| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104; and
Bristol Myers Squibb Pharmacology Research Institute, Princeton, NJ 08543
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent
mechanism of resistance to Toxoplasma gondii. These mice
were resistant to infection with the ME49 strain of T.
gondii. Analysis of the immune response of acutely infected
CD28-/- mice revealed that IL-12 was required for T cell
production of IFN- and this was independent of the CD40/CD40 ligand
interaction. A similar mechanism of IL-12-dependent, CD28/B7
independent production of IFN- by T cells was also observed in
wild-type mice. Interestingly, although chronically infected wild-type
mice were resistant to rechallenge with the virulent RH strain of
T. gondii, chronically infected CD28-/-
mice were susceptible to rechallenge with the RH strain. This
deficiency in the protective memory response by CD28-/-
mice correlated with a lack of IL-2 and IFN- in recall responses and
reduced numbers of CD4+ T cells expressing a memory
phenotype. Together, our findings demonstrate that CD28 is not required
for the development of a protective T cell response to T.
gondii, but CD28 is required for an optimal secondary immune
response. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(14). The role of the CD28/B7 interaction in regulating the development of protective T cell-mediated immune responses to infection is still not well understood. Initial studies in mice infected with Leishmania major reported that treatment with CTLA4-Ig inhibited the development of a Th2-type response and led to the development of a protective Th1-type response (15). Subsequent studies with CD28-/- mice showed that CD28 had a limited role in the development and differentiation of either Th1 or Th2 subsets during leishmaniasis (16). In addition, Gause and colleagues reported that in mice infected with the nematode Heligmosomoides polygyrus the development of a Th2-type response was comparable in CD28-/- and wild-type (WT) mice (17). Similarly, infection of CD28-/- mice with lymphocytic choriomeningitis virus (LCMV) resulted in the generation of a functional T cell response (18). In those studies, the role of CD28 in the development of T cell responses to viral infection was shown to vary with the "strength" of the antigenic stimulus because CD28-/- mice developed anergic CD8+ T cells when stimulated with LCMV peptide. In contrast to the studies with H. polygyrus, King and colleagues demonstrated that CD28 was required for the development of a Th2-type response in mice infected with Schistosoma mansoni (19). These previous studies examined the role of CD28 in the activation of T cell responses during infection. Few studies have analyzed the role of CD28 in the generation of memory cells following infection. However, studies with influenza virus and H. polygyrus have led to the idea that the induction of T cell memory is not dependent on CD28 (20, 21).
Toxoplasma gondii is an important opportunistic infection in
patients with defects in T cell function (22). Infection
with T. gondii results in an acute phase of disease during
which NK and T cells produce IFN-, the major mediator of resistance
to T. gondii (23). Normally, this response
leads to the control of the infection; however, T. gondii
has a latent cyst stage that ensures that the host remains chronically
infected (22). Resistance to this chronic phase of
infection is dependent on CD4+ and
CD8+ T cells (24), as is immunity to
rechallenge with a virulent strain of T. gondii (25, 26). The role of the CD28/B7 interaction in the regulation of
protective T cell responses to infection with T. gondii is
not clear. Our previous studies demonstrated that the CD28/B7
interaction was required for maximal NK cell responses to T.
gondii in SCID mice (27). More recent studies have
shown that in humans serologically negative for T.
gondii, infection of their monocytes with T. gondii
results in increased expression of B7-1 and B7-2 and that these
costimulatory ligands were required for the ability of their T cells to
proliferate and produce IFN- in response to T. gondii in
vitro (28).
Given the importance of costimulation for T cell activation and the
role of T cell production of IFN- for resistance to T.
gondii, we decided to analyze the role of the CD28/B7 interaction
in the generation of protective T cell responses during toxoplasmosis.
We assessed T cell responses in WT and CD28-/-
BALB/c mice during the acute and chronic phase of infection with
T. gondii. Our studies demonstrate that the activation of T
cells following infection is independent of the CD28/B7 interaction.
Rather, it appears that IL-12 is a critical factor that allows T cell
activation and resistance to this pathogen to develop independently of
the CD28/B7 interaction. Moreover, our studies reveal that CD28 is
required for the ability of chronically infected mice to mount a
protective secondary response against a virulent strain of T.
gondii. These latter studies suggest an important role for CD28 in
either the generation or maintenance of memory cell populations.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female Swiss Webster, CBA/CaJ, and male BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). BALB/c CD28-/- (10) were obtained from The Jackson Laboratory and were bred and maintained within Thoren caging units (Thoren Caging Systems, Hazleton, PA) within the animal facilities at the University of Pennsylvania. 129/B6 CD40 ligand (CD40L)-deficient mice (CD40L-/-) (29) were provided by Immunex (Seattle, WA) and were bred and maintained within Thoren caging units at the animal facilities at the University of Pennsylvania. Mice were between 6 and 8 wk of age when used for experiments.
