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* Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Bristol-Myers Squibb Pharmacology Research Institute, Princeton, NJ 08543; and
Amgen Inc., Thousand Oaks, CA 91320
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. CD28 is a costimulatory molecule important for optimal
activation of T cells, but CD28-/- mice are resistant to
T. gondii, demonstrating that CD28-independent
mechanisms regulate T cell responses during toxoplasmosis. The
identification of the B7-related protein 1/inducible costimulator
protein (ICOS) pathway and its ability to regulate the
production of IFN- suggested that this pathway may be involved in
the CD28-independent activation of T cells required for resistance to
T. gondii. In support of this hypothesis, infection of
wild-type or CD28-/- mice with T. gondii
resulted in the increased expression of ICOS by activated
CD4+ and CD8+ T cells. In addition, both
costimulatory pathways contributed to the in vitro production of
IFN- by parasite-specific T cells and when both pathways were
blocked, there was an additive effect that resulted in almost complete
inhibition of IFN- production. Although in vivo blockade of the ICOS
costimulatory pathway did not result in the early mortality of
wild-type mice infected with T. gondii, it did lead to
increased susceptibility of CD28-/- mice to T.
gondi associated with reduced serum levels of IFN-,
increased parasite burden, and increased mortality compared with the
control group. Together, these results identify a critical role for
ICOS in the protective Th1-type response required for resistance to
T. gondii and suggest that ICOS and CD28 are parallel
costimulatory pathways, either of which is sufficient to mediate
resistance to this intracellular pathogen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the major mediator of resistance to infection
(1). Given the critical role of T cells in the initiation
and maintenance of immunity to T. gondii
(2, 3, 4), it is important to understand the events that
regulate their responses. Since the description of the two signal
hypothesis of lymphocyte activation, it has been recognized that
costimulation is an important process involved in the development and
regulation of T cell responses. The B7/CD28 pathway is an important
costimulatory interaction involved in T cell activation
(5). However, previous studies from this laboratory have
shown that CD28-/- mice infected with
T. gondii have a reduced CD4+ T cell
response, but do mount a T cell response that is sufficient to provide
resistance to this infection (6, 7). These results suggest
that other costimulatory pathways are likely involved in the regulation
of T cell responses during toxoplasmosis, or may reflect the ability of
T. gondii infection to provide a TCR signal that is
sufficient to overcome the requirement for CD28-mediated costimulation
(8, 9).
Costimulatory functions have been ascribed to molecules such as ICAM-1,
CD2, CD30, CD40 ligand, CD44, CD137, and 4-1BB
(10, 11, 12, 13), but our studies have suggested that neither
ICAM-1, CD40 ligand, or CD44 are critical for the development of T cell
responses required for resistance to T. gondii (7, 14). Recently, inducible costimulator protein
(ICOS),4 a molecule
that is homologous to CD28 has been shown to be a potent costimulatory
molecule in T cell activation (15, 16). Unlike CD28, which
is constitutively expressed on T cells, ICOS is expressed only on
activated CD4+ and CD8+ T
cells and a subpopulation of memory T cells (15, 16). The
ligand for ICOS is B7-related protein 1 (B7RP-1; B7 h, GL50, ICOSL),
which is structurally related to members of the B7 family, and is
expressed on macrophages, B cells, TNF-
-activated fibroblast cells,
as well as on other nonlymphoid tissues (15, 17, 18, 19).
Early studies demonstrated that ICOS was involved in the production of
IL-4, IL-10, and IFN-, but not IL-2 by T cells, and subsequent
studies reported that ICOS is expressed preferentially on Th2-type T
cells and is important during Th2-type immune responses
(20, 21, 22, 23, 24, 25). Additional studies have shown that ICOS is
involved in the regulation of Th1 and Th2 responses to the parasites
Nippostrongylus brasiliensis and Leishmania
mexicana as well as the viral pathogens lymphocytic
choriomeningitis virus and vesicular stomatitis virus but the
importance of ICOS in the outcome of these infections is unclear
(26, 27). Nevertheless, since ICOS is capable of providing
a costimulatory signal to activate T cells, this was a candidate
costimulatory molecule for the CD28-independent regulation of Th1-type
T cell responses during toxoplasmosis. The studies presented here
reveal that ICOS expression is up-regulated on activated T cells
following infection with T. gondii and that this is
independent of CD28-mediated signaling. Moreover, ICOS and CD28 are
both required for optimal production of IFN- by parasite-specific T
cells and the in vivo blockade of the B7RP-1-ICOS interaction results
in increased susceptibility of CD28-/- mice to
infection. Together, these results identify a critical role for ICOS in
the protective Th1-type response to T. gondii and suggest
that ICOS and CD28 are parallel costimulatory pathways, either of which
is sufficient to mediate resistance to T. gondii.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, Swiss Webster, and CBA/CaJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 CD28-deficient animals (28) were bred and housed in the animal facilities at the University of Pennsylvania (Philadelphia, PA). All mice used were maintained within microisolator caging units at the University Laboratory Animal Resource facilities at the University of Pennsylvania. Six- to 12-wk-old C57BL/6 or CD28-/- mice were used for all experiments with three to six mice per group.
