INFECTION AND IMMUNITY, Aug. 2010, p. 3484–3492
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 8
CD4 T-Cell Suppression by Cells from Toxoplasma gondii-Infected
Retinas Is Mediated by Surface Protein PD-L1?
Elizabeth Charles,1† Sunil Joshi,1† John D. Ash,2Barbara A. Fox,3A. Darise Farris,4
David J. Bzik,3Mark L. Lang,1and Ira J. Blader1*
Department of Microbiology and Immunology1and Department of Ophthalmology,2University of Oklahoma Health Sciences Center,
and Arthritis and Immunology Research Program, Oklahoma Medical Research Foundation,4Oklahoma City, Oklahoma 73104,
and Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire 037563
Received 4 February 2010/Returned for modification 23 February 2010/Accepted 17 May 2010
In the inflamed retina, CD4?T cells can cause retinal damage when they are not properly regulated. Since
tissue expression of major histocompatibility complex (MHC) class II and costimulatory molecules is a key
mechanism for regulating effector T cells, we tested the hypothesis that upregulation of these proteins in the
retina contributes to the regulation of CD4 T cells. Here we report that in retinas infected with the protozoan
parasite Toxoplasma gondii, MHC class II is upregulated on infiltrating leukocytes as well as on resident retinal
cells, including photoreceptors. Flow cytometric analysis indicated that B7 costimulatory family members
(CD80, CD86, ICOS-L, and programmed death ligand 2 [PD-L2]) were not expressed on class II?cells. In
contrast, PD-L1 (also named B7-H1 or CD274) was expressed on the majority of both hematopoietic and
resident retinal MHC class II-expressing cells. Retinal cells from Toxoplasma-infected animals were able to
suppress T-cell activation in a PD-L1-dependent manner. Finally, we demonstrate that the expression of MHC
class II and PD-L1 was critically dependent on gamma interferon (IFN-?) expression. These data suggest that
retinal MHC class II and PD-L1 expression is a novel mechanism by which the retina protects itself from CD4
T-cell-mediated immune damage in ocular toxoplasmosis and other types of retinal immune responses.
Toxoplasma gondii is an obligate intracellular protozoan par-
asite that infects humans and animals either congenitally or
postnatally (38, 42). There are no vaccines to prevent this
infection, and currently prescribed therapies are poorly toler-
ated and have severe side effects. In addition, the parasite
responds to these drugs as well as to the host’s immune re-
sponse by transforming into quiescent, asymptomatic cysts in
tissues such as the eye, brain, and muscle (54). These cysts
occasionally reactivate, and the released parasites will cause
disease unless a properly regulated immune response is
mounted. The retina is a common site for cyst reactivation and
causes a disease, called ocular toxoplasmosis, that is one of the
most common infections of the posterior retina (21).
The ability of a host to control a reactivated Toxoplasma
infection is dependent largely on CD8?T cells and, to a lesser
extent, on CD4?T cells (13, 43, 52). However, CD4?T cells
must be tightly regulated, because these cells cause immune-
mediated tissue destruction in the eye and other tissues (23, 32,
36). Because damaged retinal cells cannot be repaired or re-
generated, it is imperative to understand how CD4?T cells are
regulated in the retina in ocular toxoplasmosis. As central
regulators of CD4?T cells, major histocompatibility complex
(MHC) class II-expressing cells are likely to be critical in con-
trolling CD4-based T-cell responses.
MHC class II expression and function in the retina have
been the source of some debate. This is because retinal MHC
class II expression is low as a result of the high expression of
immunosuppressive cytokines and the blood-ocular barrier
(49). Regardless, retinal microglia, endothelial cells, retinal
pigment epithelial (RPE) cells, and other, less well character-
ized cells can express MHC class II under certain conditions
(18, 25, 57). But the identity and function of MHC class II-
expressing cells in Toxoplasma-infected retinas remain un-
At sites of inflammation, CD4?T cells are regulated by two
distinct signals from their target cells. First, they must come in
contact with a cell expressing peptide-loaded MHC class II.
Second, costimulatory molecules dictate the response of the
engaged T cells. One major group of costimulatory molecules
is the B7 protein family, which includes CD80, CD86, and
ICOS-L (16). These proteins, which bind CD28 (CD80 and
CD86) or ICOS (ICOS-L), are important for resistance to
Toxoplasma (51, 56). Two additional B7 family members, pro-
grammed death ligand 1 (PD-L1) and PD-L2, bind the T-cell
surface protein PD-1 and downregulate activated T cells (16).
While PD-L1 and PD-L2 are functionally redundant, they have
distinct expression patterns; PD-L2 is expressed primarily by
cells derived from hematopoietic cells, and PD-L1 is expressed
on hematopoietic and nonhematopoietic cells. In mice and
humans, PD-L1 but not PD-L2 is expressed in various regions
of the eye, including the cornea, ciliary body, iris, and retina
(22, 48, 58). Ocular PD-L1 expression is important for the
prevention of corneal allograft rejection, suggesting that it is a
key player in maintaining immune privilege in the anterior
chamber of the eye (22, 48). However, the importance of
PD-L1 in the posterior chamber in inflammatory events such as
Toxoplasma infections is unknown.
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, BMSB 1019, University of Oklahoma Health
Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK
73104. Phone: (405) 271-2133. Fax: (405) 271-3117. E-mail: iblader
† E.C. and S.J. contributed equally to this work.
?Published ahead of print on 24 May 2010.
Here we report that CD4 recall responses to Toxoplasma
antigen are blocked by retinal cells isolated from intravitreally
infected mice. Flow cytometric and immunohistochemical
analyses demonstrate that both infiltrating leukocytes and res-
ident retinal cells expressed MHC class II and that MHC class
II expression was dependent on gamma interferon (IFN-?).
Surprisingly, an overwhelming majority of the MHC class II-
expressing cells did not express positively acting costimulatory
molecules but rather expressed PD-L1. Finally, the suppressive
effect of parasite-infected retinal cells on T cells was PD-L1
MATERIALS AND METHODS
Parasites. The RH and cpsII pyrimidine auxotrophic Toxoplasma strains were
grown in human foreskin fibroblasts as previously described (7, 11). To prepare
parasites for injections, infected monolayers were scraped and parasites were
mechanically released from their host cells by being passed three times through
a 27-gauge syringe needle. The parasite suspension was filtered through a 3-?m-
pore-size polycarbonate filter to separate parasites from host cells. Parasites were
extensively washed in phenol red-free Dulbecco’s modified Eagle medium
(DMEM) and were then resuspended in phenol red-free DMEM. Soluble
tachyzoite antigen (STAg) was prepared in the presence of a protease inhibitor
cocktail (Calbiochem) as previously described (27), except that the lysate was
centrifuged at 16,000 ? g for 20 min at 4°C.
