for 25 min (Vector Laboratories). The tissue was then washed and incubated with an avidin-HRP complex (Vector Laboratories) for 30 min, washed, and the color was developed with diaminobenzidine (DAB; Dako, Carpinteria, CA). The Ag-positive cells are visualized as a reddish brown, which is the precipitate caused by the HRP cleaving the DAB.
Stastical analysis
Survival rates were calculated according to the method of Kaplan and Meier and compared by log rank analysis. The program used for the analysis was S-Plus guide to statistical data analysis (MathSoft, Seattle, WA). The p values are indicated for each analysis in Results.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Engagement of the OX-40R causes a potent costimulatory response
leading to T cell proliferation, increased cytokine production, and
enhanced survival of effector T cells (23, 24, 39, 40, 41). If
as we predict, the OX-40R+ cells at the tumor
site or draining lymph nodes are the tumor-specific T cells in vivo
(27, 28, 29), then engagement of the OX-40R in vivo should
lead to an enhanced antitumor-specific response. Therefore, we
investigated whether engagement of the OX-40R in vivo during tumor
priming would lead to an increase in tumor-free survival or a delay in
tumor development. Fig. 1 depicts the
results when mice that were injected with a lethal inoculum of MCA 303
(methylcholanthrene-induced sarcoma (32)) s.c. and were
treated 3 and 7 days later with either mOX-40L:Ig, DR3:Ig, or saline.
Saline-treated mice and mice treated with DR3:Ig had to be sacrificed
at the same time because of progressive tumor growth. In contrast, all
of the mice that received mOX-40L:Ig experienced delayed tumor growth
and 60% were free of tumor for >70 days. The mOX-40L:Ig-treated mice
were rechallenged with MCA 303 tumor s.c. The mice failed to develop
tumors, as all the control mice did, suggesting they had developed a
tumor-specific memory T cell response (data not shown).
|
). It should be noted that the MCA 303
tumor line becomes more tumorigenic and less immunogenic the more times
that it is passaged in vivo. The MCA 303 tumor line in Fig. 2 had been
passaged more times in vivo than in Fig. 1, accounting for the
observation that the OX-40L:Ig treatment was a bit less effective at
the 100-µg dose.
|
shows the fate of mice inoculated
with in vitro passaged MCA 303 that were then treated with mOX-40L:Ig.
In all experiments, in vitro passaged MCA 303 was easier to treat than
in vivo passaged tumor. All of the mice were inoculated with tumor s.c.
and injected with mOX-40L:Ig on days 3 and 7 after tumor inoculation
survived the initial tumor challenge, whereas all of the mice injected
with saline had to be sacrificed due to excessive tumor burden (Fig. 3A). The mOX-40L:Ig-treated mice that survived the initial
tumor challenge from Fig. 3A were then rechallenged with MCA
303. All of the mice survived the second challenge and were tumor free
for 53 days (data not shown). The surviving mice were then reinoculated
with MCA 303 s.c. and depleted of CD8 cells by i.p. injection of
anti-Lyt-2 Ab 10 days later. Three days later, the mice were
sacrificed and shown to be devoid of splenic CD8 cells (<2%).