Parasites
Soluble Ags of T. gondii (STAg) were prepared from RH strain tachyzoites as previously described (30). STAg was titrated to determine the optimal concentration for splenocyte proliferation and was used at 25–40 µg/ml for these experiments. Virulent RH or Ts-4 tachyzoites were maintained in vitro at 37°C or 34°C, respectively, using human foreskin fibroblasts cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 1% FCS (HyClone, Logan, UT), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (50 µg/ml). Cysts of the ME49 strain of T. gondii were harvested from brains of CBA/CaJ mice infected for 1–2 mo. For experimental infections, mice were given 20 or 100 ME49 cysts, 10,000 RH tachyzoites, or PBS (BioWhittaker, Walkersville, MD) i.p. in a volume of 0.2 ml.
Vaccination
BALB/c WT and CD28-/- mice were vaccinated with 2 x 104 Ts-4 tachyzoites two times at 2-wk intervals (25). Two weeks after the last vaccination, mice were challenged with 10,000 tachyzoites of the RH strain of T. gondii and survival was monitored.
Histology
At different times postinfection, samples of lung, liver, and brain were removed from each mouse, fixed in 4% formaldehyde/70% ethanol/0.8 N acetic acid, and embedded in paraffin. Organs were sectioned and stained with hematoxylin and eosin for visualization of pathological changes. T. gondii parasites and Ags were detected in tissues of infected mice using polyclonal rabbit Abs against T. gondii (31). Cytospins of peritoneal exudate cells (PECs) were prepared as previously described and used to estimate the percentage of cells infected with T. gondii (27).
Reagents
Anti-mouse CD3
(145-2C11) was prepared from hybridoma
supernatants. Hamster anti-CD40L (M158) was provided by Immunex.
The hybridoma for rat anti-mouse IL-12 p40 (C17.8) was provided by
Dr. G. Trinchieri (Wistar Institute, Philadelphia, PA). These Abs were
administered i.p. to mice on days 0 and 3 postinfection at a dose of 1
mg/mouse for anti-IL-12 and 200 µg/mouse for anti-CD40L per
treatment. Rat mAbs specific for ß-galactosidase were used as isotype
controls. Human CTLA4-Ig, a fusion protein comprised of the human
CTLA-4 extracellular domain and Fc portion of human IgG, was supplied
by Bristol Myers Squibb Research Institute (Princeton, NJ) and used at
a concentration of 10–20 µg/ml. The chimeric control fusion protein
L6 (Chi-L6) was supplied by Bristol Myers Squibb. Monoclonal Abs
specific for CD4 (GK1.5) and CD8 (2.43) were purified from ascites and
administered i.p. to mice at a concentration of 200 µg/mouse 24
h before infection and again on days 0 and 3 of infection. Murine IL-12
(5.6 x 106 U/mg) was supplied by the
Immunology Department of Genetics Institute (Cambridge, MA).
Recombinant human IL-2 (1.6 x 107 U/mg) was
purchased from Chiron (Emmeryville, CA). Recombinant murine IL-2 was
purchased from Genzyme (Cambridge, MA).
Analysis of T and NK cell responses
Spleens from uninfected or infected animals were harvested and
dissociated into single-cell suspension in complete RPMI 1640 medium
(Life Technologies) containing 10% heat-inactivated FCS (HyClone),
penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (0.25
µg/ml) (BioWhittaker). Erythrocytes were depleted using 0.83% w/v
ammonium chloride (Sigma, St. Louis, MO). Cells were then washed three
times and resuspended in complete RPMI 1640 before being plated at a
cell density of 4 x 105 cells per well in a
final volume of 200 µl in 96-well plates (Costar, Cambridge, MA).
Cells were stimulated with soluble anti-CD3 (1–10 µg/ml) or
parasite Ag (10–50 µg/ml), and supernatants were harvested
after 24 and 48 h, respectively. IFN-, IL-2, and IL-4 levels
were measured using two site ELISAs as previously described (32, 33). IL-12 (p40) levels were measured using mAb C17.8 and
biotinylated C15.6 (grown from hybridomas provided by G. Trinchieri,
Wistar Institute). Cytolysis of 51Cr-labeled
YAC-1 cells (American Type Culture Collection, Manassas, VA) was used
to measure NK cell cytolytic activity as described previously
(34).
