Activation of OX40 prolongs and exacerbates autoimmune experimental uveitis.
ABSTRACT T cells are essential for the development of autoimmune uveitis. Although the costimulatory molecule OX40 promotes T-cell function and expansion, it is unclear whether OX40 is implicated in ocular inflammation. The purpose of this study was to examine the role of OX40 in uveitis.
Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein peptide 161-180 (IRBP(161-180)). Some mice received an intravenous administration of OX40-activating antibody on days 0 and 4 after IRBP(161-180) sensitization or on days 10 and 14 of uveitis onset. The severity of EAU was evaluated by histology at different time points. In addition, ocular inflammatory cytokine expression was determined by real time-PCR, and peripheral activated CD4(+)CD44(+)CD62L(-) T cells and IL-7Rα expression were analyzed by flow cytometry. The activated CD4(+)CD44(+) lymphocytes were rechallenged with IRBP(161-180) in vitro to assess their antigen recall response.
The authors demonstrated a marked OX40 expression by infiltrating lymphocytes in enucleated human eyes with end-stage inflammation. In addition, the administration of OX40-activating antibody prolonged and exacerbated the disease course of EAU. Moreover, activation of OX40 not only increased CD4(+)CD44(+)CD62L(-) lymphocyte number, it upregulated IL-7Rα expression in the activated T-cell population. Lastly, these cells exhibited a stronger interferon-γ response to IRBP(161-180) restimulation in vitro.
The results reveal a pathogenic role of OX40 in uveitis. Furthermore, the upregulation of IL-7R in CD4(+)CD44(+) lymphocytes suggests that the activation of OX40 promotes the generation or expansion of uveitogenic memory T cells.
Article: The Future of Uveitis Treatment.[Show abstract] [Hide abstract]
ABSTRACT: Uveitis is a heterogeneous collection of diseases with polygenic and environmental influences. This heterogeneity presents challenges in trial design and selection of end points. Despite the multitude of causes, therapeutics targeting common inflammatory pathways are effective in treating diverse forms of uveitis. These treatments, including corticosteroids and immunomodulatory agents, although often effective, can have untoward side effects, limiting their utility. The search for drugs with equal or improved efficacy that are safe is therefore paramount. A mechanism-based approach is most likely to yield the future breakthroughs in the treatment of uveitis. We review the literature and provide examples of the nuances of immune regulation and dysregulation that can be targeted for therapeutic benefit. As our understanding of the causes of uveitis grows we will learn how to better apply antibodies designed to block interaction between inflammatory cytokines and their receptors. T-lymphocyte activation can be targeted by blocking co-stimulatory pathways or inhibiting major histocompatibility complex protein interactions. Furthermore, intracellular downstream molecules from cytokine or other pathways can be inhibited using small molecule inhibitors, which have the benefit of being orally bioavailable. An emerging field is the lipid-mediated inflammatory and regulatory pathways. Alternatively, anti-inflammatory cytokines can be provided by administering recombinant protein, and intracellular "brakes" of inflammatory pathways can be introduced potentially by gene therapy. Novel approaches of delivering a therapeutic substance include, but are not limited to, the use of small interfering RNA, viral and nonviral gene therapy, and microparticle or viscous gel sustained-release drug-delivery platforms. The author(s) have no proprietary or commercial interest in any materials discussed in this article.Ophthalmology 10/2013; · 5.56 Impact Factor
Activation of OX40 Prolongs and Exacerbates
Autoimmune Experimental Uveitis
Xiumei Wu,1James T. Rosenbaum,*,2,3Grazyna Adamus,2Gary L. Zhang,1Jie Duan,1
Andrew Weinberg,4and Zili Zhang*,1
PURPOSE. T cells are essential for the development of autoim-
mune uveitis. Although the costimulatory molecule OX40 pro-
motes T-cell function and expansion, it is unclear whether
OX40 is implicated in ocular inflammation. The purpose of this
study was to examine the role of OX40 in uveitis.
METHODS. Experimental autoimmune uveitis (EAU) was in-
duced in B10.RIII mice by subcutaneous injection of inter-
photoreceptor retinoid-binding protein peptide 161–180
(IRBP161–180). Some mice received an intravenous adminis-
tration of OX40-activating antibody on days 0 and 4 after
IRBP161–180sensitization or on days 10 and 14 of uveitis onset.
