Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells
Journal of Neuroinflammation
Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells
Scott W McPherson 0
Neal D Heuss 0
Mark J Pierson 0
Dale S Gregerson 0
0 Department of Ophthalmology and Visual Neurosciences, University of Minnesota , Rm. 310, Lion's Research Bldg.,2001 6th St. SE., Minneapolis, Minnesota 55455-3007 , USA
Background: We previously reported that the peripheral regulatory T cells (pTregs) generated 'on-demand' in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo. Methods: Transgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (gal) as retinal neo-self antigen and gal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination. Results: Local depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in gal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal gal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local gal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after gal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation. Conclusions: Our results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.
regulatory T cells; peripheral regulatory T cells; autoimmunity; dendritic cells
While various types of T cells possess regulatory activity
[1,2], it is well established in mice that CD4+CD25+
FoxP3+ T cells are a dominant regulatory T cell (Treg)
and indispensible for immune homeostasis [3,4]. FoxP3+
Tregs are broadly categorized by their origin as either
thymically-derived Tregs (tTregs) or as
derived Tregs (pTregs) . Most FoxP3+ Tregs are
tTregs. Their generation, along with negative selection
of strongly autoreactive T cells in the thymus, provides a
foundation against autoimmunity. pTregs are thought to
assist tTregs in limiting autoimmune inflammation .
In addition, pTregs are important in regulating immune
responses to external antigens (Ag) encountered in the
gut or airway, providing maternal-fetal tolerance [7-9],
as well as tolerance to commensal microbiota [10-12].
Analysis of pTregs for their overall contribution to the
Treg population, as well as their exact sites of generation
and action (within a specific tissue or nearby secondary
lymphoid tissue), is a matter of ongoing investigation.
Limiting these studies is the lack of unique marker(s)
distinguishing tTregs and pTregs. Some reports suggested
the transcription factor Helios  and neuropilin-1 
specifically marked tTregs. Other studies argued that
Helios was upregulated in pTregs and activated T cells
[15,16] and was not expressed in all tTregs . Likewise,
neuropilin-1 might not be a definitive Treg subset marker
as pTregs expressed it during inflammation , and its
expression was influenced by the local cytokine milieu,
particularly TGF- , a factor known to be critical for
pTreg development. Nonetheless, it is well established that
Tregs are generated in secondary lymphoid tissue as a
result of interaction between Ag-bearing dendritic cells
(DC) and T cells, and exert their regulatory action by
limiting the priming of T cells . Tregs are also known to
reside and accumulate in non-lymphoid tissue, especially
in response to inflammation. However, it has been difficult
to determine if these tissue Tregs resulted from
immigration of tTreg or were pTregs generated in secondary
The accumulation of Tregs in non-lymphoid tissues
also allows for the possibility that they might be
generated directly within the tissue from nave or effector T
cells (Teffs). A recent study showed pTreg development
dependent on resident lung macrophages . Although
the macrophages were isolated, Ag-pulsed, and then
reintroduced in recipient mice by intra-tracheal transfer
along with FoxP3 T cells, the resulting pTregs and
macrophages were primarily found in lung tissue and not in
the mediastinal lymph nodes. The retina, because of its
apparent lack of lymphatic drainage  and high
concentration of TGF-  and retinoic acid , might
also be a site for tissue-specific pTreg generation.
Indeed, injection of nave T cells specific for the retinal Ag
interphotoreceptor retinoid binding protein (IRBP) into
the posterior segment of the eye resulted in their
conversion to FoxP3+ Tregs . Subsequently, we expanded
on this result demonstrating that FoxP3+ Tregs specific
for retinal Ag were generated within the retina from
circulating Ag-specific FoxP3 T cells . Further, these
pTregs provided a local, specific protection against
experimental autoimmune uveoretinitis (EAU) induced by
Ag immunization or adoptive transfer of activated,
retinal Ag-specific T cells.
Given that central tolerance (negative selection and
tTreg generation) to self-Ags is not always complete
, our results implied that local, on demand
generation and action of pTregs for tissue specific self-Ags
provided a crucial secondary mechanism for immune
homeostasis that functioned in immune privileged
tissues. Critical to the development and activation of
pTregs is interaction with appropriate antigen presenting
cells (APC). Microglia (MG) have long been considered
to be the primary APC of CNS tissue [28-33]. In
quiescent CNS, particularly the retina, there is controversy
about the existence, function, and significance of DC as
APC [34-37]. However, there is a growing body of
evidence for DC in the CNS . We recently demonstrated
the presence of DC in quiescent retina, their expansion in
response to neural injury, and their capability as APC
in vitro to generate Teffs and Tregs [39,40].
In this study, we investigated the role of locally
generated, locally acting pTregs in immune homeostasis and
the ability of retinal DC to support T cell activation and
expansion within the retina. Using mice that express E.
coli beta-galactosidase (gal mice) as a retinal neo-self
Ag along with CD4+ T cell receptor transgenic (TCR-Tg)
mice specific for gal, and mice with selectively
depletable Tregs or DC, we found that local depletion of Tregs
from the retina was sufficient to permit development of
spontaneous EAU and that local DC, not MG, were
necessary to generate Ag-specific T cell responses within
the retina that included pTreg generation.
