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Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, respectively. The preferential interaction of B7-2 with its
ligand(s) in the induction of these responses correlates with its
constitutive expression relative to that of B7-1. However, B7-1 can
equally mediate costimulation for the production of either IL-4 or
IFN- when expressed at high levels. Thus, in leishmaniasis,
costimulation involving B7-1 or B7-2 can result in the production of
either Th1 or Th2 cytokines, rather than a preferential induction of
one type of response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies both in vivo and in vitro have suggested that B7-1 and B7-2
have distinct roles in Th cell differentiation (10, 11). For example,
during experimental allergic encephalomyelitis, a disease mediated by a
dominant Th1 response, treatment of mice with anti-B7-1 mAb
ameliorated disease, whereas anti-B7-2 mAb treatment resulted in
exacerbated disease (10). These results suggested that while B7-1
signaling leads to the induction of Th1 responses, B7-2-mediated
costimulation promotes Th2 development. In contrast, in vivo blocking
studies demonstrated that B7-2 is the dominant molecule functioning in
the induction of diabetes in nonobese diabetic mice, another
Th1-mediated disease (12). Other studies using B7-1- or B7-2-expressing
cell lines have provided evidence that neither B7-1 nor B7-2
differentially regulate Th cell development (13, 14, 15). Moreover, studies
in which TCR transgenic cells were stimulated with Ag in the presence
of B7-1-/- or B7-2-/- APCs demonstrate
that either B7-1 or B7-2 could contribute to IFN- and IL-4
production (16).
Experimental infection with the intracellular parasite Leishmania
major is a well-established model for studying Th1 and Th2
development. In experimental murine leishmaniasis, infections in
resistant mice (C3H, C57BL/6) are associated with Th1 responses,
whereas infections in susceptible mice (BALB/c) are associated with a
Th2 response (17, 18, 19, 20). It is well documented that the early production
of IL-12 and IFN- after L. major infection promotes Th1
differentiation in resistant mice (19, 21, 22, 23). In BALB/c mice, the
early IL-4 burst after infection promotes Th2 development and prevents
IL-12 responsiveness (20, 24, 25, 26). Furthermore, immunotherapies that
promote Th1 development in susceptible mice (e.g., neutralization of
IL-4, treatment with IL-12) are only effective if administered before
the development of an established Th2 response (<7–14 days
postinfection) (20, 25, 27, 28, 29). Thus, the early immune response after
infection with L. major is critical for the development of
Th1 and Th2 cells.
The effects of B7 blockade on Th1 and Th2 development during infection with L. major were initially examined using CTLA-4Ig, a soluble form of CTLA-4 that binds to both B7-1 and B7-2 (30, 31). CTLA-4Ig treatment of resistant C57BL/6 mice had no effect on the outcome of disease (30). In contrast, treatment of BALB/c mice with a single dose of CTLA-4Ig at the time of infection promoted resistance in these mice, which was associated with decreased levels of IL-4 mRNA. These results suggested that B7, presumably through its interaction with CD28, was required for the development of a Th2, but not a Th1, response during leishmaniasis. More recently, blockade of B7-2 (but not B7-1) in WT3 BALB/c mice was shown to affect disease outcome by decreasing parasite burden and the production of Th2 cytokines measured 4 wk postinfection with L. major (32). Anti-B7-2 treatment of C57BL/6 mice decreased the parasite load, but did not alter cytokine production. B7-1 blockade did not affect the resistant phenotype. These results led to the conclusion that B7-2 was critical for Th2 differentiation, but that during L. major infection, a Th1 response does not depend upon B7-1 signaling. An alternative interpretation of these results is that the differential effects of anti-B7-1 and anti-B7-2 mAbs reflect the differences in the kinetics or relative levels of tissue expression of these molecules. Thus, instead of differentially promoting Th1 and Th2 development, it has been proposed that B7-1 and B7-2 may differentially contribute to immune responses due to the nature of their expression patterns (33). Whether B7-1 and B7-2 preferentially promote Th1 and Th2 development, respectively, or are differentially expressed during L. major infection has not been determined. Furthermore, while the studies described above demonstrated the effects of B7 blockade on disease outcome, the effect of B7 blockade on the early immune response has not been examined.