Parasites and infection
Swiss Webster and CBA/CaJ mice were used to maintain the ME49 strain of T. gondii and used as sources of tissue cysts for these experiments. For infection with ME49 tissue cysts, brains of chronically infected CBA/CaJ mice were harvested and prepared as previously described (29, 30). Mice were inoculated i.p. with 20 ME49 cysts in a volume of 0.2 ml. Serum samples were collected at different time points and survival was monitored. The RH strain of T. gondii was maintained in human foreskin fibroblast as previously described (7) and used to prepare soluble Toxoplasma Ag (STAg) as previously described (31). STAg activity was titrated to determine the optimal concentration for splenocyte proliferation and cytokine production (25 µg/ml).
Cytological analysis
At the time of sacrifice, peritoneal exudate cells were harvested with 5 ml of ice-cold 1x PBS without Ca2+/Mg2+ (BioWhittaker, Walkersville, MD) and 5 x 105 cells/100 µl were used to prepare cytospins to quantitate parasite burden. Cells were fixed and stained using Protocol Hema3 (Biochemical Sciences, Swedesboro, NJ) as described in the manufacturer’s manual, then mounted and sealed using Cytoseal (Stephens Scientific, Kalamazoo, MI).
Reagents
Complete RPMI 1640 (Life Technologies, Gaithersburg, MD) medium
containing 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT),
sodium pyruvate, nonessential amino acids, penicillin (100 U/ml),
streptomycin (1 mg/ml), and amphotericin B (25 ng/ml) (BioWhittaker).
The 145-2C11 hybridoma for anti-CD3
was provided by Dr. J.
Bluestone (University of Chicago, Chicago, IL) and used to prepare
purified anti-CD3. HuCTLA-4Ig, a fusion protein comprised of the
human CTLA-4 extracellular domain and Fc portion of human IgG, and the
chimeric control fusion protein L6 (Chi-L6) was supplied by
Bristol-Myers Squibb Research Institute (Princeton, NJ) and used at a
concentration of 20 µg/ml. B7RP-1Fc, a fusion protein consisting of
the extracellular portion of the mouse B7RP-1 and the Fc portion of
human IgG, was used to detect expression of ICOS and was supplied by
Amgen (Thousand Oaks, CA). mAb specific to B7RP-1 (1B7; Amgen) was used
at a concentration of 20 µg/ml. Infected mice were treated with 1 mg
of anti-B7RP-1 or rat Ig (Sigma-Aldrich, St. Louis, MO) at days 0
and 5 postinfection.
Isolation of brain mononuclear cells
Isolation of brain mononuclear cells (BMNCs) were performed as previously described (6). Briefly, mice were anesthetized with 200 mg/kg Kitaset (Fort Dodge Animal Health, Fort Dodge, IA) and 10 mg/kg Xyla-Ject (Phoenix Pharmaceuticals, St. Joseph, MO). Mice were perfused through the left ventricle with 50–60 ml of ice-cold PBS to remove peripheral blood. Brains were then collected, minced, resuspended in complete RPMI 1640, and digested with 100 µg of collagenase/dispase (Roche Diagnostics, Indianapolis, IN) and 300 µg of DNase (Roche Diagnostics) for 45 min at 37°C. Brain digests were then pelleted at 2000 x g for 10 min at 4°C and resuspended in 60% isotonic Percoll solution and overlaid with 30% Percoll solution. The Percoll gradient was centrifuged at 1000 x g for 25 min at 25°C and the top myelin layer was carefully removed before harvesting BMNCs at the 60 and 30% interphase layer. Cells were washed with complete RPMI 1640 before further analysis. Due to the low number of BMNCs obtained per animal, cells from at least three mice were pooled in each experiment.