CD8-depleted spleen cells (1.45 x 107) from
the tumor-immune mice were transferred into naive mice, and 15 days
later the mice were challenged with MCA 303 s.c. Fig. 3B shows that the mice receiving the CD8-depleted spleen
cells were resistant to tumor challenge, whereas the control mice had
to be sacrificed due to tumor burden. As a control for the adoptive
transfer experiments described above, we transferred the same amount of
CD8-depleted spleen cells or total splenocytes isolated from naive mice
into naive mice and challenged them with MCA 303 15 days after adoptive
transfer. As opposed to the adoptive transfer from the OX-40R-treated
mice, all of the naive splenocytes conferred no adoptive immunity to
the MCA 303 tumor, and all of the mice were sacrificed due to excessive
tumor burden (data not shown). Tumor-draining lymph node OX-40R+ cells have enhanced tumor-specific responses
To understand the therapeutic effect that the OX-40L:Ig reagent
has in tumor-bearing mice, we sorted OX-40R+
cells from tumor-draining lymph nodes and assessed their ability to
respond in a tumor Ag-specific assay. Our previous data in EAE showed
that the OX-40R+ T cells isolated from the CNS or
Ag-primed lymph nodes had enhanced responses to the "local" Ag
where the T cells were isolated from (27). Therefore, we
wanted to assess the ability of OX-40R+ cells to
respond to tumor Ag(s). Since all of the tumors within this study were
MHC class II negative and could not directly present Ag to
CD4+ T cells, we devised a scheme to assay
tumor-specific CD4+ T cell responses from
tumor-primed APC. Mice were immunized with D5G6 (a B16 melanoma variant
(31)), and 10 days later the tumor draining lymph nodes
were isolated. The OX-40R was expressed solely on the
CD4+ T cells and represented 3% of the total
lymph node population or 13% of the CD4+
population (data not shown) (these numbers were similar for all
separations that were performed). The tumor-draining lymph node cells
were sorted by magnetic separation and the
OX-40R+ T cells were >70% of the
CD4+ population, whereas the
OX-40- fraction was 4% of the
CD4+ population. The cells were cultured in IL-2
for 7 days and then restimulated with irradiated tumor-draining lymph
node cells isolated from mice inoculated with D5G6 or MCA 205 (both
tumors were derived on the Blck/6 background). Fig. 4A presents IFN- production
from the D5G6 OX-40R+ vs the
OX-40R- cells and shows a 3-fold enhanced
response in the OX-40R+, whereas neither group
responded to the MCA 205-draining lymph node cells. There were some
contaminating cells in our magnetic separation of the
OX-40R+ cells (mostly B cells and a small
percentage of CD8 T cells); therefore, we wanted to assess whether
CD4+ T cells within the
OX-40R+ fraction were responsible for the
cytokine production. The OX-40R+ T cell fraction
was stimulated with either D5G6 tumor-draining lymph nodes cells from
normal mice or D5G6 tumor-draining lymph nodes from MHC class II ko
mice. Fig. 4B shows that the OX-40R+ T
cells responded preferentially to the D5G6-draining lymph node cells
from normal but not MHC class II ko mice. The data confirm that most of
the response (IFN- production) is due to a
CD4+ T cell interacting with an MHC class
II+ APC and not from CD8 T cell contamination. It
should be noted that the MHC class II ko mice were able to prime D5G6
tumor-specific CD8+ cells that could eradicate
3-day established lung metastases (B. Fox, unpublished data).
OX-40R-specific treatment in a poorly immunogenic tumor model (B16/F10)
The F10 variant of the B16/Bl6 melanoma line does not elicit a
protective immune response when injected as an irradiated vaccine s.c.
(data not shown), and has therefore been characterized as a poorly
immunogenic tumor. We wanted to determine whether OX-40R engagement
during tumor priming would enhance immunity to this aggressive tumor.
Fig. 5A shows that treatment
with murine OX-40L (mOX-40L):Ig on days 3 and 7 after inoculation with
F10 also exhibited antitumor effects. Approximately 25% of treated
mice survived tumor challenge long term. An Ab to OX-40R (mAb OX-86
(37)) delivered at the same dose and time schedule
enhanced tumor-free survival to a level comparable to treatment with
mOX-40L:Ig (Fig. 5B). The percentage of tumor-free mice
following Ab treatment was very similar to OX-40L:Ig treatment. Both
reagents resulted in a statistically significant level of tumor
protection as assessed by log rank analysis (p
= 0.007 (Ab) and 0.05 (mOX-40L:Ig)). We have now used the
m-anti-OX-40R Ab in four separate tumor models, and it has been as
therapeutic as the OX-40L:Ig construct in all of the models tested
so far.
