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Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, 19104; and
Bristol Myers Squibb Pharmacology Research Institute, Princeton, NJ 08543
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and mediate resistance to the
parasite. Our studies revealed that expression of B7.1 and B7.2 was
up-regulated in brains of mice with toxoplasmic encephalitis (TE).
Because CD28/B7-mediated costimulation is important for T cell
activation, we assessed the contribution of this interaction to the
production of IFN- by T cells from brains and spleens of mice with
TE. Stimulation of splenocytes with Toxoplasma Ag or
anti-CD3 mAb resulted in production of IFN-, which was inhibited
by 90% in the presence of CTLA4-Ig, an antagonist of B7 stimulation.
However, production of IFN- by T cells from the brains of these mice
was only slightly reduced (20%) by the addition of CTLA4-Ig. To
address the role of the CD28/B7 interaction during TE, we compared the
development of disease in C57BL/6 wild-type (wt) and
CD28-/- mice. Although the parasite burden was similar in
wt and CD28-/- mice, CD28-/- mice developed
less severe encephalitis and survived longer than wt mice. Ex vivo
recall responses revealed that mononuclear cells isolated from the
brains of chronically infected CD28-/- mice produced less
IFN- than wt cells, and this correlated with reduced numbers of
intracerebral CD4+ T cells in CD28-/- mice
compared with wt mice. Taken together, our data show that resistance to
T. gondii in the brain is independent of CD28 and
suggest a role for CD28 in development of immune-mediated pathology
during TE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is
critical for resistance to TE. Thus, in mice chronically infected with
T. gondii, depletion of both CD4+ and
CD8+ T cells results in increased severity of TE
and death of mice within 2 wk (4). Moreover,
administration of anti-IFN- to mice with TE results in increased
parasite numbers and death of mice within 2 wk (4).
Interestingly, although CD4+ T cells in
combination with CD8+ T cells are involved in
resistance to TE, there are contradictory reports on the effects of
CD4+ T cells alone. Vollmer and colleagues
(5) reported that depletion of CD4+
T cells in C3H/HeN mice resulted in an increase in severity of
TE. However, Israelski et al. (6) suggested that depletion
of CD4+ T cells ameliorated the severity of TE.
Thus, it appears that CD4+ T cells play a dual
role during TE; they mediate resistance to T. gondii in the
brain, but may also contribute to the development of immunopathology.
In the last decade, we have gained an understanding of the role of
cytokines, in particular IL-12, in the events that lead to the
generation of parasite-specific T cells that produce high levels of
IFN- and that mediate resistance to T. gondii
(7). However, the role of costimulation in this process is
less clear. Activation of T cell responses normally requires two
signals. Signal one is provided by the MHC/TCR interaction, which alone
fails to activate T cells, while an additional costimulatory signal
allows the activation of T cells to progress (8). The
interaction of CD28 on T cells with B7 molecules expressed on APC is
one of the most important second signals required for T cell
activation. The CD28/B7 interaction lowers the threshold of T cell
activation and enhances proliferative and effector cell responses of T
cells (8). In addition, costimulation via CD28 leads to
increased production of growth factors like IL-2 and up-regulates
levels of anti-apoptotic factors like Bcl-xL
and thereby enhances survival of T cells (9). The
importance of the CD28/B7 interaction in the regulation of T cell
responses is illustrated by studies in which T cells from mice
deficient in CD28 were shown to have severe defects in proliferation
and cytokine production when stimulated with alloantigens, mitogen, or
anti-CD3 mAb (10, 11). Moreover, blockade of the
CD28/B7 interaction has also been shown to inhibit T cell-mediated
reponses during graft rejection (12, 13), experimental
autoimmune encephalomyelitis (EAE) (14, 15), diabetes
(16, 17), and during infection (18, 19).