The severity of EAU was evaluated by histology at different time
points. In addition, ocular inflammatory cytokine expression was
determined by real time-PCR, and peripheral activated
CD4?CD44?CD62L?T cells and IL-7R? expression were
analyzed by flow cytometry. The activated CD4?CD44?
lymphocytes were rechallenged with IRBP161–180in vitro to
assess their antigen recall response.
RESULTS. The authors demonstrated a marked OX40 expression
by infiltrating lymphocytes in enucleated human eyes with
end-stage inflammation. In addition, the administration of
OX40-activating antibody prolonged and exacerbated the dis-
ease course of EAU. Moreover, activation of OX40 not only
increased CD4?CD44?CD62L?lymphocyte number, it up-
regulated IL-7R? expression in the activated T-cell population.
Lastly, these cells exhibited a stronger interferon-? response to
IRBP161–180restimulation in vitro.
CONCLUSIONS. The results reveal a pathogenic role of OX40 in
uveitis. Furthermore, the upregulation of IL-7R in CD4?CD44?
lymphocytes suggests that the activation of OX40 promotes
the generation or expansion of uveitogenic memory T cells.
(Invest Ophthalmol Vis Sci. 2011;52:8520–8526) DOI:
with many systemic immune-mediated diseases (e.g., sarcoid-
osis, ankylosing spondylitis, inflammatory bowel disease). Uve-
itis has a high prevalence (115.3/100,000) in the United States
and is comparable to diabetes as a major cause of visual loss.1,2
Although the etiology of uveitis is multifactorial, CD4?T lym-
phocytes play an important role in the pathogenesis of uveitis
by recognizing uveitogenic antigen and orchestrating the im-
During T-cell activation, costimulatory molecules provide
a pivotal signal to the T-cell response. OX40 (CD134) is a
well-recognized costimulatory molecule in the TNF receptor
superfamily. It is induced in activated T cells.4,5By interact-
ing with OX40L on antigen-presenting cells, OX40 triggers
the phospho-inositide 3-kinase (PI3K)-AKT signaling path-
way, leading to NF-?B translocation.6Unlike constitutively
expressed CD28, which is responsible for the initial T-cell
activation, OX40 provides a second wave of costimulation to
enhance T-cell effector response, proliferation, and sur-
Many forms of uveitis and autoimmune diseases display a
chronic and relapsing clinical course. Both effector and
memory T cells contribute to the recurrent inflammatory
response in these disorders. After antigen encounter and
T-cell receptor activation, T lymphocytes differentiate into
subsets with phenotypic and functional distinction. Short-
lived effector T cells orchestrate and maximize the immune
response, whereas some antigen-experienced T cells be-
come long-lasting memory cells that are responsible for the
antigen recall response. Many studies have shown that OX40
promotes the development of effector and memory T
cells.9,10Although OX40 has been involved in a number of
clinically common and important autoimmune diseases,10,11
little is known of the role of OX40 in uveitis. Recently, we
reported12that blocking OX40 signaling using anti-OX40
ligand antibody attenuated inflammatory cell infiltration in
mouse uveitis models. In addition, the activation of OX40
augmented the effector function of T cells in acute ocular
inflammation.12However, it remains to be further defined
whether OX40 is implicated in the pathology of human
uveitis and other more completely characterized models,
such as experimental autoimmune uveitis (EAU).
In this study, we demonstrated a robust infiltration of
OX40?cells in the human eye with end-stage inflammation. In
addition, OX40-activating antibody treatment augments EAU.
Furthermore, enhanced OX40 activation in EAU not only ex-
pands the CD4?CD44?CD62L?T-cell population, it increases
IL-7R? and Bcl-6 expression. Thus, these findings suggest that
OX40 may play an instrumental role in the upregulation of
activated/memory T cells during the course of ocular inflam-
veitis is a serious ophthalmologic disorder characterized
by intraocular inflammation. It is commonly associated
From the Departments of1Pediatrics,2Ophthalmology, and3Med-
icine, Oregon Health & Science University, Portland, Oregon; and the
4Earle A. Chiles Research Institute, Providence Portland Medical Cen-
ter, Portland, Oregon.