Analysis of T cell responses
Individual spleens from animals were harvested, dissociated into
single-cell suspensions, and depleted of erythrocytes using 0.83% w/v
ammonium chloride (Sigma-Aldrich). Cells were washed and resuspended in
complete RPMI 1640 before further analysis. Splenocytes or BMNCs were
plated at a cell density of 2 x 105
cells/well in a final volume of 200 µl in 96-well plates (Costar,
Costar, NY). Cells were stimulated with soluble anti-CD3 (145-2C11)
at a final concentration of 1 µg/ml or STAg for 48 h at
37°C/5% CO2 in the presence of CTLA-4Ig and/or
anti-B7RP-1. Levels of IFN- from splenocyte cultures were
measured using ELISA as previously described (32).
Flow cytometric analysis
To assess surface expression of ICOS on T cells, purified
splenocytes were resuspended in FACS buffer (1x PBS, 0.2% BSA
fraction V, and 4 mM sodium azide) at a concentration of 1 x
107 cells/ml. A total of
106 cells was preincubated with saturating
concentrations of Fc Block for 20 min on ice and stained with PE or
PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8 (BD
PharMingen, San Diego, CA), and B7RP-1Fc (1 mg/ml) for 30 min on ice.
Cells were washed with FACS buffer and then incubated with biotinylated
goat anti-human IgG (Vector Laboratories, Burlingame, CA) for 30
min on ice. Cells were washed with FACS buffer and incubated with
allophycocyanin-conjugated streptavidin (BD PharMingen) for an
additional 20 min on ice. Cells were then washed, resuspended in FACS
buffer, and analyzed using a FACSCalibur flow cytometer (BD
Biosciences, San Jose, CA). For intracellular detection of IFN-,
purified splenocytes were plated in a 96-well plate (Costar) at a
density of 4 x 105 cells/well in a final
volume of 200 µl. Cells were stimulated with STAg (25 µg/ml) for
72 h, then PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A
(10 µg/ml; Sigma-Aldrich) were added to cultures during the last
5 h of stimulation. Cells were harvested, washed, resuspended in
FACS buffer, and then stained with various fluorochrome-conjugated Abs
as described above. Cells were then washed with FACS buffer, fixed with
4% w/v paraformaldehyde, washed again, and permeabilized with 0.1%
w/v saponin in FACS buffer. After permeabilization, cells were stained
with allophycocyanin-conjugated anti-IFN- (BD PharMingen) for 30
min on ice. Cells were washed once with 0.1% saponin buffer and then
with FACS buffer. Analysis of the cells was performed using a
FACSCalibur flow cytometer (BD Biosciences). Results were analyzed
using CellQuest software (BD Biosciences). Ab concentrations were
empirically determined to give optimal staining for flow cytometric
analyses.
Statistics
INSTAT software (GraphPad, San Diego, CA) was used for unpaired or paired two-tailed Student’s t test. A value of p < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recent identification of the B7RP-1-ICOS interaction and its
role in providing a costimulatory signal for T cell activation
suggested a possible alternative costimulatory signal involved in the
CD28-independent mechanism of T cell-mediated resistance to T.
gondii (6, 7). To determine whether ICOS was involved
in the immune response to T. gondii, experiments were
performed to compare the expression of ICOS in uninfected and infected
mice. C57BL/6 mice were infected i.p. with 20 cysts of the ME49 strain
of T. gondii and at 7 days after infection, splenocytes were
harvested, then analyzed for expression of ICOS. In uninfected C57BL/6
animals, only a small percentage of T cells expressed ICOS and all of
these T cells were CD44high (Fig. 1
A). By day 7 after infection,
there was small increase in the percentage of
CD8+ T cells which expressed ICOS and a marked
expansion in the percentage of CD4+ T cells
expressing ICOS (Fig. 1B). Although a slight variation in
the magnitude of ICOS up-regulation on CD4+ and
CD8+ T cells was observed in four independent
experiments, the levels of ICOS expressed on T cells from infected
animals was consistently and reproducibly higher than uninfected
C57BL/6 mice. In contrast, at 6 wk postinfection, ICOS expression on
splenic CD4+ T cells had decreased compared with
splenic T cells from 7-day infected animals, albeit ICOS expression on
splenic T cells from 6-wk infected animals remained higher than those
of uninfected mice (Fig. 1C).
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D).