Given the critical role of T cells in the control of T.
gondii in the brain and the importance of costimulation for T cell
activation, we analyzed the role of the CD28/B7 interaction in the
regulation of T cell responses during TE. Our studies demonstrate a
marked up-regulation of the costimulatory molecules B7.1 and B7.2 in
the brain during TE. However, production of IFN- by T cells present
in the brain during TE and control of the parasite in the brain is
largely independent of the CD28/B7 interaction. In addition, we found
that mice lacking CD28 have reduced numbers of
CD4+ T cells in the brain during TE, and this is
associated with a less severe encephalitis and delayed time to death.
These findings suggest that although CD28 plays little, if any, role in
priming T cells for resistance against T. gondii, it may
contribute to the development of T cell-mediated pathology during
TE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female CBA/CaJ and C57BL/6 mice were obtained from The Jackson Laboratories (Bar Harbor, ME). C57BL/6 CD28-deficient (CD28-/-) mice (10) originally obtained from Jackson Laboratories were bred within the University Laboratory Animal Resources facilities of the University of Pennsylvania. For experiments, 6- to 10-wk-old mice were inoculated i.p. with 15 cysts of the ME49 strain of T. gondii, which had been prepared from the brains of chronically infected CBA/CaJ mice. At the time of sacrifice, mice to be used were chosen randomly.
Reagents
Soluble Toxoplasma Ag (STAg) was prepared from in
vitro cultured tachyzoites of T. gondii strain RH. Purified
parasites were suspended in distilled water and repeatedly
freeze-thawed. Following centrifugation (20 min, 800 x
g; 15 min, 10,000 x g), the Ag-containing
supernatant was stored at -70°C. STAg was titrated to determine the
optimal concentration for induction of cytokines and was used at 30
µg/ml. Anti-CD3
mAb (145-2C11) and anti-IL-2 mAb (JES 6-1A12)
were prepared from hybridoma supernatant. CTLA4-Ig, a fusion protein
comprising the extracellular domain of human CTLA-4 plus the Fc portion
of human IgG, and the chimeric Ig L6 (Chi-L6) were supplied by Bristol
Myers Squibb Research Institute (Princeton, NJ). Anti-IL-12 p40 mAb
(C17.8) was provided by Dr. Giorgio Trinchieri (Wistar Institute,
Philadelphia, PA). Rat IgG was obtained from Sigma (St. Louis, MO). In
titration experiments, the use of 3, 10, or 30 µg/ml of CTLA4-Ig and
5, 10, 20, or 40 µg/ml of anti-IL-2 mAb had similar effects on
the production of IFN- by brain-associated mononuclear cells (BMNC).
Thus, CTLA4-Ig was routinely used at a concentration of 30 µg/ml and
anti-IL-2 mAb at 20 µg/ml. In previous studies (C.A.H.,
unpublished observations), we have found that 5 µg/ml of
anti-IL-12 will completely abolish the effects of 10 ng/ml of IL-12
on NK cells, and this mAb was routinely used at a concentration of 20
µg/ml.
Immunohistochemistry
Brains from C57BL/6 mice, either uninfected or infected for 10 wk with T. gondii, were mounted in OCT compound (Miles Scientific, Naperville, IL), snap-frozen in cooled 2-methylbutane, and stored at -70°C. Cryostat sections (5 µm) were mounted on poly-L-lysine-coated microscope slides and fixed in ice-cold acetone for 10 min. Sections were incubated for 30 min with 0.3% H202/0.2 M NaN3 to quench endogenous peroxidase activity, followed by blocking with 10% goat serum (Vector Laboratories, Burlingame, CA) in HBSS. Sections were then stained for 1 h with primary mAb against B7.1 (16-10A1; PharMingen, San Diego, CA), B7.2 (GL1; PharMingen) or isotype control mAb (PharMingen). After washing in HBSS, sections were incubated with biotinylated anti-hamster IgG Ab or anti-rat IgG Ab (Vector Laboratories). Subsequent incubation of the slides with peroxidase-conjugated avidin-biotin complex (Vectastain Elite ABC kit, Vector) was performed according to the manufacturer’s instructions. Sections were developed with 3,3'-diaminobenzidine (Vector Laboratories), counterstained with hematoxylin, dehydrated, and mounted.