Supported by National Institutes of Health Grants EY016788 (ZZ),
EY013093 (JTR), and EY006484 (JTR), the Stan and Madelle Rosenfeld
Family Trust, the William and Mary Bauman Foundation, Research to
Prevent Blindness, and the William C. Kuzell Foundation.
Submitted for publication April 1, 2011; revised July 15, 2011;
accepted August 12, 2011.
Disclosure: X. Wu, None; J.T. Rosenbaum, None; G. Adamus,
None; G.L. Zhang, None; J. Duan, None; A. Weinberg, None; Z.
*Each of the following is a corresponding author: Zili Zhang,
Department of Pediatrics, Oregon Health & Science University, 707
Gaines Street, CDRCP, Portland, OR 97239; email@example.com.
James T. Rosenbaum, Department of Ophthalmology, Oregon Health &
Science University, L467Ad, Portland, OR 97239;
Immunology and Microbiology
Investigative Ophthalmology & Visual Science, October 2011, Vol. 52, No. 11
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
Six-week-old female B10.RIII mice (Jackson Laboratory, Bar Harbor,
ME) were used for the experiments. The animal experimental proto-
cols were in accordance with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and were approved by our
institutional animal care and use committee.
Experimental Autoimmune Uveitis
EAU was induced in B10.RIII mice by subcutaneous immunization
(near the base of the tail) with 40 ?g interphotoreceptor retinoid-
binding protein peptide 161–180 (IRBP161–180) (Ser-Gly-Ile-Pro-Tyr-Ile-
Ile-Ser-Tyr-Leu-His-Pro-Gly-Asn-Thr-Ile-Leu-His-Val-Asp) (AnaSpec, Fre-
mont, CA) in 200 ?L complete Freund’s adjuvant (Sigma-Aldrich, St.
Louis, MO) with Mycobacterium tuberculosis strain H37RA. The eyes
were harvested for histology at different time points during the exper-
Activation of OX40
Some B10.RIII mice were also treated with OX40-activating antibody
(Clone OX86; 100 ?g/mouse) by tail vein injection on days 0 and 4 or
days 10 and 14 after IRBP161–180immunization. The OX40-activating
antibody was produced in the laboratory of one of the authors (AW)
from hybridomas and was affinity purified on protein G columns. This
monoclonal antibody is a rat IgG1 that specifically interacts with
mouse OX40, leading to the enhancement of T-cell activation and
function.4Furthermore, this antibody promotes a T-cell response in
wild-type mice but not in OX40 knockout animals, suggesting that this
agonistic antibody specifically activates OX40.4
Cell Culture, Isolation, and Stimulation
After B10.RIII mice were euthanized, their submandibular draining
lymph nodes and spleens were removed. Single-cell suspensions were
prepared by passing the tissue through a 70-?m cell strainer (BD
Biosciences, Mountain View, CA). Splenic red blood cells (RBCs) were
lysed with 1? RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) at room
temperature for 5 minutes. The cell suspension was washed twice with
RPMI 1640 and then cultured in RPMI 1640 with 10% fetal bovine
serum (FBS) in an atmosphere of 95% air and 5% CO2at 37°C with 4
?g/mL IRBP161–180peptide for 72 hours.
For histologic evaluation, the eyes were fixed in 3% paraformaldehyde.
Then the tissues were embedded in paraffin, sectioned, and stained
with hematoxylin and eosin. Ocular inflammation was assessed by light
microscopy, and the severity of EAU was graded on a four-point scale
based on inflammatory cell infiltration, retinal folding, and destruc-
Immunohistochemistry of OX40 Staining
Paraffin-embedded sections of human eye globes were rehydrated and
then steamed in pressure cooker for 20 minutes in 1? EDTA/Trip
buffer (pH 9.0). After incubation in 3% H2O2in methanol quench
solution for 10 minutes, these slides were stained with 1:100 dilution
of isotype IgG or anti-human OX40 antibody (PharMingen, San Diego,
CA) for 1 hour at room temperature. Next, the slides were rinsed with
Tris-buffered saline with Tween, followed by peroxidase-conjugated
anti-mouse IgG (ImmPress Reagent; Vector Laboratories, Burlingame,
CA). Finally, diaminobenzidine was added to detect OX40 staining, and
the slides were counterstained with hematoxylin.