|
A similar protocol was designed to treat mice with colorectal
tumors (CT26). The strategy was exactly as described above (mOX-40L:Ig,
two-dose regimen). Human OX-40L:Ig was used as a negative control
because it does not signal the murine OX-40R (42). The
two-dose regimen significantly enhanced tumor-free survival
(p = 0.04; data not shown). Fig. 6A shows that multiple
injections of OX-40L:Ig was beneficial as assayed by tumor-free
survival with a p value of higher confidence
(p = 0.01) than the two-injection dose scheme. Multiple
injections (days 2, 7, 14, 21, 27, and 40) appeared to be better
(greater numbers of survivors), but no statistical difference was
observed when directly compared with the two-dose regimen as assessed
by log rank analysis. Seven of the surviving mice from the
mOX-40L:Ig-treated group were then rechallenged with CT26. All of the
mOX-40L:Ig mice resisted the tumor challenge and remained tumor free,
whereas all of the naive control mice succumbed to the tumor challenge
(Fig. 6B). On day 30, the seven tumor-free mice were then
rechallenged with a syngeneic tumor of different histology in the
opposite flank from the CT26 injections (Renca, renal origin and this
tumor does respond to anti-OX-40R-mediated therapy (data not
shown)) to test for a tumor-specific T cell memory response. Six of
seven of the CT26-resistant mice had to be sacrificed due to tumor
burden associated with the Renca tumor, which suggests that the
CT26-resistant mice had specificity for tumor Ags associated with colon
cancer.
Summary of OX-40R engagement during tumor priming
Table I summarizes the data in four
different tumor models in which the antitumor effects of OX-40R
engagement during tumor priming have been examined. Although the data
suggest that more immunogenic tumors respond better to the therapy, we
have therapeutic results even in the poorly immunogenic melanoma model
(F10). Data for all of the tumor lines, except for the SM1 breast
cancer line, have been presented in the previous figures. The SM1 tumor
cell line is weakly immunogenic (data not shown). Mice that have been
injected with the SM1 tumor and treated with OX-40L:Ig on days 3 and 7
after tumor inoculation had enhanced antitumor activity as shown by the
increase of tumor-free survival. The SM1 data were subjected to log
rank statistical analysis and shown to be significant with a
p = 0.01.
|
The existence of OX-40R+ T cells within
breast cancer samples would suggest the possibility of treating breast
cancer patients with anti-OX-40R-specific reagents. To determine
the spatial relationship between OX-40R+ T cells
and tumor cells, we examined several human breast cancer biopsies by
immunohistochemistry. Both primary tumors and tumor-involved lymph
nodes were analyzed for CD4+ and
OX-40R+ cells. Fig. 7 is a representative sample from two
separate patients (from a study of 44 patients), both with infiltrating
duct carcinoma. Fig. 7A depicts the tumor-infiltrating
lymphocytes within a primary tumor, whereas Fig. 7B depicts
a tumor-infiltrated lymph node. In Fig. 7A, the
CD4+ cells are seen infiltrating the outer edge
of the tumor. At higher magnification, one can see that the
OX-40R+ T cells represent a subset of the
invading lymphocytes and are in close proximity to the tumor cells. A
number of the OX-40R+ cells appear to be larger
(blasts) and some appear to be undergoing mitosis. Fig. 7B
depicts a lymph node where more than half of the node architecture has
been invaded by the tumor. There is an abundance of
CD4+ cells surrounding the invading tumor. The
OX-40R+ cells are found concentrated in areas
directly adjacent to the invading tumor cells (lower right
panel). OX-40R+ cells are also found in
uninvolved areas, but the greatest number was found closest to the site
of tumor infiltration. Based on our work in autoimmune disease and
the data in Fig. 4, we hypothesize that the
OX-40R+ cells within these tissue sections
represent the tumor Ag-specific T cells (27, 28).
|
. All of the tumors tested in
this study were MHC class II negative; therefore, we are not exactly
sure why the OX-40R+ T cells would be in close
proximity to the tumors. We hypothesize that an APC (such as a
dendritic cell) may be near the tumor T cell boarder and may present Ag
to CD4+ T cells, causing up-regulation of the
OX-40R. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Engaging the OX-40R in vivo during tumor priming led to 20–60% cure
rates for mice challenged with four different solid tumors with varying
immunogenicity. The data suggest that OX-40R-based therapy could be a
universal enhancer of the immune system, not only for tumor immunity,
but also as an immunologic adjuvant for other types of vaccines (e.g.,
viral, bacterial, etc.). The human OX-40L:Ig protein is a form of a
potential immune adjuvant that is available now for use in human
clinical trials and has been shown to stimulate human T cells in vitro.