Cell preparations
Spleens were harvested and dissociated into single-cell suspension in complete RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 50 µM 2-ME, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml fungizone (Life Technologies). Erythrocytes were depleted using 0.83% ammonium chloride (Sigma), and cells were washed twice in complete RPMI 1640 before further analysis. For the preparation of mononuclear cells from the brain, mice were anesthetized using methoxyfluorane and perfused through the left cardiac ventricle with 40 ml of ice-cold PBS to remove peripheral blood. Brains were removed, minced with scissors, and then digested for 1 h at 37°C with 300 µg/ml collagenase/dispase (Boehringer Mannheim, Indianapolis, IN) and 600 µg/ml DNase I (Boehringer Mannheim) in complete RPMI. The dissociated brain tissue was pelleted at 200 x g for 10 min, resuspended in a 60% isotonic Percoll solution (Sigma), and overlayed with a 30% Percoll solution. Discontinuous gradients were centrifuged for 25 min at 1000 x g. After removal of the myelin layer on top of the gradient, BMNC were harvested from the 30% (1.035 g/ml) to 60% (1.07 g/ml) interphase and washed twice in complete RPMI before further analysis. Due to the low number of BMNC obtained per animal, cells from at least three mice were pooled per experiment unless otherwise stated.
Flow cytometric analysis
Cells were stained directly after their isolation using the following primary mAb (PharMingen or Caltag, San Francisco, CA): rat anti-mouse mAb, FITC-conjugated anti-F4/80 (IgG2b), anti-B220 (RA3-6B2, IgG2a), anti-CD4 (RM4-5, IgG2a), anti-CD8 (53-5.8, IgG1), biotinylated anti-CD62 ligand (CD62L) (Mel14, IgG2a), anti-CD44 (IM7, IgG2b), anti-CD25 (7D4, rat IgM), PE-labeled anti-B7.2 (RMMP-1, IgG2a), and PE-conjugated hamster anti-mouse B7.1 (16-10A1). Appropriate isotype control mAb were obtained from PharMingen or Caltag and included in each experiment. To block nonspecific binding via Fc receptors, cells were incubated for 15 min on ice with 50 µg/ml rat IgG (Sigma) plus 50 µg/ml Fc Block (PharMingen) in FACS buffer (PBS, 0.2% BSA, 4 mM NaN3). Cells were then stained for 30 min on ice with primary mAb. After one wash, appropriate samples were incubated for 30 min with PE-conjugated streptavidin (PharMingen). After a further wash, propidium iodide (5 µg/sample) was added. Cells were immediately measured on a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) and analyzed with the CELLQuest software (Becton Dickinson). Samples were gated on live leukocytes based on forward and side scatter and by exclusion of propidium iodide-positive cells. Per sample, 10,000 events in this gate were acquired.
In vitro recall response
Spleen cells and BMNC prepared at 8–12 wk postinfection were
plated in complete RPMI at 1 x 106 cells/ml
in a final volume of 200 µl. Cultures were left untreated or were
stimulated with 30 µg/ml STAg or 1 µg/ml anti-CD3 mAb. Where
indicated, CTLA4-Ig (30 µg/ml), anti-IL-2 mAb (20 µg/ml), or
anti-IL-12 mAb (20 µg/ml) was added, while control cultures were
incubated with Chi-L6 (30 µg/ml) or rat IgG (20 µg/ml).
Supernatants were collected after 48 h of incubation at 37°C.
Cytokines were determined by sandwich ELISA as previously described
(20) using the following mAb pairs: IFN-, R4-6A2 and
biotinylated AN-18; IL-2, JES6-1A12 and biotinylated JES6-5H4; IL-12,
C17.8 and biotinylated C15.6. Cytokine concentrations were determined
from the appropriate standard curves using recombinant cytokines
(Genzyme, Cambridge, MA).
