Journal of Autoimmunity 45 (2013) 7e14

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Journal of Autoimmunity
journal homepage: www.elsevier.com/locate/jautimm

The eye: A window to the soul of the immune system

V.L. Perez a, b, *, A.M. Saeed a, Y. Tan a, M. Urbieta a, F. Cruz-Guilloty a, b
a
b

Laboratory of Ocular Immunology and Transplantation, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
Department of Microbiology & Immunology, University of Miami Miller School of Medicine, Miami, FL 33136, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 18 June 2013
Accepted 18 June 2013

The eye is considered as an immune privileged site, and with good reason. It has evolved a variety of
molecular and cellular mechanisms that limit immune responses to preserve vision. For example, the
cornea is mainly protected from autoimmunity by the lack of blood and lymphatic vessels, whereas the
retinaeblood barrier is maintained in an immunosuppressive state by the retinal pigment epithelium.
However, there are several scenarios in which immune privilege is altered and the eye becomes susceptible to immune attack. In this review, we highlight the role of the immune system in two clinical
conditions that affect the anterior and posterior segments of the eye: corneal transplantation and agerelated macular degeneration. Interestingly, crosstalk between the innate and adaptive immune systems is critical in both acute and chronic inammatory responses in the eye, with T cells playing a central
role in combination with neutrophils and macrophages. In addition, we emphasize the advantage of
using the eye as a model for in vivo longitudinal imaging of the immune system in action. Through this
technique, it has been possible to identify functionally distinct intra-graft motility patterns of responding
T cells, as well as the importance of chemokine signaling in situ for T cell activation. The detailed study of
ocular autoimmunity could provide novel therapeutic strategies for blinding diseases while also
providing more general information on acute versus chronic inammation.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Allo-transplantation
Cornea
Retina
Inammation
Oxidative stress

1. Introduction
The great English poet and playwright William Shakespeare
once said: The Eyes are the window to your soul. Little did he
know that this organ would become a true window to the
mammalian immune system in centuries to come. Our laboratory
has used this window to visualize in real time the development of
ocular immune responses to allo and autoantigens to dissect
pathways of ocular immune regulation. This is of importance to us,
as the eye has being considered as an immune privilege site and
understanding the immune-regulatory pathways of this organ
could lead to novel observations in the eld of immunology. The
immune system is known to be either directly or indirectly implicated in a large array of ocular pathologies involving both the
anterior and posterior segments of the eye. Anterior segment pathologies with established immune system intervention include,
but may not be limited to, the following: viral and bacterialinduced keratitis, infectious and non-infectious (autoimmune)
* Corresponding author. Ocular Surface Center, Microbiology & Immunology,
Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638
10th Ave NW, Miami, FL 33136, USA.
E-mail address: Vperez4@med.miami.edu (V.L. Perez).
0896-8411/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jaut.2013.06.011

uveitis, dry-eye syndromes, oncogenic events such as uveal melanoma, and corneal allograft rejection following transplantation.
Diseases of the posterior segment in which inammation is
thought to play a role include age-related macular degeneration
(AMD), glaucoma, chorioretinal disorders and autoimmune retinopathy. In line with the interests of our laboratory, the rst half of
this review will primarily focus on the innate and adaptive immune
responses that occur following allogeneic corneal transplantation,
whereas the second half will review the current state of immune
responses to auto-antigens found in the retina or posterior
segment.
The rst description pertaining to the phenomenon known
today as ocular immune privilege was made by Dutch ophthalmologist J.C. van Dooremaals over a century ago after observing
that tumor cell inoculums injected in the anterior chamber of the
eye successfully proliferated and formed tumors [1]. However, it
was Sir Peter Medawar and his student Ruppert Everett Billingham
who, several decades later, coined the term immune privilege,
after conducting a series of transplant experiments utilizing
genetically disparate rabbit strains. These experiments led to the
observation that, regardless of genetic disparities, skin grafts
transplanted into the brain or anterior chamber (AC) of the eye
of recipient rabbits would survive for a longer period of time

V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14

compared to grafts transplanted elsewhere in the body [2].