Together, these data reveal that following infection, the activation of
T cells in the spleen is accompanied by increased expression of ICOS
during the acute phase of infection. As the infection is controlled in
the periphery, the numbers of ICOS+ T cells in
the spleen return to near-normal levels. However, T cells isolated from
the brains of mice with TE continued to express high levels of ICOS.
Together, these results suggest that the infection-induced expression
of ICOS is restricted to parasite-specific effector T cells.
CD28 and ICOS are both required for parasite-specific production of
IFN-
Since the ability of T cells to produce IFN- is essential for
resistance to T. gondii (1), the role of ICOS
and CD28 in the ability of splenocytes from naive and acutely infected
mice to produce IFN- was compared. Stimulation of naive splenocytes
with anti-CD3 resulted in the production of IFN- and, as
previously reported (33), this response was almost
completely blocked by CTLA-4Ig (p < 0.0004).
In contrast, anti-B7RP-1 had no effect on the production of IFN-
by these resting T cells (Fig. 2A, p = 0.7).
These results indicate that although CD28 is critical in activation and
production of IFN- by resting T cells, ICOS plays a minimal role in
this initial activation process. When splenocytes from acutely infected
mice were stimulated with STAg, the production of IFN- by
parasite-specific T cells was partially inhibited by the addition of
CTLA-4Ig (Fig. 2B, p < 0.003). Similarly,
the addition of anti-B7RP-1 also had an inhibitory effect on the
production of IFN- in these cultures (Fig. 2B,
p < 0.001). Moreover, the addition of CTLA-4Ig plus
anti-B7RP-1 resulted in an additive effect and reduced the levels
of IFN- production to background (Fig. 2B,
p < 0.0001). Similar results were observed when
infected splenocytes from C57BL/6 mice were stimulated with
anti-CD3 (data not shown). Further analysis of Ag-stimulated
splenocytes from acutely infected animals revealed that few
CD8+ T cells were IFN-+
(data not shown) but that >75% of the CD4+ T
cells that expressed IFN-+ also expressed ICOS
(Fig. 2C).
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compared with
unstimulated cells. The addition of CTLA-4Ig or anti-B7RP-1 only
resulted in a small, but significant, reduction in the levels of
IFN- produced in response to Ag (Fig. 2D,
p < 0.005 and 0.003, respectively). However, when both
costimulatory pathways were blocked, there was a 50% reduction in
levels of IFN- produced (Fig. 2D, p <
0.001). Together, these results indicate that following infection with
T. gondii, costimulation through ICOS, in combination with
CD28, is required for optimal production of IFN-. Thus, in WT mice
there are distinct CD28- and ICOS-dependent components required for
optimal production of IFN-. Infection-induced expression of ICOS is independent of CD28
The data presented in the previous section support the hypothesis
that ICOS has a role in the regulation of IFN- responses during
toxoplasmosis and that its effects may be independent of CD28. However,
previous in vitro studies have reported that the ability of activated T
cells to express ICOS was largely dependent on costimulation through
CD28 (20, 34). Therefore, to help determine whether ICOS
was involved in CD28-independent pathways of resistance to T.
gondii, studies were performed to assess whether the
infection-induced increase in expression of ICOS occurred in
CD28-/- mice. Analysis of ICOS expression on T
cells revealed that a small percentage of CD4+
(3%) and CD8+ T cells (1%) express ICOS and
expression was restricted to CD44high T cells
(Fig. 3A). However, as
described previously, infection of CD28-/- mice
results in normal numbers of activated CD8+ T
cells, but there are decreased numbers of activated
CD4+ T cells (6, 7). This defect, as
well as the infection-induced increase in the number of splenocytes,
makes it difficult to directly compare the percentage of activated
CD4+ T cells that express ICOS in
CD28-/- and WT mice. Therefore, the data
presented in Fig. 3B show the total numbers of
CD4+ and CD8+ T cells which
express ICOS in the spleens of uninfected and infected mice. At day 7
postinfection, analysis of ICOS expression revealed that there was no
significant difference (p = 0.26) in the total
numbers of ICOS+CD8+ T
cells in WT and CD28-/- mice, and although
uninfected and infected CD28-/- mice had fewer
ICOS+CD4+ T cells than WT
controls (uninfected, p < 0.02; infected,
p < 0.01), there was a 2-fold increase in the numbers
of ICOS+CD4+ T cells in the
infected CD28-/- mice (Fig. 3B). In
contrast to the spleen, analysis of CD4+ and
CD8+ T cells isolated from the brains of
chronically infected CD28-/- mice revealed that
the majority of these T cells were CD44high and
expressed high levels of ICOS (Fig. 3C). These results
demonstrate that although there are fewer ICOS+ T
cells in the spleens of acutely infected
CD28-/- mice compared with WT mice, there is
still a significant increase in the number of
CD28-/- T cells which have up-regulated ICOS
during the acute phase of infection. Moreover, by the chronic phase of
the infection, expression of ICOS by T cells from the brains of
infected CD28-/- mice was comparable to that
observed in chronically infected WT mice. Thus, the ability of T cells
to increase expression of ICOS in response to infection can occur
independently of CD28.