B10.RIII splenocytes were suspended in phosphate-buffered saline
(PBS) containing 2% FBS. Anti-CD4, anti-CD44, anti-CD62L and anti–
IL–7R antibodies conjugated with different fluorescent colors (eBiosci-
ence, San Diego, CA) were used to label these cell surface markers for
60 minutes on ice. After PBS wash, the cells were fixed with 1?
fixation solution at 4°C.
For intracellular staining of IFN-?, the lymphocytes of submandib-
ular draining lymph nodes and spleen were harvested from IRBP161–
180-immunized mice and further cultured with 4 ?g/mL IRBP161–180in
vitro for 24 hours. Then these cells were stimulated with phorbol
myristate acetate (PMA; 50 ng/mL) and ionomycin (1 ?g/mL) for 5
hours. Brefeldin A (1:1000) was added for 2 hours. The cells were
collected and stained with fluorescein isothiocyanate–labeled anti-
mouse CD4 antibody for 30 minute. After PBS wash, the cells were
fixed and permeabilized overnight with 1? fixation/permeabilization
solution (eBioscience) at 4°C. Then they were stained intracellularly
with phycoerythrin (PE)-labeled monoclonal antibody against IFN-?
(eBioscience) for 1 hour at 4°C. Data acquisition was performed on a
flow cytometer, and data were analyzed using acquisition and analysis
Fifty microliters of culture media of isolated B10.RIII splenocytes
stimulated with IRBP161–180for 72 hours from various experimental
groups were collected for ELISA to measure IFN-? and IL-17A levels
according to the manufacturer’s protocols (BioLegend, San Diego, CA).
Total RNA from cultured CD4?cells was isolated with a purification kit
(RNeasy Mini Kit; Qiagen, Valencia, CA). First-strand cDNA synthesis
was accomplished with an oligo (dT)-primed reverse transcriptase kit
(Omniscript; Qiagen). Gene-specific cDNA was amplified by PCR using
mouse-specific primer pairs (IFN-?: sense 5?-TCA AGT GGCATA GAT
GTG GAA GAA-3? and antisense 5?-TGG CTC TGC AGG ATT TTC
ATG-3?; IL-17A: sense 5?-GTG GCG GCT ACA GTG AAG GCA-3? and
antisense 5?-GAC AAT CGA GGC CAC GCA GGT-3?; Bcl-6: sense 5?-TCA
GAG TAT TCG GAT TCT AGC TGT GA-3? and antisense 5?-TGC AGC
GTG TGC CTC TTG-3?; Blimp-1: sense 5?-ACA GAG GCC GAG TTT
GAA GAG A-3? and antisense 5?-AAG GAT GCC TCG GCT TGA A-3?;
?-actin: sense 5?-ATG CCA ACA CAG TGC TGT CT-3? and antisense
5?-AAG CAC TTG CGG TGC ACG AT-3?). Real-time PCR was performed
using a master mix (RT2Real-Time PCR Master Mix; SABiosciences,
Frederick, MD), running for 40 cycles at 95°C for 15 seconds and at
55°C for 40 seconds. The mRNA levels of investigated genes in each
sample were normalized to ?-actin mRNA and quantified using the
following formula: 2 [(Ct/?-actin ? Ct/gene of testing gene)]. The
result was expressed as the fold difference in the groups stimulated
with OX40-activating antibody compared with the group without ad-
ditional OX40 activation.
Data are expressed as the average ? SD. For EAU scoring, the median
difference between control and experimental groups was compared
using the Mann-Whitney U test. Other statistical probabilities were
evaluated by Student’s t-test or ANOVA, with a value of P ? 0.05
Expression of OX40 in Human Nonseeing Eyes
with Chronic End-Stage Inflammation
One reason OX40 has not been extensively studied in uveitis is
likely because of the limited availability of human eye tissue
with inflammation. To investigate whether OX40 is involved in
human ocular inflammation, we recently acquired four surgical
specimens of human nonseeing eyes with chronic end-stage
inflammation. The demographic information (age and sex) and
diagnoses of these patients are summarized in Table 1. After
IOVS, October 2011, Vol. 52, No. 11
OX40 Exacerbates Uveitis8521
confirming marked lymphocytic inflammation in the anterior
and posterior segments by histology, we performed immuno-
histochemistry staining to examine OX40 expression in these
eye specimens. As illustrated in representative tissue (Fig. 1A),
intense infiltration of lymphocytes was present in the ciliary
body region, and a large percentage of these cells strongly
expressed OX40. In addition, clusters of OX40?lymphocytes
were observed in the choroid in these human eye specimens.