Despite these findings that demonstrate a role for B7-mediated costimulation in the development of a Th2 response, subsequent findings using CD28-/- BALB/c mice showed that CD28 was not an absolute requirement for Th2 development after L. major infection (34). These observations raised the possibilities that either alternative ligands for B7 or compensatory costimulation pathways may exist that promote Th2 development in the absence of CD28. Therefore, it remains to be determined whether CD28-/- mice, in addition to their capacity to mount a Th2 response, exhibit an early IL-4 response during L. major infection that is independent of B7-mediated costimulation. In this study, we investigated: 1) whether B7-1 and B7-2 differentially contribute to Th cell development during the early (day 3) immune response to L. major infection, 2) whether the relative contribution of each molecule reflects levels of their expression, and 3) if B7-independent costimulation contributes to the early IL-4 response after L. major infection of CD28-/- BALB mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. major (MHOM/IL/80/Friedlin) parasites were maintained in Grace’s insect cell culture medium (Life Technologies, Grand Island, NY) containing 20% FBS (HyClone, Logan, UT), 100 U/ml penicillin (Sigma, St. Louis, MO), 100 µg/ml streptomycin sulfate (Sigma), and 2 mM L-glutamine (Sigma). Stationary phase metacyclic promastigotes were isolated by negative selection using peanut agglutinin (Sigma), as described previously (35). Female BALB/cByJ and C3HeB/FeJ mice (obtained between 5 and 6 wk of age from The Jackson Laboratory (Bar Harbor, ME)) were infected in the hind footpads with 2 x 106 metacyclic parasites. CD28-/- BALB/c breeder pairs (backcrossed 6 times with BALB/c WT mice) were obtained from The Jackson Laboratory and bred and maintained in pathogen-free facilities.
Reagents
Human and murine CTLA-4Ig, a B7-1-specific mutant CTLA-4Ig (Y100F), and the control L6 fusion protein were generously provided by Drs. R. Peach and P. Linsley (Bristol-Myers Squibb Research Institute, Seattle, WA). Rat anti-mouse B7-2 (GL1) was protein G-purified from ascites by Harlan Bioproducts for Science (Madison, WI). Purified anti-mouse B7-1 was purchased from PharMingen (San Diego, CA). Control rat IgG was purchased from Sigma. Control human IgG and hamster IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Animals were injected with 200 µg of reagent i.p. and with 200 µg in the footpads mixed together in the parasite inoculum in a volume of 50 µl at the time of infection. In some experiments, a single dose (200 µg) of reagent was given i.p. at the time of infection.
Flow cytometry
Popliteal lymph nodes (LNs) were isolated from normal or
infected mice 3 days postinfection, and single cell suspensions were
prepared. Cells (5 x 105) were transferred to 12
x 75-mm polystyrene tubes and washed with 2 ml FACs buffer (PBS
containing 0.1% BSA and 0.1% sodium azide). Pelleted cells were
resuspended in the residual volume of FACs buffer and incubated with Fc
block (10 µg anti-FcRIII/II (2.4G2) and 10 µg rat IgG) for
5–10 min to block nonspecific binding of Ab to FcR. Cells were then
stained with fluorochrome-labeled mAbs for 25 min on ice in the dark in
the presence of the Fc block, then washed in FACs buffer. Abs used were
FITC-labeled anti-mouse Mac-1, CD4, B220, or rat IgG isotype
control, and PE-labeled anti-mouse B7-1, B7-2, or rat or hamster
IgG isotype control (all from PharMingen). Propidium iodide (Sigma) was
added just before acquisition to allow the exclusion of dead cells.
Acquisition of live, gated Mac-1+, CD4+, or
B220+ cells was performed using a FACScalibur flow
cytometer (Becton Dickinson, San Jose, CA). A minimum of 10,000
live/CD4+ or live/B220+ events was acquired for
analysis of B7 expression. Fewer events (2500–5000
live/Mac-1+) representative of the Mac-1+
population were acquired due to the lower frequency of these cells in
the draining LNs. Expression of B7-1 or B7-2 on gated cell populations
was analyzed with CELLQUEST software (Becton Dickinson).
