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,
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Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, Portland, OR 97213;
Department of Neuroimmunology, Veterans Affairs Medical Center, Portland, OR 97201; and
Department of Neurology, Oregon Health Sciences University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The OX-40R is a membrane-bound protein that is expressed primarily on activated CD4+ T cells (6). It is part of a growing family of proteins in the TNF receptor superfamily, many of which are expressed by lymphocytes, for example, FAS, CD30, CD27, the TNF receptors, DR3, CD40, 4-1BB, and lymphotoxin-ß receptor. All of these proteins have several cysteine residues in the extracellular N-terminal domain, and the disulfide bonds formed within these proteins are thought to create a tertiary structure involved with ligand binding. These receptors appear to be involved in either cell growth and differentiation or cell death (apoptosis). The 4-1BB, CD30, and OX-40 receptors are all involved in T cell activation and have been shown to deliver costimulatory signals when engaged by their corresponding ligands or specific Abs (7, 8, 9, 10, 11, 12).
Recently, a membrane-bound protein with homology to TNF was cloned and
found to be the ligand for OX-40R (7, 8). The OX-40 ligand
(OX-40L)4 is a type II
membrane protein of 
34,000–40,000 m.w. (13) with homology to
TNF, which is expressed on activated B cells (14) and activated
endothelial cells (15) but not expressed on normal tissue. In addition
to delivering a costimulatory signal, OX-40R/OX-40L interaction also
mediates an adhesion event between endothelial cells and T cells (15).
The OX-40R is selectively expressed on T cells isolated from the site
of inflammation in a number of disease states, including experimental
allergic encephalomyelitis (EAE) (16, 17), rheumatoid arthritis (18),
and graft-vs-host disease (GVHD) (19), and on tumor-infiltrating
lymphocytes (20). OX-40+ T cells isolated from the
inflammatory site in EAE (rat spinal cord) exhibited T cell receptor
CDR3 binding motifs that are characteristic of those described for
myelin basic protein (MBP) reactivity (17) and produced proinflammatory
cytokines (IL-2 and IFN-
) (21). Thus, Abs to the OX-40R T cells can
be used to identify and isolate autoantigen-specific T cells from the
site of inflammation without prior knowledge of the autoantigen. In
addition, MBP-reactive CD4+ T cells are activated following
costimulation with Abs specific for OX-40R, and this activation leads
to the generation of EAE effector T cells capable of transferring EAE
into naive Lewis rats (9). These data imply that blocking OX-40R
signaling in vivo should inhibit activation and/or proliferation of
autoantigen-specific T cells at the site of inflammation and may dampen
clinical signs of EAE.
In this manuscript we follow the time course of OX-40R expression on CD4+ T cells and OX-40L expression on CD11b+ cells isolated from the central nervous system (CNS) of SJL mice with relapsing EAE (R-EAE). Disease onset correlated with the appearance of OX-40R+ T cells, which peaked at disease onset, declined as the disease regressed, and increased during relapses. Similarly, OX-40L+ CD11b+ cells were present at disease onset, appeared to remain during the acute phase of disease, disappeared at disease recovery, and reappeared during relapses. These results suggested that OX-40R:Ig-Fc chimeric protein might be useful in blocking stimulation of OX-40R+ T cells by interfering with the ability of the OX-40L+ CD11b+ APC to costimulate via OX-40L/OX-40R interaction. We show that OX-40R:Ig was able to inhibit peptide-specific proliferation of proteolipid protein (PLP)-specific T cell lines when stimulated by either peptide-pulsed thymocytes or CNS-isolated CD11b+ cells. The combination of OX-40R:Ig and CTLA-4:Ig completely inhibited T cell proliferation in vitro. In vivo administration of OX-40R:Ig was able to dampen clinical signs of acute disease and inhibit relapses if administered during the clinical signs of the relapse. Our data are suggestive of a critical costimulatory role for OX-40R/OX-40L interaction in EAE. We hypothesize that the OX-40R is engaged by the OX-40L on CD11b+ cells during Ag-specific T cell priming in the CNS of animals with EAE and inhibition of this process leads to dampening of an ongoing autoimmune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice (5–20 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the Animal Resource Facility at the Portland Veterans Affairs Medical Center under pathogen-free conditions according to institutional guidelines.