Mice and intravitreal infections. Injections were performed as described pre-
viously (7). Needles with a tip size of no more than 50 ?m were loaded with a
parasite suspension using a pneumatic pump delivery system. Six- to 12-week-old
C57BL/6 or IFN-??/?mice (purchased from Jackson Labs, Bar Harbor, ME)
were anesthetized with ketamine-xylazine, and then the needle was inserted
immediately behind the limbus-parallel conjunctival vessels. After the needle was
inserted, 0.5 ?l of the parasite suspension or phenol red-free DMEM (as a mock
control) was injected. Mice were intraperitoneally vaccinated with 104irradiated
cpsII parasites. All protocols adhered to University of Oklahoma Health Sciences
Center’s IACUC and to ARVO’s Statement on the Use of Animals in Ophthal-
mic and Vision Research.
Flow cytometry. Mice were sacrificed by CO2asphyxiation, and their eyes were
immediately harvested, placed in phosphate-buffered saline (PBS), and mechan-
ically disrupted with a stomacher (Tekmar, Cincinnati, OH). The cell suspension
was then passed through a 40-?m-pore-size mesh to remove the lens and eye cup.
Cells were incubated first in Fc block (BD Biosciences, San Jose, CA) and then
with the following antibodies (or with appropriate isotype controls): (i) fluores-
cein isothiocyanate (FITC)-conjugated antibodies against Ly6C (clone AL-21;
BD Biosciences), CD80 (clone 16-10A1; eBiosciences), CD86 (clone RMMP-2;
Invitrogen), Siglec-H (clone eBIO440C; eBioscience), CD45 (clone 30-F11; In-
vitrogen), CD11c (clone N418; Invitrogen) (conjugated with Alexa Fluor 488), or
I-Ab(clone AF6-120.1; BD Biosciences); (ii) phycoerythrin (PE)-conjugated
antibodies against Ly6G (clone 1A8; BD Biosciences), PD-L1 (clone M1H5;
eBiosciences), PDCA (clone eBio927; eBiosciences), ICOS-L (clone HK5.3;
eBiosciences), or B220 (clone RA3-6B2; eBiosciences); (iii) a Tri-Color-conju-
gated antibody against CD8 (clone CT-CD8?; Invitrogen); (iv) a peridinin chlo-
rophyll protein (PerCP)-Cy5.5-conjugated antibody against CD11b (clone M1/
70; BD Biosciences); and (iv) allophycocyanin-conjugated antibodies against
I-Ab(clone M5/114.15.2; eBiosciences) or CD45 (clone 30-F11; BD Bio-
sciences). Antibody dilutions were optimized using total splenocytes from unin-
fected mice. Single-color controls were used for compensation and gating.
Immunocytochemistry. Mock- and parasite-infected eyes were harvested and
fixed for 30 min at room temperature in 3% paraformaldehyde in PBS. Eyes were
then placed in increasing concentrations of sucrose (10 to 30%) in PBS at room
temperature, followed by overnight incubation at 4°C in PBS containing 30%
sucrose. The cryoprotected eyes were then embedded in Tissue-Tek OCT com-
pound (Sakura Fintek, Torrance, CA) and were rapidly frozen in liquid nitrogen.
Then 15-?m-thick sections were prepared with a cryostat. Sections were placed
on glass slides, incubated briefly in ice-cold methanol, and then stored at ?20°C
until use. Sections were quenched with 20 mM glycine in PBS, blocked with 10%
horse serum, and then incubated overnight at 4°C with antibodies against MHC
class II (I-Ab) or mouse IgG as a control, or against SAG1 or rabbit IgG as a
control. The slides were washed and then incubated with Alexa Fluor 594-
conjugated anti-mouse IgG to detect anti-I-Abor with an Alexa Fluor 488-
conjugated anti-rabbit antibody to detect anti-SAG1. Sections were imaged with
an Olympus FluoView FV500 confocal laser scanning microscope (Olympus,
Center Valley, PA). Laser power, pinhole settings, photomultiplier tube settings,
and intensity thresholds were kept constant for each antibody and its isotype
T-cell proliferation assay. Splenic T cells from cpsII-immunized mice were
enriched by negative selection using an AutoMACS cell sorter and antibodies
against MHC class II?, CD11c?, NK1.1?, and B220?cells conjugated to mag-
netic microbeads (Miltenyi Biotec, Auburn, CA). Antigen-presenting cells
(APCs) used in the assay were enriched CD11c?dendritic cells (DCs) prepared
by digesting spleens from naïve mice with 5 mg/ml collagenase IV (Worthington
Biochemical, Lakewood, NJ) and 0.1 mg/ml DNase (Sigma Chemical, St. Louis,
MO) in Hanks balanced salt solution (HBSS) for 30 min at 37°C. The single-cell
suspension was treated with ACK lysis buffer (Cambrex, East Rutherford, NJ) to
lyse the red blood cells. The splenocytes were then incubated with magnetic-
bead-conjugated antibodies against TCR, CD3, and NK1.1 (Miltenyi Biotech)
and were passed through an AutoMACS cell sorter to negatively select for the
CD11c?DCs. The purity of enriched DCs and T cells was always ?85% as
determined by flow cytometry. Enriched APCs were loaded with STAg protein
(1.0 mg/ml) in serum-free RPMI medium by incubation at 37°C for 1 h. T-cell
proliferation was measured in each well of a 96-well plate by plating 105purified
T cells with 5 ? 104STAg-loaded APCs (or 200 ?g of STAg alone as a negative
control). In addition, retinal cells prepared from mice 6 days after they were
either mock infected or intravitreally infected with 104RH parasites were added
to the T cells. Plates were incubated for 66 h, and then 1 ?Ci of [3H]thymidine
was added to each well for 6 h. Cells were harvested on glass fiber filters (PhD
Cell Harvester; Cambridge Technology, Inc., Cambridge, MA) and counted by
IFN-? cytokine measurement. Mock-infected and parasite-infected eyes were
harvested from euthanized mice. The eyes were then enucleated, and posterior
FIG. 1. MHC class II expression in Toxoplasma-infected eyes. Mice
were either mock infected (solid black lines) or intravitreally injected
(gray lines) with 104parasites. Four and 6 days later, eyes were har-
vested and analyzed by flow cytometry for MHC class II expression.