The expression of OX40 within the diseased human eye was
prominent, consistent with the potential clinical importance
and relevance of studying OX40 in ocular inflammation.
Exacerbation of EAU by OX40 Activating Antibody
In light of this finding, we used the B10.RIII EAU model to
further characterize the role of OX40 in uveitis. Recent re-
search14,15has linked OX40L polymorphism to susceptibility
to systemic lupus erythematosus and atherosclerosis. We pos-
tulated that enhanced OX40 function by aberrant OX40L en-
gagement or stimulation contributes to inflammation in the
eye. OX40-activating antibody has been widely used in OX40
research.16,17This approach is especially helpful to mimic the
gain-in-function change of OX40 signaling in many pathologic
conditions. Therefore, we asked whether enhancement of
OX40 activation would exacerbate ocular inflammation primar-
ily by augmenting antigen sensitization or amplifying effector
To this end, we first compared the severity of EAU between
the groups with and without further OX40 activation. Some
B10.RIII mice received 100 ?g OX40-activating antibody
(OX86) by tail vein injection on days 0 and 4 of IRBP161–180
immunization, EAU (inflammatory cell infiltration, vasculitis,
retinal folding, and destruction) was scored on days 14 and 21,
respectively. As shown in Figure 2, the mice developed marked
ocular inflammation in response to IRBP161–180priming, and
the uveitis receded on day 21 in the control group without
further OX40 stimulation. However, the mice treated with
OX40-activating antibody exhibited persistent and severe pos-
terior uveitis on day 21 (Fig. 2). This result suggested that
activation of OX40 enhances and prolongs the ocular immune
response to antigen challenge.
Next, we asked whether further activation of OX40 during
the onset of EAU could affect the outcome of ocular inflamma-
tion. Thus, we treated B10.RIII mice with 100 ?g OX40-acti-
vating antibody during IRBP161–180sensitization (days 0 and 4)
or at the time of disease onset (days 10 and 14), and EAU was
assessed on day 25 (10 days after the completion of OX40-
activating antibody treatment during EAU onset). Compared
with controls with untreated EAU, the activation of OX40 early
TABLE 1. Patient Demographic Information
Patient Age (y)SexClinical Diagnosis
enucleated human eye with end-
specimen was stained with isotype
IgG and anti-human OX40 anti-
body. (A) Invasion of numerous
of the ciliary body. (B) Clusters of
OX40?lymphocytes (arrows) in the
choroid (representative image of four
OX40 expression in an
bates EAU in B10.RIII mice with IRBP161–180peptide. OX40-activating
antibody (100 ?g/mouse) was administered intravenously during
IRBP161–180sensitization (days 0 and 4). The mice were euthanized on
days 14 and 21, respectively. Eyes were harvested at these time points
for histologic EAU evaluation.
OX40-activating antibody treatment prolongs and exacer-
8522Wu et al.
IOVS, October 2011, Vol. 52, No. 11
stage resulted in exacerbated and protracted EAU (Fig. 3A). In
either OX40-activating antibody-treated group, the mice con-
sistently exhibited more severe retinal destruction, vasculitis,
and marked retro-retinal hemorrhage/separation (Figs. 3B, 3C).
In addition, more severe choroid inflammation was seen in the
mice treated with OX40-activating antibody (Fig. 3D).
The fact that treatment with OX40-activating antibody at
disease onset augmented EAU suggested that stimulation of
OX40 also enhances effector T-cell function. Recent stud-
ies18,19have shown that Th1 and Th17 T cells are both capable
of inducing EAU. To further characterize OX40-enhanced uve-
itis, we examined the impact of OX40 activation on ocular
IFN-? and IL-17A transcript expression. Total RNA from whole
eye was isolated on day 25 after EAU induction. Real time-PCR
revealed a marked increase of IFN-? and IL-17A transcripts in
the group that received OX40-activating antibody during
IRBP161–180sensitization or uveitis onset compared with the
control group with EAU (Fig. 4).