Immunological privilege is thought to have evolved in part to
protect organs such as the brain, the eye, and the testis from loss of
function secondary to overwhelming inammatory responses.
Subsequent observations concerning ocular immune privilege led
to the hypothesis that the absence of lymphatic vessels in the AC of
the eye, as well as in other immune privileged sites, precluded the
immune system from detecting foreign antigens and therefore
initiating a response [3]. This hypothesis weakened in the late
1970s after Kaplan and Streilein documented the presence of
hemagglutinating antibodies in the serum of inbred rats shortly
after inoculation of allogeneic lymphoid cells in the AC and
demonstrating that intra-ocular antigens do elicit a systemic form
of immune-regulation, which was named anterior chamber associated immune deviation or ACAID [4e6]. Since then, this concept
has evolved and other mechanisms of immune regulation have
been shown to play a critical role in ocular disorders.
2. Immune responses in the anterior segment of the eye
2.1. Ocular immune responses to allo-antigens after corneal
transplantation
Immune reactions triggered by the presence of histoincompatible antigens are not exclusively limited to the protective ACAID
responses. This is evidenced by the fate of most corneal allografts
transplanted into tissue-incompatible recipients with highly vascularized corneal beds, referred to as high-risk recipients. Corneal
transplantation is the most common form of solid organ transplantation in the United States and is also the treatment of choice to
restore vision in patients with scarred and/or opacied corneas [7].
Despite its high rate of success in recipients with non-vascularized
corneal beds, corneal transplants performed in high-risk (vascularized beds) patients have a poor chance of survival [8]. Substantial
evidence now exists indicating that these corneal allografts undergo immunological rejection, a process in which allospecic T
cells are instrumental [9e11]. Our laboratory has a long standing
interest in utilizing real time imaging of uorescently labeled immune cells to elucidate the mechanisms that govern immune cell
recruitment to the site of transplantation, as well as how the tempo
and magnitude of cell migration impinge on activation and corneal
graft destruction.
The upregulation and expression of immune cell chemoattractants at the site of transplantation is a key feature of vascularized solid organ transplants [12]. Chemokines are small proteins
characterized by the presence and particular arrangement of four
conserved cysteine residues (C, CC, CXC, and CX3C) in their N-terminal region. More than 50 chemokines have been identied and
characterized, making this cytokine superfamily one of the largest
studied to date [13,14]. In addition to orchestration of directed cell
migration or chemotaxis, many roles have been ascribed to chemokines, including their participation in wound repair, inammation, angiogenesis, viral entry, tumor metastasis, and solid allograft
rejection [14]. The impact of chemokines in solid organ transplantation may extend beyond in-situ production of these molecules following transplantation. Studies by Brouard et al.
demonstrated that pre-transplant serum levels of specic T cell
chemokines such as CXCL9/MIG could be used as biomarkers of
acute allograft rejection [15].
We have previously documented the emergence of a specic
chemokine prole following corneal transplant surgery in murine
models of high-risk corneal transplantation [16]. This chemokine
expression pattern begins early on (days 1e7 post-transplant) with
upregulation of CXCL1/KC and CCL2/MCP-1, coinciding with the
peak of intragraft neutrophil inltration, and culminates with a