The gate includes all MHC class II?events as determined by isotype
staining controls (dashed line). The y axes of the 4-day histograms were
decreased to highlight class II?cells. Shown are three representative
histograms from independent experiments.
VOL. 78, 2010TOXOPLASMA AND RETINAL CLASS II AND PD-L1 EXPRESSION3485
segments were dissected away from the rest of the eye. The posterior segments
were then placed in lysis buffer and homogenized with a hand-held homogenizer.
Singleplex mouse IFN-? kits were purchased from Bio-Rad (Hercules, CA) and
were used according to the manufacturer’s instructions in conjunction with Bio-
Plex instrumentation (Bio-Rad). IFN-? concentrations in tissue supernatants
were measured in duplicate and determined by interpolation from standard
curves using Bio-Plex software.
Three distinct populations of cells express MHC class II in
Toxoplasma-infected eyes. Dysregulated CD4?T cells can
cause retinal tissue damage in a variety of instances, including
Toxoplasma infections and experimental autoimmune uveitis.
Since CD4?T-cell binding to peptide-loaded MHC class II on
the target cell surface is a key mechanism for the regulation of
effector CD4?T-cell activity, MHC class II expression was
examined in the retinas of Toxoplasma-infected mice. Thus,
mice were either mock infected or intravitreally infected with
104tachyzoites, and 4 or 6 days later, eyes were isolated and
processed for flow cytometry to detect MHC class II. While
?1% of cells in mock-infected eyes were MHC class II?(and
these were class IIlo), more than 10% of the cells were MHC
class II?4 days postinfection (Fig. 1, left panels). By 6 days
postinfection, the numbers of MHC class II?cells in parasite-
infected eyes increased to ?46%, while the numbers of class
II?cells in the mock-infected samples remained constant (Fig.
1, right panels). Similar time-dependent increases in staining
were observed when eyes were analyzed 6 and 8 days after they
were intravitreally injected with 102parasites (not shown).
Hematoxylin-and-eosin (H&E)-stained sections from para-
site-infected eyes revealed that large numbers of leukocytes
with nuclear morphologies typical of monocytes and neutro-
phils were present in the retinas and, to a lesser extent, in the
vitreous of Toxoplasma-infected eyes (7). To determine
whether the infiltrating leukocytes were MHC class II?, intra-
vitreally infected eyes were harvested, processed for flow cytom-
etry, and stained with antibodies against MHC class II, CD11b,
Ly6G, and CD45. This cocktail of antibodies was used because it
can discriminate between neutrophils (CD45?CD11b?Ly6G?)
and inflammatory monocytes (CD45?CD11b?Ly6G?) (15) as
well as between retinal microglia (CD45lo) and resident neuro-
retinal cells (CD45?). Three distinct populations of MHC class
II-expressing cells were found in Toxoplasma-infected retinas:
populations I (CD45?CD11b?Ly6G?), II (CD45?CD11b?
Ly6G?), and III (CD45?CD11b?Ly6G?) (Fig. 2A and B). In
addition, populations I and II stained strongly with anti-Ly6C,
suggesting that the Ly6G?cells in population II are inflammatory
monocytes (Fig. 2C). These data indicated that populations I, II,
and III consisted of neutrophils, monocytes, and resident retinal
The histograms in Fig. 1 indicate that MHC class II expres-
sion levels were widespread in Toxoplasma-infected retinas.
We therefore tested whether all cells expressed heterogenous
FIG. 2. Three distinct populations of MHC class II-expressing cells in Toxoplasma-infected eyes. Mice were intravitreally injected with 104
parasites, and 6 days later, eyes were harvested and analyzed by using flow cytometry. (A) FACS plot of CD11b and Ly6G expression on MHC
class II?-gated cells. The distinct populations are highlighted as I (CD11b?Ly6G?), II (CD11b?Ly6G?), and III (CD11b?Ly6G?). (B through
D) Histograms of CD45 (B), Ly6C (C), and MHC class II (D) expression levels in each of the 3 populations. The dashed lines in panels B and
C represent isotype control staining levels. MFI, mean fluorescence intensity of each population. Representative results from three independent
experiments are shown.
3486 CHARLES ET AL.INFECT. IMMUN.
amounts of MHC class II or whether each population ex-
pressed class II at specific levels. The data indicated that neu-
trophils (population I) and resident retinal cells (population
III) expressed low levels of MHC class II, while monocytes
(population II) expressed it at much higher levels (Fig. 2D).
Dendritic cell marker staining of MHC class II?cells in
Toxoplasma-infected retinas. DCs are a large group of APCs
that, in general, can be placed into 1 of 3 major classes: conven-
tional DCs (cDCs); inflammatory, blood-derived DCs (iDCs);
and plasmacytoid DCs (pDCs). Because CD11c is expressed at
high levels on cDCs and iDCs and at low levels on pDCs, we
assessed CD11c expression on class II?cells 6 days after intra-
vitreal injection of mice with 104parasites. We found that
CD11c was expressed on cells in population II at levels com-
parable to those of splenic CD11c?DCs (not shown). CD11c
expression was, however, undetectable on cells in populations
I and III (Fig. 3A). In addition, none of the class II?cells
stained with anti-CD8? or with the dendritic cell marker 33D1
(not shown). These data, combined with the fact that popula-
tion II cells were Ly6C?, suggested that in parasite-infected
retinas, blood-derived monocytes differentiate into iDCs. In
addition, the CD45?class II?cells do not appear to be derived
from the 33D1-expressing resident retinal dendritic cells re-
cently identified by Xu and coworkers (57).
To determine whether pDCs were present in Toxoplasma-
infected retinas, PDCA1 expression on MHC class II?cells
was analyzed. We used a PE-conjugated anti-PDCA1 antibody,
which precluded us from distinguishing the 3 populations of
cells as shown in Fig. 2. However, we defined the class II?