Currently, we are in the process of producing a panel of humanized
anti-OX-40 mAbs. Although the Ab and the soluble OX-40L fusion
protein worked with similar potency in our tumor models (Fig. 5 and
data not shown), we feel that the Ab may be less immunogenic and would
potentially have a longer half-life in vivo. Both the fusion protein
and anti-OX-40R Abs are likely candidates for human vaccine
adujvant therapies in the future. Currently, we do not have a mouse
reagent that recognizes the mouse OX-40R; therefore, we cannot make a
direct comparison between the reagents we have been using (rat
anti-mouse or mouse OX-40L:human Ig) in mice vs a mouse
anti-OX-40R reagent. It could be possible that when multiple
injections of the OX-40L chimeric fusion protein (mouse/human) are
administered (Fig. 6) that Abs may be produced that enhance the
response by cross-linking the fusion protein in vivo. Currently, we are
synthesizing the mouse/mouse OX-40L:Ig fusion protein to assess
multiple injections use of this reagent for long-term experiments
in vivo.
Enhancement of tumor immunity with Abs such as anti-4-1BB or anti-CTLA-4 are other examples of T cell activation Ags which when triggered or blocked enhance tumor-specific immunity (8, 43). Like the OX-40R, the 4-1BB receptor, also a member of the TNFR family, was originally described as a T cell activation Ag that has potent costimulatory properties (44). The 4-1BB receptor is expressed on CD8 and CD4 T cells as well as NK cells (44). 4-1BB receptor costimulatory function appears to primarily affect CD8+ T cells (45), and engagement of this receptor during tumor priming led to a 50-fold increase in tumor-specific CD8+ T cell cytolytic function and led to enhanced tumor-free survival (8). The CTLA-4 protein is expressed on both CD8 and CD4 T cells and when engaged by its ligand(s) (B7.1 or B7.2) delivers an inhibitory signal to the T cell. Abs that block CTLA-4/B7 interaction enhance Ag-specific T cell function (22) and can enhance tumor-specific immunity (43). The OX-40R-specific therapy although potent, never led to 100% tumor-free mice. Therefore, combination therapy with anti-CTLA-4 or anti-4-1BB and anti-OX-40R may synergize to accentuate Ag-specific T cell therapy leading to a higher percentage of tumor-free mice. Future experiments will attempt to maximize tumor-specific T cell therapies by combining two or more of these Abs during tumor priming with the hope of enhancing both CD4 and CD8 Ag-specific effector/memory T cell responses.
Recently, we have shown that in vivo engagement of the OX-40R during
Ag-specific priming increases the number and life span of Ag-specific
CD4+ T cells (see Ref. 24 and data
not shown). Most T cells become susceptible to activation-induced cell
death after encountering Ag at the effector T cell stage, and only a
few go on to become memory T cells (46). We hypothesize
that engagement of the OX-40R during tumor priming targets the
tumor-reactive CD4+ T cells and rescues them from
activation-induced cell death. An increase in the number of Ag-specific
T cells may allow the mice to remain tumor-free and fight a secondary
tumor challenge. Fig. 3B shows that the adoptive transfer of
CD8-depleted spleen cells from OX-40R-treated tumor-immune mice can
confer antitumor immunity. These data suggest that there is an increase
and/or enhancement of tumor Ag-specific memory
CD4+ T cells, and that they are able to transfer
adoptive protection. Because the tumor cells in all four models do not
express MHC class II, we do not feel that the
CD4+ T cells are the ultimate effector cells that
interact with the tumor. Instead, we hypothesize that enhanced cytokine
production by the tumor Ag-specific CD4+ T cells
help to activate CD8+ T cells, NK cells, and/or
macrophages, which in turn may directly interact with and destroy
tumors. We have some direct evidence for the indirect model of tumor
irradication in the Renca model (Renal cell carcinoma). We found the
Renca tumor to respond to anti-OX-40R therapy, but if either the
CD4 or CD8 T cells were deleted the therapeutic effect was diminished
(data not shown).