Materials and methods
The gal mice (B6-arrgal mice, MHC haplotype I-Ab or
B10.A-arrgal, MHC haplotype I-Ak) have been described
in detail elsewhere [26,41-43]. Briefly, rod photoreceptor
cell expression of gal mimics that of endogenous arrestin,
producing approximately 150 ng gal/retina and < 0.5 ng
gal/pineal gland. Analysis of tissue for gal expression
was done as described  with the following
modifications: the 12 M cryostat sections of OCT-embedded
tissue were fixed in PBS with 4% paraformaldehyde
and 0.25% gluteraldehyde for 7 min and then incubated
with X-gal substrate (5-bromo-4-chloro-3-indolyl
-Dgalactoside) for 2 to 24 h. The galTCR mice (B10.A) and
the BG2 mice (C57BL/6 J) mice carry MHC class II
restricted (CD4+) T cells that recognize gal protein,
specifically epitopes YVVDEANIETHGMV (galTCR) or
SVTLPAASHAI (BG2), and have been described
elsewhere [44,45]. The FG mice, which express green
fluorescent protein (GFP) only under control of the endogenous
FoxP3 promoter, and FDG mice, which express diphtheria
toxin receptor (DTR) and GFP under control of the
endogenous FoxP3 promoter, have been described [4,46];
both are C57BL/6 J. The CDG mice (C57BL/6 J) express a
chimeric GFP and DTR under control of a transgenic
CD11c promoter . Rag/ mice (RAG-2/ mice, stock
# 008449) were obtained from breeding stock purchased
from Jackson Laboratory (Bar Harbor, ME, USA). All mice
were negative for the rd8 mutation associated with retinal
degeneration . All mice were handled in accordance
with the Association for Research in Vision and
Ophthalmology (ARVO) Statement for the Use of Animals in
Ophthalmic and Vision Research, and the University of
Minnesota Institutional Animal Care and Use Committee
guidelines. Mice were housed under specific pathogen-free
conditions on lactose-free chow.
Induction and transfer of regulatory T cells
For induction of pTregs in response to soluble Ag, FG
mice were injected intravenously (i.v.) with 100 g of
gal or bovine serum albumin (BSA) solubilized in
phosphate buffered saline (PBS) at 1 mg/mL. At 8 days
post-injection, CD4+ T cells were isolated from pooled
lymph nodes (LN) and spleens by magnetic separation
(Miltenyi, San Diego, CA, USA) and then sorted by
fluorescence-activated cell sorting (FACS) for GFP+
(FoxP3+ Tregs) cells. The cells were washed and
resuspended in PBS to 5 106/mL. For induction of pTregs
in response to retinal gal, magnetically purified CD4+ T
cells from normal FG-BG2 double transgenic mice were
sorted by FACS for GFP cells. The cells were washed
and resuspended to 5 105/mL. Cells were transferred
i.v. with recipient mice and number of cells transferred
Diphtheria toxin (DTx), gal, and saline injections into
the eye were done by trans-corneal deposition into the
anterior chamber (AC) as previously described . One
microliter doses containing saline or the indicated amount
of DTx or gal were given. Systemic depletion of Tregs or
DC was done by intraperitoneal (i.p.) injections of DTx
with dose and timing indicated.
Analysis of the delayed-type hypersensitive response and
Analysis of the delayed-type hypersensitive (DTH)
response (ear swelling assay) was done by injection of
gal (50 g in 10 L) into the ear pinna as previously
described . Enucleations were done as previously
Induction and analysis of autoimmune disease
EAU was induced by subcutaneous (s.c.) immunization
of mice with a single 200-L dose containing a total of
200 g of mouse IRBP peptides 1 to 20 (kindly provided
in part by Dr. R. Caspi) and 461 to 480 (100 g
each peptide) emulsified in complete Freunds adjuvant
(CFA) containing 5 mg/mL Mycobacterium tuberculosis
(H37Ra, Sigma, St. Louis, MO, USA) followed by 0.5 g
pertussis toxin (Sigma) per mouse given in 100 L saline
i.p. At 21 days post-immunization, the eyes were
harvested, fixed in 10% buffered formalin, paraffin
embedded, sectioned (5 M), and stained with hematoxylin
and eosin. The slides were examined in a masked
fashion and the induced EAU was scored from 0 (no disease)
to 5 (complete loss of photoreceptor cells plus damage
to the inner layers of the retina) based on
histopathological changes in the retina .
Pooled spleen and LN cell suspensions from the
indicated mice were prepared by tissue homogenization
followed by filtration through a 70 M cell strainer.
Lymphocytes were also prepared from whole blood. Red
blood cells were lysed using 0.17 M NH4Cl, and the
remaining cells were washed twice in PBS with the final
suspension made in FACS buffer (PBS with 2% FCS and
0.02% sodium azide). 0.25 to 2.0 L/106 cells of the
appropriate fluorescent-labeled antibodies (BD Biosciences,
San Jose, CA, USA or eBioscience, San Diego, CA, USA)
were added to the cell suspension and incubated on ice
for 30 min. The cells were washed, resuspended in FACS
buffer, and analyzed on FACSCalibur or FACSCanto flow
cytometers using CellQuest (BD Biosciences) or FlowJo
(Tree Star, Ashland, OR, USA) software. CD4+ T cells
from immunized FG mice and normal BG2 x FG mice
were sorted into regulatory (GFP+) or effector (GFP)
populations using a FACSAria flow cytometer (BD
Bioscience). For analysis of retinal cells by flow cytometry,
mice were euthanized, perfused, and the retinas removed
as described . Control experiments showed that the
perfusion procedure effectively removed passenger cells
from the retinal vasculature so that their contribution
was not significant (data not shown). The retinas were
dissociated using a solution of 0.2 g/mL Liberase/TM
(Roche, Indianapolis, IN, USA) and 0.05% DNase in
PBS, and stained with the indicated antibodies. Gating
strategy and analysis of retinal mononuclear cells and
lymphocytes has been described [26,40]. For the purpose
of analysis, a single sample comprised all cells collected
from a single retina.