CD11b?cells as infiltrating leukocytes and class II?CD11b?
cells as resident retinal cells, since ?95% of CD11b?cells were
CD45?(not shown). Surprisingly, more than 65% of cells from
either group were PDCA1?(Fig. 3B and C). Since IFN-? is
upregulated in parasite-infected retinas (see Fig. 7A) and reg-
ulates PDCA1 expression (2), we examined the expression of a
second pDC marker, Siglec-H (1). Fewer than 6% of PDCA1?
cells in either the CD11b?or the CD11b?population were
also Siglec-H?, strongly suggesting that the class II?cells were
not pDCs. Collectively, these data indicate that iDCs are the
major class of dendritic cells in Toxoplasma-infected retinas
and that the majority of class II?cells in infected eyes are not
MHC class II expression is promiscuous throughout all
layers of Toxoplasma-infected retinas. Resident retinal cells,
including microglia, endothelial cells, Mu ¨ller cells, and retinal
pigment epithelial cells, can express MHC class II in vitro and
in some cases in vivo. In parasite-infected retinas, the CD45?/lo
MHC class II?cells were CD11b?, suggesting that they were
not retinal microglia, which are CD11b?myeloid-derived cells
(18, 25). To determine whether MHC class II?CD45?cells
were endothelial cells, expression of the CD31 endothelial
cell marker was assessed by flow cytometry. The data indicated
that class II?cells were CD31?, indicating that in Toxoplasma-
infected retinas, endothelial cells were not MHC class II?(not
Due to a lack of established flow cytometry markers for
other types of retinal cells, we used immunohistochemistry to
identify the CD45?MHC class II?cells. Thus, frozen sections
FIG. 3. Dendritic cell staining in Toxoplasma-infected eyes. Mice were intravitreally injected with 104parasites. Six days later, eyes were
harvested and analyzed using flow cytometry. (A) Histogram of CD11c expression on the three populations of MHC class II-expressing cells from
Fig. 2A. (B) FACS plots of PDCA1 and Siglec-H staining of class II?CD11b?and class II?CD11b?cells.
VOL. 78, 2010TOXOPLASMA AND RETINAL CLASS II AND PD-L1 EXPRESSION3487
from eyes harvested 6 days after they were intravitreally in-
jected with 104parasites were stained with antibodies against
MHC class II and the Toxoplasma tachyzoite surface protein
SAG1, as well as with 4?,6-diamidino-2-phenylindole (DAPI)
to identify the retinal layers (Fig. 4). SAG1 staining, which was
localized primarily to necrotic regions of the retina, indicated
the presence of large numbers of parasites in parasite-infected
retinas. MHC class II staining was not detectable in mock-
infected retinas (not shown), which was consistent with our
flow cytometry data as well as with other reports that retinas
normally express very few class II?cells (4). In contrast, there
was widespread, strong MHC class II expression in all regions
of the infected retina that were SAG1?(Fig. 4). High class II
expression was noted in cells containing bean-shaped nuclei,
suggesting that these were the class IIhiinflammatory mono-
cytes (Fig. 4, boxed area). We also noted class II staining,
which was greater than that in isotype control-stained retinas
(not shown), in regions with little SAG1 staining (Fig. 4, ar-
rowheads). These included both the outer nuclear layer
(ONL), which contains photoreceptors, and the inner nuclear
layer (INL), which contains several neurons, such as bipolar
and horizontal cells. Thus, MHC class II expression in Toxo-
plasma-infected retinas is not restricted to cells derived from
hematopoietic cells but is also present on multiple types of
resident retinal cells.
Costimulatory-molecule expression on MHC class II?cells
in parasite-infected retinas. The outcome of an interaction
between a T cell and its target cell is dependent on costimu-
latory molecules, which may either stimulate or inhibit a T cell.
We therefore examined the expression of members of the B7
family of costimulatory molecules. Single-cell suspensions pre-
pared from retinas infected with 104parasites were stained
with antibodies against positive-acting (CD80, CD86, and
ICOS-L) and negative-acting (PD-L1 and PD-L2) coinhibitory
FIG. 4. MHC class II is expressed on resident retinal cells in Toxoplasma-infected retinas. Mice were intravitreally injected with 104parasites. Six days
later, eyes were harvested, fixed, and processed for immunocytochemistry. Sections were stained to detect either DAPI (blue), MHC class II (red), or
SAG1 (green). (Top) Low-magnification (?40) image of the posterior retina from an infected eye. (Bottom) High-magnification (?400) images of the
area highlighted in the yellow box in the top image. Note the SAG1 staining and the intense class II staining in necrotic regions of the retina. Arrowheads
point to class II?nuclear cells in inner and outer nuclear layers. The boxes highlight a crescent-shaped, class IIhimonocyte. Bars, 100 ?m. Abbreviations:
C, choroid; RPE, retinal pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
3488 CHARLES ET AL.INFECT. IMMUN.
members. Expression of CD86 or PD-L2 could not be detected
on class II?cells, and only a small number of CD11b?class II?
cells expressed CD80 or ICOS-L (Fig. 5). In contrast, PD-L1
was expressed at high levels on ?80% of both CD11b?class
II?cells and CD11b?class II?cells. Similar results were
obtained using retinal cells analyzed 4 days after infection
(data not shown). These data indicate that the majority of class
II?cells express PD-L1 but not other B7 family members.
Toxoplasma-infected retinal cells suppress T-cell activation
via a PD-L1-dependent mechanism. To examine the functional
significance of MHC class II and PD-L1 expression in parasite-
infected retinas, we first tested what effect the addition of cells
from Toxoplasma-infected retinas had on the recall response of
T cells to Toxoplasma antigen-loaded dendritic cells by mea-
suring [3H]thymidine incorporation. Thus, purified splenic T
cells from cpsII-vaccinated mice were stimulated for 72 h with
STAg-loaded CD11c?dendritic cells together with retinal cells
from mock-infected or intravitreally infected mice. T-cell pro-
liferation was increased when cells were cultured with STAg-
loaded APCs and retinal cells from mock-infected mice (Fig.
6A, filled bars). In contrast, a significant, dose-dependent re-
duction in T-cell proliferation was observed when the T cells
were exposed to increasing numbers of retinal cells from in-
travitreally infected mice (Fig. 6A, open bars). This effect on
T-cell proliferation was not a consequence of Toxoplasma rep-
lication within T cells and lysis of T cells, since the addition of
1 ?M pyrimethamine, a significant inhibitor of parasite repli-
cation, had no significant affect on the ability of parasite-in-
fected retinal cells to suppress T-cell responses (Fig. 6B).
We next tested whether the inhibition of T-cell recall re-
sponses by parasite-infected retinal cells was PD-L1 depen-
dent. Thus, the T-cell recall assay was repeated, but anti-
PD-L1 or an isotype control antibody was added to the T-cell
cultures incubated with retinal cells from parasite-infected
mice. The data indicated that the isotype control antibody had
no significant effect on T-cell inhibition by infected retinal cells
(Fig. 6C). In contrast, the anti-PD-L1 antibody significantly
abrogated the ability of the infected retinal cells to suppress
T-cell proliferation. These data indicate that PD-L1 is func-
tionally expressed in Toxoplasma-infected eyes.