Antigens
Mouse spinal cord homogenates were prepared from SJL spinal cord as previously described (22). High-pressure liquid chromatography-purified PLP139–151 (amino acid sequence HCLGKWLGHPDKF) was purchased from the Beckman Center (Stanford, CA) or synthesized on an ABI Synergy 432A peptide synthesizer (Perkin-Elmer, Foster City, CA).
Induction of R-EAE
To induce active EAE, female SJL/J mice were inoculated s.c. in the flanks at four sites with a total of 0.2 ml of emulsion of saline containing 150 µg of PLP139–151 and an equal volume of CFA containing 200 µg of Mycobacterium tuberculosis H37RA. Mice were examined daily by an investigator blinded to treatment for the development of neurological deficits. Degrees of hindlimb and forelimb weaknesses were assessed as previously outlined (22, 23).
Animals with minimal hindlimb weakness were easily flipped onto their backs with a twist of the tail but could easily right themselves. With mild hindlimb weakness, mice had difficulty righting themselves after being flipped onto their backs. Animals with moderate hindlimb weakness could walk with no difficulty but could not right themselves after being flipped onto their backs. Mice with moderately severe hindlimb weakness could walk upright only with difficulty. Mice exhibiting severe hindlimb weakness could not walk upright but were still able to move hindlimbs. Animals with hindlimb paraplegia displayed no volitional leg movement.
With mild forelimb weakness, mice walked with chests close to the ground. With moderate forelimb weakness, mice could not lift their chests off the ground but could move around the cages with difficulty. With severe forelimb weakness, mice could not place forelimbs under their chests and were unable to move around the cage.
Clinical severity of disease was graded as follows using a modification of a previously published grading scale (24): 0, normal; 1, minimal hindlimb weakness; 2, mild hindlimb weakness; 3, moderate hindlimb weakness; 4, moderately severe hindlimb weakness; 5, severe hindlimb weakness; 6, hindlimb paraplegia; 7, hindlimb paraplegia with mild forelimb weakness; 8, hindlimb paraplegia with moderate forelimb weakness; and 9, hindlimb paraplegia with severe forelimb paralysis.
Gelatin cubes were placed in cages of animals with severe clinical disease. In addition to gelatin cubes, animals involved in treatment studies also received injections of sterile saline or human Ig in sterile saline (0.2 ml) as a diluent that also served to rehydrate animals.
Induction of adoptively transferred EAE
Adoptive transfer of EAE was accomplished by stimulating Ag-specific T cell lines for 3 days in culture with PLP139–151 peptide (5 µg/ml) and irradiated syngeneic thymocytes as APCs. Cells were then washed and resuspended in RPMI 1640 and transferred to naive recipient SJL/J mice by i.p. injection of 5 x 106 viable blasts.
Generation of PLP peptide-specific T cell lines
To generate Ag-specific T cell lines, mice were inoculated s.c. in the flanks at four sites on days 0 and 7 with a total of 0.3 ml of emulsion containing 1.0 mg of mouse spinal cord homogenate and an equal volume of CFA with 30 µg of M. tuberculosis H37RA (Difco, Detroit, MI). Draining inguinal lymph node cells (LNC) were obtained 14 days after immunization and stimulated with PLP139–151 to produce Ag-specific T cell lines as previously described (25). Briefly, LNC (7 x 106/ml) were cultured in RPMI 1640 and 1% normal mouse serum (stimulation medium) for 3 days with Ag (50 µg/ml of PLP139–151) and then expanded in RPMI 1640 and 10% FCS (growth medium) containing human rIL-2 (National Cancer Institute, Frederick, MD) for 4–10 days. The T cell lines (5 x 105 cells/ml) were restimulated with Ag (5 µg/ml of PLP139–151) and a 10-fold excess of irradiated (2400 rad) syngeneic thymocytes as APCs and then alternately cycled between growth medium and stimulation medium.