surge in T cell chemokine expression, namely CXCL9/MIG and

CXCL10/IP-10, concomitant with the peak of CD4 T cell inltration
and allograft rejection (days 11e14 post-transplantation) [16]. The
notion of chemokine regulatory cascades and their involvement in
other models of solid organ allograft rejection has been previously
examined [17]. Other relevant chemokine studies performed by
other groups have demonstrated an important role for chemokine
receptors that control immune cell migration from the site of
inammation to secondary lymphoid organs. A critical example of
such receptors is CCR7, which is expressed by resident antigen
presenting cells (APCs) of the cornea. CCR7 has been implicated in
promoting direct alloantigen recognition in the context of high-risk
corneal transplantation by facilitating migration of alloantigenloaded donor APCs into the draining lymph nodes [18].
Our current hypothesis sustains that early phase chemokines
such as CXCL1/KC are responsible for the recruitment of neutrophils
into the graft; once activated by the inammatory milieu within the
graft, these cells are capable of interferon gamma (IFNg) production, which would result in the upregulation of late phase chemokines (CXCL9/MIG and CXCL10/IP-10) responsible for
alloreactive T cell recruitment. We have found ample evidence in
support of this hypothesis. Experiments involving in vivo neutralization of CXCL1/KC with rabbit anti-sera resulted in prolonged
allograft survival in high-risk recipients. In line with our hypothesis, the same anti-KC treated animals showed signicantly
decreased numbers of CD4 T cell inltrates upon histological examination of corneal allografts [16]. Future studies in our laboratory
will explore the possibility of promoting long-term corneal allograft survival by administering anti-chemokine combination therapies aimed at reducing the number of innate and adaptive
immune cell inltrates.
2.2. The eye as the perfect setting for in vivo imaging of the immune
system
In vivo imaging has emerged as an indispensable tool in biological research, and a variety of imaging techniques have been
developed to noninvasively monitor tissues under living conditions. By virtue of its easy access, avascular and transparent structure, the cornea has been used as a natural body window for
noninvasive imaging to study the physiology of pancreatic islets
transplanted into the AC of the mouse eye in vivo [19]. More
recently, intraocular transplantation was used to noninvasively
study immune responses with single cell-resolution during rejection of islet allografts in the living animal [20].
Although corneal graft rejection is multifactorial where various
mechanisms and cell types are likely to be involved, it is well
accepted that CD4 T-cells play a crucial role in this process. In vivo
depletion of CD4 T-cells resulted in improved corneal allograft
survival [11,21e23]. Adoptive transfer of activated CD4 T-cells into
immune compromised mice induced rejection in 100% of corneal
allografts [23]. We reasoned that using high-resolution in vivo
imaging to study T-cell motility within corneal allografts could
provide a cellular mechanism that contributes to effector T-cell
function during allorejection. In order to visualize T cells in vivo, we
employed Bonzo mice as recipients for corneal transplantation.
Bonzo or CXCR6 (also known as CD186, STRL33, or TYMSTR) is a
putative chemokine receptor with unclear biologic function [24].
However, it is preferentially expressed on activated and memory Tcells [25e27]. In Bonzo mice, expression of the green uorescent
protein (GFP) is driven by the Bonzo gene promoter, allowing for
in vivo visualization of T cells [28,29]. Our group successfully used
uorescence confocal microscopy to obtain longitudinally images
after corneal transplantation. As early as post-operative day (POD)
1, Bonzo recipient GFP (both CD4 and CD8) T-cells appeared in

V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14

the host cornea of both syngeneic and allogeneic recipients, but not
within the grafts [30]. While syngeneic grafts remained relatively
clear of inltrating GFP T-cells throughout the follow up period, Tcells progressively inltrated the allogeneic grafts starting on POD7
and signicantly increased between POD14 and POD21 [30]. As
shown in Fig. 1, T cells can be seen throughout the ocular surface,
from host to the grafts, at POD21. Furthermore, 3-D time-lapse
recordings (20 min) enabled us to understand the dynamic
behavior of the inltrating T cells, which display different phenotypes (round, elongated, and rufed) with distinct morphological
and dynamic features [30]. Round cells appeared predominantly
spherical with low net translational movement. Fast moving elongated cells displayed ameboidal-type movement with a large
leading edge and a thin, long trailing tail (uropod) and traveled for
long distances (30 mm/20 min). Rufed cells, however, moved
vividly within shorter distances and tended to form clusters as they
engaged in simultaneous contacts with neighboring T-cells. In
syngeneic grafts, T-cells appear predominantly round with markedly low motility, while we found all cell types in allografts (Fig. 2).
The round and ruffed cells actively moved and continuously
changed shape as they migrated within the allograft tissue [30].
Since our previous work demonstrated an orchestrated production of chemokines after transplantation, we tested the hypothesis that CCL5/RANTES, CXCL9/MIG, and CXCL10/IP-10 direct
leukocyte migration and trafcking into the ocular tissue and shape
immune responses to allo-antigens by neutralizing them with
systemic administration of TAK-779, a highly selective antagonist of
CCR5 and CXCR3 [31,32]. Corneal allograft survival was signicantly
improved [30]. To determine the acute and local effects of CCR5/
CXCR3 blockade on cellular motility, we injected TAK-779 directly
into the stroma of corneal allografts during ongoing rejection. Local
TAK treatment resulted in signicant phenotypic and dynamic
changes in graft-inltrating T-cells; the majority of cells converted
from the predominant rufed to the round phenotype [30]. Such
changes associated with signicantly reduced displacement and
velocity of the cells (Fig. 3). More importantly, injection of a CXCL9/
CXCL10 mix subsequent to TAK-779 increased the proportion of
elongated and rufed T-cells and recovered the movement dynamics of the overall population to a higher motility state [30].
Our ndings demonstrate that, in addition to their involvement
in immune cell recruitment into corneal allografts, specic chemokines played an important role in mediating T-lymphocyte local
motility patterns, which signicantly inuenced T-cell activation
and effector function, and impacted on corneal graft survival. This