IFN-? is required for MHC class II and PD-L1 expression
in Toxoplasma-infected eyes. We next assessed whether IFN-?
was required for MHC class II and PD-L1 expression in par-
asite-infected eyes, since IFN-? is a critical regulator of these
proteins (8, 28, 29, 35, 41). IFN-? protein levels were measured
in the eyes of mice 4 and 6 days after the mice were mock-
infected or intravitreally infected with 104parasites. IFN-? was
upregulated approximately 20-fold 4 days postinfection and
500-fold 6 days postinfection (Fig. 7A).
We next compared MHC class II expression 6 days after
wild-type and IFN-? knockout (IFN-?KO) mice were intravit-
really infected. We found that ?85% of CD11b?cells were
class II?in wild-type mice, while only 26% of these cells were
class II?in IFN-?KO mice. In CD11b?cells, class II expres-
sion was also reduced from approximately 43% in wild-type
mice to 6% in IFN-?KO mice (Fig. 7B). Similarly, PD-L1
expression in both CD11b?and CD11b?cells was also criti-
cally dependent on IFN-?. However, a distinct population of
cells that strongly expressed both MHC class II and PD-L1 was
refractory to the loss of IFN-?, suggesting that other signals
regulate these cells. These data indicate that MHC class II
expression and PD-L1 expression in Toxoplasma-infected eyes
are largely dependent on IFN-?.
Immune privilege is important for the retina, because most
damaged neural retinal cells cannot be repaired or replaced. The
retina maintains its immune-privileged state, in part, by a paucity
of endogenous MHC class II expression in the retina (49, 50).
Recently, however, cells that do express MHC class II in inflamed
retinas have been identified, and the majority of these cells are
33, 34, 44, 57). In Toxoplasma-infected eyes, MHC class II was
expressed on a much more heterogenous population of resident
retinal cells, including retinal neurons such as photoreceptors. It
is not clear why other models of inflammation have yet to detect
MHC class II expression on these retinal cells. One reason could
be that one of the major retinal inflammation models is experi-
mental autoimmune uveoretinitis, which is stimulated by inducing
autoimmune responses to a photoreceptor antigen (6). Thus,
FIG. 5. B7 family member expression on MHC class II?cells. Mice
were intravitreally injected with 104parasites. Six days later, eyes were
harvested and stained with anti-MHC class II and the indicated antibod-
ies. Representative FACS plots from three independent experiments are
VOL. 78, 2010TOXOPLASMA AND RETINAL CLASS II AND PD-L1 EXPRESSION3489
Our work, therefore, further highlights the need to study retinal
inflammation with both autoimmune and infection models.
Toxoplasma infections result in the development of long-
lived tissue cysts in numerous tissues, including immune-priv-
ileged sites such as the retina. Studying the activation and
regulation of retinal anti-Toxoplasma immune responses after
a cyst reactivates has been difficult for several reasons. First,
the number of cysts that develop in the retina is highly variable
(12, 39). Second, the timing and location of spontaneous cyst
reactivation is difficult to predict as well as to detect. Finally,
chronically infected mice develop retinal damage only after
becoming immune compromised by either genetic or pharma-
cological intervention. For these reasons, we used the
tachyzoite intravitreal infection model to mimic the events that
take place after a cyst ruptures and parasites convert to
tachyzoites. The utility of this model is highlighted by the
observations in this report (functional MHC class II and
PD-L1 expression in the retina). Intravitreal infections do,
however, have some caveats, as do all infection models. These
include the fact that intravitreal injection of tachyzoites by-
passes any impact that chronic Toxoplasma tissue cysts and
bradyzoites may have on host surveillance and immunity. Nev-
ertheless, the physiological relevance of our findings is sup-
ported by recent work demonstrating that PD-L1 mRNA is
upregulated in the brains of mice chronically infected with
Toxoplasma (20). Moreover, the PD-L1 receptor, PD-1, is ex-
pressed on T cells recruited to Toxoplasma-infected brains (55)
and retinas (our unpublished results). Another potential
caveat is that our experiments were performed primarily by
injecting relatively high numbers (104) of parasites. But class II
and PD-L1 upregulation also occurred with lower doses of
parasites (103), approximating the number that would be re-
leased after as few as 1 to 2 cyst bursts (9).
Our data clearly establish IFN-? as a critical regulator of MHC
class II and PD-L1 expression on both infiltrating leukocytes and
resident retinal cells. However, IFN-? does not appear to be
sufficient for class II expression, since intravitreal or subretinal
IFN-? injection upregulated MHC class II in the cornea, in the
choroid, and on retinal pigment epithelial cells, but not on cells of
the inner retina (4, 19). This suggests that Toxoplasma infection
induces retinal cells to become responsive to IFN-?, perhaps by
upregulating IFN-? receptor expression.
Another important question is what cells are the source for
IFN-? in intravitreally infected mice. During reactivated ocular
FIG. 6. T-cell suppression by retinal cells from Toxoplasma-infected retinas is PD-L1 dependent. (A) Splenic T cells isolated from cpsII-
vaccinated mice were incubated with STAg alone (shaded bar) or STAg-loaded splenic APCs in the presence of increasing numbers (1 ? 105, 5 ?
104, or 1 ? 104) of retinal cells from either mock-infected (filled bars) or parasite-infected (open bars) mice. Shown are the averages for
quadruplicate samples repeated two independent times.*, P ? 0.005 by a two-tailed, unpaired Student t test. (B) Splenic T cells from vaccinated
mice were incubated with STAg alone (shaded bar) or with STAg-loaded DCs in the absence or presence of parasite-infected retinal cells.
Pyrimethamine (1 ?M) was included as indicated. Shown are results of a representative experiment repeated three times in quadruplicate.*, P ?
0.005 by Student’s t test. (C) Splenic T cells from vaccinated mice were incubated with the indicated cells (5 ? 104parasite-infected retinal cells
were used where indicated) and antibodies. Shown are results of a representative experiment carried out in quadruplicate. Asterisks indicate
significance by Student’s t test (**, P ? 0.01;*, P ? 0.05).
3490 CHARLES ET AL.INFECT. IMMUN.
toxoplasmosis, CD8?and CD4?T cells are important IFN-?-
secreting cells. However, significant numbers of retinal T cells
are not detectable 6 days after the injection of 104parasites.