In vitro recall responses
Single cell suspensions were prepared from the draining
popliteal LNs of infected mice at 3 days postinfection. In some
experiments, LNs were harvested at 14 days postinfection. Cells were
resuspended in complete tissue culture medium (DMEM containing 10% FBS
(HyClone), 100 U/ml penicillin, and 100 µg/ml streptomycin, 2 mM
L-glutamine (Sigma), 25 mM HEPES (Sigma), and 5 x
10-5 M 2-ME (Sigma)) and cultured in 96-well tissue
culture plates at 8 x 105 cells/well, in the presence
or absence of 50 µg/ml soluble leishmanial Ag (SLA) (prepared as
described previously; 36). Cells were cultured with or without 2.5
µg/ml anti-IL-4R mAb (M1; generously provided by Dr. F. Finkelman
(University of Cincinnati, Cincinnati, OH)) to block consumption of
IL-4. Blocking reagents (anti-B7-1, anti-B7-2, CTLA-4Ig, Y100F,
or controls) were added at a final concentration of 5–20 µg/ml.
Supernatants were collected 3 days later, and IL-4 and IFN- were
measured by ELISA.
Proliferation assays
Cell division was assessed by flow cytometry using the dye carboxyfluorescein diacetate succinimydlester (CFSE; Molecular Probes, Eugene, OR), as described previously (37). Briefly, LN cells were washed in PBS, resuspended in PBS at 8 x 106 cells/ml, and incubated at room temperature with an equal volume of 2.5 µM CFSE diluted in PBS. After 5 min, 0.5 ml of FBS was added to stop the labeling, and the cells were washed once in PBS followed by one wash in complete tissue culture medium. Cells were cultured in 96-well U-bottom plates in the presence or absence of SLA as described above. After 3 days, the cells were harvested from each well, washed in FACs buffer, and surface stained with PE-labeled anti-CD4 (PharMingen). Assessment of cell division within the CD4+ population was performed with a FACScalibur flow cytometer on 20,000 propidium iodide-/CD4+ events.
ELISAs
IFN- and IL-4 were measured by ELISA, as previously described
(20, 38), using R46A2 and polyclonal rabbit anti-IFN- as the
coating and detecting Abs, respectively, for IFN-, and 11B11 and
BVD6 for IL-4. IFN- produced from a Th1 clone stimulated with
plate-bound anti-CD3 was used to generate a standard curve,
from which the levels of IFN- in culture supernatants were
calculated. IL-4 levels were calculated from a standard curve using
murine rIL-4 (generously provided by Dr. F. Finkelman).
CD4+ T cell purification
CD4+ T cells were purified by negative selection
using magnetic cell separation (MACS) (Miltenyi Biotec, Sunnyvale, CA),
according to the manufacturer’s instructions. Cells were incubated
with biotinylated anti-FcRIII/II, -CD8, -B220, and -MHC class II,
followed by washing in PBS and subsequent incubation with MACS
streptavidin microbeads. Labeled cells were applied to the separation
column and the CD4-enriched cell population collected. Purity was
assessed by flow cytometry (
97% CD4+).
Stimulation of CD4+ T cells using B7-transfected P815 cell lines
CD4+ T cells were incubated with irradiated (100 Gy)
B7-1-transfected P815 cells, B7-2-transfected P815 cells, or
nontransfected parent P815 cells (Dr. L. Lanier, DNAX, Palo Alto, CA;
and Dr. M. Azuma, National Children’s Medical Research Center, Tokyo,
Japan) at a 1:2 ratio (T cell:transfectant). Anti-CD3 (145-2C11) was
added at a final concentration of 100 ng/ml, along with M1 (2.5 µg/ml
final concentration). Supernatants were harvested 48 h later and
assayed for IFN- and IL-4 by ELISA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the effect of B7 blockade on the early immune
response to L. major infection, BALB/c and C3H mice were
injected with CTLA-4Ig or control Ig at the time of infection. At 3
days postinfection, cells isolated from the draining LN of infected
BALB/c mice treated with control Ig produced IL-4 when restimulated
with SLA in vitro (Fig. 1
A).
In contrast, IL-4 production by LN cells from CTLA-4Ig-treated mice was
markedly reduced. In a total of four experiments, the average reduction
in IL-4 after CTLA-4Ig treatment of BALB/c mice was 71 ± 19.8%.