novel effect could only be studied by the in vivo imaging techniques

we used, which cannot be readily modeled in vitro. Therefore, the
orchestration of chemokine production is a critical feature of ocular
immune regulation of adaptive immune responses to allo-antigens.
3. Immune responses in the posterior segment of the eye
3.1. Inammation and the innate immune system in age-related
macular degeneration
Moving away from the anterior portion of the eye, the posterior
segment of the eye also houses an immunologically unique structure: the retina. The retina contains photoreceptor cells which
detect light and generate electrical impulses that eventually form
vision. Like the cornea, the retina also exhibits mechanisms of
immune regulation. Many mechanisms contribute toward retinal
immune protection, but relevant for our discussion are those
associated with a very important cell layer in the retina known as
the retinal pigment epithelium (RPE). RPE cells have a variety of
functions that maintain the health of the retina and its precious
photoreceptor cells (e.g., phagocytosis of damaged photoreceptors,
supply and transport of nutrients, recycling of retinol, etc.). The RPE
also aids in the retinas immune privilege by active and passive
mechanisms. The RPE is responsible for forming the blooderetinabarrier (BRB), which physically sequesters away retinal antigens
and keeps the systemic immune system from entering the retinal
space. The RPE also actively inhibits immune cells through
expression of surface ligands and secretion of cytokines that inhibit,
for instance, T cells and macrophages [33,34]. Thus, the healthy
retina is inherently averse to inammation.
However, the aging process disrupts the normal functioning of
the retina and especially the RPE. One the most prevalent diseases
of the retina is age-related macular degeneration (AMD), which is
the leading cause of blindness in the US and other industrialized
countries, largely due to a rise in the aging population. As its name
implies, AMD is a disease of the macula. The macula, the central
portion of the retina, contains the highest concentration of
photoreceptor cells and is responsible for central vision. A key
clinical nding in patients with AMD or in patients at-risk for AMD
development is drusen, which is photoreceptor cellular debris that
accumulates below the RPE and clinically manifests as small yellow
clumps during a funduscopic examination. Drusen is the biggest
risk factor that predicts AMD progression. Mechanistically, drusen
develops because with age, RPE cells have an impaired ability to

Fig. 1. In vivo imaging of T cell responses in allogeneic versus syngeneic cornea grafts. Inltrating T cells in the corneal grafts at post-operative day (POD) 14. Images were taken in
the center of the grafts. Many GFP T cells migrated into the allografts, displaying distinct morphological and dynamic phenotypes. Only a few GFP T cells were observed in
syngeneic grafts at POD14.