Cytofluorometric analysis
After dissociation and lysis of erythrocytes, cells were resuspended at a final concentration of 1 x 107 cells/ml in FACS buffer composed of 1x PBS, 0.2% BSA fraction V (Sigma), and 4 mM sodium azide. Then, 106 cells were stained with various conjugated mAbs specific for CD4, CD8, CD44, CD45RB, or CD62L for 20 min on ice in the presence of saturating amounts of Fc Block (PharMingen, San Diego, CA). Cells were then washed and analyzed using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). For biotinylated Abs, cells were stained and washed as described above, then incubated with FITC- or PE-conjugated streptavidin (PharMingen) for 20 min on ice. Cells were then washed with FACS buffer and analyzed. Each Ab and streptavidin reagents were used at dilutions empirically determined to give optimal staining for flow cytometric analyses. Results were analyzed using Cell Quest software (Becton Dickinson).
Intracellular cytokine staining
Erythrocyte-depleted splenocytes from chronically infected WT or
CD28-/- mice were plated in a 24-well plate (Costar) at a
density of 5 x 106 cells per well in a final volume
of 1 ml. Cells were then stimulated with STAg (25 µg/ml) for 72
h. PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma) and brefeldin A
(10 µg/ml; Sigma) were added to the cultures during the last 5 h
of stimulation. Cells were then harvested and resuspended in FACS
buffer at a final concentration of 1 x 107
cells per ml. Then, 106 cells were stained with
either FITC-labeled anti-CD4 or anti-CD8 mAbs (PharMingen) for
20 min on ice. Cells were washed with FACS buffer and fixed with 1%
w/v paraformaldehyde. Cells were washed again and permeablized with
0.1% saponin in FACS buffer. After permeablization, cells were stained
with PE-conjugated anti-IFN- (PharMingen) for 30 min on ice.
Cells were washed with 0.1% w/v saponin and then with FACS buffer.
Cells were analyzed using a FACScalibur flow cytometer (Becton
Dickinson).
Statistics
INSTAT software (GraphPad, San Diego, CA) was used for unpaired two-tailed Student’s t test or paired t test evaluations. Values of p < 0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Infection of WT and CD28-/- male BALB/c
mice i.p. with 20 cysts of the ME49 strain of T. gondii did
not result in death of these mice for a period of at least 4 mo. When a
higher parasite dose (100 cysts) was used to infect BALB/c
CD28-/- mice, we saw increased mortality of
these mice during the acute phase of the infection in two of three
experiments (data not shown). These results are similar to a previous
report from Suzuki and colleagues in which BALB/c mice, infected i.p.
with increasing numbers of parasites, were more susceptible to the
acute phase of infection (i.e., 80 cysts resulted in 75% mortality)
(35). Nonetheless, histological analysis and cyst counts
of the brains of these chronically infected BALB/c WT and
CD28-/- mice revealed that there were no
differences in parasite burden or pathology (G. Reichmann, E. N.
Villegas, and C. A. Hunter, unpublished observations). Given the
important role for CD28 in the activation of T cell responses in many
experimental systems, these results were surprising. Therefore, we
decided to investigate why CD28 was not required for resistance to
T. gondii. As a measure of the initial response to T.
gondii, we assayed parasite burden, serum levels of IFN- and
IL-12, as well as the ability of NK cells to lyse the YAC-1 tumor cell
line. At day 5 postinfection, there were no significant differences in
the numbers of infected cells in the peritoneum (<1% of cells
infected for WT and CD28-/- mice) or of the
serum levels of IFN- and IL-12 (Fig. 1
A). However, there was a 2-
to 4-fold decrease in the levels of NK cell cytotoxic activity for the
YAC-1 tumor cell target (Fig. 1B). These latter results are
consistent with our previous studies, which showed that blockade of the
CD28/B7 interaction in SCID mice infected with T. gondii
results in decreased NK cell activity (27).
|
. As
shown in Fig. 2A, T cells from
uninfected WT mice produced IFN- in response to stimulation with
soluble anti-CD3. This effect was dependent on B7 molecules because
the addition of CTLA4-Ig to the cultures inhibited the production of
IFN- (Fig. 2B). When the responses of WT mice were
assessed, we observed that infection for 5 days resulted in enhanced
production of IFN- in response to anti-CD3 (Fig. 2A).
However, in contrast to splenocytes from uninfected WT mice, a
significant proportion of the IFN- produced was not inhibited by
CTLA4-Ig (Fig. 2C). Thus, in WT mice infected with T.
gondii a significant portion of the IFN- produced is
independent of the CD28/B7 interaction. In contrast to WT mice, T cells
from uninfected CD28-/- mice failed to produce
appreciable levels of IFN- in response to anti-CD3 and CTLA4-Ig
did not alter this response (Fig. 2, A and B).
However, by day 5 of infection, stimulation of splenocytes from
CD28-/- mice with anti-CD3 resulted in the
production of elevated levels of IFN- in comparison to uninfected
CD28-/- mice (Fig. 2A). Moreover,
these levels of IFN- were not affected by the addition of CTLA4-Ig
to the cultures (Fig. 2C). Thus, infection of
CD28-/- mice with T. gondii results
in enhanced production of IFN- by T cells stimulated with
anti-CD3.
|
in response to STAg.