IFN- production was also reduced as a result of CTLA4-Ig treatment.
Similarly, IFN- production by LN cells from infected C3H was reduced
when these mice were injected with CTLA-4Ig compared with infected
control mice (Fig. 1A). In six experiments, IFN-
production was reduced by an average of 59.8 ± 39.7% when C3H
mice were treated with CTLA-4Ig.
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B) and
>40% when stimulated with soluble anti-CD3. In contrast, few
(
2%) dividing CD4+ cells were detected when cells from
CTLA-4Ig-injected mice were restimulated with SLA. This percentage was
similar to that observed when cells from infected control animals were
cultured in medium alone, or when cells from uninfected animals were
cultured in the presence of SLA (Fig. 1B). Effect of B7 blockade after infection of BALB/c CD28-/- mice
CTLA-4Ig inhibited both T cell proliferation and cytokine
production in infected BALB/c and C3H mice, which suggests that CD28/B7
interactions are critical in the early immune response during
Leishmania infection. However, the requirement for CD28 in
generating a Th2 response has been questioned recently since
CD28-/- BALB/c mice are still susceptible to
leishmaniasis (34). These results seemingly contradict previous studies
in which treatment of WT BALB/c mice with CTLA-4Ig or anti-B7-2
significantly affected disease outcome (30, 32). One explanation for
these divergent results could be that B7 binds to CTLA-4 (or another
ligand) in CD28-/- mice and promotes T cell activation
(39). To assess this possibility, we tested whether CTLA-4Ig treatment
would have the same effect in CD28-/- mice as it did in
conventional animals. As shown above, treatment of BALB/c WT mice with
CTLA-4Ig resulted in a marked reduction of IL-4 (and IFN-)
production (Fig. 2A), as well
as proliferation by LN cells isolated 3 days postinfection (Fig. 2B). In contrast, CTLA-4Ig did not block cytokine production
or proliferation of SLA-stimulated LN cells from infected
CD28-/- mice (Fig. 2, A and B).
Thus, it appears that CD28-/- mice may have developed a
compensatory costimulatory pathway, which we are investigating
currently. However, in normal mice, this compensatory pathway does not
seem to play a significant role in establishing early immune responses,
since CTLA-4Ig treatment blocks T cell responses in WT mice (Figs. 1 and 2).
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Next, we examined the relative contribution of B7-1 and B7-2 to
cytokine production following L. major infection. LN cells
were isolated from WT or CD28-/- BALB/c mice 3 days after
infection and restimulated in vitro with SLA in the absence or presence
of blocking mAbs specific for B7-1, B7-2, or both. Additional wells
contained SLA with CTLA-4Ig, Y100F (a B7-1-specific CTLA-4Ig mutant;
40), or the control fusion protein, L6. Anti-B7-1 mAb had little
effect on IL-4 production compared with cells that were cultured in the
presence of control Ab (Fig. 2C). Y100F also had no effect
on IL-4 production by SLA-stimulated LN cells. In contrast, blocking
B7-2 alone, or blocking both B7-1 and B7-2, resulted in a marked
decrease in the production of IL-4 by WT BALB/c LN cells. While
CTLA-4Ig, anti-B7-2, or both anti-B7-1 and anti-B7-2
reduced IL-4 production by LN cells from WT mice, all of the treatments
shown had the same apparent lack of effect on cytokine production by LN
cells from CD28-/- mice (Fig. 2C). These
results are consistent with those obtained after in vivo B7 blockade
(Fig. 2, A and B).
It has been suggested previously that B7-1 may function to prolong
primary T cell responses, since it has been found to be up-regulated
later during an immune response (33, 41). Therefore, we examined
whether B7-1 contributed to IL-4 production in WT or
CD28-/- mice by measuring recall responses by LN cells
isolated from mice 14 days after infection (Fig. 3). IL-4 production by WT LN cells was
decreased in the presence of anti-B7-2, a combination of
anti-B7-1/anti-B7-2, or CTLA-4Ig (Fig. 3). In contrast, B7-1
blockade had no effect on IL-4 production. Similar to previous findings
by Brown et al. (34), IL-4 production in CD28-/- mice
several weeks after L. major infection is markedly reduced
compared with that of WT mice. Nevertheless, blocking B7-1, B7-2, or
both combined had no effect on Th2 responses in CD28-/-
mice up to 14 days postinfection.