Evaluation of histopathology and parasite burden
At the time of sacrifice, samples of lung, liver, and brain were removed from C57BL/6 wt and CD28-/- mice, fixed in 10% neutral buffered formalin (Sigma), and embedded in paraffin. Organs were sectioned (5 µm) and stained with hematoxylin and eosin for visualization of pathological changes. To score pathological changes, a blinded analysis was performed using a score of 0 for no pathlogical changes; 1 for mild disease characterized by few lymphocytic infiltrates no perivascular cuffs and no meningitis; 2 for widespread lymphocytic infiltration with localized perivascular cuffs and meningitis; 3 for widespread lymphocytic infiltration, perivascular cuffing and meningitis, local gliosis, occasional necrosis, and neutrophils; and 4 for inflammation throughout the brain with prominent perivascular cuffs and meningitis, widespread areas of necrosis, large numbers of neutrophils, and a prominent gliosis. Slides were graded blindly by a single individual. To determine the cyst number, half of each brain was homogenized in 2 ml PBS by repeated passage through a 21-gauge needle. For each brain, three aliquots of 30 µl of the homogenate were scanned microscopically, and the number of cysts were counted and used to estimate the total number of cysts per brain.
Statistical analyses
Statistical analyses were performed using the INSTAT or PRISM software (GraphPad, San Diego, CA). Unless otherwise stated, unpaired two-tailed Student’s t tests were performed. Survival curves were analyzed using a logrank test, and pathology scores were analyzed using the Mann-Whitney U test as previously described (21). Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because immune-mediated control of T. gondii infection
in the brain is T cell dependent (4) and optimal T cell
stimulation requires costimulatory signals in addition to Ag
recognition through the TCR, we investigated the expression of the
costimulatory molecules B7.1 and B7.2 in the brains of mice with TE.
Immunohistochemical analysis revealed that these molecules were not
expressed in the brains of uninfected C57BL/6 mice. However, in brains
from mice infected for 10 wk with T. gondii, expression of
B7.1 and B7.2 was up-regulated. Both molecules were expressed in
perivascular cuffs and in inflammatory foci within the brain parenchyma
and were associated with areas of infiltration (data not shown). We
were unable to detect any differences in the patterns of B7 expression
from mice that differed in their severity of TE. Flow cytometric
analysis of mononuclear cells isolated from the brain confirmed these
results (Fig. 1
). BMNC from
uninfected mice expressed very low levels of B7.1 (mean fluorescence
intensity (MFI) = 12.3) and B7.2 (MFI = 10.3). In contrast,
BMNC isolated at 10 wk postinfection expressed higher levels of B7.1
(MFI = 65.75) and B7.2 (MFI = 115). Two-color analysis
revealed that the majority of the cells expressing B7.1 or B7.2 were
F4/80+ macrophages/microglia (50–60%), while
only 2% were B220+ B cells (data not shown).
Together, the immunohistochemical staining plus the flow cytometric
analysis revealed a marked up-regulation of B7 expression in the brain
during TE.
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Because the expression of B7 molecules in the brain was
up-regulated during TE, we assessed the role of B7-mediated
costimulation in the regulation of intracerebral T cell responses
during TE. BMNC were isolated from wt mice at 10 wk postinfection and
were stimulated with STAg or anti-CD3 mAb in the presence or
absence of CTLA4-Ig, a B7 antagonist. Because IFN- is the major
mediator of resistance against T. gondii (22),
we measured the production of IFN- by BMNC and compared it to the
production of IFN- by splenocytes from the same mice. As shown in
Fig. 2, stimulation of splenocytes with
STAg or anti-CD3 resulted in production of IFN-, and this was
almost completely inhibited (85–95%) in the presence of CTLA4-Ig
(p = 0.001 and <0.0001, respectively).
Stimulation of BMNC from these mice with STAg or anti-CD3 also
induced the production of high levels of IFN-, but the addition of
CTLA4-Ig only resulted in a 15–30% reduction in the production of
IFN- by cells stimulated with STAg (p =
0.0013) and failed to significantly reduce the production of IFN- by
cells stimulated with anti-CD3 (p = 0.16).