Splenocytes from infected WT mice produced significant levels of
IFN- in response to STAg, and this was reduced by 
60% in the
presence of CTLA4-Ig (Fig. 3A). Splenocytes from infected
CD28-/- mice produced reduced levels of
Ag-specific IFN- in comparison to infected WT mice (Fig. 3A). Interestingly, the recall responses of cells isolated
from the peritoneum of WT mice that had been infected i.p. revealed
that the levels of IFN- produced in response to STAg were only
inhibited by 25–30% in the presence of CTLA4-Ig (Fig. 3B).
Furthermore, the levels of IFN- produced by peritoneal cells
isolated from infected CD28-/- mice were
similar to the levels produced by WT mice. The source of these
responses in the peritoneum of CD28-/- mice
were shown to involve both CD4+ and
CD8+ T cells as in vivo depletion of either of
these subsets resulted in reduced production of IFN- (Fig. 3C). Similar results were observed with WT mice (data not
shown). Thus, at the local site of infection the T cell response to
infection was comparable in WT and CD28-/-
mice, and it is likely that the serum levels of cytokines that were
detected (see Fig. 1
) are a function of the immune response at the
local site of infection rather than in the spleen. Together, these
observations demonstrate a requirement for CD28-mediated costimulation
for the ability of splenocytes to produce optimal levels of parasite
specific IFN- in vitro. However, our results identify a
CD28-independent component to these T cell responses, which is observed
in WT and CD28-/- mice and which is sufficient
to protect against infection with T. gondii.
|
Because of the important role of IL-12 in the generation of
protective T cell responses to T. gondii (36, 37) and the identification of a role for the CD40/CD40L
interaction in costimulation of T cell responses (38, 39, 40)
and resistance to the intracellular parasite Leishmania
(29, 41, 42), we chose to study the role of these factors
in the CD28-independent activation of T cells to produce IFN-
following infection. The addition of anti-IL-12 to splenocytes from
CD28-/- mice infected for 5 days reduced the
production of IFN- in response to anti-CD3, whereas
anti-CD40L had no significant effect (Fig. 4A). Similarly, treatment of
CD28-/- mice with a neutralizing mAb specific
for IL-12, but not an Ab specific for CD40L, before infection inhibited
the infection-induced activation of T cells, as measured by production
of IFN- (Fig. 4B). Moreover, splenocytes from
CD28-/- mice that were infected and treated
with anti-IL-12 in vivo and were stimulated in vitro with
anti-CD3 or STAg in the presence of anti-IL-12 produced minimal
levels of IFN- (data not shown). The significance of these studies
was illustrated by the fact that administration of anti-IL-12
resulted in the death of the CD28-/- mice
within 10 days of infection. In contrast, mice treated with
anti-CD40L or deficient in CD40L survived for at least 4 wk after
infection (Fig. 4C).
|
after infection with T. gondii, we then tested
whether IL-12 was capable of enhancing the responses of splenocytes
from uninfected CD28-/- mice stimulated with
anti-CD3. The addition of IL-12 to WT splenocytes stimulated with
anti-CD3 resulted in a 5-fold increase in the levels of IFN-
produced (Fig. 4D), while the addition of IL-12 to
splenocytes from uninfected CD28-/- mice
resulted in a 10- to 20-fold increase in the levels of IFN-. Similar
results were observed with splenocytes from infected WT and CD28
-/- mice stimulated with anti-CD3 alone or
in combination with IL-12 (data not shown). Although the levels of
IFN- produced by splenocytes from CD28-/-
mice are reduced compared with those obtained with cells from WT mice,
these data support our in vivo results that, following infection, IL-12
can stimulate T cell production of IFN- independently of
CD28. CD28 is required for the generation of protective recall responses to T. gondii
To determine the requirements for CD28-mediated costimulation
during memory T cell-mediated immune responses, we assessed the ability
of chronically infected mice to resist a challenge with the virulent RH
strain of T. gondii. Although the RH strain of T.
gondii stimulates high systemic levels of IL-12 and IFN-, it is
still a lethal infection, and protective immunity in immunized mice is
dependent on CD4+ and CD8+
T cell responses (25, 26). Whereas naive WT and
CD28-/- mice succumbed to challenge with RH
between days 8–10 postinfection (Fig. 5A), WT mice chronically
infected with ME49 for 12 wk were resistant to rechallenge with the RH
strain and remained alive for >30 days postchallenge (Fig. 5B). In contrast, chronically infected
CD28-/- mice rechallenged with RH died between
12–14 days after rechallenge (Fig. 5B). Similar results
were obtained when a lower dose of RH (2000 tachyzoites i.p.) were used
to challenge these mice or if WT and CD28-/-
mice were immunized with the temperature-sensitive mutant of T.