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production.
Similar to the effects of B7-2 blockade on IL-4 production by BALB/c LN
cells, IFN- production by LN cells from C3H mice infected for 3 days
was inhibited by B7-2 blockade (Fig. 4A). In contrast,
anti-B7-1 mAb had little or no effect on either IFN- or IL-4
production. In the presence of CTLA-4Ig, or both anti-B7-1 and
anti-B7-2, cytokine production by BALB/c and C3H LN cells was
further reduced compared with cultures that contained anti-B7-2
mAbs only. Furthermore, B7-2 blockade did not result in a concomitant
increase in IFN- production by LN cells from BALB/c mice, but
instead resulted in a marked reduction of IFN- as well (data not
shown). Similar results were obtained with LN cells obtained from
infected C57BL/6 mice (data not shown).
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B). Similarly,
blockade of B7-2 in C3H mice resulted in decreased IFN- production
when LN cells were restimulated with SLA (Fig. 4). In contrast,
blockade of B7-1 with Y100F had little or no effect on cytokine
production by LN cells from both infected BALB/c and C3H mice. Expression of B7-1 and B7-2 during L. major infection
To determine whether the differential effects of B7-1 vs B7-2
blockade on early cytokine responses reflected the relative levels of
expression of these molecules, we examined B7-1 and B7-2 expression in
the draining LN cells of uninfected, as well as infected, BALB/c and
C3H mice at 3 days postinfection. Flow cytometric analysis of gated
Mac-1+, B220+, and CD4+ cell
populations revealed that, consistent with previous findings (42), B7-2
is constitutively expressed on all three cell populations (Fig. 5, A and B). The
level of expression is increased on B220+ cells in both
BALB/c and C3H mice during infection and on CD4+ cells in
C3H mice. In contrast, little or no detectable B7-1 is present on
B220+ or CD4+ cells from uninfected or infected
mice. B7-1 is expressed on Mac-1+ cells from both BALB/c
and C3H mice, although expression of this molecule does not appear to
increase during infection. Similar patterns of expression were observed
on LN cell populations from infected and uninfected C57BL/6 mice (data
not shown).
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Our data suggest that B7-2 is the dominant ligand for CD28/CTLA-4
during early immune responses to L. major infection. Since
its function appears to correlate with its expression, we wanted to
determine whether B7-1 could provide equal costimulation for
Leishmania-reactive cells if expressed at levels comparable
with B7-2. To address this question, CD4+ T cells were
purified from infected BALB/c and C3H mice and stimulated with
anti-CD3 in the presence of P815 cells transfected with B7-1 or
B7-2. Flow cytometric analysis confirmed similar levels of B7
expression in these transfectants (Ref. 13, and data not shown).
Coculturing anti-CD3-stimulated CD4+ T cells from
infected BALB/c mice with B7-1 transfectants resulted in increased IL-4
production when compared with anti-CD3-stimulated CD4+
T cells cultured in the presence of nontransfected P815 cells (Fig. 6). Costimulation provided by the
transfectants was mediated by B7, because cytokine production was
inhibited by the addition of B7-specific mAbs. Both B7-1 and B7-2
transfectants provided costimulation for IL-4 production by BALB/c
CD4+ T cells; the amount of cytokine produced in the
presence of B7-2 transfectants was comparable to that obtained with the
B7-1 transfectants. Low levels of cytokines were measured in cultures
that contained transfectants and T cells without anti-CD3 (Fig. 6),
or in cultures that contained anti-CD3 and transfectants without T
cells (data not shown). Similarly, either B7-1- or B7-2-transfected
cells were capable of costimulating anti-CD3-induced IFN-
production by CD4+ T cells from C3H mice (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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produced by BALB/c LN cells, rather than a concomitant
increase of IFN-. These observations indicate that blocking
CD28/B7-2 interactions prevents efficient T cell priming and activation
rather than resulting in immune deviation.