Thus, a non-T-cell source for IFN-? is most likely responsible
for the early upregulation of MHC class II and PD-L1. Inflam-
matory monocytes and neutrophils are the two major types of
leukocytes in parasite-infected eyes. The monocytes are not
predicted to express significant amounts of IFN-? in parasite-
infected retinas, since a failure to recruit these cells to Toxo-
plasma-infected peritoneal cavities or mucosae did not result
in reduced IFN-? expression (10, 46). Although a failure to
recruit neutrophils during an intraperitoneal infection leads to
decreased IFN-? release, this is most likely due to a loss of
neutrophil-derived interleukin 12 (IL-12) (3). However, other
studies have demonstrated that neutrophils can express and
release IFN-? (26, 30), indicating that in parasite-infected ret-
inas, neutrophil-derived IFN-? may contribute to MHC class II
and PD-L1 expression. Brain microglia (which stain as CD45lo
CD11blocells) are important early sources of IFN-? after cyst
reactivation in the brain (53), and it is therefore possible that
retinal microglia respond similarly to Toxoplasma. Finally, a
third minor population (?5%) of CD45?cells that were
CD11b?and Ly6G?was present in parasite-infected eyes and
could be expressing IFN-? (not shown). The identity of these
cells is unknown, since they did not stain with anti-NK1.1 or
anti-B220 antibodies, suggesting that they were not NK cells or
B cells, respectively.
The importance of IFN-? in controlling Toxoplasma repli-
cation and in upregulating retinal PD-L1 expression suggests
that IFN-? triggers a negative feedback loop to downregulate
recruited effector T cells in an effort to limit immune-mediated
pathology. IL-10 is another cytokine demonstrated to down-
regulate T-cell responses to Toxoplasma in the periphery and
the retina (14, 37). However, PD-L1 expression is not depen-
dent on IL-10 (5). Moreover, PD-L1 and IL-10 act in parallel
to regulate the immune response during chronic lymphocytic
choriomeningitis virus infection (5). Similarly, we propose that
in the retina, IL-10 and PD-L1 act in concert to prevent im-
mune-mediated tissue damage. Such a model is supported by
the well-documented role of PD-L1 in downregulating T-cell-
based responses during viral, bacterial, and parasitic infections
(see, e.g., references 5, 24, and 31) as well in various autoim-
mune models (see, e.g., references 27, 40, 45, and 47). In
addition, retinal-cell suppression of T-cell responses was not
fully abrogated by the anti-PD-L1 antibody, suggesting the
presence of other T-cell-suppressing factors. Our future work
will focus on identifying and defining the contributions of these
various factors to the immunological responses during ocular
We thank John Boothroyd for providing the SAG1 antisera, Paul
Kincade for the Siglec-H antibody, and Jerry Niederkorn for critical
reading of the manuscript.
This work is supported by grants from the Oklahoma Center for the
Advancement of Science and Technology (HR05-138S) and by NIH
grants A069986 to I.J.B., AI048097 to A.D.F., EY016459 to J.D.A.,
AI078993 to M.L.L., and AI41930 to D.J.B.
1. Blasius, A. L., M. Cella, J. Maldonado, T. Takai, and M. Colonna. 2006.
Siglec-H is an IPC-specific receptor that modulates type I IFN secretion
through DAP12. Blood 107:2474–2476.
2. Blasius, A. L., E. Giurisato, M. Cella, R. D. Schreiber, A. S. Shaw, and M.
Colonna. 2006. Bone marrow stromal cell antigen 2 is a specific marker of
type I IFN-producing cells in the naive mouse, but a promiscuous cell surface
antigen following IFN stimulation. J. Immunol. 177:3260–3265.
3. Bliss, S. K., B. A. Butcher, and E. Y. Denkers. 2000. Rapid recruitment of
neutrophils containing prestored IL-12 during microbial infection. J. Immu-
4. Brandt, C., P. Knupfer, G. Boush, R. Gausas, and J. Chandler. 1990. In vivo
induction of Ia expression in murine cornea after intravitreal injection of
interferon-gamma. Invest. Ophthalmol. Vis. Sci. 31:2248–2253.
5. Brooks, D. G., S. J. Ha, H. Elsaesser, A. H. Sharpe, G. J. Freeman, and M. B.
Oldstone. 2008. IL-10 and PD-L1 operate through distinct pathways to
suppress T-cell activity during persistent viral infection. Proc. Natl. Acad. Sci.
U. S. A. 105:20428–20433.
6. Caspi, R. R., F. G. Roberge, C. C. Chan, B. Wiggert, G. J. Chader, L. A.
Rozenszajn, Z. Lando, and R. B. Nussenblatt. 1988. A new model of auto-
immune disease. Experimental autoimmune uveoretinitis induced in mice
with two different retinal antigens. J. Immunol. 140:1490–1495.
7. Charles, E., M. C. Callegan, and I. J. Blader. 2007. The SAG1 Toxoplasma
surface protein is not required for acute ocular toxoplasmosis in mice. Infect.
8. Dong, H., S. E. Strome, D. R. Salomao, H. Tamura, F. Hirano, D. B. Flies,
P. C. Roche, J. Lu, G. Zhu, K. Tamada, V. A. Lennon, E. Celis, and L. Chen.
2002. Tumor-associated B7–H1 promotes T-cell apoptosis: a potential mech-
anism of immune evasion. Nat. Med. 8:793–800.
9. Dubey, J. P., D. S. Lindsay, and C. A. Speer. 1998. Structures of Toxoplasma
gondii tachyzoites, bradyzoites, and sporozoites and biology and develop-
ment of tissue cysts. Clin. Microbiol. Rev. 11:267–299.
10. Dunay, I. R., R. A. Damatta, B. Fux, R. Presti, S. Greco, M. Colonna, and
L. D. Sibley. 2008. Gr1?inflammatory monocytes are required for mucosal
resistance to the pathogen Toxoplasma gondii. Immunity 29:306–317.
11. Fox, B. A., and D. J. Bzik. 2002. De novo pyrimidine biosynthesis is required
for virulence of Toxoplasma gondii. Nature 415:926–929.
12. Gazzinelli, R. T., A. Brezin, Q. Li, R. B. Nussenblatt, and C. C. Chan. 1994.
Toxoplasma gondii: acquired ocular toxoplasmosis in the murine model,
protective role of TNF-? and IFN-?. Exp. Parasitol. 78:217–229.