gondii, Ts-4, and then challenged with 10,000 RH strain of
T. gondii (data not shown).
|
and IL-12 7 days after
challenge of naive WT mice with the RH strain revealed high levels of
IFN- and IL-12 (Fig. 6, A
and B) associated with a high parasite burden (Fig. 6E). In contrast, chronically infected WT mice rechallenged
with RH produced low levels of IL-12 and IFN- (Fig. 6, A
and B), had a low parasite burden (Fig. 6E), no
detectable parasites in the liver, and low numbers of parasites present
in the lungs (Fig. 7C). We
interpret these data as showing that chronically infected WT mice mount
a prompt secondary response that controls early parasite replication
and as a consequence fails to induce high levels of systemic IL-12 and
IFN-. In contrast, chronically infected
CD28-/- mice rechallenged with RH displayed a
different pattern of responses compared with WT mice. Analysis of serum
levels of IFN- and IL-12 7 days after challenge of
CD28-/- mice with the RH strain revealed that
naive mice produced high levels of IFN- and IL-12 (Fig. 6, C and D) associated with a high parasite burden
(Fig. 6E). Similarly, chronically infected
CD28-/- mice rechallenged with RH produced
comparable levels of IL-12 and IFN- (Fig. 6, C and
D) on day 7 postinfection. At this time point, parasites
were readily detected in peritoneal cells (Figs. 6E and
7B), and large numbers of parasites were detected using
immunohistochemistry in the livers of these mice (data not shown). By
14 days after infection, the numbers of parasites in the peritoneum of
these mice had been reduced (Fig. 6E), and a similar
reduction was also observed in the liver (data not shown). However,
large numbers of parasites were still present in the lungs (Fig. 7D). We interpret these data as showing that chronically
infected CD28-/- mice do not fully control the
initial challenge with RH and as a consequence mount a response that is
similar in strength to the primary response seen in naive
CD28-/- mice. However, although chronically
infected CD28-/- mice are susceptible to
rechallenge with RH, they do have elements of a protective secondary
response that is illustrated by the decreased parasite burden compared
with naive CD28-/- mice and the ability to
clear parasites from the peritoneum and liver.
|
|
and IL-2
after stimulation with STAg (Fig. 8,
A and C). At this time point, the addition of
CTLA4-Ig to the cultures almost completely ablated this recall
response. In chronically infected CD28-/- mice,
we could not detect the production of IFN- or IL-2 in response to
STAg (Fig. 8, B and D). Furthermore, the addition
of CTLA4-Ig to these cultures did not alter the production of these
cytokines. Thus, unlike the acute phase of infection (see Fig. 3A), the ability of memory cells to produce IFN- is
dependent on CD28. The addition of IL-2 to splenocytes from chronically
infected WT or CD28-/- mice did not stimulate
the production of IFN-. However, IL-2 did enhance the production of
IFN- by these splenocytes from chronically infected mice stimulated
with STAg (Fig. 8, E and F).
|
, A, B, and
E). There were comparable numbers of "memory"
CD8+ T cells present in the spleens of
chronically infected CD28-/- and WT mice (Fig. 9, C–E). Analysis of age-matched, uninfected controls for
memory cell populations revealed that in the absence of CD28 there was
a reduction in the percentage of CD4+ T cells expressing a
memory phenotype (WT, 29% CD62Llow;
CD28-/-, 15% CD62Llow).
Thus, the defect in protective recall responses that we observed in
CD28-/- mice correlates with decreased numbers
of memory T cells, specifically the CD4+ T cell
response.