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To determine the possible role of ICOS as an alternative
costimulatory molecule in the CD28-independent resistance to T.
gondii (6, 7), splenocytes from infected
CD28-/- mice were stimulated with STAg and
production of IFN- by T cells in the presence of CTLA-4Ig,
anti-B7RP-1, or in combination were measured by ELISA. Splenocytes from
CD28-/- mice infected for 7 days and stimulated
with STAg produced low levels of IFN- and the addition of CTLA-4Ig
did not alter IFN- levels (7) (data not shown).
However, the addition of anti-B7RP-1 resulted in a marked decrease
(p < 0.002) in the production of IFN- (Fig. 4A). To further explore the in
vitro role of ICOS in regulating effector T cell function in the
absence of CD28, BMNCs from the brains of chronically infected
CD28-/- animals were stimulated with toxoplasma
Ag, and IFN- production in the presence or absence of
anti-B7RP-1 was measured. BMNCs from chronically infected
CD28-/- mice stimulated with STAg produced
significantly higher levels of IFN- compared with unstimulated cells
(p < 0.001), and anti-B7RP-1 significantly
inhibited (p < 0.001) the response to STAg
(Fig. 4B). These results strengthen the hypothesis that the
B7RP-1/ICOS pathway is required for the CD28-independent mechanisms
involved in the production of IFN- by parasite-specific T cells
during the acute and chronic phases of infection.
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To determine the in vivo role of ICOS, WT mice were infected with
T. gondii and treated with anti-B7RP-1 at days 0 and 5
after infection. Initial studies revealed no significant difference in
mortality, serum levels of IFN-, or parasite burden in treated WT
mice compared with control treated mice (data not shown). However,
CD28-/- mice treated with anti-B7RP-1 on
days 0, 3, and 5 of infection showed increased susceptibility to
infection compared with infected CD28-/- mice
that received the sham treatment (Fig. 5A). The ability of
anti-B7RP-1 treatment to increase susceptibility of
CD28-/- mice to infection correlated with a
significant reduction in serum levels of IFN- (Fig. 5B,
p < 0.03) and an increase in the parasite burden
compared with sham-treated CD28-/- mice (Fig. 5C, p < 0.005). Analysis of splenocytes
from infected CD28-/- mice treated with
anti-B7RP-1 revealed that they produced reduced levels of IFN-
when stimulated with STAg compared with control
CD28-/- mice. Although these results failed to
reach statistical significance (p = 0.17) a
similar trend (7.9 vs 4.1 ng; n = 3 mice/group) was
also observed in a repeat experiment. However, for both experimental
groups, the addition of anti-B7RP-1 in vitro resulted in a
significant reduction in the levels of IFN- (Fig. 5D,
p < 0.03 for both experimental groups). Taken
together, these results demonstrate that in the absence of CD28, ICOS
has an important role in the production of IFN- and resistance to
T. gondii.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during the acute phase of toxoplasmosis. In addition, although
blockade of the B7RP-1-ICOS interaction did not affect the overall
susceptibility of WT mice to T. gondii,
CD28-/- mice treated with anti-B7RP-1
became susceptible to infection. In agreement with these findings,
previous studies have shown that ICOS is involved in the regulation of
CD28-dependent and CD28-independent Th1 and Th2 responses to the
nematode N. brasiliensis and the viral pathogens lymphocytic
choriomeningitis virus and vesicular stomatitis virus
(26). However, the importance of ICOS in the outcome of
these infections was unclear and blockade of ICOS in
CD28-/- mice did not alter the time of
expulsion of N. brasiliensis. Similarly,
ICOS-/- mice are slightly more resistant to
infection with L. mexicana than WT mice, although the Th1
and Th2 responses specific for L. mexicana in these mice are
compromised (27). Thus, the data presented here provide
the first demonstration that ICOS has a critical role in resistance to
a pathogen.