Isolation of brain mononuclear cells
Mononuclear cells were isolated from SJL/J brains as previously described, (21, 26, 27). Mice were not perfused before removal of CNS tissue. Briefly, brains were removed from donor animals, and a single cell suspension was prepared by passage through a wire mesh. Cells were washed in RPMI 1640, resuspended in 7 ml of 80% Percoll (Pharmacia, Uppsala, Sweden), and then overlaid with 8 ml of 40% Percoll to form a discontinuous gradient in a 15-ml centrifuge tube. The gradient was centrifuged at 500 x g for 35 min, and the 40–80% interface was harvested. The cells were washed three times with RPMI 1640 and stained for FACS analysis or used as APC for proliferation assays.
Enrichment of macrophage/microglia populations from the CNS
The CNS-isolated leukocytes were enriched for CD11b+ cells in a two-step process. Macrophages/microglia were stained with Mac-1 FITC for 20 min at 4°C and stained with a secondary anti-FITC-labeled microbead (Miltenyi Biotec, Auburn, CA). The CD11b+-labeled cells were then purified by positive selection using a magnetic bead separation column (Miltenyi Biotec). Following enrichment, the CD11b+ cells were analyzed by FACS analysis and found to be >80% pure.
Proliferation assays
The in vitro proliferative responses of PLP139–151-specific T cell lines were determined in 96-well microtiter plates as reported previously (26, 27). Cells were incubated for 72 h with Ag with the following cell concentrations: PLP139–151-specific T cell lines 2 x 104 cells per well with 2 x 105 syngeneic irradiated thymocytes per well or 6000 syngeneic CD11b+ positive brain macrophage/microglia per well as APC. Tritiated thymidine was added for the last 18 h of culture, and the cpm of incorporated label were determined in triplicate wells by standard liquid scintillation techniques.
Flow microfluorometry analysis
Immunofluorescence analysis of cells was performed on a FACScan (Becton Dickinson, Mountain View, CA) as described previously (21, 25). T cell lines and cells isolated from different organs were incubated with the specific mAbs for 15 min at 4°C in staining buffer (PBS with 3% newborn calf serum and 0.1% sodium azide), washed twice, resuspended in 300 µl of staining buffer containing 5 µg/ml of propidium iodide for dead cell discrimination, and directly analyzed. Each sample was stained with an isotype-matched control.
Monoclonal Abs
mAbs used for FACS analysis were as follows: CD4 (clone GK1.5, rat IgG2b), Mac-1 (CD11b) (clone M1/70, rat IgG2b), and anti-IAs (clone OX-6, mouse IgG1) were purchased from PharMingen (San Diego, CA). The goat anti-human IgG (Fc-specific) secondary Ab used to detect T cells stained with recombinant human-Ig fusion proteins was obtained from Sigma (St. Louis, MO).
Recombinant Ig fusion proteins for FACS staining and in vivo treatment
The soluble human OX-40R:Fc-Ig recombinant protein was produced
by deleting the membrane-spanning portion of the OX-40R molecule and
fusing the gene to the Fc portion of human Ig. The human OX-40R:Ig
protein has been shown to bind to the murine OX-40L (8). This protein
was supplied by Cantab Pharmaceuticals (Cambridge, U.K.). The soluble
murine OX-40L:Fc-Ig was used to detect the murine OX-40R, and the
cell-staining procedure was as described for the OX-40R:Ig construct.