|
production in chronically infected CD28-/-
mice, intracellular staining for IFN- was performed. Results showed
a high percentage of CD8+ T cells that were
positive for IFN- in unstimulated cultures from WT mice. However,
this percentage was increased when these cultures were stimulated with
STAg (Table I). In these cultures, only a
small percentage of CD4+ T cells were positive
for IFN-, and even following stimulation with STAg alone or STAg
plus IL-2 this percentage remained small (7–8%). When splenocytes
from CD28-/- mice were examined, the percentage
of CD8+ T cells that were positive for IFN- in
unstimulated cultures was lower than that observed in WT mice (9% vs
18%), and this percentage was not increased when cultures were
stimulated with STAg. However, the addition of IL-2 alone, or in
combination with STAg, resulted in an increase in the percentage of
CD8+ T cells that were positive for IFN-
(17–20%). When the response of CD4+ T cells
from CD28-/- mice were compared with WT mice,
there were fewer cells positive for IFN-, and this percentage
remained low even after stimulation with STAg plus IL-2 (3%). These
data have to be interpreted with care because the percentage of
IFN--positive cells does not always correlate with levels of IFN-
protein produced in these cultures (see Fig. 8). Nevertheless, in
combination with the IFN- protein data shown in Fig. 8, we interpret
the intracellular staining as showing that CD8+ T
cells from chronically infected CD28-/- mice
are defective in their ability to produce IFN- unless provided with
a source of exogenous IL-2.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our findings that IL-12 can act, independently of CD28, as a signal for
T cell production of IFN- confirms the central role of IL-12 in
resistance to T. gondii (36, 37, 46). The
activation of lymphocytes to produce IFN- can occur in several ways:
the classical MHC/TCR interaction in the presence of costimulation for
either naive or educated Th1 cells in the presence of IL-12 is probably
the best characterized pathway (47). There is also the
"innate" ability of activated T cells to produce IFN- in
response to IL-12 and costimulation, independently of stimulation
through the TCR (48). Our studies show that the induction
of high levels of IL-12, following infection with ME49, is sufficient
to generate protective effector T cells. These findings are in
agreement with recent studies that demonstrated that stimulation of
human PBMCs with maximal concentrations of PHA to produce IFN- was
IL-12 dependent, whereas at lower levels of T cell activation a
CD28-dependent pathway was observed (49). Together with
our in vivo studies, it appears that, in the presence of high levels of
IL-12, CD28 is not required for activation of T cells to produce
IFN- or to protect mice during the acute phase of toxoplasmosis.
Moreover, because CD28-/- mice survive for >4
mo, resistance to the chronic phase of infection appears to be CD28
independent. This is supported by our analysis of the immune response
of C57BL/6 CD28-/- mice during toxoplasmic
encephalitis, which revealed that there are sufficient numbers of
Ag-specific effector T cells present in the brain that are able to
inhibit parasite
replication.5
Recent studies have reported that naive T cells express low levels of
the IL-12R, and that maximal responsiveness to IL-12 is dependent on
stimulation through CD28, which results in up-regulation of the IL-12R
(50, 51). Our results show that the ability of T cells to
produce IFN- in response to IL-12 is independent of CD28, but is
reduced in comparison to cells that can receive costimulation through
CD28. However, it should be noted that stimulation through CD28 does
not simply up-regulate IL-12R expression but also results in increased
stability of IFN- mRNA, which enhances production of IFN- protein
(52). Thus, the reduced levels of IFN- produced by
CD28-/- T cells in our experiments may not be a
result of reduced responsiveness to IL-12 but rather may be a
consequence of decreased stability of IFN- mRNA.
Whether IL-12 alone is sufficient to provide a second signal required
for T cell production of IFN- or if other costimulatory molecules
are involved in this process is not known. We have partially addressed
this issue by examining the possible role of the CD40/CD40L interaction
in this process. This costimulatory pathway can regulate the ability of
accessory cells to provide costimulation through B7 (53, 54), as well as direct activation of T cells
(55, 56, 57). Our in vitro and in vivo studies with
anti-CD40L as well as CD40L-deficient mice demonstrate that this
interaction is not required for initial resistance to T.
gondii. These observations are similar to recent studies with
Histoplasma capsulatum and Mycobacterium
tuberculosis (58, 59) but differ from studies with
Leishmania species, Pneumocystis carinii, and
Cryptosporidium parvum, in which CD40L is required for the
generation of protective T cell responses (29, 41, 42, 60, 61). Interestingly, mice deficient in CD40L are more susceptible
than WT mice to the chronic phase of infection with T.
gondii (A. Sher, unpublished observations, and our own unpublished
data).
There are few reports on the role of CD28 in the generation of memory T
cell responses. Based on studies that examined T cell responses to
influenza virus and H. polygyrus in
CD28-/- mice, it has been proposed that
induction of T cell memory does not require CD28 (20, 21)
but may be important for the generation of effector T cells from either
naive or memory T cells (20). However, our studies with
CD28-/- mice chronically infected with
T. gondii reveal a defective memory response.
Thus, CD28 is required for optimal memory responses in this
experimental system, and other costimulatory molecules do not
substitute for this interaction. Nevertheless, it is important to note
that although chronically infected CD28-/- mice
were susceptible to rechallenge with the RH strain of T.
gondii, and had a reduced number of T cells expressing a memory
phenotype as well as defective production of IFN-, they did display
partial resistance to the RH strain compared with naive mice. This was
illustrated by the delay in time to death of chronically infected mice
(14 days) vs naive mice (8 days). In addition, the parasite burden seen
in rechallenged mice at day 7 postinfection (10–15% infected PECs)
was less than that observed in naive mice (>90% infected PECs) at the
same time point. Moreover, although the chronically infected
CD28-/- mice died with prominent inflammatory
lesions in the lungs associated with large numbers of parasites, there
had been a reduction in the numbers of parasites found in the
peritoneum and liver between day 7 and 14 postchallenge. Thus, even in
the absence of CD28, there are signs of a parasite-specific memory T
cell response in vivo, and this can be revealed in vitro by addition of
IL-2 to the cultures. These results are consistent with previous
studies that demonstrated that immunization-induced resistance against
challenge with the RH strain of T. gondii was due to the
ability of CD4+ T cells to produce IL-2 and
thereby provide help for CD8+ T cell production
of IFN- (25). Indeed, studies with LCMV have also shown
that there is a requirement for CD4+ T cell
production of IL-2 for sustained production of IFN- by
CD8+ T cells following infection
(62).