Beta-galactosidase (gal)-specific regulatory T cells are
made in the periphery and modulate CD4+ T cell
responses to gal
The lack of gal expression in the thymus of gal mice
by reverse transcription PCR (RT-PCR)  and X-gal
staining (Figure 1A) suggested that most gal-specific
Tregs are likely pTregs. This was supported by our
studies that showed that injection of Ag into the nave
mouse eye led to local generation of pTregs that
inhibited EAU . In further support, we sought additional
direct evidence for the peripheral generation of
galspecific Tregs, evidence for their function both in the
retina and systemically, and evidence that the presence
of the retina affected the generation of circulating
To demonstrate that functional, gal-specific, FoxP3+
pTregs could be generated in vivo, we asked if Tregs
induced by administration of soluble Ag  and then
transferred into BG2 mice could suppress the DTH
response of gal-specific CD4+ BG2 T cells. FG mice were
Figure 1 Regulatory T cells (Tregs) specific for beta-galactosidase
(gal) were generated in the periphery and modulated a CD4+ T
cell mediated delayed-type hypersensitivity (DTH) response. (A)
X-gal staining of retina and thymus from a gal mouse and control
B6 mouse thymus. Photoreceptor (PC) layer of gal mouse retina
indicated showing intense X-gal staining in the outer plexiform layer
(top) and outer segments (bottom). Retinas were incubated for 2 h and
thymus for 24 h with X-gal. (B) Inhibition of CD4+ T cell (BG2)-induced
DTH to gal in mice receiving gal specific Tregs. Transferred mice
received 5 x 105 Tregs intravenously (i.v.) from mice injected with
bovine serum albumin (BSA) or gal. Recipient mice were given gal in
the ear and then measured for ear swelling at the indicated times
post-gal injection. (C) Percent of pooled CD4+ T cells that are Tregs
(GFP+) in control and antigen (Ag)-inoculated FG mice. (D) Enhancement
of BG2-induced DTH following systemic depletion of Tregs. Mice were
depleted by 250 ng DTx given intraperitoneally (i.p.) on days 0, 3, and 6,
followed by gal in the ear on day 7. Ear swelling was measured at
indicated times post-gal injection. (E) Analysis of Treg levels in the
blood of control mice and FDG-BG2 mice given DTx i.p. at the time of
gal injection in the ear (day 7). Results are given as mean SD with
P values determined by t test.
injected i.v. with gal or BSA. After 8 days, GFP+ Tregs
from the gal and BSA treated mice were purified by
FACS and transferred into separate groups of nave BG2
mice. The recipient BG2 mice were then analyzed for
their DTH response to gal. Control BG2 mice had a
significant DTH response to gal compared to nave B6
mice (Figure 1B). After transfer, BG2 mice that received
Tregs from BSA treated mice had no reduction in ear
swelling compared to control mice, while mice receiving
Tregs from gal treated mice showed a significant
reduction in ear swelling (Figure 1B). FACS analysis
showed that the size of the overall Treg population
measured as a percent of circulating CD4+ T cells was
unaffected by Ag injection (Figure 1C), a finding not
unexpected given that homeostatic mechanisms tightly
regulate the overall size of the T cell pool and its subsets,
including Tregs [53-56]. However, the results indicated
that Ag-specific, gal-induced pTregs were generated in
sufficient numbers to have a significant effect on the
DTH response of gal-specific CD4+ BG2 T cells.
Conversely, we also examined whether removal of Tregs would
enhance the DTH response to gal. FDG-BG2 double Tg
mice were depleted of Tregs by systemic injections of DTx
and then assayed for their DTH response to gal. Treg
depleted FDG-BG2 mice had significantly increased ear
swelling compared to control FDG-BG2 mice (Figure 1D,
top). To show that the increase ear swelling was specifically
due to Treg depletion, we also compared the DTH
response in DTx and non-DTx treated BG2 mice (Figure 1D,
bottom) and found it was similar to each other and to the
undepleted FDG-BG2 mice. Analysis of blood for
circulating Tregs showed that DTx treated FDG-BG2 mice
were substantially depleted of Tregs compared to control
FDG-BG2 mice (Figure 1E). Although the mice in these
experiments do not carry the gal transgene, the results show
a detectable level of control of the BG2-mediated DTH
response by spontaneously generated Tregs, and that addition
(Figure 1B) or depletion (Figure 1D) of even a small
number of circulating, gal-specific Tregs can modulate the
immune response to gal in a site not thought to be immune
Since greater than 99.8% of the gal in gal mice is
expressed in the retina, a comparison of mice with or
without the principal gal source removed by
enucleation would test the role of the retinal Ag in
galspecific pTreg development. Mature, nave CD4+ T cells
from FG-BG2 double Tg mice were sorted by flow
cytometry for GFP (FoxP3) cells and transferred into normal
or enucleated B6-gal x Rag/ mice. Since Rag/ mice
lack T cells, including endogenous Tregs, all new Tregs
must be pTregs derived from the transferred FoxP3 T
cells. When challenged with Ag to determine specificity,
recipient mice totally lacking gal (Rag/ mice) or lacking
retinal gal (enucleated B6-gal x Rag/ mice) had an
equivalent elevated DTH response compared to gal+
recipients (Figure 2A), suggesting that the retinal gal
promotes the generation of functional retinal Ag-specific
pTregs from nave, mature precursor T cells in the
periphery. To assess the stability of retina-dependent pTregs,
DTH responses were also analyzed in B10.A-gal x Rag/
mice where precursor T cells were provided in the mice by
their also having a galTCR transgene. At 4 months
postenucleation, we observed similar levels of DTH inhibition
in both normal and enucleated galTCR x B10.A-gal x
Rag/ mice compared to similarly treated galTCR x
Rag/ mice (Figure 2B). Together, these results show
retinal gal was the primary Ag source for gal-specific
pTreg formation and that once formed, functional
galspecific Tregs persisted in circulation even if the source of
Ag was removed.