These results suggest that, during TE, T cell production of IFN- in
the brain is largely CD28 independent.
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during
T. gondii infection (23), it seemed likely that
IL-2 was required for the production of IFN- by intracerebral T
cells during TE. At 10 wk postinfection, splenocytes cultured in medium
produced little IL-2, but the production of IL-2 was enhanced upon
stimulation with STAg or anti-CD3 mAb (Fig. 3A). Neutralization of IL-2 in
these cultures led to a strong reduction (65–70%) in the STAg- or
anti-CD3-induced production of IFN- (p =
0.037 and 0.0017, respectively; Fig. 3B). In contrast, no
IL-2 was detected in the supernatants of BMNC, and the addition of
anti-IL-2 mAb to these cultures had no significant effect on the
production of IFN- by unstimulated cells or cells stimulated with
STAg or anti-CD3 (p = 0.7, 0.08, and 0.2,
respectively; Fig. 3C).
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by T cells (24, 25) and is required for the
CD28-independent production of IFN- by splenic T cells during acute
toxoplasmosis (5). Therefore, we tested if IL-12 was
required for the CD28-independent production of IFN- by
intracerebral T cells during chronic toxoplasmosis. At 10 wk
postinfection, stimulation of splenocytes with STAg resulted in a
4-fold increase in the production of IL-12 compared with no
stimulation or stimulation with anti-CD3 mAb (Fig. 4A). BMNC isolated from these
chronically infected mice produced high levels of IL-12 when cultured
in medium alone, and these IL-12 levels were not significantly
increased in the presence of STAg or anti-CD3 mAb (Fig. 4A). Neutralization of IL-12 in cultures of splenocytes
stimulated with STAg or anti-CD3 did not significantly affect the
production of IFN- (p = 0.4 and 0.5,
respectively; Fig. 4B). Similar results were observed with
unstimulated BMNC or cultures stimulated with STAg or anti-CD3
(p = 0.8, 0.2, and 0.6, respectively; Fig. 5C). Together, our results
suggest that, in contrast to T cell responses in the periphery, the
response of T cells in the brain during TE, as measured by the
production of IFN-, is largely CD28 independent and that neither
IL-2 or IL-12 are required for the production of IFN- by these
cells.
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To test our hypothesis that the intracerebral
T cell response during TE is independent of the CD28/B7 interaction, we
inoculated wt and CD28-/- mice with 15 cysts of
the ME49 strain of T. gondii and monitored the survival of
these mice. In accordance with our previous results in BALB/c
mice,5 both wt and CD28-/- mice
were resistant to acute toxoplasmosis and developed a chronic
infection. C57BL/6 wt mice, which are susceptible to TE, started to die
at 
2 mo postinfection, with 80% mortality by 6 mo postinfection
(Fig. 5
A). However, CD28-/- mice
survived significantly longer, with 60% of the mice surviving at 6 mo
postinfection. Similar survival patterns were observed in two
additional experiments in both female and male mice. Statistical
analysis of the survival curves from these experiments revealed
a p < 0.0001 for each of the three survival
curves.