13. Gazzinelli, R. T., F. T. Hakim, S. Hieny, G. M. Shearer, and A. Sher. 1991.
FIG. 7. IFN-? is important for MHC class II and PD-L1 expression in
Toxoplasma-infected eyes. (A) Mice were intravitreally injected with 104
parasites (Toxo). After 4 and 6 days, eyes were harvested and enucleated,
and IFN-? levels were measured in lysates prepared from posterior seg-
ments. P values were determined using an unpaired, nonparametric Stu-
dent t test. (B) Wild-type and IFN-?KO mice were intravitreally injected
with 104parasites. Six days later, eyes were harvested and processed for
flow cytometry. Shown are representative FACS plots of MHC class II
and PD-L1 expression from three independent experiments.
VOL. 78, 2010TOXOPLASMA AND RETINAL CLASS II AND PD-L1 EXPRESSION3491
Synergistic role of CD4?and CD8?T lymphocytes in IFN-? production and Download full-text
protective immunity induced by an attenuated Toxoplasma gondii vaccine.
J. Immunol. 146:286–292.
14. Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, R.
Kuhn, W. Muller, G. Trinchieri, and A. Sher. 1996. In the absence of
endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to
a lethal immune response dependent on CD4?T cells and accompanied by
overproduction of IL-12, IFN-? and TNF-?. J. Immunol. 157:798–805.
15. Geissmann, F., S. Jung, and D. R. Littman. 2003. Blood monocytes
consist of two principal subsets with distinct migratory properties. Immu-
16. Greenwald, R. J., G. J. Freeman, and A. H. Sharpe. 2005. The B7 family
revisited. Annu. Rev. Immunol. 23:515–548.
17. Gregerson, D. S., N. D. Heuss, K. L. Lew, S. W. McPherson, and D. A.
Ferrington. 2007. Interaction of retinal pigmented epithelial cells and CD4
T cells leads to T-cell anergy. Invest. Ophthalmol. Vis Sci. 48:4654–4663.
18. Gregerson, D. S., and J. Yang. 2003. CD45-positive cells of the retina and
their responsiveness to in vivo and in vitro treatment with IFN-? or anti-
CD40. Invest. Ophthalmol. Vis Sci. 44:3083–3093.
19. Hamel, C. P., B. Detrick, and J. J. Hooks. 1990. Evaluation of Ia expression
in rat ocular tissues following inoculation with interferon-gamma. Exp. Eye
20. Hermes, G., J. W. Ajioka, K. A. Kelly, E. Mui, F. Roberts, K. Kasza, T. Mayr,
M. J. Kirisits, R. Wollmann, D. J. Ferguson, C. W. Roberts, J. H. Hwang, T.
Trendler, R. P. Kennan, Y. Suzuki, C. Reardon, W. F. Hickey, L. Chen, and
R. McLeod. 2008. Neurological and behavioral abnormalities, ventricular
dilatation, altered cellular functions, inflammation, and neuronal injury in
brains of mice due to common, persistent, parasitic infection. J. Neuroin-
21. Holland, G. N. 2003. Ocular toxoplasmosis: a global reassessment. Part I:
epidemiology and course of disease. Am. J. Ophthalmol. 136:973–988.
22. Hori, J., M. Wang, M. Miyashita, K. Tanemoto, H. Takahashi, T. Takemori,
K. Okumura, H. Yagita, and M. Azuma. 2006. B7–H1-induced apoptosis as
a mechanism of immune privilege of corneal allografts. J. Immunol. 177:
23. Israelski, D. M., F. G. Araujo, F. K. Conley, Y. Suzuki, S. Sharma, and J. S.
Remington. 1989. Treatment with anti-L3T4 (CD4) monoclonal antibody
reduces the inflammatory response in toxoplasmic encephalitis. J. Immunol.
24. Jurado, J. O., I. B. Alvarez, V. Pasquinelli, G. J. Martinez, M. F. Quiroga, E.
Abbate, R. M. Musella, H. E. Chuluyan, and V. E. Garcia. 2008. Pro-
grammed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell
effector functions during human tuberculosis. J. Immunol. 181:116–125.
25. Kaneko, H., K. M. Nishiguchi, M. Nakamura, S. Kachi, and H. Terasaki.
2008. Characteristics of bone marrow-derived microglia in the normal and
injured retina. Invest. Ophthalmol. Vis Sci. 49:4162–4168.
26. Kirby, A. C., U. Yrlid, and M. J. Wick. 2002. The innate immune response
differs in primary and secondary Salmonella infection. J. Immunol. 169:
27. Latchman, Y. E., S. C. Liang, Y. Wu, T. Chernova, R. A. Sobel, M. Klemm,
V. K. Kuchroo, G. J. Freeman, and A. H. Sharpe. 2004. PD-L1-deficient mice
show that PD-L1 on T cells, antigen-presenting cells, and host tissues neg-
atively regulates T cells. Proc. Natl. Acad. Sci. U. S. A. 101:10691–10696.
28. Lazar-Molnar, E., A. Gacser, G. J. Freeman, S. C. Almo, S. G. Nathenson,
and J. D. Nosanchuk. 2008. The PD-1/PD-L costimulatory pathway critically
affects host resistance to the pathogenic fungus Histoplasma capsulatum.
Proc. Natl. Acad. Sci. U. S. A. 105:2658–2663.
29. Lee, S. K., S. H. Seo, B. S. Kim, C. D. Kim, J. H. Lee, J. S. Kang, P. J. Maeng,
and J. S. Lim. 2005. IFN-? regulates the expression of B7–H1 in dermal
fibroblast cells. J. Dermatol. Sci. 40:95–103.
30. Li, L., L. Huang, S.-S. J. Sung, P. I. Lobo, M. G. Brown, R. K. Gregg, V. H.
Engelhard, and M. D. Okusa. 2007. NKT cell activation mediates neutrophil
IFN-? production and renal ischemia-reperfusion injury. J. Immunol. 178:
31. Liang, S. C., R. J. Greenwald, Y. E. Latchman, L. Rosas, A. Satoskar, G. J.
Freeman, and A. H. Sharpe. 2006. PD-L1 and PD-L2 have distinct roles in
regulating host immunity to cutaneous leishmaniasis. Eur. J. Immunol. 36:
32. Liesenfeld, O., J. Kosek, J. S. Remington, and Y. Suzuki. 1996. Association
of CD4?T cell-dependent, interferon-gamma-mediated necrosis of the
small intestine with genetic susceptibility of mice to peroral infection with
Toxoplasma gondii. J. Exp. Med. 184:597–607.