Local regulatory T cell depletion from the retina induces
spontaneous experimental autoimmune uveoretinitis
B6 mice are minimally permissive for experimental
autoimmune uveoretinitis (EAU) [57,58]. Recently, we
demonstrated that cells expressing DTR could be locally
eliminated from the retina by AC injections of DTx
[39,40] and that depletion of retinal Tregs in B6-gal
mice enhanced gal-mediated EAU induced by adoptive
transfer or immunization . To confirm the
importance of local Tregs within the retina, we sought evidence
that they were also protective against spontaneous
autoimmunity. Control B6 mice and strains expressing DTR
in FoxP3+ cells were given various regimens of DTx into
the right AC and analyzed after 3 weeks of treatments
(Figure 3A). Control B6 mice given AC DTx did not
exhibit any signs of retinal autoimmunity. FDG and
galFDG mice each exhibited a rare incident of retinal
autoimmune disease (1/71 and 1/77, respectively) following
AC depletion of Tregs (Figure 3A). Given that FDG and
gal-FDG mice highly express a number of known,
endogenous, immunopathogenic, retinal auto-antigens (for
example, IRBP, opsin, arrestin, and recoverin), but do not
have an elevated frequency of one particular retinal-Ag
specific Teff population, the resulting T cell-mediated
autoimmunity could be directed against any of several
retinal self-Ags. As expected, FDG-BG2 mice did not develop
retinal autoimmunity, as their Teffs are largely
galspecific thus having only a limited repertoire against
selfAgs . However, mice having a high frequency of Teffs
specific for gal plus cognate gal Ag expression in the
retina (gal-FDG-BG2 mice) did develop EAU at a
significant rate (15/82, 18.3%) following retinal Treg depletion,
with a general trend of increased incidence with higher
DTx doses (Figure 3A). The left eyes of most of the
gal-FDG-BG2 mice were also examined and found to be
negative for EAU (0/74, data not shown). Further,
galFDG-BG2 mice that were given AC saline injections did
not develop retinal autoimmunity in that eye (0/22,
Figure 3A, bottom). The lack of disease in the left eyes of
DTx treated mice and in the saline injected eyes
demonstrates that the autoimmunity in DTx treated eyes was
specifically due to the local depletion of Tregs from the
retina and that Tregs need to be present within the retina
to be protective. Analysis of circulating Tregs showed no
difference between nave and AC DTx treated
gal-FDGBG2 mice (Figure 3C), again supporting the idea that
circulating Tregs play a secondary role in protection of
the retina from autoimmunity relative to the contribution
of local Tregs.
Prolonged, systemic regulatory T cell depletion in
gal-FDG-BG2 mice induces spontaneous experimental
Although DTx given systemically can penetrate the
retina and deplete DTR+ cells from the retina , it was
of interest to observe in our previous studies  that
EAU induced by gal immunization or adoptive transfer
of activated, gal-specific T cells could not be enhanced
by systemic DTx treatment. Since immunization and
adoptive transfer protocols result in only a brief window
for a limited number of activated T cells to elicit EAU,
we hypothesized that systemic DTx treatment of
galFDG-BG2 mice might generate EAU at a high rate since
the retinas in these mice would be exposed to a very
high number of Ag-specific Teffs, as depletion of Tregs
would lead to polyclonal T cell activation. The highest
dose of AC DTx (25 ng, 3x per week, 3 weeks) when
delivered systemically (systemic low dose) did not alter
circulating Treg levels compared to nave mice (Figure 4A)
and failed to induce EAU (0/40, Figure 4B), again
supporting the importance of local Tregs against
autoimmunity. While it is known that systemic Treg depletion
results in polyclonal activation of autoreactive T cells
leading to progressive multi-organ autoimmune disease , a
high dose of systemic DTx that resulted in near total Treg
depletion (Figure 4A) only induced EAU in
gal-FDGBG2 mice at a rate similar to the local depletion (4/22,
18.2%, Figure 4B), and not at all in the other strains
lacking the combination of gal-specific TCR-Tg T cells,
retinal gal, and DTx-depletable Tregs (Figure 4B).