Following the identification of ICOS, a consensus quickly emerged that
the B7RP-1-ICOS interaction was more important in Th2- than Th1-type
responses. Thus, blockade of the B7RP-1-ICOS interaction inhibited Th2-
but not Th1-mediated lung inflammation (21), and several
studies have shown that Th2 cells express higher levels of ICOS than do
Th1 cells (20, 26). Moreover, ICOS-deficient mice have
severe defects in Th2 responses, CD40-mediated Ab class switching, and
germinal center formation but produced normal levels of IFN-
(22, 23, 24). However, this paradigm has been challenged by
studies which demonstrated that blockade of the B7RP-1-ICOS interaction
ameliorated experimental allergic encephalomyelitis and graft
rejection, which are both mediated by Th1 cells (25, 35).
The studies presented here extend those findings and identify an
important role for ICOS in Th1-type responses required for resistance
to an intracellular pathogen.
Analysis of ICOS expression following infection with T.
gondii revealed distinct patterns associated with different phases
of infection. During the acute phase of infection, high levels of ICOS
were expressed on activated CD4+ T cells from the
spleen, consistent with the model that CD4+ T
cells are the major source of T cell-derived IFN- during acute
infection (36). In contrast, during toxoplasmic
encephalitis, when the brain is the site most affected, expression of
ICOS by splenic T cells had decreased, while CD4+
and CD8+ T cells isolated from the brains of
infected mice expressed high levels of ICOS. Since ICOS can provide
costimulation for both CD4+ (15, 23, 25, 35, 37) and CD8+ (38) T cells
to make IFN-, this finding is consistent with their role in
producing IFN- required for resistance to toxoplasmic encephalitis
(3, 39). However, in the studies with T cells from the
brains of mice with TE, blockade of ICOS alone had a small but
reproducible inhibitory effect on their ability to produce IFN-.
When the blockade was combined with CTLA4-Ig, there was an additive
effect, although significant levels of IFN- were still produced.
Nonetheless, the simultaneous blockade of CD28 and ICOS led to a 50%
reduction in levels of IFN- produced by these T cells and suggest a
role for these costimulatory pathways in the pathogenesis of
toxoplasmic encephalitis. Similarly, although the administration of
anti-B7RP-1 to infected CD28-/- mice
resulted in reduced serum levels of IFN- and reduced production of
IFN- in recall responses, there were still significant levels of
IFN- and some of these mice did survive the acute phase of
infection. There are several possible explanations for these findings
and perhaps the blockade of the B7RP-1-ICOS interaction in these
studies was incomplete and studies with ICOS-/-
mice would be needed to address this issue. Alternatively, the studies
presented here have focused on the role of ICOS in the regulation of T
cell responses but have not addressed whether ICOS has a role in the
regulation of NK cell production of IFN- which provides an innate
mechanism of resistance to T. gondii (40, 41).
Our previous studies have shown that infection of severe combined
immunodeficient mice with T. gondii stimulates NK cells to
express CD28 and that the CD28-B7 interaction is involved in the
regulation of NK cell production of IFN- (42). Related
to the studies presented here, although the NK cell responses to
T. gondii are blunted in CD28-/-
mice (7), these NK cells may still contribute to the low
systemic levels of IFN- seen in infected
CD28-/- mice treated with anti-B7RP-1.
Alternatively, it is also possible that the administration of
anti-B7RP-1 may inhibit NK cell responses since preliminary studies
indicate that although resting NK cell do not express ICOS,
IL-2-activated NK cells can express low levels of ICOS (our
unpublished observations). Whether this has any functional significance
for NK cell responses during toxoplasmosis is unclear and is the
subject of ongoing studies.
Initial in vitro studies demonstrated that the stimulation of T cells with anti-CD3 resulted in ICOS expression which was largely dependent on the B7-CD28 interaction (20) and suggested that some of the effects attributed to CD28 could be due to signaling through ICOS. Since CD28 is expressed constitutively, whereas ICOS expression is inducible, a model in which stimulation through CD28 was required for the activation of T cells and increased expression of ICOS was proposed. However, when human CD4+ T cells are stimulated with suboptimal levels of anti-CD3, ICOS can provide costimulation (34) and has also been shown to have a role in the costimulation of CD28-/- T cells (26). The in vivo data presented here show that in the absence of CD28 there is a marked reduction in the total number of CD4+ T cells that express ICOS. Although these data confirm the important role of CD28 for optimal CD4+ T cell responses during toxoplasmosis, we interpret the reduced levels of ICOS as a result of reduced numbers of activated CD4+ T cells in these mice. However, the high levels of ICOS observed on CD4+ and CD8+ T cells isolated from brains of CD28-/- mice with TE demonstrate that stimulation through CD28 is not required for expression of ICOS during toxoplasmosis.