Increase of CD4?CD44?IL-7R??T Cells by OX40
Previously, we showed that the activation of OX40 enhanced
effector T-cell function in the ovalbumin-induced acute uveitis
model.12In this study, we investigated whether the stimulation
of OX40 could expand activated T cells while exacerbating
EAU severity and augmenting ocular inflammatory cytokine
expression. Three weeks after IRBP161–180priming, T-cell acti-
vation markers CD44 and CD62L of splenic CD4?T lymphocytes
were analyzed by flow cytometry in the mice treated with and
without OX40-activating antibody. As illustrated in Figure 5, the
activation of OX40 during IRBP161–180sensitization signifi-
cantly increased the CD4?CD44?CD62L?population.
Given that uveitis often displays a chronic and recurrent
clinical course, we asked whether OX40 also promotes mem-
ory T-cell development in EAU. IL-7 is essential to the long-term
survival of naive and memory CD4?T cells, and the cellular
response to IL-7 is significantly influenced by IL-7R expres-
sion.20It has been shown that the surface level of IL-7R is
downregulated when naive T cells are activated and IL-7R
reappears in the lymphocytes that commit to memory lin-
eage.20The expression of IL-7R enhances memory T-cell sur-
vival.21To study the effect of OX40 on memory T cells in
uveitis, we examined whether OX40 activation affects IL-7R?
expression in naive CD4?CD44?and activated CD4?CD44?T
cells. Flow cytometry showed that control EAU mice had an
average of 9.28% IL-7R?cells in splenic CD4?CD44?lympho-
cytes. The OX40-activating antibody administered on days 0
and 4 or at uveitis onset augmented IL-7R??cells to 12.81%
and 14.84%, respectively (Fig. 6A). Nevertheless, the activation
ofOX40 didnot increase
CD4?CD44?population (Fig. 6A). In addition, the mean fluo-
treatment at disease onset exacerbates
EAU in B10.RIII mice with IRBP161–180
peptide. OX40-activating antibody (100
?g/mouse) was administered intrave-
nously during IRBP161–180sensitization
(days 0 and 4) or at EAU onset (days 10
and 14) after IRBP161–180immunization.
On day 25, the mice were euthanized.
Eyes were harvested for histologic EAU
evaluation (A). Representative histology
of OX40-activating antibody-enhanced
age, retro-retinal separation, and hemor-
rhage (closed arrows). (C) Retinal vasc-
ulitis (open arrows). (D) Inflammatory
infiltrates in the choroid (arrowheads).
scription of IFN-? and IL-17 in EAU. EAU and OX40-activating antibody
treatment during IRBP161–180sensitization or early uveitis onset are
described. Three weeks later, the eyes were harvested, and the expres-
sion of IFN-? and IL-17A was assessed by real time-PCR. The level of
investigated mRNA was normalized to ?-actin, and the relative quantity
was further compared with the EAU group without OX40-activating
antibody treatment (n ? 3 mice/group).
OX40-activating antibody treatment enhances ocular tran-
IOVS, October 2011, Vol. 52, No. 11
OX40 Exacerbates Uveitis8523
CD4?CD44?T cells was compared between the control group
and the group treated with OX40 antibody. Treatment with
OX40-activating antibody during antigen sensitization or at
uveitis onset showed an increase in IL-7R? MFI by 14.68% ?
8.57% and 21.58% ? 9.48%, respectively. When we restimu-
lated these lymphocytes with IRBP161–180in vitro, the total
IFN-? production was significantly higher in the splenocytes
from OX40-activating antibody-treated mice than in the control
group (Fig. 6B). However, it was unclear whether the aug-
mented cytokine expression was caused by the increase in
total number of IRBP-reactive lymphocytes, cytokine produc-
tion in single cells, or both. To address this question, we
examined intracellular IFN-? expression by flow cytometry.
Splenocytes were harvested from EAU mice with and without
OX40-activating antibody treatment during IRBP sensitization.
These cells were further cultured with IRBP for an additional
36 hours, followed by PMA and ionomycin stimulation. Then
intracellular IFN-? production was analyzed in CD4?lympho-
cytes. Compared with control EAU mice, we found a minimal
increase of IFN-? expression per individual CD4?T cells in the
splenocytes of the animals that received OX40-activating anti-
body in vivo. The MFI of intracellular IFN-? in control and
OX40-activating antibody-treated groups was 138.05 ? 2.09
and 143.73 ? 1.55, respectively. This result suggested that the
augmented inflammatory cytokine expression was primarily
caused by expansion of the IRBP-reactive T-cell population.