Flow cytometric analysis suggested that the differential effects of
B7-1 vs B7-2 blockade reflect the different patterns of expression of
these molecules on LN cell populations. Although both B7-1 and B7-2 are
expressed at high levels on Mac-1+ cells, these cells
constitute a small percentage (
1–3%) of the cells present in the
draining LN. The vast majority of the remaining cell types
(B220+, CD4+, collectively representing 75%
of an uninfected LN and 90% of an infected LN) express constitutive
levels of B7-2, but little to no detectable levels of B7-1. While not
all of these cells are capable of presenting Ag, it has been shown that
CD4+ T cells are capable of responding to B7 costimulatory
signals delivered in "trans" (i.e., signals provided by cells other
than the APC, or bystander cells) (43). Considering the relative
expression of B7-2 and B7-1 on LN cell populations, our observation
that B7-2, but not B7-1, blockade affects early immune responses
induced by Leishmania infection would suggest that
B7-2-expressing bystander cells may contribute to the induction of
these responses.
Although B7-1 appears to play a minor role in promoting early immune
responses in L. major infected BALB/c or C3H mice, B7-1 can
provide sufficient costimulation for cytokine production in the absence
of B7-2 or if expressed at high levels. Maximal inhibition of cytokine
production was obtained with CTLA-4Ig or in the presence of both
anti-B7-1 and anti-B7-2 mAbs compared with either mAb alone.
Moreover, B7-1-transfected cell lines induced comparable levels of
cytokines as B7-2 transfectants. Furthermore, since costimulation
provided by B7-1 or B7-2 transfectants led to both IFN- and IL-4
production by C3H and BALB/c LN cells, respectively, it appears that
during the immune response to infection with L.
major, signaling by B7-2 does not preferentially promote Th2
responses, nor does signaling by B7-1 preferentially promote Th1
responses.
Whereas our studies are focused on the role of B7-mediated costimulation during the early immune response after L. major infection, previous findings have demonstrated the effects of B7 blockade on disease outcome (30, 32). Our observations that B7 blockade inhibited the early immune responses in BALB/c mice are consistent with the findings that B7 blockade promotes a resistant phenotype in BALB/c mice that is associated with decreased IL-4 production and a decrease in parasite burden (30, 32). Therefore, it would appear that B7 (B7-2) blockade affects both early immune responses and Th2 development following L. major infection of susceptible mice. In contrast, while our results suggest that B7-mediated costimulation is important during the early immune response in C3H mice, the absence of B7-2-mediated costimulation does not appear to influence either the capacity of resistant mice to resolve L. major infection or Th1 development (30, 32). Thus, although B7 blockade affects early immune responses in both resistant and susceptible mice, subsequent Th1 and Th2 development are differentially affected in the absence of B7-mediated costimulation.
Several studies have demonstrated that the role for CD28 in primary T cell activation is to prolong cell survival and sustain clonal expansion (44, 45, 46). Therefore, blocking CD28/B7 interactions results in unresponsiveness, or clonal anergy, that ultimately limits the size of the developing Th cell population. Previous reports have shown that Th2 development after L. major infection is influenced by the magnitude of the early immune response. Treatments that diminish the magnitude of the initial T cell response after infection of BALB/c mice (e.g., sublethal irradiation (47), treatment with anti-CD4 (27), anti-IL-2 (48), or anti-IL-4 (20, 28) Abs) affect disease outcome and result in the development of a Th1, rather than a Th2, response. It is conceivable that B7 blockade early after L. major infection would decrease the magnitude of the developing T cell response, thereby affecting Th2 development. In contrast, altering the level of B7-mediated costimulation, which can affect clonal expansion and the magnitude of the early immune response, would have less impact on Th1 development. This contention is consistent with previous findings where relatively fewer numbers of responding cells during infection are necessary to promote Th1 development (49). Thus, although clonal expansion of T cells in infected C3H mice may be limited in the absence of CD28, the development of a Th1 response will proceed largely unaffected. These observations may be further explained by recent findings by Malvey et al. (50), which demonstrated that the induction of unresponsiveness to Ag limits the clonal expansion of responding cells, but does not prevent effector cell function. Although anergy induction resulted in a lack of clonal expansion, differentiation into Th1 cells capable of mediating delayed-type hypersensitivity responses and providing help for IgG2a production was unaffected.