33. Liversidge, J., H. F. Sewell, A. W. Thomson, and J. V. Forrester. 1988.
Lymphokine-induced MHC class II antigen expression on cultured retinal
pigment epithelial cells and the influence of cyclosporin A. Immunology
34. Liversidge, J. M., H. F. Sewell, and J. V. Forrester. 1988. Human retinal
pigment epithelial cells differentially express MHC class II (HLA, DP, DR
and DQ) antigens in response to in vitro stimulation with lymphokine or
purified IFN-?. Clin. Exp. Immunol. 73:489–494.
35. Loke, P., and J. P. Allison. 2003. PD-L1 and PD-L2 are differentially regu-
lated by Th1 and Th2 cells. Proc. Natl. Acad. Sci. U. S. A. 100:5336–5341.
36. Lu, F., S. Huang, and L. H. Kasper. 2004. CD4?T cells in the pathogenesis
of murine ocular toxoplasmosis. Infect. Immun. 72:4966–4972.
37. Lu, F., S. Huang, and L. H. Kasper. 2003. Interleukin-10 and pathogenesis
of murine ocular toxoplasmosis. Infect. Immun. 71:7159–7163.
38. Luft, B. J., and J. S. Remington. 1992. Toxoplasmic encephalitis in AIDS.
Clin. Infect. Dis. 15:211–222.
39. Lyons, R. E., J. P. Anthony, D. J. Ferguson, N. Byrne, J. Alexander, F.
Roberts, and C. W. Roberts. 2001. Immunological studies of chronic ocular
toxoplasmosis: up-regulation of major histocompatibility complex class I and
transforming growth factor beta and a protective role for interleukin-6.
Infect. Immun. 69:2589–2595.
40. Martin-Orozco, N., Y. H. Wang, H. Yagita, and C. Dong. 2006. Programmed
death (PD) ligand-1/PD-1 interaction is required for CD8?T cell tolerance
to tissue antigens. J. Immunol. 177:8291–8295.
41. Mazanet, M. M., and C. C. Hughes. 2002. B7–H1 is expressed by human
endothelial cells and suppresses T cell cytokine synthesis. J. Immunol. 169:
42. Montoya, J. G., and O. Liesenfeld. 2004. Toxoplasmosis. Lancet 363:1965–
43. Parker, S. J., C. W. Roberts, and J. Alexander. 1991. CD8?T cells are the
major lymphocyte subpopulation involved in the protective immune re-
sponse to Toxoplasma gondii in mice. Clin. Exp. Immunol. 84:207–212.
44. Percopo, C. M., J. J. Hooks, T. Shinohara, R. Caspi, and B. Detrick. 1990.
Cytokine-mediated activation of a neuronal retinal resident cell provokes
antigen presentation. J. Immunol. 145:4101–4107.
45. Reynoso, E. D., K. G. Elpek, L. Francisco, R. Bronson, A. Bellemare-Pelle-
tier, A. H. Sharpe, G. J. Freeman, and S. J. Turley. 2009. Intestinal tolerance
is converted to autoimmune enteritis upon PD-1 ligand blockade. J. Immu-
46. Robben, P. M., M. LaRegina, W. A. Kuziel, and L. D. Sibley. 2005. Recruit-
ment of Gr-1?monocytes is essential for control of acute toxoplasmosis. J.
Exp. Med. 201:1761–1769.
47. Schreiner, B., S. L. Bailey, T. Shin, L. Chen, and S. D. Miller. 2008. PD-1
ligands expressed on myeloid-derived APC in the CNS regulate T-cell re-
sponses in EAE. Eur. J. Immunol. 38:2706–2717.
48. Shen, L., Y. Jin, G. J. Freeman, A. H. Sharpe, and M. R. Dana. 2007. The
function of donor versus recipient programmed death-ligand 1 in corneal
allograft survival. J. Immunol. 179:3672–3679.
49. Streilein, J. W. 2003. Ocular immune privilege: the eye takes a dim but
practical view of immunity and inflammation. J. Leukoc. Biol. 74:179–185.
50. Streilein, J. W., K. Ohta, J. S. Mo, and A. W. Taylor. 2002. Ocular immune
privilege and the impact of intraocular inflammation. DNA Cell Biol. 21:
51. Subauste, C. S., R. de Waal Malefyt, and F. Fuh. 1998. Role of CD80 (B7.1)
and CD86 (B7.2) in the immune response to an intracellular pathogen.
J. Immunol. 160:1831–1840.
52. Suzuki, Y., and J. S. Remington. 1988. Dual regulation of resistance against
Toxoplasma gondii infection by Lyt-2?and Lyt-1?, L3T4?T cells in mice.
J. Immunol. 140:3943–3946.
53. Wang, X., and Y. Suzuki. 2007. Microglia produce IFN-? independently from
T cells during acute toxoplasmosis in the brain. J. Interferon Cytokine Res.
54. Weiss, L. M., and K. Kim. 2000. The development and biology of bradyzoites
of Toxoplasma gondii. Front. Biosci. 5:D391–D405.
55. Wilson, E. H., T. H. Harris, P. Mrass, B. John, E. D. Tait, G. F. Wu, M.
Pepper, E. J. Wherry, F. Dzierzinski, D. Roos, P. G. Haydon, T. M. Laufer,
W. Weninger, and C. A. Hunter. 2009. Behavior of parasite-specific effector
CD8?T cells in the brain and visualization of a kinesis-associated system of
reticular fibers. Immunity 30:300–311.
56. Wilson, E. H., C. Zaph, M. Mohrs, A. Welcher, J. Siu, D. Artis, and C. A.
Hunter. 2006. B7RP-1-ICOS interactions are required for optimal infection-
induced expansion of CD4?Th1 and Th2 responses. J. Immunol. 177:2365–
57. Xu, H., R. Dawson, J. V. Forrester, and J. Liversidge. 2007. Identification of
novel dendritic cell populations in normal mouse retina. Invest. Ophthalmol.
Vis Sci. 48:1701–1710.
58. Yang, W., H.-C. Li, P. W. Chen, H. Alizadeh, Y. He, R. N. Hogan, and J. Y.
Niederkorn. 2009. PD-L1 expression on human ocular cells and its possible
role in regulating immune-mediated ocular inflammation. Invest. Ophthal-
mol. Vis. Sci. 50:273–280.
Editor: J. H. Adams
3492CHARLES ET AL.INFECT. IMMUN.