Together, these data indicate that OX40 promotes the devel-
opment and expansion of uveitogenic memory lymphocytes.
Recent studies22–24have shown that decreased Blimp-1 and
increased Bcl-6 transcription regulators are instrumental in the
generation of memory CD4?precursors, respectively. To fur-
ther correlate OX40 activation with the development of mem-
ory lymphocytes during uveitis, we examined the ocular tran-
scription of IL-7R?, Bcl-6, and Blimp-1, markers associated with
memory T-cell differentiation in EAU (Fig. 7). Compared with
control mice, the ocular expression of IL-7R? and Bcl-6 tended
to be enhanced in both OX40-activating antibody-treated
groups regardless of the timing of OX40 activation (Fig. 7), and
the increase of ocular Bcl-6 transcription became statistically
significant in the mice that received OX40 activation during
IRBP sensitization (P ? 0.05). In contrast, a reduction of
Blimp-1 transcription (41.07 ? 16.28-fold) occurred in the eyes
of mice that received OX40-activating antibody during IRBP
priming. Activation of OX40 at EAU onset did not suppress
Blimp-1expression (data not shown), indicating that OX40 sig-
naling influences Blimp-1 regulation in a time-dependent man-
ner. Thus, this result suggests that further activation of OX40
may skew the memory T-cell response in the process of uveitis
development by regulating the expression of IL-7R, Bcl-6, and
In this study, we implicate OX40 in the severe ocular inflam-
mation of human patients. Moreover, further activation of
OX40 significantly exacerbates the severity of EAU. In addition
to expanding activated T cells, OX40 can potentially exert its
immunologic impact on memory T cells through the signaling
of IL-7R, Bcl-6, and Blimp-1.
CD62L?T cells and reduces CD4?CD44-CD62L?lymphocytes. EAU and
OX40-activating antibody treatment during IRBP161–180sensitization or at
uveitis onset are described. Three weeks later, the spleens were har-
vested, and peripheral T cells were assessed by flow cytometry for surface
markers of CD4, CD44, and CD62L. The change of naive and activated
CD4?cells was compared between control EAU and OX40-activating
antibody-treated groups (n ? 7 mice/group). *P ? 0.05.
OX40-activating antibody treatment increases CD4?CD44?
treatment augments IL-7R? expression
of peripheral CD4?CD44?T cells and
their response to IRBP161–180restimu-
lation. EAU and OX40-activating anti-
sitization or at uveitis onset are
described. Three weeks later, the
splenocytes were harvested and la-
beled with anti-CD4, anti-CD44, and
anti-IL-7R? antibodies. (A) IL-7R? ex-
pression in the CD4?CD44?popula-
tion was analyzed by flow cytometry
(n ? 7 mice/group). *P ? 0.05. (B)
Stimulation of splenocytes in vitro
with IRBP161–180(4 ?g/mL) for 3 days.
The IFN-? level in the culture media
was measured by ELISA (n ? 7 mice/
group). *P ? 0.05.
8524Wu et al.
IOVS, October 2011, Vol. 52, No. 11
OX40 is a key costimulatory molecule that is expressed 24
hours after T-cell activation. It has been shown to enhance
effector lymphocyte function and to promote memory T-cell
development.5,6,9,10In the B10.RIII EAU model, we found that
activation of OX40 during the IRBP161–180priming phase or at
disease onset markedly augments ocular inflammation. This
suggests that OX40 not only boosts the antigen priming pro-
cess but also amplifies the pathologic T-cell response.
It has been shown that both activated effector T cells and
Treg express OX40.25In contrast to our observations, Wein-
berg et al.17recently reported that OX40-activating antibody
ameliorates experimental autoimmune encephalopathy by ex-
panding Treg numbers during the antigen-sensitization period
before the disease onset. We also observed a potentially unique
effect of OX40 in the pathogenesis of uveitis. Activation of
OX40 at the time of IRBP161–180immunization markedly ex-
tended the disease course of EAU. These findings suggest that
aberrant OX40 signaling in uveitis may augment the effector
function and longevity of uveitogenic T cells.