The studies presented here do not clearly distinguish whether the
defect in recall responses observed in chronically infected
CD28-/- mice is due to failure to generate a
complete memory cell response and/or if costimulation is required for
the ability of memory cells to provide effector functions. The ability
of CTLA4-Ig to block the in vitro recall responses observed with
chronically infected WT mice suggests that CD28 is needed for the
ability of these memory cells to produce IFN-. In contrast, our
studies that identified a reduction in the numbers of
CD4+ "memory" phenotype cells in chronically
infected CD28-/- mice support the idea that
CD28 is required for the generation or maintenance of memory T cell
responses. There are several possible mechanisms whereby CD28 may
affect memory cell responses. In particular, stimulation through CD28
leads to the production of cytokines (such as IL-2) that may be
required for proliferation and subsequent differentiation of memory
cells from either Ag-specific naive or effector cell populations.
Alternatively, the anti-apoptotic effects induced via stimulation
through CD28 (63) may be important in the maintenance and
expansion of long-lived memory cell populations. Gaining an
understanding of the role of CD28 in the generation/maintenance of
memory cells and their effector functions may help in the design of
vaccines to protect against infection as well as the design of
intervention strategies to manage chronic immune-mediated diseases.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Institute for Medical Microbiology and Virology, Heinrich-Heine-University, Dusseldorf, Germany.
3 Address correspondence and reprint request 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: CD28-/-, CD28-deficient mice; WT, wild type; LCMV, lymphocytic choriomeningitis virus; CD40L, CD40 ligand; CD40L-/-, CD40L-deficient mice; STAg, soluble toxoplasma Ag; PECs, peritoneal exudate cells.
5 G. Reichmann, E. N. Villegas, L. Craig, R. Peach, and C. A. Hunter. 1999. The CD28/B7 interaction is not required for resistance to Toxoplasma gondii in the brain but contributes to the development of immunopathology. Submitted for publication.
Received for publication January 19, 1999. Accepted for publication June 16, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
: 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]
interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor 
. Infect. Immun. 62:2818. synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533.[Abstract]
production. Eur. J. Immunol. 26:1621.[Medline]
production by mouse T helper cell clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223. from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner. J. Immunol. 160:1701. and IL-4 responses to viral infections: requirements for IL-2. J. Immunol. 160:5007.This article has been cited by other articles:
|
|
 |
|
  L. Chen, W. Cheng, P. Shivshankar, L. Lei, X. Zhang, Y. Wu, I-T. Yeh, and G. Zhong Distinct Roles of CD28- and CD40 Ligand-Mediated Costimulation in the Development of Protective Immunity and Pathology during Chlamydia muridarum Urogenital Infection in Mice Infect. Immun., July 1, 2009; 77(7): 3080 - 3089. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Gigley, B. A. Fox, and D. J. Bzik Cell-Mediated Immunity to Toxoplasma gondii Develops Primarily by Local Th1 Host Immune Responses in the Absence of Parasite Replication J. Immunol., January 15, 2009; 182(2): 1069 - 1078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fuse, W. Zhang, and E. J. Usherwood Control of Memory CD8+ T Cell Differentiation by CD80/CD86-CD28 Costimulation and Restoration by IL-2 during the Recall Response J. Immunol., January 15, 2008; 180(2): 1148 - 1157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M. Wuthrich, T. Warner, and B. S. Klein CD28 Is Required for Optimal Induction, but Not Maintenance, of Vaccine-Induced Immunity to Blastomyces dermatitidis Infect. Immun., November 1, 2005; 73(11): 7436 - 7441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
L. A. Lieberman, E. N. Villegas, and C. A. Hunter Interleukin-15-Deficient Mice Develop Protective Immunity to Toxoplasma gondii Infect. Immun., November 1, 2004; 72(11): 6729 - 6732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 J. H. Bream, D. L. Hodge, R. Gonsky, R. Spolski, W. J. Leonard, S. Krebs, S. Targan, A. Morinobu, J. J. O'Shea, and H. A. Young A Distal Region in the Interferon-{gamma} Gene Is a Site of Epigenetic Remodeling and Transcriptional Regulation by Interleukin-2 J. Biol. Chem., September 24, 2004; 279(39): 41249 - 41257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Lieberman, F. Cardillo, A. M. Owyang, D. M. Rennick, D. J. Cua, R. A. Kastelein, and C. A. Hunter IL-23 Provides a Limited Mechanism of Resistance to Acute Toxoplasmosis in the Absence of IL-12 J. Immunol., August 1, 2004; 173(3): 1887 - 1893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