We now have direct evidence that engagement of the OX-40R dramatically increases memory T cell development (47). The experiments were performed with T cells that expressed the transgenic TCR for OVA (DO11.10) and could be detected with an anti-idiotypic Ab (48). Soluble Ag was injected into mice seeded with TCR transgenic T cells and given five daily injections of anti-OX-40R in a proinflammatory environment (LPS injection) during the priming phase. The mice were sacrificed 60 days after immunization, and there was a 60-fold increase in Ag-specific cells compared with mice receiving Ag alone or Ag plus LPS. The Ag-specific cells were phenotyped and shown to be small resting memory cells (by forward scatter and high CD44 staining).
We have found that the OX-40R is only expressed on CD4+ T cells isolated from the "inflammatory sites" in cancer and autoimmune disease and is turned over quite rapidly (within 24–48 h) (26, 29). However, it has been shown that both CD4 and CD8 T cells can express the OX-40R if stimulated in vitro with Con A or PHA (42). It appears that the only way to up-regulate OX-40R expression on T cells is through TCR engagement. Even in highly inflammatory situations, such as superantigen stimulation, there appears to be no bystander up-regulation of the OX-40R on Ag-nonspecific cells (24, 47). In superantigen-injected mice, the OX-40R is solely expressed on Vß3/CD4+ T cells, which is the target TCR for this superantigen (24). Therefore, we believe that engaging the OX-40R during tumor priming in vivo would target the most recently Ag-activated T cells.
It has been shown that the inflammation associated with superantigen
stimulation and clinical signs of EAE involves the production of Th1
cytokines (49). We have shown that engaging the OX-40R on
Th1 lines accentuates T cell proliferation by up-regulating
transcription and translation of IL-2 (39). We have also
shown that effector T cells appear to be more sensitive to
OX-40R-specific costimulation than naive T cells (23).
Effector T cells that have differentiated to produce either Th1 or Th2
cytokines are both sensitive to OX-40R-specific costimulation
(23). Engaging the OX-40R on Th2 effector cells increased
translation and secretion of IL-4 and IL-5 and enhanced their
proliferation. Two reports recently showed that engaging the OX-40R
polarizes cells to the Th2 phenotype (40, 41). Our data
suggest that T cell polarization is dependent on the cytokine milieu
present during T cell differentiation and engagement of the OX-40R will
accentuate both a Th1 or Th2 response. It has been shown that a
Th2-immune response can be detrimental to tumor eradication in vivo,
but a Th1 response is beneficial (50). Therefore, it might
be important to enhance Th1 responses during tumor priming (with IL-12,
IFN-, and/or anti-IL-4) to get an effective antitumor-immune
response when administering reagents that engage the OX-40R in vivo. In
contrast, other groups have shown that tumor-specific Th2 cells can
play a role in activating eosinophils and might be beneficial to an
antitumor immune response (51).
The OX-40L is expressed only on activated APC such as B cells, dendritic cells, endothelial cells, and macrophages (19, 20, 52, 53). In vivo expression of the OX-40L appears to occur in highly inflammatory situations such as infection of mice with mouse mammary tumor virus (draining lymph nodes) or in mice with EAE on macrophages isolated from the inflamed organ (brain) (31, 53). Even in normal primary T cell responses such as immunization with Ag in CFA, OX-40L expression was quite low on spleen macrophages (50). The OX-40R is expressed every time a T cell is triggered through the TCR; therefore, the potent OX-40R costimulatory effects might be regulated by the inexcessability of the OX-40L on APC. The immune system has evolved to generate an immune response to clear foreign entities rapidly and then readily down-regulate itself. Since OX-40L-mediated costimulation is quite potent at the effector T cell stage, it may only be necessary in cases where a massive invasion occurs, which in turn causes a long-lasting inflammatory response. Because aggressive tumors have been shown to down-regulate immune responses through immunosuppressive mechanisms (54), APC near the tumor site would probably not express the OX-40L. We hypothesize that engagement of the OX-40R in vivo enhances a beneficial tumor-specific immune response by adding a signal that is absent in the tumor-bearing mice. This would explain the observation that a percentage of the tumor-challenged mice injected with OX-40R:Ig were able to remain tumor free.