Figure 2 New, peripherally generated beta-galactosidase (gal)-specific regulatory T cells (Tregs) inhibit delayed-type hypersensitivity
(DTH). (A) Ear swelling assay showing that retinal gal is required to generate gal-specific Tregs that can modulate the DTH response to gal.
Mice were analyzed 80 days post-transfer of 5 x 104 CD4+GFP cells from FG-BG2 mice. (B) Ear swelling assay showing that the presence of the
retina is not necessary for maintaining gal-specific Tregs. Mice were enucleated and compared to non-enucleated mice 4 months post-enucleation.
Ear swelling measured at indicated time post-gal injection. Results are given as mean SD with P values determined by t test.
Retinal dendritic cells are necessary for retinal T cell
Small numbers of DC in the quiescent retina can be
identified by GFP expression in CDG mice and in vitro
these retinal DC act as APC [39,40]. To assess whether
retinal DC function as APC locally within the retina,
we crossed FG-BG2 mice with DC-depletable CDG
mice (FG-BG2-CDG mice). While large doses of DTx
(200 ng) are highly effective in short-term assays ,
they are eventually lethal to CDG mice. We found that
GFP+ DC can be depleted from the retina by serial i.p.
administration of small doses of DTx (25 ng) for up to
10 days . This, combined with our other finding that
AC injection of gal induces a T cell response within the
retina of FG-BG2 mice  provided a way to examine
the role of retinal DC in the retinal T cell response
(Figure 5A). Although in FG-BG2-CDG mice both Tregs
and DC are GFP+, we distinguished the cells by CD4
and CD11b staining. gal-specific BG2 T cells are also
positive for TCR-V11.
Injection of gal into the AC of FG-BG2-CDG mice
stimulated a retinal T cell response that included BG2
(V11+) and nonspecific Teffs (V11), as well as Tregs
that were BG2 or nonspecific (Figure 5B). In contrast,
non-TCR-Tg (BG2) controls (FG mice) did not have a
significant T cell response when given AC injections of
gal (Figure 5B), suggesting that the response of Ag
nonspecific V11 T cells within the retina of BG2
mice is dependent on generating the gal-specific
V11+ T cell response that produces cytokines and
chemokines that can support the recruitment of Ag
nonspecific T cells into the retina. If FG-BG2-CDG
mice were also treated with DTx to deplete DC, the
entire T cell response (Teff and Treg, BG2 and
nonspecific) to gal was eliminated, yielding T cells
numbers similar to nave FG-BG2-CDG mice and FG-BG2
mice given AC saline (Figure 5B). When given gal
and DTx, control FG-BG2 mice lacking depletable DC
had a similar T cell response to gal stimulated
Figure 3 Local depletion of regulatory T cells (Tregs) induces spontaneous autoimmune disease. (A) Incidence and severity of experimental
autoimmune uveoretinitis (EAU) in the eyes of mice following anterior chamber of the eye (AC) injections of diphtheria toxin (DTx) or saline. Amount,
timing, and duration of AC injection protocols are indicated. Statistical set A compares gal-FDG-BG2 mice treated with saline or DTx, P values
determined by Fishers exact test. Statistical set B compares mice with or without BG2 T cells following DTx treatment, P values determined by Fishers
exact test. (B) Representative histology of eyes from gal-FDG-BG2 mice that received DTx or saline into the right AC. (C) Fluorescence-activated cell
sorting (FACS) analysis of peripheral blood mononuclear cells showing AC injection of DTx does not alter circulating Treg levels. Results are given as
mean SD with P values determined by t test.
Figure 4 Systemic depletion of regulatory T cell (Tregs) induces
spontaneous autoimmune disease in the retina. (A) Analysis of
circulating Tregs levels following low and high dose systemic
depletion protocols. Results are given as mean SD with P values
determined by t test. (B) Incidence and severity of experimental
autoimmune uveoretinitis (EAU) following systemic injections of
diphtheria toxin (DTx). Amount, timing, and duration of systemic
DTx injection protocols are indicated. P values determined by
Fishers exact test. Analysis of EAU and Treg levels were done at
21 days post-initial DTx injection.
We also analyzed the retinal mononuclear cell response
(CD11b+ cells, but not Ly6G+ polymorphonuclear
granulocytes). FG-BG2-CDG mice given AC gal had an
elevated number of DC (CD11b+GFP+ cells) compared to
nave controls, but MG numbers were similar (Figure 5C).
However, when these mice were also given DTx, DC
numbers were reduced to background while MG numbers
were unchanged. Total mononuclear cell numbers in gal/
DTx treated control FG-BG2 mice lacking GFP-labeled,
depletable DC were similar to gal treated FG-BG2-CDG
mice (Figure 5C). Together, this data suggested that there
is a DC-dependency in the retinal Teff and Treg responses
to specific Ag, and that neither MG nor GFP recruited
macrophages contributed to the T cell response.
Beta-galactosidase in the retina primes it toward
Previously, we established that there is a robust T cell
response in the retinas of FDG-BG2 mice to locally
Figure 5 Depletion of retinal dendritic cell (DC) eliminates the
local immune response. (A) Schematic showing timing and
location of diphtheria toxin (DTx) (25 ng), beta-galactosidase (gal)
(10 g), and saline injections. (B) Fluorescence-activated cell sorting
(FACS) analysis showing reduction in T cell numbers in retinas
depleted of DC. T cells were analyzed as being effector (GFP) or
regulatory (GFP+) and gal-specific (V11+) or nonspecific (V11).