To analyze the ability of wt and CD28-/- mice
to control parasite growth in the brain, we determined the
number of cysts at this site. At 8 and 12 wk postinfection, large
numbers of cysts were detected in the brains of wt and
CD28-/- mice (Table I). However, at both time points, cyst
numbers were not significantly different in wt and
CD28-/- mice (p >
0.5). These results were confirmed by immunohistochemical staining of
T. gondii in brain sections. Between 8 and 12 wk
postinfection in both wt and CD28-/- mice,
parasites were rarely seen within inflammatory foci, but intact cysts
were scattered throughout otherwise normal areas of the brain (data not
shown). In addition, lung, liver, and brain were assessed for
histopathology between 8 and 12 wk postinfection. In both mouse
strains, only mild inflammation was observed in lung and liver. As
expected, the brain was the main organ affected during chronic
toxoplasmosis, and both wt and CD28-/- mice
developed TE. Wt mice developed moderate to severe encephalitis and
meningitis characterized by perivascular cuffs, random foci of necrosis
and gliosis within the parenchyma, and multifocal infiltrates of
inflammatory cells within the meninges (Fig. 5, B and
C). The inflammatory infiltrates within the perivascular
cuffs and in the meninges consisted primarily of lymphocytes, with
fewer plasma cells, macrophages, and neutrophils. The parenchymal foci
of necrosis contained a higher proportion of neutrophils and often
times nuclear debris. In the brains of infected
CD28-/- mice, we also observed perivascular
cuffs, random foci of necrosis and gliosis, as well as meningitis;
however, the lesions were less severe (Fig. 5, D and
E). In addition, the cell population within inflammatory
foci around the vessels and in the parenchyma contained more
neutrophils and macrophages and fewer lymphocytes, while, similar to wt
mice, inflammatory infiltrates in the meninges contained primarily
lymphocytes. When chronically infected mice (wt, n =
14; CD28-/-, n = 15) were
graded blindly for severity of TE and grades analyzed using a Wilcoxin
Mann-Whitney U test, there was a significant
(p = 0.0036) difference between the wt and
CD28-/- mice. Taken together, these data
show that immune-mediated control of T. gondii in the
brain does not require CD28. In contrast, these findings suggest that
stimulation through CD28 during chronic toxoplasmosis results in
increased severity of TE and associated mortality.
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Because CD28-/- mice infected with
T. gondii survived significantly longer and showed less
severe brain pathology than wt mice, we compared the intracerebral T
cell responses of chronically infected wt and
CD28-/- mice. In accordance with the reduced
brain pathology in CD28-/- mice, fewer
mononuclear cells were obtained from the brains of
CD28-/- mice than from wt mice between 10 and
12 wk postinfection. In a representative experiment with four mice per
group, 4.17 x 106 ± 0.49 x
106 mononuclear cells were isolated from
CD28-/- brains and 5.66 x
106 ± 0.62 x 106
mononuclear cells from wt brains (p < 0.01).
Similar results were observed in four experiments. Analysis of the
recall response of intracerebral T cells showed that stimulation of
BMNC from wt mice with STAg or anti-CD3 mAb resulted in the
production of high levels of IFN- (Fig. 6). In contrast, stimulation of BMNC from
infected CD28-/- mice induced 40–50% less
IFN- (p < 0.05).
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by BMNC
isolated from CD28-/- mice, we compared the
expression of CD62L, CD44, and CD25 by intracerebral and splenic T cell
populations in C57BL/6 wt and CD28-/- mice.
Naive T cells express CD62Lhigh,
CD44low; however, upon stimulation, expression of
CD62L is down-regulated while expression of CD44 is increased
(26). At 10 wk postinfection, splenocytes from chronically
infected CD28-/- mice had fewer
CD4+ T cells with a
CD62Llow, CD44high
phenotype than splenocytes from chronically infected wt mice (Fig. 7A). In contrast, splenocytes
from infected CD28-/- and wt mice had similar
percentages of CD8+ T cells with a
CD62Llow, CD44high
phenotype. We have also shown similar results for splenic T cells from
BALB/c CD28-/- mice with chronic
toxoplasmosis.5 Further analysis showed that, at 10 wk
postinfection, none of the splenic CD8+ T cells
and only 5% of the splenic CD4+ T cells
expressed the early activation marker CD25 (Fig. 7A),
suggesting that the predominant T cell populations in the spleens of
mice with chronic toxoplasmosis are resting cells. Phenotypic analysis
of T cells isolated from brains of wt mice with TE revealed that almost
all CD4+ and CD8+ T cells
expressed the CD62Llow,
CD44high phenotype (Fig. 7B).
Similarly, all CD4+ and
CD8+ T cells from the brains of
CD28-/- mice with TE expressed this phenotype.