This protein was supplied by Cantab Pharmaceuticals. CTLA-4:Fc-Ig was
supplied by Smith Kline-Beecham (King of Prussia, PA) and was used to
detect CD80 and/or CD86 in Fig. 3
A and for some of the in
vivo experiments discussed in detail below. The lymphotoxin-ß
receptor:Fc-Ig fusion protein was used to detect membrane-bound
lymphotoxin-ß and was supplied by Carl Ware (La Jolla for Allergy and
Immunology, San Diego, CA). All fusion protein staining was
accomplished using 1 µg of fusion protein to stain 1 x
106 cells and a goat anti-human FITC (Caltag, South San
Francisco, CA) was used to detect staining of the recombinant fusion
proteins. Where specified, human Ig (IgG1 isotype) was used as a
control for staining as well as in the in vivo treatment experiments
(Smith-Kline Beecham). For the in vivo experiments, OX-40R:Fc-Ig,
CTLA-4:Fc-Ig, and human Ig were given i.p. in 0.2 ml of saline at the
specified concentrations for the individual experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has previously been reported that the OX-40R was
preferentially expressed on autoantigen-specific CD4+ T
cells isolated from the CNS of Lewis rats with EAE (16, 17, 21).
Therefore, we used a soluble murine OX-40L:Ig chimeric protein to stain
T cells isolated from the brains of SJL mice with R-EAE for the
presence of OX-40R. Fig. 1 depicts a time
course of OX-40R expression on CD4+ T cells isolated from
the spleen and brain of mice with actively induced EAE, compared with T
cells from naive mice. Naive animals had very few CD4+ T
cells in the brain, and they expressed little or no OX-40R. By
comparison, CD4+ T cells isolated from the brains of mice
with actively induced EAE expressed significant levels of OX-40R. In a
representative experiment (Fig. 1), at disease onset (day 1 of disease,
13 days after immunization) 25% of CD4+ cells expressed
OX-40R, which increased to 40% at peak disease (day 3 of disease, 13
days after immunization), decreased to 18% at disease recovery (17–20
days after immunization), and increased to 34% at severe disease
relapse (21–28 days after immunization) (relapse time point was part
of a different experiment; data not shown). It should be noted that at
peak disease 10–15% of the CD11b+ cells also express
the CD4 Ag but do not express the OX-40R. Therefore, at peak disease
the percentages of CD4+ T cells expressing OX-40R may be
underestimated due to contaminating CD4+ macrophages. T
cells isolated from the spinal cord showed similar trends to those
observed in the brain (data not shown). However, the number of
infiltrating cells obtained from a diseased spinal cord was 10-fold
less than that obtained from a diseased brain. Therefore, brains from
mice with EAE were used in preference to spinal cord in subsequent
experiments. By comparison, in nonneural tissue such as the spleen,
examination of CD4+/OX-40R+ T cells obtained
from naive spleens and spleens from animals with active EAE
demonstrated only a slight increase in OX-40R expression with disease
onset (11.7% vs 6.7%). Interestingly, there was little variation of
OX-40R expression in the spleen during the time course of disease
(onset, 11.7%; peak of disease, 9.8%; and disease recovery,
14.4%).
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To characterize the cell type within the CNS capable of
presenting Ag to the CD4+/OX-40R+ T cells, we
first examined MHC class II expression at various time points of
actively induced EAE. In normal brain, 93% of the cells expressed
CD11b. However, <2% of these CD11b+ cells expressed MHC
class II (Fig. 2). The CD11b+
cells found in the naive brain were CD45low, which is a
phenotype consistent with the presence of brain microglia and not bone
marrow derived macrophages (data not shown) (28, 29). Upon examination
of the brain infiltrates obtained from animals at various time points
of active EAE, it is clear that with disease onset a significant
population of CD11b negative cells appear (40% at disease onset (see
Fig. 2), most of which are CD4+ T cells). However, in the
CD11b+ cell population there is a dramatic increase in the
percentage of cells expressing MHC class II. At disease onset, 15% of
the CD11b+ cells express MHC class II, which increased to
83% at peak disease (Fig. 2). Interestingly, there is no decrease of
MHC class II expression by CD11b+ cells following recovery
from active disease (93%). At peak disease, 62% of the
CD11b+ cells also expressed the CD45high
phenotype, a characteristic staining profile of bone marrow-derived
macrophages (data not shown) (28, 29).
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, the CD11b+ cells isolated
from the brain of animals with severe disease expressed CD80 and/or
CD86 (70%) and OX-40L (67%) but no surface lymphotoxin-ß. Others
have also reported selective up-regulation of B7.1 (CD80) expression
within the CNS of mice with EAE (30).
