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*
Immunobiology Program, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and
Departments of Internal Medicine and Microbiology, University of Iowa, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by CTLA-4
engagement. Following CD4+ T cell activation, intracellular
growth of Leishmania chagasi in cocultured splenic
macrophages required both CTLA-4 function and TGF-ß1 secretion.
Cross-linkage of CTLA-4 markedly increased L. chagasi
replication in cocultures of infected macrophages and activated
CD4+ T cells, and parasite growth could be completely
blocked with neutralizing anti-TGF-ß1 Ab. Exogenous addition of
rTGF-ß1 restored parasite growth in cultures protected from
parasitism by CTLA-4 blockade. These results indicate that the negative
costimulatory receptor CTLA-4 is critically involved in TGF-ß
production and in intracellular parasite replication seen in murine
kalaazar. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Visceral leishmaniasis
(VL),3 or kalaazar, is
a life-threatening human parasitic infection caused by Leishmania
chagasi in the New World and characterized by a failure of host
CD4+ T cells to produce IFN- and to eradicate
the parasite (11) that grows unchecked in macrophages
(M
). Recent studies in murine models of VL demonstrated a
deleterious role for CTLA-4 engagement, resulting in the
down-regulation of host T cell responses to visceralizing
Leishmania (12, 13). Blockade of CTLA-4
enhances clearance of Leishmania donovani in vivo
(12). Moreover, both proliferative unresponsiveness and
deficient cytokine secretion by CD4+ T cells from
chronic L. chagasi infection can be restored by B7-1 or
CTLA-4 blockade, leading to parasite clearance (13).
It is well established that TGF-ß plays an important regulatory role
in experimental cutaneous leishmanisis, enhancing parasite virulence
and replication in M (8, 14). Moreover, in vivo
treatment of mice with anti-TGF-ß promotes healing of
Leishmania major infection and enhances NO production by
M (15). Recently, studies in both mouse
(16) and hamster (17) VL have suggested a
pathogenic role for TGF-ß in the course of visceralizing infection.
However, a direct correlation between CTLA-4 regulation of T cell
responses and TGF-ß production has not been demonstrated in any
experimental disease model. In the present study, we investigated
whether there is such direct link between TGF-ß secretion and CTLA-4
down-regulation of anti-parasite CD4+ T cell
responses in chronic murine VL. We demonstrate that CTLA-4 engagement
induces CD4+ T cells from murine kalaazar to
secrete several-fold more TGF-ß than naive CD4+
T cells, and that this increased TGF-ß secretion is responsible for
CTLA-4-mediated arrest of anti-parasite defense in the
infected host.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice of both sexes (6–8 wk of age) were used. An isolate of L. chagasi (MHOM/Br/72/strain 46; Ref. 18) was provided by Dr. C. Corbett (University of São Paulo, São Paulo, Brazil). L. chagasi amastigotes were purified from the spleens of infected Syrian hamsters, as described (19). Mice were infected i.v. with 2 x 107 amastigotes in 100 µl of saline (PBS). All animals used in this study were between 64 and 110 days of infection. Mice of both sexes gave comparable splenomegaly and splenic parasite burden at the chronic stage.
Materials
The following mAbs against murine spleen cells (PharMingen, San
Diego, CA) were used in cultures: purified anti-CTLA-4 mAb 4F10-11,
purified anti-CD4 mAb GK1.5, control hamster IgG mAb, control rat
IgG2b mAb, and purified anti-CD3
mAb 145-2C11. The following
mAbs (PharMingen) were used to isolate CD4+ T
cells by negative selection: anti-B220, anti-MHC class II,
anti-MAC-1, and anti-CD16/CD32. In addition, the following Abs,
also used in the negative selection mixture, were kindly donated by Dr.
Ethan Shevach (National Institutes of Health, Bethesda, MD):
anti-
TCR mAb 13D5, anti-CD8 mAb 53.6.7, and anti-rat
Ig 
-chain mAb MAR 18.5. In some experiments, a neutralizing
anti-human TGF-ß1 IgY (the avian equivalent of IgG; R&D Systems,
Minneapolis, MN; catalogue no. AB-101-NA), cross-reactive with mouse
TGF-ß, but not with unrelated cytokines, and a control chicken IgY
(R&D Systems; catalogue no. AB-101-C), were used. Recombinant cytokines
used were: mouse rIL-10 (Sigma, St. Louis, MO), mouse rIL-2, mouse
rIFN- (both from PharMingen), and human rTGF-ß1 (R&D Systems).
Isolation and purification of CD4+ T cells from normal and infected mice
Three different splenic cell populations containing
CD4+ T cells were used, depending on the type of
experiment: highly purified CD4+ T cells,
CD8- splenocytes, and a mixture of
CD4+ T cells plus infected M. Highly purified
splenic CD4+ T cells from control or infected
donors were first passed through a nylon wool column. Nonadherent cells
were subjected to negative selection using an Ab mixture, followed by
anti-rat Ig plus complement, as described (20).
Briefly, nonadherent cells were treated with anti-CD8,
anti-B220, anti-MHC class II, anti-MAC-1,
anti-CD16/CD32, and anti- TCR (all at 10 µg/ml),
followed by a second step incubation with mouse anti-rat Ig
-chain mAb MAR 18.5 (10 µg/ml), plus rabbit Low-Tox complement
(Cedarlane, Hornby, Ontario, Canada). CD8-depleted
(CD8-) spleen cells (C-mediated lysis after
treatment with anti-CD8 and anti-rat Ig) were also used as a
source of CD4+ T cells containing B cells and
M. To obtain a mixture of CD4+ T cells and
M, splenocytes from infected mice were depleted of
CD8+ T cells, B cells, T cells, and NK
cells by means of incubation with specific mAbs, followed by treatment
with MAR 18.5 plus complement. M were not depleted and served as a
source of both L. chagasi-infected cells and endogenous
accessory cells.