Effector CD4?T cells can differentiate to Th1, Th2, and
Th17 subsets on the basis of distinctive transcription factor and
cytokine expression and function. These unique T-cell subsets
undertake special immunologic tasks and responsibilities. Add-
ing to the complexity of our immune system, some T cells are
found to coexpress cytokines representative of more than one
subset. Although we simplistically conceptualize that one dis-
tinctive T-cell subset mediates one particular disease, in reality
multiple T-cell lineages are often involved in uveitis and other
disease processes. Recently, Caspi et al.18demonstrated that
Th1 and Th17 cells are each capable of inducing EAU, depend-
ing on different antigen stimulation conditions. OX40 has been
shown to promote Th1 and Th2 differentiation.26,27We have
recently reported12that OX40 also augments Th17 effector
function. In this study, we demonstrated that the activation of
OX40 enhances the ocular expression of mRNA for IFN-? and
IL-17 in EAU, which suggests that OX40 promiscuously acti-
vates different T-cell subsets during inflammation.
After antigen encounter, some activated T cells become
long-lasting memory cells that are responsible for the antigen
recall response. Both effector and memory T cells contribute to
the chronic and relapsing course of uveitis. Consistent with
recent published studies,28,29we found that OX40 agonistic
antibody treatment significantly expands CD4?CD44?CD62L?
lymphocytes in the EAU model. In addition, the stimulation of
OX40 increases IL-7R? expression in this activated T-cell pop-
ulation. IL-7 is a common ? (?c) cytokine that plays an indis-
pensable role in memory T-cell development. IL-7 enhances
antiapoptotic gene Bcl-2 expression and inhibits proapoptotic
factors BAX and BAD.30,31In addition, the cellular response to
IL-7 is regulated by the expression of IL-7R. IL-7R consists of
IL-7R? and the ?c chain subunit. Distinct from other ?c chain
cytokine receptors that are upregulated in activated effector T
cells, IL-7R is primarily expressed by naive and memory lym-
phocytes, suggesting its critical role in supporting these two
T-cell populations. Indeed, studies32,33have demonstrated the
dependence of memory T-cell survival on IL-7 and IL-7R. Our
study has shown that OX40 primarily upregulates IL-7R? in
CD4?CD44?T cells, suggesting that these activated lympho-
cytes become memory T cells or memory precursors.
In addition to unique cytokine milieus, T-cell differentiation
requires intrinsic signals from master transcription factors.
Bcl-6 and Blimp-1 are reciprocal transcription factors that play
key roles in determining lymphocyte destiny.34They were
initially found to regulate B- and T-follicle helper cell differen-
tiation. However, the latest studies22–24demonstrate that Bcl-6
and Blimp-1 ubiquitously control the development of effector
and memory CD4?T cells. Bcl-6 promotes memory T-cell
development, whereas Blimp-1 enhances effector T-cell prolif-
eration and function. In addition, ?c cytokines have been
shown to induce the expression of Bcl-6 and Blimp-1.35Here,
we have shown that the activation of OX40 results in a recip-
rocal change of Bcl-6 and Blimp-1 in the eyes of the mice
developing EAU, thus further supporting the notion that OX40
promotes memory T-cell development in uveitis.
In summary, this study underscores the role of OX40 in the
pathogenesis of uveitis. It also implicates OX40 in the devel-
opment of uveitogenic memory T cells. Although OX40 could
directly upregulate IL-7R and Bcl-6 to facilitate the generation
of memory lymphocytes, at this time we cannot exclude the
possibility that the increase of IL-7R and Bcl-6 levels is second-
ary to the expansion of memory T cells that express these
molecules. This provides a rationale to further study how
OX40 regulates memory T-cell development. Further research
in this field is important not only for understanding the molec-
ular mechanism of T-cell regulation by OX40 but also for
identifying downstream therapeutic targets of OX40 signaling
to treat uveitis.
The authors thank Narsing Rao (Doheny Eye Institute) for providing
two surgical specimens of human nonseeing eyes, Isabella Phan for
evaluating the histopathology of human eye tissue, and Christopher
Corless and Cara Poage for technical assistance with the immunohis-
tochemistry of OX40 staining.
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EAU and OX40-activating antibody
treatment during IRBP161–180sensiti-
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