In summary, engagement of the OX-40R in vivo during tumor priming
significantly delayed and prevented the appearance of tumors compared
with control-treated mice. This OX-40R effect was dose dependent and
was observed in both immunogenic and nonimmunogenic tumor models.
OX-40R expression was found on T cells localized at the tumor site in
several different human cancers (melanoma, head and neck, and breast
cancer (Ref. 29 and Fig. 7). In this manuscript, we
examined the physical relationship of the OX-40R+
T cells to breast cancer cells in both primary tumors and a
tumor-invaded lymph node. The OX-40R+ T cells
were concentrated in areas surrounding the tumor, and we hypothesize
(from Fig. 4 and our work in EAE (27, 28)) that they are
the tumor-specific T cells. The combination of the OX-40R therapeutic
data in the mouse tumor model and the appearance of
OX-40R+ in tumor-bearing patients suggests that
we will be able to enhance immune tumor reactivity with reagents
designed to engage the OX-40R in patients with cancer. The data also
suggest that engaging OX-40R may be a useful immune adjuvant to enhance
Ag-specific responses in a wide variety of vaccine settings.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Andrew Weinberg, Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, 4805 North East Glisan, Portland, OR 97213. E-mail address:
3 Abbreviations used in this paper: OX-40R, a membrane bound protein that binds to the OX-40 Ab; OX-40L, a membrane protein that binds to the OX-40R; EAE, experimental autoimmune encephalomyelitis; ko, knockout; DAB, diaminobenzidine; m, murine; DR3, the extracellular domain of death receptor 3 fused to human Ig Fc.
Received for publication September 20, 1999. Accepted for publication December 1, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
elicits antitumor CD4+ MHC class II-restricted T cells that are cytolytic and tumoricidal in vivo. J. Immunol. 154:4613.[Abstract]
and IL-2. Am. J. Pathol. 4:747.
This article has been cited by other articles:
|
|
 |
|
  A. M. Keller, Y. Xiao, V. Peperzak, S. H. Naik, and J. Borst Costimulatory ligand CD70 allows induction of CD8+ T-cell immunity by immature dendritic cells in a vaccination setting Blood, May 21, 2009; 113(21): 5167 - 5175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
R. Houot and R. Levy T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy Blood, April 9, 2009; 113(15): 3546 - 3552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Krause, M. Bruckner, C. Uermosi, E. Singer, M. Groettrup, and D. F. Legler Prostaglandin E2 enhances T-cell proliferation by inducing the costimulatory molecules OX40L, CD70, and 4-1BBL on dendritic cells Blood, March 12, 2009; 113(11): 2451 - 2460. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Ruby and A. D. Weinberg OX40-Enhanced Tumor Rejection and Effector T Cell Differentiation Decreases with Age J. Immunol., February 1, 2009; 182(3): 1481 - 1489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Gough, C. E. Ruby, W. L. Redmond, B. Dhungel, A. Brown, and A. D. Weinberg OX40 Agonist Therapy Enhances CD8 Infiltration and Decreases Immune Suppression in the Tumor Cancer Res., July 1, 2008; 68(13): 5206 - 5215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
C. E. Ruby, R. Montler, R. Zheng, S. Shu, and A. D. Weinberg IL-12 Is Required for Anti-OX40-Mediated CD4 T Cell Survival J. Immunol., February 15, 2008; 180(4): 2140 - 2148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