Results from Fig. 1 suggested that, in the brain, the percentage of
CD4+ T cells expressing OX-40R correlated with active EAE
disease progression. Therefore, in the same mice, we determined if
OX-40L expression by CD11b+ cells also correlated with
disease progression. As demonstrated in Fig. 4, brain microglia cells obtained from
naive brain were negative for OX-40L expression. By comparison,
CD11b+ cells isolated from the brains of animals exhibiting
actively induced EAE were shown to express significant levels of
OX-40L. Expression of OX-40L by CD11b+ cells dramatically
increased at disease onset (day 1 of disease, 13 days after
immunization; 58% of CD11b+ cells) and stayed relatively
the same at the peak of disease (day 3 of disease, 13 days after
immunization; 52%). OX40L expression was undetectable at disease
recovery (17–20 days after immunization; < 1%), in contrast to MHC
class II expression, which remained elevated. Interestingly, OX-40L
expression on CD11b+ brain APC reappeared during clinical
relapses (43% express OX-40L) (relapse time point was part of a
different experiment; data not shown). There was little or no
expression of OX-40L in spleens of animals with active disease (Fig. 4).
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OX-40R-specific inhibition of in vitro T cell proliferation
Results from Figs. 1 and 4 suggest that OX-40L may be
important for costimulating myelin-specific T cells found within the
CNS of animals with actively induced EAE. To determine whether OX-40L
expression by CD11b+ cells isolated from the CNS of mice
with EAE could act as APC and provide costimulation, we isolated these
cells from the brains of animals exhibiting active EAE and used them in
a T cell proliferation assays. Brain APC were assessed for their
ability to present Ag to PLP139–151-specific T cells in
the presence or absence of soluble costimulatory blocking reagents
(CTLA-4:Ig and OX-40R:Ig). Irradiated thymocytes (used as control APC)
or brain macrophages were placed in culture in the presence of
CTLA-4:Ig, OX-40R:Ig, or CTLA-4:Ig and OX-40R:Ig for 1 h. The APC
were added to PLP139–151-specific T cells in the presence
of specific peptide. The proliferation results are shown in Fig. 5.
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A), soluble CTLA-4 inhibited T cell
proliferation by 22% when present at 2 µg/ml and by 71% when
present at 40 µg/ml. T cell proliferation was not inhibited in the
presence of 2 µg/ml of OX-40R:Ig but was inhibited at 40 µg/ml
(48%). The combination of CTLA-4:Ig and OX-40R:Ig at 2 µg/ml
inhibited T cell proliferation by 22%, and when present at 40 µg/ml
it decreased proliferation by 95%. Human IgG was added at 80 µg/ml
to the assay and did not significantly inhibit proliferation.
Using irradiated brain CD11b+ cells (> 80% positive)
(Fig. 5B) as APC (at an APC:T cell ratio of 0.3:1), soluble
CTLA-4 at 2 µg/ml inhibited Ag-specific T cell proliferation by 26%
and by 32% at 40 µg/ml. Soluble OX-40R:Ig, when present at 2
µg/ml, inhibited T cell proliferation by 15% and at 40 µg/ml
inhibited T cell proliferation by 71%. The combination of CTLA-4:Ig
and OX-40R:Ig at 2 µg/ml inhibited T cell proliferation by 44% and
higher concentrations (40 µg/ml) inhibited proliferation by 91%.
When Abs directed against MHC class II (OX-6) were added, T cell
proliferation was completely inhibited.
In vivo therapy of actively induced EAE with soluble OX-40R:Ig
The in vitro proliferation data, as well as FACS analysis
(
Figs. 1–5), suggested that it might be possible to dampen an ongoing
immune response within the CNS of animals with EAE by blocking
OX-40R/OX-40L interaction. Therefore, we treated mice with OX-40R:Ig to
bind the OX-40L present on APC and prevent OX-40R-specific signaling.