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V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14

Fig. 2. T cell distribution within allografts. Images were taken during ongoing rejection at POD21 spanning from central graft to the host bed, including the graft interphase. GFP T
cells with different morphological and functional patterns were seen throughout.

phagocytose damaged photoreceptor cells and cellular debris accumulates as a result. It has been recognized that RPE cell
dysfunction is closely associated with AMD [35].
The etiologic mechanisms behind AMD are not fully understood,
but mounting evidence supports a role involving retinal inammation and autoimmunity, at least in a subset of AMD patients. This
is best evidenced by the presence of complement proteins in drusen and the fact that polymorphisms in complement genes are
highly associated with AMD [35]. A striking 50e70% of AMD cases
are associated with single-nucleotide polymorphisms in complement factor H, factor B, or C2 genes [36e39]. Evidence for a role of
autoimmunity in AMD comes from the presence of autoantibodies
against retinal proteins detected in the serum of AMD patients
[35,40,41].
In addition to inammatory processes, oxidative stress has long
been recognized to play a pathological role in AMD. For example,

Fig. 3. Acute inhibition of chemokine receptor signaling alters T cell motility patters in
corneal allografts. TAK-779 (which inhibits CCR5 and CXCR3) was administered
through local injection in the cornea stroma at POD21. Flower plot representation of
movement trajectories of individual cells tracked within the allografts before and
14 min after intrastromal TAK-779 injection during ongoing rejection.

cigarette smoking and light exposure increase the risk of AMD,

while zinc and antioxidant vitamins are known to be protective
factors for AMD [42]. The aging process in general is associated with
the accumulation of oxidative damage. The retina, in particular, is
subjected to high levels of oxidative stress because of its exposure
to light, high oxygenation and continual RPE phagocytic activity
[43,44]. Thus, the retina likely accumulates a great deal of oxidative
insults over time and a persons intrinsic or environmental exposures will impact the degree of oxidative damage, dictating their
probability of developing AMD.
Oxidative stress, inammation, and autoimmunity are not
separate etiologic factors in AMD pathogenesis. In fact, work from
our group and others has shown that these factors are actually
linked in the context of AMD. Two examples involving lipid peroxidation products are highlighted: malondialdehyde (MDA) and
carboxyethylpyrrole (CEP). MDA and CEP have pro-inammatory
activity and interact with both the innate and the adaptive immune systems, although there are specic differences for each of
these immune responses in the process of AMD development.
As oxidative stress builds up in the retina, lipids found in the cell
membrane undergo lipid peroxidation. Oxidation of phosphatidylcholine, for instance, gives rise to the reactive degradation
product MDA, which is actually found in drusen [45]. Weismann
et al. recently discovered that MDA interacts with complement
factor H (CFH) [46]. CFH is centrally important in AMD and a
particular CFH polymorphism (yielding a missense mutation
[Y402H]) is highly associated with the AMD disease state [36e38].
Interestingly, this mutant version of CFH has an impaired ability to
bind MDA. This has very important immunological consequences
with respect to retinal inammation. As inammation and light
exposure induce retinal oxidative stress over decades, levels of
MDA become elevated. This MDA serves as a docking site for (wildtype) CFH, which acts to minimize inammation, and lessens tissue
damage. Mutant CFH, on the other hand, cannot localize to these
areas of oxidative stress and as a result inammation and tissue