The close structural and functional relationship between ICOS and CD28
raises the question of whether these molecules are functionally
redundant or whether they have distinct roles in the regulation of T
cell responses required for resistance to infection. For example, in
mice chronically infected with T. gondii, the CD28-mediated
production of IL-2 is important for the ability of
CD8+ T cells to make IFN- in recall responses
and more recently studies have indicated an important role for IL-2
during ICOS-mediated costimulation (34). Thus, although
these may be parallel pathways, both may be required for optimal T cell
responses in this particular experimental system. Nevertheless, during
toxoplasmosis, WT mice treated with anti-B7RP-1 are resistant to
T. gondii, but in the absence of CD28, ICOS is critical for
resistance. These findings correlate with the presence of
CD28-dependent and ICOS-dependent pathways for the production of
IFN- during toxoplasmosis and suggest that ICOS and CD28 are
parallel costimulatory pathways, either of which is sufficient to
mediate the production of IFN- and resistance to T.
gondii. Several questions about the role of ICOS in the regulation
of immunity to T. gondii remain unresolved. Our studies have
not addressed whether the B7RP-1-ICOS interaction is restricted to the
regulation of IFN- production or whether these molecules may also
regulate other T cell functions necessary for resistance to T.
gondii. Thus, further studies are required to delineate the
functional relationship between CD28 and ICOS and how each pathway
integrates to provide an optimal T cell response necessary for
resistance to T. gondii.
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Acknowledgments |
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Footnotes |
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2 Current address: Cancer Research Division, Molecular and Cellular Biology Department, Immunology Division, University of California, 441 LSA, Berkeley, CA 94720-3200.
3 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008. E-mail address: chunter{at}phl.vet.upenn.edu
4 Abbreviations used in this paper: ICOS, inducible costimulator protein; B7RP-1, B7-related protein 1; BMNC, brain-associated mononuclear cell; Chi-L6, chimeric protein L6; STAg, soluble Toxoplasma Ag; TE, toxoplasmic encephalitis; WT, wild type.
Received for publication February 1, 2002. Accepted for publication May 7, 2002.
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: the major mediator of resistance against Toxoplasma gondii. Science 240:516. production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J. Immunol. 146:286.[Abstract]
and immunopathology during infection with Toxoplasma gondii. J. Immunol. 166:5726.. Immunity 11:423.[Medline]
-chain (c) reveals an unexpected role for CD4+ T lymphocytes in early IFN--dependent resistance to Toxoplasma gondii. J. Immunol. 160:2565. for prevention of toxoplasmic encephalitis in mice. J. Immunol. 143:2045.[Abstract]
by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.. Infect. Immun. 62:2818.This article has been cited by other articles:
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  J. M. Rojo, E. Pini, G. Ojeda, R. Bello, C. Dong, R. A. Flavell, U. Dianzani, and P. Portoles CD4+ICOS+ T lymphocytes inhibit T cell activation 'in vitro' and attenuate autoimmune encephalitis 'in vivo' Int. Immunol., April 1, 2008; 20(4): 577 - 589. [Abstract] [Full Text] [PDF]
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 A. H.-M. Tan, S. Y.-P. Goh, S.-C. Wong, and K.-P. Lam T Helper Cell-specific Regulation of Inducible Costimulator Expression via Distinct Mechanisms Mediated by T-bet and GATA-3 J. Biol. Chem., January 4, 2008; 283(1): 128 - 136. [Abstract] [Full Text] [PDF]
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 B. P. Iliopoulou, J. Alroy, and B. T. Huber CD28 Deficiency Exacerbates Joint Inflammation upon Borrelia burgdorferi Infection, Resulting in the Development of Chronic Lyme Arthritis J. Immunol., December 15, 2007; 179(12): 8076 - 8082. [Abstract] [Full Text] [PDF]