While CD28/B7 interactions appear to be critical to Th2 responses in WT mice, BALB/c mice lacking CD28 exhibit no demonstrable defect in Th2 development after infection with L. major (34). It has been speculated that this discrepancy may be due to developmental compensatory mechanisms that have arisen in the CD28-/- BALB/c mice as a result of their genetic defect (34). Gause and his colleagues (51, 52, 53) have suggested that B7-mediated costimulation in the absence of CD28 contributes to IL-4 production, since B7 blockade in WT mice inhibits the Th2 response after infection with the nematode Heligmosomoides polygyrus, yet CD28-/- mice infected with H. polygyrus develop a Th2 response. Similarly, in leishmaniasis, CTLA-4 may be signaling in a positive fashion (54). However, the inability of CTLA-4Ig or anti-B7-1/anti-B7-2 mAb to inhibit IL-4 production in CD28-/- mice suggests that a B7-independent compensatory pathway is operating in these animals. Moreover, previous reports have demonstrated a role for CTLA-4 in negatively regulating immune responses during leishmaniasis (55, 56). Recently, it has been shown that heat stable Ag (HSA) can provide costimulation for IL-4 production in response to keyhole limpet hemocyanin in the absence of CD28 (57). IL-4 production, however, is ablated in mice that are deficient in both CD28 and HSA. Whether this and/or other costimulatory pathways compensate in CD28-/- mice during leishmaniasis is currently under investigation.
In summary, this study demonstrates that B7-mediated costimulation is
critical in promoting the early immune responses after L.
major infection in both resistant and susceptible mice. However,
this observation does not appear to be true in CD28-deficient mice;
these mice have developed B7-independent pathways that enable the
development of early IL-4 responses after L. major
infection. Consistent with its level of expression, B7-2 is the
dominant molecule that contributes to early immune responses in BALB/c
and C3H mice. However, B7-1 or B7-2, when expressed at comparable
levels, can provide equal costimulation leading to either early IFN-
or IL-4 production, which suggests that B7-1 and B7-2 do not mediate
distinct signaling pathways that promote early immune responses during
Leishmania. Immunoprophylaxis designed to preferentially
promote a particular Th response for controlling leishmaniasis
therefore may not necessitate the exclusive use of B7-1 vs B7-2 for
providing costimulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Phillip Scott, School of Veterinary Medicine, Department of Pathobiology, 216 Rosenthal Building, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: WT, wild-type; LN, lymph node; SLA, soluble leishmanial Ag; CFSE, carboxyfluorescein diacetate succinimydlester.
Received for publication September 11, 1998. Accepted for publication March 11, 1999.
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References |
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or IL-4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59. modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis. J. Immunol. 147:3149.[Abstract]
, interleukin 2, interleukin 4, and interleukin 10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc. Natl. Acad. Sci. USA 88:7011.-independent mechanism. J. Exp. Med. 171:115.This article has been cited by other articles:
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  S. P. Vagvala, L. G. Thebeau, S. R. Wilson, and L. A. Morrison Virus-Encoded B7-2 Costimulation Molecules Enhance the Protective Capacity of a Replication-Defective Herpes Simplex Virus Type 2 Vaccine in Immunocompetent Mice J. Virol., January 15, 2009; 83(2): 953 - 960. [Abstract] [Full Text] [PDF]
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 G. E. Kaiko, S. Phipps, D. K. Hickey, C. E. Lam, P. M. Hansbro, P. S. Foster, and K. W. Beagley Chlamydia muridarum Infection Subverts Dendritic Cell Function to Promote Th2 Immunity and Airways Hyperreactivity J. Immunol., February 15, 2008; 180(4): 2225 - 2232. [Abstract] [Full Text] [PDF]
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L. G. Thebeau, S. P. Vagvala, Y. M. Wong, and L. A. Morrison B7 Costimulation Molecules Expressed from the Herpes Simplex Virus 2 Genome Rescue Immune Induction in B7-Deficient Mice J. Virol., November 15, 2007; 81(22): 12200 - 12209. [Abstract] [Full Text] [PDF]