V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14

damage proceed unchecked. The discovery of the interaction of CFH

and MDA is a striking example of where oxidative stress and immunity intersect and this interaction has a profound clinical
implication since a homozygotic CFH mutation carries a 6-fold increase of AMD development [47].
Oxidative damage to retinal tissues induces inammation and is
capable of creating novel self-antigens, which trigger a pathological
autoimmune process targeting the macula/retina. In terms of possible
self-antigens, our lab has previously explored the role of oxidatively
modied lipideprotein adducts as potential initiating signals. Evidence for oxidatively modied proteins in AMD pathogenesis stems
from the analysis of drusen. Crabb et al. analyzed drusen from AMD
patient donor eyes with mass spectrometry and identied over a
hundred different proteins [48]. Interestingly, a signicant number of
the proteins had oxidative protein modications. In particular, proteins were covalently cross-linked with a reactive lipid moiety called
carboxyethylpyrrole (CEP). CEP is formed from its precursor docosahexanoic acid (DHA) under conditions of oxidative stress [49]. Once
generated from DHA, CEP then condenses with amino acids of proteins (lysine, cysteine, histidine) forming covalent adducts. Interestingly, DHA-containing lipids are highly abundant in the RPE and
photoreceptor cells and DHA also happens to be the most oxidizable
fatty acid in the body [49]. Since the retina is inherently a highly
oxidizing environment, it provides an optimized setting for the
generation of CEP-adducted proteins. Over time, the aging process
likely drives the accumulation of CEP-adducts.
Work from Gu et al. further implicates CEP-adducted proteins
with AMD. They observed greater amounts of CEP-adducts in the
RPE and photoreceptor cells of AMD patient donor eyes, when
compared to healthy donor eyes [50]. Furthermore, they found CEPadducted proteins in the blood of AMD patients and found higher
levels of CEP auto-antibodies in AMD patient sera (versus agematched healthy controls) [50,51]. Importantly, this last result
demonstrates that CEP-adducts are immunogenic and the host
mounts an adaptive immune response against CEP. Our group
demonstrated that CEP is not only immunogenic, but that CEP can
induce an immune-mediated attack upon the retina [52]. Young
healthy mice were immunized with CEP-adducted self protein
(albumin) and their eyes were analyzed to determine if AMD-like
lesions developed (Fig. 4). The rationale for this approach is that
CEP-immunization would greatly enhance the sensitivity of the
immune system to react to endogenously generated CEP. This high
sensitivity could conceivably promote immune recognition of even
small, basal levels of CEP-adducts that may be present in the eyes of
otherwise healthy mice. Therefore, immunized mice would
generate a pathological auto-immune response targeting the
retina. The results from these experiments show that CEPimmunized mice develop an antibody immune response. Not
only do CEP-immunized mice develop anti-CEP antibodies, but

11

these mice also develop retinal lesions and RPE damage. The pathology observed in CEP-immunized mice mirrors that of human
AMD pathology. Furthermore, it was found that the degree of
retinal pathology directly correlated with the degree of CEP antibody production [52].
In addition to retinopathy, CEP-immunized mice were characterized by retinal deposition of complement proteins (C3d) and the
appearance of retinal inltrating macrophages [52]. The role of
macrophages in the pathogenesis of AMD remains controversial,
due to contradictory ndings from transgenic and retinal injurymediated animal models [53]. However, emerging evidence
points towards a mechanism by which macrophages accumulate
within the retina with aging. Progression towards AMD does not
depend on the simple absence or presence of macrophages, but
depends on the type of macrophages present. Macrophages can
adopt different polarization states with distinct effector functions
and this may dictate disease outcome. Data from our laboratory
demonstrated that the macrophages observed in the CEPimmunization model are inammatory (IL-12 and TNF-a-producing) macrophages [54]. Inammatory (also known as M1) macrophages have a tissue destructive role, as opposed to other types of
macrophages (like tissue remodeling M2 macrophages) [55]. In
support of this, Cao et al. demonstrated a general accumulation of
retinal macrophages associated with age [56]. Interestingly, they
found an increased M1 to M2 ratio in patients with AMD, when
compared with retinal samples from age-matched controls [56]. It
is possible that these M1 macrophages (perhaps in response to
complement deposition or chemokine-regulated recruitment)
mediate RPE-injury.
Retinal inltrating macrophages could be initiating destruction
or responding to the tissue damage. Evidence from our lab suggests
that these macrophages might be actually inducing the observed
retinal damage. For instance, it is possible that the observed complement deposition on the RPE may prime these cells for opsonization by macrophages. Additional evidence for macrophages
inducing/causing damage is that macrophage inltration temporally precedes lesion development [54]. Furthermore, mice with
impaired macrophage-recruitment ability (CCR2 knockout mice),
failed to develop CEP-induced retinopathy [54]. Therefore, macrophages seem to have a causative role in retinal injury in conditions
associated with M1 polarization.
3.2. The adaptive immune system in age-related macular
degeneration
Overall, the CEP model of AMD points to the conclusion that
AMD is an autoimmune disease in which a lipid peroxidation
product coordinates both a pathological innate (as evidenced by
complement and macrophages) and adaptive (as evidenced by

Fig. 4. Immunization with CEP-adducted mouse serum albumin (CEP-MSA) leads to macrophage inltration into the retina and AMD-like pathology in mice. Eyes were harvested at
day 150 post-immunization from CEP-MSA immunized (versus nave) C57BL/6 mice, and histology was performed as described [52]. Nave retinas look normal and devoid of
inltrating cells (left image), whereas CEP immunization results in the recruitment of macrophages to the outer retina (center image) and focal lesions of the RPE and photoreceptor
outer segments (right image). The RPE is located at the lower part of each image.