In contrast to splenic T cells, 20% of the
CD4+ and 10% of the CD8+ T
cells isolated from the brains expressed CD25, indicating that
intracerebral T cells are effector cells rather than resting memory
cells. Although intracerebral T cells from chronically infected wt and
CD28-/- mice were phenotypically similar, there
was an 50% reduction in the percentage of
CD4+ T cells in the brains of
CD28-/- mice (p <
0.01, paired t test) compared with wt mice (Fig. 8), while the percentage of intracerebral
CD8+ T cells was similar in
CD28-/- and wt mice. This decreased percentage
of CD4+ T cells was accompanied by an increased
percentage of macrophages. For example, in one experiment the
percentage of F4/80+ cells in wt BMNC was 19.5%,
whereas the BMNC from CD28-/- mice contained
29.6% F4/80+ cells (p =
0.001). Interestingly, FACS analysis of spleens from chronically
infected wt and CD28-/- mice did not reveal any
significant difference in the percentages of CD4+
and CD8+ T cells (p =
0.77 and 0.73, respectively). Thus, the decreased susceptibility of
CD28-/- mice to TE is accompanied by decreased
numbers of CD4+ T cells in the brain and is
associated with reduced levels of IFN- at this site.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in recall responses by parasite-specific T
cells in the periphery, but has a less significant contribution to the
production of IFN- by parasite specific T cells from the brain.
These results indicate that T cell responses to T. gondii in
the brain are largely independent of the CD28/B7 interaction, and this
is supported by our studies, which showed that, in the absence of CD28,
mice were able to control parasite growth in the brain and did not
become more susceptible to the infection. Given the important role of the CD28/B7 interaction in the regulation of T cell responses and the importance of T cells for resistance to TE, it was surprising to us that the CD28/B7 interaction does not contribute significantly to the protective T cell response in the brains of mice with chronic toxoplasmosis. Phenotypic characterization of T cells isolated from the brains of mice with TE revealed that they exhibited an effector phenotype characterized by the expression of CD62Llow, CD44high and low levels of the activation marker CD25. Because effector T cells are less dependent on costimulation through CD28 than naive T cells (27, 28), it is possible that the minimal role of CD28/B7 in activation of intracerebral T cells during TE simply reflects the activation status of these T cells. Whether other costimulatory molecules play a role in the CD28-independent activation of these effector T cells during TE is not known, but we have ruled out a role for endogenous IL-12 or IL-2 as second signals involved in T cell activation at this site. This contrasts with the role of these cytokines in promoting T cell responses in the periphery during toxoplasmosis (29, 30, 31). Nevertheless, there are many molecules, including heat-stable Ag (32), 4-1BB ligand (33, 34), ICAM-1 (35), or VCAM-1 (36), that have costimulatory activity for T cells and that may provide a second signal for effector T cell responses in the brain. The CD40/CD40 ligand interaction is also an important costimulatory pathway (37, 38), and in our preliminary studies the absence of CD40 ligand did not affect the activation of T cells during TE (G.R. and C.A.H., manuscript in preparation). Further studies are required to define the nature of the signals that regulate T cell responses in the brain during TE.
Although we have demonstrated that resistance to TE is independent of CD28 we did detect elevated levels of B7.1 and B7.2 in the brain during TE. The consequences of the up-regulation of these stimulatory molecules in the brain, an immunoprivileged site (39) that normally expresses negligible levels of B7.1 or B7.2, is unclear. The B7 molecules also interact with CTLA-4, which provides a negative regulatory signal for T cell activation (40, 41). However, the addition of CTLA4-Ig (which would also block B7/CTLA-4 interactions) to cultures of BMNC from wt and CD28-/- mice did not augment cytokine production. Thus, these results suggest that the B7/CTLA-4 interaction has a limited role in the regulation of T cell responses during TE. Nevertheless, our immunohistochemical studies localized the increased expression of B7 during TE to inflammatory foci that contain T cells. This colocalization of T cells and B7-expressing accessory cells suggests that the CD28/B7 interaction would occur at these sites. Other studies that have examined the expression of B7 in the brain during inflammation have reported increased expression of B7.1 and B7.2 in multiple sclerosis lesions (42) and in murine EAE (15, 43). Although systemic administration of CTLA4-Ig or anti-B7.1 plus anti-B7.2 did not affect the severity of established EAE (44, 45), the intracerebral administration of CTLA4-Ig following the onset of clinical EAE ameliorated disease (46). Thus, the CD28/B7 pathway has a role in the local regulation of this autoimmune inflammatory disease of the brain. Our findings that in the absence of CD28 (and presumably B7-mediated activation of T cells) there is a reduction in the severity of TE, but no change in parasite burden, suggests that CD28/B7 interactions may be involved in the inflammatory process within the brain during TE.