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M. D. Leech and R. K. Grencis Induction of Enhanced Immunity to Intestinal Nematodes Using IL-9-Producing Dendritic Cells J. Immunol., February 15, 2006; 176(4): 2505 - 2511. [Abstract] [Full Text] [PDF]
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 D. J. Costa, C. Favali, J. Clarencio, L. Afonso, V. Conceicao, J. C. Miranda, R. G. Titus, J. Valenzuela, M. Barral-Netto, A. Barral, et al. Lutzomyia longipalpis Salivary Gland Homogenate Impairs Cytokine Production and Costimulatory Molecule Expression on Human Monocytes and Dendritic Cells Infect. Immun., March 1, 2004; 72(3): 1298 - 1305. [Abstract] [Full Text] [PDF]
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P. Zhang, M. Martin, Q.-B. Yang, S. M. Michalek, and J. Katz Role of B7 Costimulatory Molecules in Immune Responses and T-Helper Cell Differentiation in Response to Recombinant HagB from Porphyromonas gingivalis Infect. Immun., February 1, 2004; 72(2): 637 - 644. [Abstract] [Full Text] [PDF]
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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]
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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]
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 X.-F. Bai, J. Liu, K. F. May Jr, Y. Guo, P. Zheng, and Y. Liu B7-CTLA4 interaction promotes cognate destruction of tumor cells by cytotoxic T lymphocytes in vivo Blood, April 15, 2002; 99(8): 2880 - 2889. [Abstract] [Full Text] [PDF]
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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]
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M. M. L. Pompeu, C. Brodskyn, M. J. Teixeira, J. Clarencio, J. Van Weyenberg, I. C. B. Coelho, S. A. Cardoso, A. Barral, and M. Barral-Netto Differences in Gamma Interferon Production In Vitro Predict the Pace of the In Vivo Response to Leishmania amazonensis in Healthy Volunteers Infect. Immun., December 1, 2001; 69(12): 7453 - 7460. [Abstract] [Full Text] [PDF]
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L. Li, K. L. Legge, B. Min, J. J. Bell, R. Gregg, J. Caprio, and H. Zaghouani Neonatal Immunity Develops in a Transgenic TCR Transfer Model and Reveals a Requirement for Elevated Cell Input to Achieve Organ-Specific Responses J. Immunol., September 1, 2001; 167(5): 2585 - 2594. [Abstract] [Full Text] [PDF]
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C. I. Brodskyn, G. K. DeKrey, and R. G. Titus Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro Infect. Immun., February 1, 2001; 69(2): 665 - 672. [Abstract] [Full Text] [PDF]
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M. A. Marovich, M. A. McDowell, E. K. Thomas, and T. B. Nutman IL-12p70 Production by Leishmania major-Harboring Human Dendritic Cells Is a CD40/CD40 Ligand-Dependent Process J. Immunol., June 1, 2000; 164(11): 5858 - 5865. [Abstract] [Full Text] [PDF]
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A. N. Vallejo, L. O. Mugge, P. A. Klimiuk, C. M. Weyand, and J. J. Goronzy Central Role of Thrombospondin-1 in the Activation and Clonal Expansion of Inflammatory T Cells J. Immunol., March 15, 2000; 164(6): 2947 - 2954. [Abstract] [Full Text] [PDF]
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N. A. Gomes, C. R. Gattass, V. Barreto-de-Souza, M. E. Wilson, and G. A. DosReis TGF-{beta} Mediates CTLA-4 Suppression of Cellular Immunity in Murine Kalaazar J. Immunol., February 15, 2000; 164(4): 2001 - 2008. [Abstract] [Full Text] [PDF]
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H.-W. Mittrucker, A. Kohler, T. W. Mak, and S. H. E. Kaufmann Critical Role of CD28 in Protective Immunity Against Salmonella typhimurium J. Immunol., December 15, 1999; 163(12): 6769 - 6776. [Abstract] [Full Text] [PDF]
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F. P. Heinzel and R. A. Maier Jr. Interleukin-4-Independent Acceleration of Cutaneous Leishmaniasis in Susceptible BALB/c Mice following Treatment with Anti-CTLA4 Antibody Infect. Immun., December 1, 1999; 67(12): 6454 - 6460. [Abstract] [Full Text] [PDF]
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