Y. Miyahira, M. Katae, S. Kobayashi, T. Takeuchi, Y. Fukuchi, R. Abe, K. Okumura, H. Yagita, and T. Aoki Critical Contribution of CD28-CD80/CD86 Costimulatory Pathway to Protection from Trypanosoma cruzi Infection Infect. Immun., June 1, 2003; 71(6): 3131 - 3137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Montagnoli, A. Bacci, S. Bozza, R. Gaziano, P. Mosci, A. H. Sharpe, and L. Romani B7/CD28-Dependent CD4+CD25+ Regulatory T Cells Are Essential Components of the Memory-Protective Immunity to Candida albicans J. Immunol., December 1, 2002; 169(11): 6298 - 6308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-M. Hu, H. Winter, J. Ma, M. Croft, W. J. Urba, and B. A. Fox CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells J. Immunol., November 1, 2002; 169(9): 4897 - 4904. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Suresh, M. Vig, S. Bhatia, E. P. B. Goodspeed, B. John, U. Kandpal, S. Srivastava, A. George, R. Sen, V. Bal, et al. Pentoxifylline Functions As an Adjuvant In Vivo to Enhance T Cell Immune Responses by Inhibiting Activation-Induced Death J. Immunol., October 15, 2002; 169(8): 4262 - 4272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. N. Villegas, L. A. Lieberman, S. R. Carding, and C. A. Hunter Susceptibility of Interleukin-2-Deficient Mice to Toxoplasma gondii Is Associated with a Defect in the Production of Gamma Interferon Infect. Immun., September 1, 2002; 70(9): 4757 - 4761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. N. Villegas, L. A. Lieberman, N. Mason, S. L. Blass, V. P. Zediak, R. Peach, T. Horan, S. Yoshinaga, and C. A. Hunter A Role for Inducible Costimulator Protein in the CD28- Independent Mechanism of Resistance to Toxoplasma gondii J. Immunol., July 15, 2002; 169(2): 937 - 943. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Rajagopalan, M. K. Smart, E. V. Marietta, and C. S. David Staphylococcal enterotoxin B-induced activation and concomitant resistance to cell death in CD28-deficient HLA-DQ8 transgenic mice Int. Immunol., July 1, 2002; 14(7): 801 - 812. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Compton and J. P. Farrell CD28 Costimulation and Parasite Dose Combine to Influence the Susceptibility of BALB/c Mice to Infection with Leishmania major J. Immunol., February 1, 2002; 168(3): 1302 - 1308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Hunter, D. Yu, M. Gee, C. V. Ngo, C. Sevignani, M. Goldschmidt, T. V. Golovkina, S. Evans, W. F. Lee, and A. Thomas-Tikhonenko Cutting Edge: Systemic Inhibition of Angiogenesis Underlies Resistance to Tumors During Acute Toxoplasmosis J. Immunol., May 15, 2001; 166(10): 5878 - 5881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Blass, E. Pure, and C. A. Hunter A Role for CD44 in the Production of IFN-{{gamma}} and Immunopathology During Infection with Toxoplasma gondii J. Immunol., May 1, 2001; 166(9): 5726 - 5732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. H. Hogan, W. Markofski, A. Bock, B. Barger, J. D. Morrissey, and M. Sandor Mycobacterium bovis BCG-Induced Granuloma Formation Depends on Gamma Interferon and CD40 Ligand but Does Not Require CD28 Infect. Immun., April 1, 2001; 69(4): 2596 - 2603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Cai, T. Radzanowski, E. N. Villegas, R. Kastelein, and C. A. Hunter Identification of STAT4-Dependent and Independent Mechanisms of Resistance to Toxoplasma gondii J. Immunol., September 1, 2000; 165(5): 2619 - 2627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Subauste and M. Wessendarp Human Dendritic Cells Discriminate Between Viable and Killed Toxoplasma gondii Tachyzoites: Dendritic Cell Activation After Infection with Viable Parasites Results in CD28 and CD40 Ligand Signaling That Controls IL-12-Dependent and -Independent T Cell Production of IFN-{gamma} J. Immunol., August 1, 2000; 165(3): 1498 - 1505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. N. Villegas, U. Wille, L. Craig, P. S. Linsley, D. M. Rennick, R. Peach, and C. A. Hunter Blockade of Costimulation Prevents Infection-Induced Immunopathology in Interleukin-10-Deficient Mice Infect. Immun., May 1, 2000; 68(5): 2837 - 2844. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