The most interesting aspect of these studies was the finding that, in
the absence of CD28, mice developed less severe TE and survived for a
longer period of time. This finding correlated with a reduced
production of IFN- and a reduced number of
CD4+ T cells in the brains of
CD28-/- mice. The events that lead to the
development of pathology in the brains of mice susceptible to TE are
complex. There is a requirement for both CD4+ and
CD8+ T cells to mediate resistance to T.
gondii at this site (4, 47), but the presence of a
chronic inflammatory reaction within the brain would also result in
immunopathology and contribute to the development of disease. A role
for CD4+ T cells in mediating immunopathology
during toxoplasmosis has been described during the acute phase of
disease (48, 49, 50, 51), and, relevant to our studies, depletion
of CD4+ T cells was shown to ameliorate the
severity of TE in C3H/HeN mice chronically infected with T.
gondii (6). While our data suggest that CD28/B7
costimulation may contribute to the development of immunopathology
during TE, our studies do not distinguish whether CD28-mediated
costimulation is required in the periphery or at the local site of
inflammation, the brain. Costimulation through CD28 may directly lead
to increased production of IFN- by pathogenic T cells because CD28
signaling increases the stability of cytokine mRNA, including IFN-
(52, 53, 54). Alternatively, CD28 signaling induces the
expression of survival factors like Bcl-xL, which
protects T cells against Fas-mediated cell death (9, 55).
Thus, during TE, CD28 costimulation may prolong the survival of
activated T cells, which, in the end, may lead to increased numbers of
pathogenic T cells. Indeed, our data show that
CD28-/- mice with TE do have reduced numbers of
CD4+ T cells with a
CD62Llow, CD44high
phenotype in the spleen and, concurrently, reduced numbers of effector
CD4+ T cells in the brain.
The CD28/B7 interaction is generally regarded as one of the most important costimulatory pathways involved in the activation of T cell responses (8). However, the role of the CD28/B7 interaction in the immune response to infection remains unclear. CD28-/- mice infected with lymphocytic choriomeningitis virus could still generate cytotoxic T cells and be induced to show delayed-type hypersensitivity after infection (10). In addition, the absence of CD28 does not alter the T cell-dependent outcome of infection with Leishmania major (56) or Heligmosomoides polygyrus (57). The studies presented here add to our knowledge of the role of CD28 in resistance to an important opportunistic pathogen and suggest that during TE, this interaction has a more important contribution to the development of immunopathology than to protective responses. Strategies designed to antagonize costimulation through CD28 may prove useful in the management of the pathological consequences of TE without affecting anti-parasite effector mechanisms.
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Acknowledgments |
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Footnotes |
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2 Current address: Institute for Medical Microbiology and Virology, Heinrich-Heine-University, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
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:
4 Abbreviations used in this paper: TE, toxoplasmic encephalitis; BMNC, brain-associated mononuclear cells; CD28-/-, CD28-deficient; Chi-L6, chimeric protein L6; STAg, soluble Toxoplasma Ag; wt, wild type; CD62L, CD62 ligand; EAE, experimental autoimmune encephalomyelitis; MFI, mean fluorescence intensity.
5 Villegas, E. N., M. M. Elloso, G. Reichmann, R. Peach, and C. A. Hunter. 1999. Role of CD28 in the generation of effector and memory responses required for resistance to Toxoplasma gondii. Submitted for publication.
Received for publication March 24, 1999. Accepted for publication June 29, 1999.
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