(C) FACS analysis of retinal mononuclear cells showing that DC but
not microglia (MG) were reduced in mice treated with DTx. Results
are given as mean SD with P values determined by t test.
administered Ag and that systemic depletion of Tregs
did not affect the Ag-stimulated appearance of Tregs
within the retina, leading to our conclusion that
protective Tregs were made on-demand . Given that T cell
responses within the retina are dependent on local DC
and that DC from quiescent retina favor Treg
production , we assessed whether the T cell response within
the retina would be limited in mice expressing retinal
gal. To ascertain the effect of retinal gal on the
immune response to exogenous Ag, we compared the T
cell response in FDG-BG2 and gal-FDG-BG2 mice
following AC injection of gal with or without systemic
pre-depletion of Tregs (Figure 6A). In agreement with
our prior findings , retinal Teffs (GFPV11+ and
GFPV11 cells) increased following AC gal injection
in FDG-BG2 mice, especially with systemic Treg
depletion (Figure 6B, D-left side of panels). AC injection of
gal into FDG-BG2 mice also increased retinal Treg
numbers (GFP+V11+ and GFP+V11 cells) regardless
of whether they were systemically pre-depleted by
treatment with DTx (Figure 6C, E-left side of panels). In
contrast, AC gal injection done in mice expressing
retinal gal resulted in a significantly reduced number of
GFP and GFP+ V11+ T cells (Figure 6B, C-right side
compared to left side of panels for gal) showing that
both the Teff and Treg Ag-specific response to gal was
limited. The downregulation of the response was not
due to elevated V11+ Tregs associated with retinal gal
expression as Ag-specific Teffs and Tregs were also
reduced between FDG-BG2 and gal-FDG-BG2 mice
following systemic DTx plus AC gal (Figure 6B, C-right
side compared to left side of panels for gal/DTx).
Retinal gal expression also reduced the V11 T cell
response to AC gal injection (Figure 6D, E-right side
compared to left side of panels for gal). However, the
number of V11 Tregs and Teffs was similar between
gal-FDG-BG2 and FDG-BG2 mice following systemic
DTx plus AC gal (Figure 6D, E-right side compared to
left side of panels for gal/DTx), likely the result of
expansion of polyclonal, self-reactive V11 T cells no
longer under the control of circulating Tregs due to their
depletion. These results suggest that gal expression within
Figure 6 Retinal beta-galactosidase (gal) expression induced immunological unresponsiveness. (A) Legend and time course of the
experiments. Mice received 250 ng diphtheria toxin (DTx) and/or gal (20 g) or saline as indicated. Retinal T cell analysis was done by
fluorescence-activated cell sorting (FACS) 3 days post-injection to the anterior chamber of the eye (AC). (B-E) Number of T cells per retina. T cells
were analyzed as being effector (GFP) or regulatory (GFP+) and gal-specific (V11+) or nonspecific (V11). Results are given as mean SD with
P values determined by t test comparing FDG-BG2 to gal-FDG-BG2 mice for each type of T cell with significant differences indicated by bracket.
Local depletion of regulatory T cell enhances
interphotoreceptor retinoid binding protein-induced
experimental autoimmune uveoretinitis
B6 mice are minimally permissive for EAU [57,58].
Recently, we demonstrated that cells expressing DTR could
be locally eliminated from the retina by AC injections of
DTx [39,40], and that depletion of retinal Tregs in
B6gal mice enhanced gal-mediated EAU . To assess
whether our findings extended to an endogenous retinal
Ag, we asked if retinal depletion of Tregs could also
enhance EAU induced by IRBP, an extracellular retinal
protein with a much greater expression level then gal, and
the most common retinal self-Ag used to induce EAU in
mice . FDG mice were immunized with peptides of
IRBP, with or without injection of DTx into the right
AC. Immunized only mice, as well as the left eyes of
immunized mice that received AC injections of DTx,
developed a similarly modest incidence and severity of EAU
(Figure 7A, B). However, Treg-depleted retinas from
right eyes had a significant enhancement in the
incidence and severity of EAU. Analysis of blood showed
that the AC injections of DTx did not lower circulating
Treg levels but actually resulted in a slight increase over
the course of the experiment (Figure 7C). Given that
DTx only eliminates existing Tregs while not preventing
new Treg generation, the selective pressure to maintain
Tregs, and the low dose of DTx used, it was not
surprising to find that circulating Tregs were not lost.
Consistent with our previous findings using gal as a target
neo-self Ag, these results suggest that Tregs need to be
present within the retina to have a protective effect.
Previously we demonstrated that antigen-specific pTregs
could be generated in an on-demand manner within
the retina and be protective against experimentally
induced autoimmunity directed against the neo-self
antigen gal , suggesting that pTregs are an important
mechanism for homeostasis of immune privileged tissue.
In this study we further assessed the nature of retinal
pTregs and showed they are protective against
autoimmune disease directed at the endogenous retinal
antigen IRBP and also protective against spontaneous retinal
autoimmunity. We also assessed the role of retinal APC,
providing evidence that it is the local DC, and not MG,
within the retina that are crucial for generating both Teff
and Treg responses to retinal Ags.