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V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14

antibody production) immune response directed against the retina.

Additional evidence strengthens the idea that AMD is an autoimmune disease and stems from work in both animal models and
clinical studies implicating T-cells in the pathogenesis of AMD.
While appreciably little is known about the potential role of
adaptive immunity, specically T cells, in the initiation or regulation of AMD pathology, this is a current area of increased interest in
the eld. Most of the evidence for adaptive immunity in AMD
comes from the presence of anti-retinal autoantibodies in AMD
patients, which is recapitulated in our CEP mouse model [52,57,58].
Despite the fact that autoantibodies are associated with AMD, it is
still unknown whether they cause or protect from damage, or if
they simply arise as secondary effects and have no specic role in
disease pathogenesis. On the other hand, increased IgG ratios in
AMD patients could point to a productive immune response that
includes antigen-specic T cells [58]. In this context, oxidation
specic epitopes (OSEs), such as MDA and CEP, could provide
the signals from the outer retina that initiate inammation
and possibly involve the adaptive immune system in AMD
pathogenesis.
T cells constitute the main effector cells of the adaptive immune
response, providing the antigen specicity lacking in innate immunity. One of the primary jobs carried out by T cells is the
orchestration of appropriate responses by recruiting other immune
cells and shaping the type of response based on the cytokines they
produce. The study of experimental autoimmune uveitis (EAU), an
animal model in mice and rats of the inammatory human disease,
has greatly inuenced our understanding of T cell-mediated autoimmune responses in the retina. EAU is induced by immunization
with retinal antigens and susceptibility depends on the genetic
background of the host: C57BL/6 (B6) mice are susceptible while
BALB/c mice are resistant. It has been shown that both Th1 and
Th17 cells can mediate EAU pathology depending on the context of
initial antigen exposure, suggesting a role for antigen presenting
cells (APCs) in dictating pathology outcomes [59]. Further complicating the picture is the fact that retinal antigens can induce regulatory T cell (Treg) peripheral tolerance [60]. As for the role of T
cells in AMD, surprisingly little information is available in the
literature. Robert Nussenblatts group showed that complement
component 5a (C5a) can induce IL-22 and IL-17 expression in human CD4 T cells and that elevated levels of these cytokines were
present in AMD patients [61]. Furthermore, they also showed that
the IL-17RC promoter region is preferentially hypomethylated in
AMD patients [62]. Taken together, these data show the ability of T
cells to respond to retinal antigens, to affect tissue integrity and to
potentially be involved in AMD.
In addition to our published data showing that M1 macrophages
play a role in the onset of disease [54], additional data strongly
suggest that CEP-specic T cells play a leading role in the initiation
of AMD in our model (FCG, VLP, unpublished observations). MDAspecic T cells have been reported and shown to be involved in
the pathology of atherosclerosis [63], which proves that antigenspecic T cells can indeed recognize lipid peroxidation products
that modify proteins and serve as haptens. This antigen specicity
is provided by unique T cell receptors (TCR) on the surface of T cells,
but to date not a single TCR against lipid modications of proteins
has been isolated. In the future, the analysis of CEP and/or MDAspecic T cell clones will shed light on relevant pathways (Th1/Tbet; Th2/Stat6; Th17/Ror-gt) that mediate responses against
oxidative damage, in a context-dependent manner. Interestingly,
MDA-specic T cells adopt a Th2 phenotype [63], whereas CEPspecic T cells produce IFNg and IL-17 (FCG, VLP, unpublished observations). In the case of AMD, pro-inammatory cytokine production by antigen-specic T cells may contribute to the
polarization of macrophages toward the M1 phenotype, providing a