C. A. Williams, S. E. Murray, A. D. Weinberg, and D. C. Parker OX40-Mediated Differentiation to Effector Function Requires IL-2 Receptor Signaling but Not CD28, CD40, IL-12Rbeta2, or T-bet J. Immunol., June 15, 2007; 178(12): 7694 - 7702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Thompson, H. Dong, and E. D. Kwon Implications of B7-H1 Expression in Clear Cell Carcinoma of the Kidney for Prognostication and Therapy Clin. Cancer Res., January 15, 2007; 13(2): 709s - 715s. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Usui, Y. Okunuki, T. Hattori, M. Takeuchi, T. Kezuka, H. Goto, and M. Usui Expression of Costimulatory Molecules on Human Retinoblastoma Cells Y-79: Functional Expression of CD40 and B7H1. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4607 - 4613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Yu, F. Y. Yue, X. X. Gu, H. Schwartz, C. M. Kovacs, and M. A. Ostrowski OX40 Ligation of CD4+ T Cells Enhances Virus-Specific CD8+ T Cell Memory Responses Independently of IL-2 and CD4+ T Regulatory Cell Inhibition J. Immunol., February 15, 2006; 176(4): 2486 - 2495. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
B. Valzasina, C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, and M. P. Colombo Triggering of OX40 (CD134) on CD4+CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR Blood, April 1, 2005; 105(7): 2845 - 2851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
A. Zingoni, T. Sornasse, B. G. Cocks, Y. Tanaka, A. Santoni, and L. L. Lanier Cross-Talk between Activated Human NK Cells and CD4+ T Cells via OX40-OX40 Ligand Interactions J. Immunol., September 15, 2004; 173(6): 3716 - 3724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
S. K. Lathrop, C. A. Huddleston, P. A. Dullforce, M. J. Montfort, A. D. Weinberg, and D. C. Parker A Signal through OX40 (CD134) Allows Anergic, Autoreactive T Cells to Acquire Effector Cell Functions J. Immunol., June 1, 2004; 172(11): 6735 - 6743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. M. Finney, A. N. Akbar, and A. D. G. Lawson Activation of Resting Human Primary T Cells with Chimeric Receptors: Costimulation from CD28, Inducible Costimulator, CD134, and CD137 in Series with Signals from the TCR{zeta} Chain J. Immunol., January 1, 2004; 172(1): 104 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Martin-Orozco, Z. Chen, L. Poirot, E. Hyatt, A. Chen, O. Kanagawa, A. Sharpe, D. Mathis, and C. Benoist Paradoxical Dampening of Anti-Islet Self-Reactivity but Promotion of Diabetes by OX40 Ligand J. Immunol., December 15, 2003; 171(12): 6954 - 6960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Humphreys, L. Edwards, G. Walzl, A. J. Rae, G. Dougan, S. Hill, and T. Hussell OX40 Ligation on Activated T Cells Enhances the Control of Cryptococcus neoformans and Reduces Pulmonary Eosinophilia J. Immunol., June 15, 2003; 170(12): 6125 - 6132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Blazar, A. H. Sharpe, A. I. Chen, A. Panoskaltsis-Mortari, C. Lees, H. Akiba, H. Yagita, N. Killeen, and P. A. Taylor Ligation of OX40 (CD134) regulates graft-versus-host disease (GVHD) and graft rejection in allogeneic bone marrow transplant recipients Blood, May 1, 2003; 101(9): 3741 - 3748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M. Dechant, G. Vidarsson, B. Stockmeyer, R. Repp, M. J. Glennie, M. Gramatzki, J. G. J. van de Winkel, and T. Valerius Chimeric IgA antibodies against HLA class II effectively trigger lymphoma cell killing Blood, December 15, 2002; 100(13): 4574 - 4580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W. Chan, C. D. Hopke, S. M. Krams, and O. M. Martinez CD30 Expression Identifies the Predominant Proliferating T Lymphocyte Population in Human Alloimmune Responses J. Immunol., August 15, 2002; 169(4): 1784 - 1791. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
L. C. Ndhlovu, N. Ishii, K. Murata, T. Sato, and K. Sugamura Critical Involvement of OX40 Ligand Signals in the T Cell Priming Events During Experimental Autoimmune Encephalomyelitis J. Immunol., September 1, 2001; 167(5): 2991 - 2999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kjærgaard, J. Tanaka, J. A. Kim, K. Rothchild, A. Weinberg, and S. Shu Therapeutic Efficacy of OX-40 Receptor Antibody Depends on Tumor Immunogenicity and Anatomic Site of Tumor Growth Cancer Res., October 1, 2000; 60(19): 5514 - 5521. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