Using gal-specific CD4+ TCR Tg (BG2) mice in this
study along with gal-specific CD8+ TCR Tg (BG1) mice
Figure 7 Interphotoreceptor retinoid binding protein (IRBP)-induced autoimmune disease is enhanced in retinas depleted of
regulatory T cells (Tregs). (A) Incidence and severity of experimental autoimmune uveoretinitis (EAU) in IRBP-immunized only FDG mice and in
right and left eyes of FDG mice that were IRBP-immunized and given diphtheria toxin (DTx) into the right anterior chamber of the eye (AC). Mice
were given 25 ng of DTx 3 times per week for 3 weeks starting the day of immunization. Retinas were analyzed for EAU at 21 days post-immunization.
(B) Histology of right and left eyes from IRBP-immunized mice given DTx into the right AC. (C) Analysis of blood for Tregs in nave (day 0) and
DTx-treated FDG mice (day 21). For disease scores, mean and SD are given, P values were determined by Fishers exact test for incidence
and MannWhitney test for severity. For blood analysis, results are given as mean SD with P value determined by t test.
in our previous study , we found that the DTH
response to gal could be modulated by the addition or
subtraction of FoxP3+ Tregs. Although gal expression
was absent in these TCR Tg mice, the ability to up or
down regulate the DTH response by altering Treg levels
implies a certain baseline amount of tTreg-mediated
control in nave TCR Tg mice. It has been demonstrated
that T cells specific for foreign or neo-self Ags can be
positively selected in the thymus via cross-reactive
(Ag-mimicking) self-peptides [61,62]. This accounts for
the Ag-specific T cell response in wild-type mice
following gal immunization and allows for the formal
possibility that nave mice have a limited number of gal-reactive
tTregs, especially BG2 mice given their large number of
Ag-specific precursor T cells. Nonetheless, experiments
herein using Rag/ mice transferred with FoxP3
precursor T cells clearly demonstrated the Ag-dependent
peripheral generation of gal-specific Tregs. Combined with our
previous results , we propose that locally generated,
locally acting, Ag-specific pTregs are a crucial factor in the
contribution of Tregs towards retinal immune privilege
and that tTregs to gal generated by selection on
crossreactive self-peptides are limited in number and function.
Underscoring the importance of local Tregs in retinal
immune privilege is the observation that local Treg
depletion from the retina enhanced IRBP-induced EAU,
recapitulating the results found with retinal gal . In
addition to being highly expressed in the retina, there is
also aire promoted expression of IRBP in the thymus
[63,64] providing a certain level of tolerance to
IRBPmediated autoimmunity mediated by negative selection
and generation of Ag-specific tTregs. While these studies
clearly demonstrated the importance of central tolerance
to IRBP in mitigating autoimmunity, there are
circumstances that suggest that locally generated IRBP-specific
pTregs also contribute to retinal immune privilege. First,
based on the amount of IRBP in the human retina 
and the relative size of the human and murine retina
[66-68], we estimate endogenous IRBP in the retina to be
at least tenfold the concentration of retinal gal in the gal
mice. This high level of IRBP expression in an
environment that is primed by high concentrations of TGF- and
retinoic acid for Treg production [69-72] makes
IRBPspecific pTreg generation likely. Second, an IRBP-specific
Teff population that escaped negative selection has been
described  and thus making obvious a need for
peripheral regulation. While it has been shown that this T cell
population has limited pathogenicity [73,74], the ability to
regulate its priming and effector functions within the
retina would be an efficient method of control. Unlike
our previous studies with gal, we did not formally
demonstrate herein that IRBP-specific pTregs are
made in the retina. However, our results combined
with other investigations suggest that it is likely, if not
necessary, that there be local production of
Our retinal gal/BG2 TCR Tg/FoxP3-DTR model
system is well suited for demonstrating the role of pTregs
in tissue-specific immune privilege in that naive mice,
whether expressing one or any combination of the
transgenes, do not develop autoimmune disease. This is in
contrast to other models of autoimmune disease,
including EAU, using TCR Tg mice specific for self-Ag or
neo-self Tg Ag that actually develop a high level of
spontaneous autoimmune disease [75-77] despite thymic
expression of the Ag. In these models, autoimmunity
induced by Treg depletion might not be distinguishable
from the spontaneous autoimmunity. Since we had
demonstrated that retinal depletion of Tregs enhanced EAU
in gal-FDG mice that were either gal-immunized or
transferred with activated gal-specific T cells  it was
logical to ascertain whether the same depletion regimen
could induce autoimmunity in otherwise naive
galFDG-BG2 mice. Our finding that significant EAU was
only found in DTx-treated eyes of the triple Tg mice and
not in their contralateral eye nor in mice lacking one or
more of the transgenes further supports the idea that
local action of Ag-specific Tregs is a crucial component
of retinal immune privilege.