Animals were immunized with PLP139–151 and CFA and treated
with 400 µg of OX-40R:Ig or saline on the first day of disease onset
and for two days thereafter. Fig. 6 and
Table I (Expt. 1), demonstrate that the
group treated with OX-40R:Ig had less severe clinical signs of disease
compared with the control group and recovered more quickly from the
acute phase of disease, although both groups did relapse (Fig. 6).
Table I shows two additional treatment experiments (Expts. 2 and 3). In
experiment 2, animals were treated with OX-40R:Ig, CTLA-4:Ig,
OX-40R:Ig, and CTLA-4:Ig or saline. Treatment was initiated 10 days
after active immunization and continued daily for 5 days. Both
OX-40R:Ig and CTLA-4:Ig groups showed decreased disease severity, and
the combination showed no enhancement over OX-40R:Ig or CTLA-4:Ig
treatment alone. In addition, 80% of the animals receiving saline died
due to severity of disease, whereas all of the treatment groups
survived. Experiment 3 compared OX-40R:Ig treatment to a human Ig
isotype control. Similar to experiment 2, 50% of the control animals
died, whereas all of the OX-40R:Ig group survived, and the control
group had more severe disease in comparison to the OX-40R:Ig-treated
group.
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In vivo therapy of adoptively transferred EAE with soluble OX-40R:Ig
We next assessed whether treatment with OX-40R:Ig during the acute
phase of adoptive (passive) EAE had a therapeutic effect. Fig. 7 and Table II (Expt. A) show that the
OX-40R:Ig-treated mice had less severe disease than the human
IgG-treated mice. In experiment B (Table II), the mice were injected
with OX-40R:Ig for 5 consecutive days after disease onset. Forty
percent of the control animals died due to severe EAE, while all of the
OX-40R:Ig-treated group survived and had a lower maximum disease score.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data show that blocking OX-40R/OX-40L interaction during the acute phase of disease did not prevent animals from having clinical relapses. This suggests that the T cells that cause clinical relapses were not affected by the initial blockade of OX-40R/OX-40L-mediated T cell costimulation. There are two possible explanations for the cause of clinical relapses in EAE: 1) The T cells that are initially activated in the CNS recirculate to the periphery, become reactivated, and cross the blood-brain barrier once again leading to a clinical relapse. 2) Naive T cells from the periphery are activated by myelin Ags after the acute phase of disease and migrate to the CNS leading to the relapse. If T cells were able to circulate out of the CNS, then treating with OX-40R:Ig did not induce anergy or cause T cell death at the site of inflammation. However, if peripheral naive T cells stimulated by myelin Ags caused the relapse, then the T cells responsible for the acute phase of EAE within the CNS could have been anergized by treatment with OX-40R:Ig. We are currently exploring whether T cell activation blocked by OX-40R:Ig can cause T cell anergy or activated induced cell death.
T cells isolated from inflammatory lesions in EAE have an activated memory phenotype (IL-2R+, CD45RBlow, CD44high, Mel-14low) (32). We hypothesize that T cells invading the CNS in active EAE have been Ag primed in the draining lymph nodes, and within 10–12 days after stimulation circulate out of the lymph node and penetrate the CNS. These cells have been characterized as short-term memory "effector" T cells, and upon encountering Ag within the CNS produce a burst of cytokines that in turn leads to clinical paralysis (21). In a TCR transgenic model (33), we are currently investigating the stage of CD4+ T cell development (naive or effector) during which the OX-40R costimulatory event is biologically relevant (Gramaglia et al. (34)). Using a MHC class II transfected fibroblast (DCEK) (35) that is capable of presenting Ag to the TCR transgenic T cells, we have cotransfected the OX-40L or B7.1 gene products individually or in combination. The OX-40L transfected DCEK line did not induce proliferation or IL-2 production in naive T cells, but provided potent costimulation to both Th1 and Th2 effector T cells. In contrast, the DCEK line transfected with OX-40L and B7.1 provided a potent signal for naive T cell activation and also induced potent costimulation for effector T cells. In the Lewis rat, we have also shown that the OX-40R provides costimulation for long-term MBP-specific T cell lines (9). The data from the TCR transgenics and the Lewis rat model suggest that the OX-40R appears to be biologically active on effector or memory effector T cells and can synergize with B7.1 to stimulate naive T cells. The data presented here suggest that the T cells invading the CNS in animals with EAE (memory effector T cells) are sensitive to OX-40R costimulation and therefore responded to the OX-40R:Ig treatment. An alternative interpretation of the treatment results is that cross-linking the OX-40L on CNS APC induces inhibitory cytokines that can down-regulate T cell function.