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 F. Dzierszinski, M. Pepper, J. S. Stumhofer, D. F. LaRosa, E. H. Wilson, L. A. Turka, S. K. Halonen, C. A. Hunter, and D. S. Roos Presentation of Toxoplasma gondii Antigens via the Endogenous Major Histocompatibility Complex Class I Pathway in Nonprofessional and Professional Antigen-Presenting Cells Infect. Immun., November 1, 2007; 75(11): 5200 - 5209. [Abstract] [Full Text] [PDF]
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E. H. Wilson, C. Zaph, M. Mohrs, A. Welcher, J. Siu, D. Artis, and C. A. Hunter B7RP-1-ICOS Interactions Are Required for Optimal Infection-Induced Expansion of CD4+ Th1 and Th2 Responses J. Immunol., August 15, 2006; 177(4): 2365 - 2372. [Abstract] [Full Text] [PDF]
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M. F. Quiroga, V. Pasquinelli, G. J. Martinez, J. O. Jurado, L. C. Zorrilla, R. M. Musella, E. Abbate, P. A. Sieling, and V. E. Garcia Inducible Costimulator: A Modulator of IFN-{gamma} Production in Human Tuberculosis J. Immunol., May 15, 2006; 176(10): 5965 - 5974. [Abstract] [Full Text] [PDF]
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M. Vidric, A. T. Bladt, U. Dianzani, and T. H. Watts Role for Inducible Costimulator in Control of Salmonella enterica Serovar Typhimurium Infection in Mice Infect. Immun., February 1, 2006; 74(2): 1050 - 1061. [Abstract] [Full Text] [PDF]
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M. Vidric, W.-K. Suh, U. Dianzani, T. W. Mak, and T. H. Watts Cooperation between 4-1BB and ICOS in the Immune Response to Influenza Virus Revealed by Studies of CD28/ICOS-Deficient Mice J. Immunol., December 1, 2005; 175(11): 7288 - 7296. [Abstract] [Full Text] [PDF]
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 O. Atochina and D. Harn LNFPIII/LeX-Stimulated Macrophages Activate Natural Killer Cells via CD40-CD40L Interaction Clin. Vaccine Immunol., September 1, 2005; 12(9): 1041 - 1049. [Abstract] [Full Text] [PDF]
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 P. Loke, X. Zang, L. Hsuan, R. Waitz, R. M. Locksley, J. E. Allen, and J. P. Allison Inducible costimulator is required for type 2 antibody isotype switching but not T helper cell type 2 responses in chronic nematode infection PNAS, July 12, 2005; 102(28): 9872 - 9877. [Abstract] [Full Text] [PDF]
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R. M. Elias, L. R. Sardinha, K. R. B. Bastos, C. A. Zago, A. P. F. da Silva, J. M. Alvarez, and M. R. D. Lima Role of CD28 in Polyclonal and Specific T and B Cell Responses Required for Protection against Blood Stage Malaria J. Immunol., January 15, 2005; 174(2): 790 - 799. [Abstract] [Full Text] [PDF]
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T. Rummel, J. Batchelder, P. Flaherty, G. LaFleur, P. Nanavati, J. M. Burns, and W. P. Weidanz CD28 Costimulation Is Required for the Expression of T-Cell-Dependent Cell-Mediated Immunity against Blood-Stage Plasmodium chabaudi Malaria Parasites Infect. Immun., October 1, 2004; 72(10): 5768 - 5774. [Abstract] [Full Text] [PDF]
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N. J. Mason, H.-C. Liou, and C. A. Hunter T Cell-Intrinsic Expression of c-Rel Regulates Th1 Cell Responses Essential for Resistance to Toxoplasma gondii J. Immunol., March 15, 2004; 172(6): 3704 - 3711. [Abstract] [Full Text] [PDF]
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J. Yagi, Y. Arimura, U. Dianzani, T. Uede, T. Okamoto, and T. Uchiyama Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development J. Immunol., July 15, 2003; 171(2): 783 - 794. [Abstract] [Full Text] [PDF]
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R. V. Parry, C. A. Rumbley, L. H. Vandenberghe, C. H. June, and J. L. Riley CD28 and Inducible Costimulatory Protein Src Homology 2 Binding Domains Show Distinct Regulation of Phosphatidylinositol 3-Kinase, Bcl-xL, and IL-2 Expression in Primary Human CD4 T Lymphocytes J. Immunol., July 1, 2003; 171(1): 166 - 174. [Abstract] [Full Text] [PDF]
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) or anti-B7RP-1 (
)
and infected with T. gondii for 7 days. Cells were
stimulated with STAg alone or in combination with anti-B7RP-1 for
48 h and the production of IFN-