An interesting difference between
immunized/transferred gal-FDG mice of our previous study  and
gal-FDG-BG2 mice herein was that significant systemic
Treg depletion induced by high doses of DTx caused
EAU only in gal-FDG-BG2 mice and then only at a rate
equal to that observed with local depletion. In rodent
strains with limited susceptibility for EAU , the
highest incidence and severity of disease is associated with
situations that involve a chronic stimulation of a large T
cell population such as lymphopenia , activation by
commensal microbiota [76,78,79], antigen mimicry ,
and Treg loss or lack of production [64,81]. Given the
high frequency of gal-specific T cells in gal-FDG-BG2
mice, it is likely that the lympho-proliferation associated
with systemic Treg depletion  resulted in a significant
T cell activation that was sufficient to overcome the
ongoing, local production of pTregs in the retina. Although
higher doses of DTx or longer treatment would be lethal
to gal-FDG-BG2 mice, we speculate that it would result
in higher incidence and severity of EAU. Nonetheless,
the results herein and previously  suggest that it is the
local Tregs within the retina, and their on-going
generation, that sets the threshold for retinal autoimmune
Our retinal gal/BG2 TCR Tg/FoxP3-DTR model
system is also useful in examining the initial, critical
component of local immune regulation - namely the effect of
Ag expression within the tissue on the immune
response. Meaningful comparison of retinal T cells from
nave mice that do or do not express retinal gal is
difficult due to their intrinsically low numbers within the
retina. However, a direct and local antigenic challenge of
BG2 mice revealed that those also expressing retinal gal
were highly resistant to generating a T cell response,
even in mice whose Tregs were pre-depleted by systemic
injection of DTx. This finding is consistent with our
previous observation that DC from nave retina favor Treg
production. DC within gal expressing nave retina
generate gal-specific Tregs, which in turn create and
maintain an immunologically quiescent local environment.
Alternatively, or in conjunction with enhanced Treg
production, expression of gal within the retina could
induce T cell anergy. This would most likely occur in
quiescent retinas that have few DC. This allows other
cells with APC ability, such as MG, to present self-Ag.
However, retinal APC other than DC may lack the
proper co-stimulatory molecules to activate T cells, a
situation well understood to generate T cell anergy [82,83].
Regardless of the mechanisms, the tenacity of this
immunological unresponsiveness is evidenced by our observations
herein and previously  that unilateral treatment
generating a high rate of EAU in ipsilateral eyes of gal-FDG
mice and gal-FDG-BG2 mice fails to generate disease
within contralateral eyes.
While the presence of DC and their function as APC
within the retina is still a matter of active investigation,
recent in vitro studies have provided evidence that direct
contact between DC and T cells is necessary for retinal
T cell responses. In addition to our report showing Teff
and Treg generation mediated by purified retinal DC
, it has also been demonstrated that CD4+ T cells
specific for an ocular transgene required DC plus Ag to
become pathogenic . Further, we also demonstrated
in a model of EAU mediated by CD8+ T cells, in
conjunction with MHC class I/ mice, that pathogenicity
requires only the resident retinal cells be MHC class I+
, again suggesting local but not recruited APC are
crucial for retinal T cell responses. Use of a mouse
combining the FG transgene, which identifies Tregs but lacks
DTR, with the transgenes of the CDG and BG2 mice
allowed us to exam directly in vivo if retinal DC were
significant contributors to the APC function in retina.
Other retinal cells including endothelial cells [85,86],
retinal pigment epithelial cells [87,88], and especially
MG [28-33] have been proposed to have APC activity.
Although these putative retinal APC vastly outnumber
retinal DC and are not depleted by DTx treatment in
mice with the CDG transgene, we found that generation
of Teffs and Tregs within the retina as well as T cell
mediated pathogenicity was completely dependent on DTR
+GFP+ retinal DC. While these other cell types have
been shown to modulate T cell responses and perhaps
have secondary APC function, their apparent lack of
function as APC in the absence of DC clearly
demonstrates that retinal DC are required for initiation of
retinal T cell responses.
In conclusion, we have demonstrated that the retinal
environment is capable of Ag-specific pTreg generation
and that those pTregs act locally within the retina to
limiting both spontaneous and induced autoimmune
disease. We have also demonstrated that the presence of
gal within the retina limits the T cell response to
challenge with exogenous gal and that local DC within the
retina are critical for generating T cell responses within
AC: anterior chamber of the eye; Ag: antigen; APC: antigen presenting cell;
gal: beta-galactosidase; BSA: bovine serum albumin; CDG: mice expressing a
DTR/GFP fusion protein under control of the CD11c promoter; DC: dendritic
cell; DTH: delayed-type hypersensitivity; DTR: diphtheria toxin receptor;
DTx: diphtheria toxin; EAU: experimental autoimmune uveoretinitis;
FACS: fluorescence-activated cell sorting; FG: mice expressing GFP under
control of the FoxP3 promoter; FDG: mice expressing DTR and GFP under
control of the FoxP3 promoter; GFP: green fluorescence protein;
IRBP: interphotoreceptor retinoid binding protein; MG: microglia;
pTreg: peripherally derived Treg; Teff: effector T cell; Tg: transgenic;
Treg: regulatory T cell; tTreg: thymically derived Treg.
SM contributed to the design of all experiments, performed the
autoimmune and DTH experiments, and wrote the manuscript. NH
performed and analyzed the flow cytometry experiments. MP performed all
surgical procedures and mouse genotyping. DG conceived the study and
oversaw the experimental design. All authors contributed to the editing of
the manuscript. All authors read and approved the final manuscript.
We thank Heidi Roehrich for histology. This work was supported by NIH
research grants R01-EY021996 (DSG), R01-EY016376 (DSG), and core facility
grant P30-EY011374. Additional support was provided by Research to
Prevent Blindness and the Minnesota Lions Clubs.
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