The OX-40L was originally described as a protein that was up-regulated
by the transcriptional transactivating human T cell lymphotrophic
virus, type I (HTLV-I) gene product tax (13). Expression of the OX-40L
seems to be quite restricted to APC found within inflammatory sites or
APC that have been activated in vitro (14, 15). Therefore, it appears
that OX-40R-mediated costimulation in vivo may be limited by expression
of the OX-40L. We have found that the OX-40L is coexpressed with CD80
and/or CD86 on the CD11b+ cells isolated from the CNS of
animals with EAE (Fig. 3). This would suggest that if these
CD11b+ cells were presenting Ag to OX-40+ T
cells in the CNS, the T cells would receive a combination of
costimulatory signals through the OX-40R and CD28. Our in vitro studies
with the DCEK transfectants showed that the combination of B7 and
OX-40L provided a stronger costimulus than either signal by itself.
Blocking both signals showed the greatest inhibition of T cell
proliferation in vitro (Fig. 5), but we saw no enhanced amelioration of
EAE when both CTLA-4:Ig and OX-40R:Ig were administered together vs
OX-40R:Ig alone. Whether the combination of both costimulatory signals
are additive in vivo, or the sequential engagement of CD28 and OX-40R
leads to cytokine production and T cell proliferation, is unknown. If
sequential engagement of both costimulatory receptors is necessary in
vivo, then blocking one of the receptors would lead to decreased
inflammation, thereby making it unnecessary to block both receptors.
Others have shown that in vivo injections of an anti-OX-40R Ab in animals with chronic GVHD exacerbates the clinical signs of disease (36). In chronic GVHD, the administration of anti-OX-40R Abs converted the disease to acute GVHD (36). We have shown that OX-40R+ T cells from animals with acute GVHD were the cells that recognize alloantigen in vivo. We have also observed a similar pattern of OX-40R expression in bone marrow transplant patients who have GVHD (T. Tittle, unpublished observations). Currently, we have identified a CD11b+ cell population in the peripheral blood and lymph nodes of animals with GVHD that expresses the OX-40L (T. Tittle, unpublished observations). Future experiments will be aimed at tempering clinical signs of GVHD by targeting OX-40L+ cells by in vivo administration of OX-40R:Ig.
Our data suggest that the CD11b+/MHC class II+ cells isolated from the CNS of animals with EAE are presenting Ag to myelin-specific CD4+ T cells. Alternatively, myelin-specific T cells could be stimulated by the endothelial cells found at the blood-brain barrier. Others have shown that endothelial cells at the site of blood-brain barrier disruption have an activated phenotype and up-regulate MHC class II (37). Activated endothelial cells have recently been shown to express the OX-40L (15). Activated endothelial cells at the blood-brain barrier may be presenting Ag to T cells that are in the process of penetrating the CNS. If this is the case, then costimulation could occur through an OX-40R/OX-40L-mediated step. Inhibition of inflammation in our EAE model may occur by blocking a T cell endothelial cell interaction with the OX-40R:Ig.
The colocalization of OX-40L-bearing macrophages/microglia and OX-40R+ T cells within the inflamed CNS tissue of animals with EAE suggests an important role for OX-40R/OX-40L interaction in the immunopathology of EAE. Blockade of OX-40R/OX-40L interaction both prevented T cell activation in vitro and most likely led to the in vivo amelioration of clinical signs in EAE. A significant advantage of using OX-40R and OX-40L as targets for intervention is their transient expression restricted to activated T cells and activated APC. This feature allows targeting of T cells and macrophages at the inflammatory site without affecting the peripheral T cell repertoire, thereby avoiding global immune suppression.