possible link between adaptive and innate immunity in pathogenesis. However, the precise mechanisms of T cellemacrophage
interactions in chronic inammation (as opposed to acute infection
models) remain to be determined.
In the original publication of our model, we reported that CEPinduced AMD-like pathology does not develop in RAG/ mice,
which lack an adaptive immune system [52]. Therefore, a major
question in our model is the role of autoantibodies (produced by B
cells) in the generation of retinal lesions or whether the model
represents a T cell-mediated disease. To address this issue, C57BL/6
mice decient in mature B cells (mMT/ mice) were immunized
with our protocol. Although no CEP antibodies were detectable,
splenic T cells from immunized mice were activated in response to
in vitro stimulation with CEP and, importantly, strong retinal lesions
were observed in these B cell-decient mice (FCG, VLP, unpublished
observations), indicating that the observed pathology is independent of B cells (and their antibodies). This result clearly points to T
cells as the leading players within the adaptive immune system
associated with AMD in our model. However, it does not necessarily
mean that antibodies are not involved, in some cases and at some
capacity, in the AMD disease process. It is still possible that antiretinal antibodies can x complement in the outer retina or
contribute to macrophage-mediated destruction of retinal structures. Alternatively, at least a proportion of autoantibodies may
actually serve a protective role. For example, autoantibodies against
CFH were detected at lower levels in AMD patients compared to
age-matched controls [64]. Regardless of their individual activities,
autoantibodies are still the most likely and accessible candidates for
the development of AMD biomarkers, although each specicity will
require detailed functional analysis to uncover disease-related effects. Proling autoantibody signatures, as opposed to single antibodies, may prove useful in this regard.
The similarities between human AMD patients and the CEP
immunized mice support the notion that our model recapitulates
essential features of AMD pathogenesis and progression. Therefore,
it provides new avenues to study the onset factors involved in AMD.
Many important questions remain to fully elucidate the T cell
pathways relevant to the onset and progression of disease. Translation of ndings in our animal model into clinically relevant
strategies in human AMD patients will be crucial. Will immunological intervention be successful for novel prevention and/or
treatment protocols for AMD? Answers to these questions
regarding ocular disease from an immunological viewpoint could
lead to innovative treatments for this prevalent condition.
4. Conclusion
In conclusion, the eye is the window to the soul (of the immune
system) and can be successfully used to understand the mechanisms behind immune responses in transplantation and autoimmunity. Furthermore, the eye is an easily accessible organ to
evaluate and treat with therapies that will be developed from the
understanding of pathways that lead to ocular immune regulation.
5. Final comments
This paper is dedicated to Abul Abbas from Victor L. Perez:
A great mentor, teacher and friend. It is part of this dedicated issue
that addresses and recognizes unique people from the perspective
of teaching, research, public service and their implications for
autoimmunity and the patients who suffer from autoimmune disease [65e67]. Many people are lucky enough to have a good scientic mentor, but there are only a few that have a mentor who is
not only a great teacher, but a true friend as well. I am one of those
few individuals. Thanks to Abul, I developed the tools to become an

V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14

immunologist, tools that allowed me to fulll my dream of being a

clinician scientist that can use my laboratory to develop new concepts to treat blind patients and improve their quality of life.
Moreover, when I have doubts regarding what to do with any issue
in my life, I ask myself what would Abul do? and the path becomes very clear. Thanks Abul, for your inuence in my life and
giving me the opportunity to love and enjoy immunology.
Financial support
This work was supported by the National Eye Institute, National
Institutes of Health R01 EY018624-04 (VLP), The Edward N. & Della
L. Thome Memorial Foundation Bank of America N.A. Trustee
Award Program in Macular Degeneration Research (VLP), NIH
P30EY14801 (Center Grant), Research to Prevent Blindness (Unrestricted Grant to the Bascom Palmer Eye Institute). A.M.S. acknowledges partial support and assistance from the Sheila and
David Fuente Graduate Program in Cancer Biology, Sylvester
Comprehensive Cancer Center. FCG is a Howard Hughes Medical
Institute Fellow of the Life Sciences Research Foundation.
Acknowledgments
We thank members of the Perez lab, past and present, for their
contributions to this work. We also thank the active ocular
immunology community for applying the principles of autoimmunity to the eye to shed light on major clinical problems affecting
vision.
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