Our success in treating EAE suggests that targeting OX-40R/OX-40L interactions may be useful in treating multiple sclerosis and other autoimmune diseases. We have previously demonstrated that T cells in the joints of patients with rheumatoid arthritis selectively express OX-40R (21). As in EAE, targeting activated T cells and APCs within the sites of inflammation in human autoimmune diseases may be a promising approach to immunoselective regulation of the disease process, using reagents that recognize the OX-40L or the OX-40R.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew Weinberg, Earle A. Chiles Research Institute, 4805 NE Glisan, Providence Portland Medical Center, Portland, OR 97213. E-mail address:
3 These authors shared first authorship.
4 Abbreviations used in this paper: L, ligand; EAE, experimental allergic encephalomyelitis; GVHD, graft-vs-host disease; MBP, myelin basic protein; CNS, central nervous system; R-EAE, relapsing EAE; PLP, proteolipid protein.
Received for publication July 8, 1998. Accepted for publication October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated SJL/J astrocytes. J. Immunol. 160:4271.This article has been cited by other articles:
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  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]
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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. J. Jenkins, G. Perona-Wright, A. G. F. Worsley, N. Ishii, and A. S. MacDonald Dendritic Cell Expression of OX40 Ligand Acts as a Costimulatory, Not Polarizing, Signal for Optimal Th2 Priming and Memory Induction In Vivo J. Immunol., September 15, 2007; 179(6): 3515 - 3523. [Abstract] [Full Text] [PDF]
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 A. J. Lepisto, M. Xu, H. Yagita, A. D. Weinberg, and R. L. Hendricks Expression and function of the OX40/OX40L costimulatory pair during herpes stromal keratitis J. Leukoc. Biol., March 1, 2007; 81(3): 766 - 774. [Abstract] [Full Text] [PDF]
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 E. J.A van Wanrooij, G. H.M van Puijvelde, P. de Vos, H. Yagita, T. J.C. van Berkel, and J. Kuiper Interruption of the Tnfrsf4/Tnfsf4 (OX40/OX40L) Pathway Attenuates Atherogenesis in Low-Density Lipoprotein Receptor-Deficient Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 204 - 210. [Abstract] [Full Text] [PDF]
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J. R. Maxwell, R. Yadav, R. J. Rossi, C. E. Ruby, A. D. Weinberg, H. L. Aguila, and A. T. Vella IL-18 Bridges Innate and Adaptive Immunity through IFN-{gamma} and the CD134 Pathway J. Immunol., July 1, 2006; 177(1): 234 - 245. [Abstract] [Full Text] [PDF]
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P. Soroosh, S. Ine, K. Sugamura, and N. Ishii OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity J. Immunol., May 15, 2006; 176(10): 5975 - 5987. [Abstract] [Full Text] [PDF]
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S. Salek-Ardakani, T. So, B. S. Halteman, A. Altman, and M. Croft Protein Kinase C{theta} Controls Th1 Cells in Experimental Autoimmune Encephalomyelitis J. Immunol., December 1, 2005; 175(11): 7635 - 7641. [Abstract] [Full Text] [PDF]
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 B. Y. Ma, S. A. Mikolajczak, A. Danesh, K. A. Hosiawa, C. M. Cameron, A. Takaori-Kondo, T. Uchiyama, D. J. Kelvin, and A. Ochi The expression and the regulatory role of OX40 and 4-1BB heterodimer in activated human T cells Blood, September 15, 2005; 106(6): 2002 - 2010. [Abstract] [Full Text] [PDF]
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C. S. Schmidt, J. Zhao, J. Chain, D. Hepburn, B. Gitter, G. Sandusky, S. Chintalacharuvu, A. Glasebrook, and S. Na Resistance to Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis by Death Receptor 6-Deficient Mice J. Immunol., August 15, 2005; 175(4): 2286 - 2292. [Abstract] [Full Text] [PDF]
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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]
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