T cell cultures
All sources of CD4+ T cells were cultured
(3 x 105 in 0.2 ml, 2–4 x
106 in 1.0 ml) in DMEM supplemented with 10%
FCS, 2 mM glutamine, 5 x 10-5 M 2-ME, 10
µg/ml gentamicin, sodium pyruvate, MEM nonessential amino acids, and
10 mM HEPES, in either 96-well flat-bottom microtiter plates or 24-well
vessels (Corning Glassworks, Corning, NY) for 2–4 days at 37°C and
7% CO2. Cells were stimulated with soluble
anti-CD3 (10 µg/ml) or recombinant L. chagasi Lcr1 Ag
(10 µg/ml). Isotype controls and mAbs to CTLA-4 and CD4 surface
molecules were added at the saturating dose of 10 µg/ml, according to
the study of Krummel and Allison (21). Chicken
anti-TGF-ß1 and its control Ig were added at 20 µg/ml,
according to the study of Chen et al. (5). After 2–3
days, supernatants were collected and the contents of TGF-ß1, IL-2,
and IFN- were determined by sandwich ELISA. In several experiments,
nonadherent cells were removed at the end of 3–4 days culture, and the
adherent cell monolayer was washed and recultured in Schneider medium
for determination of parasite burden. Recombinant Lcr1 protein, cloned
from a L. chagasi amastigote cDNA library, stimulates T cell
responses in mice infected with L. chagasi (22)
and was purified from transformed bacterial extracts on a
nickel-containing column as described (22).
Induction of CTLA-4 engagement
Plastic wells in 96- or 24-well plates were coated with a
control hamster IgG mAb (10 µg/ml) with anti-CD3 plus control
hamster IgG mAb (both at 10 µg/ml final concentration) or with
anti-CD3 plus anti-CTLA-4 mAb (both at 10 µg/ml final
concentration), in serum-free medium, for 6 h at 37°C, then
washed extensively. In some experiments, wells were coated only with
anti-CTLA-4 or a control mAb, and Lcr1 Ag (10 µg/ml) was added in
soluble form to the culture. Either highly purified splenic
CD4+ T cells or CD4+ T
cells plus M (4 x 105/96-well; 4 x
106/24-well) were added in culture medium. In
some experiments, neutralizing anti-TGF-ß1 or chicken control Ab
(20 µg/ml) were included in soluble form from the beginning of the
culture. After 3 days, supernatants were collected for the
determination of TGF-ß1 by ELISA, and, after 3–4 days, nonadherent
cells were removed and parasite burden was determined in adherent
cells.
Cytokine ELISA
Culture supernatants were collected after 48 h in culture
for determination of IL-2 and IFN- and after 72 h for
determination of mouse TGF-ß1 contents. Quantitative ELISA assays for
mouse IL-2, IFN-, and TGF-ß1 were performed using pairs of mAbs
specific for the corresponding cytokine (PharMingen), one of which was
biotinylated, according to a protocol provided by the manufacturer.
Total TGF-ß1 was measured after acidification to activate latent
TGF-ß, followed by neutralization. Standard curves for TGF-ß, IL-2,
and IFN- were generated using known amounts of purified human
rTGF-ß1 (R&D Systems) or mouse rIL-2 and rIFN- (PharMingen). The
reaction was revealed with alkaline phosphatase-conjugated streptavidin
(Southern Biotechnology Associates, Birmingham, AL), using
p-nitrophenol phosphate (Sigma) as substrate. Results are
the mean and SE of triplicate cultures.
Determination of parasite burden
To determine the parasite burden at the end of cultures (3–4 days), we used a previously described (13) modification of the original method of Lima et al. (23) for quantitation of Leishmania in tissues. Briefly, nonadherent cells were removed from wells at the end of culture, adherent cells were washed, and 0.2–1.0 ml of Schneider medium containing 20% FCS and 2% human urine was added to the 96- and 24-wells, respectively. No extracellular parasite was detected at that stage. After 4–7 days at 26°C, resulting viable promastigotes were counted. Results are the mean and SE of triplicate cultures. It should be noted that there is a steep increase in the measured intracellular parasite load if primary cultures are extended from 3 to 4 days, as shown in some of the figures. Likewise, measured parasite load also greatly increases if the second culture in Schneider medium is extended from 4 to 7 days.
Statistical analysis
Parasite counts were first normalized by a log transformation. Transformed data and ELISA determinations were then compared by Student’s t test. Significance is indicated in figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated that CD4+ T
cells from chronic VL are unresponsive to activation by anti-CD3
mAb or recombinant L. chagasi ag Lcr1, and that suppression
can be reverted by CTLA-4 blockade, leading to cytokine production and
parasite killing (13). To investigate a link between
CTLA-4-mediated suppression and TGF-ß production, we measured
TGF-ß1 production in the supernatants of splenic
CD4+ T cells derived from chronic VL (containing
endogenous accessory cells), and activated by soluble anti-CD3 mAb
(Fig. 1
A). Stimulation of
CD4+ T cells from chronic (
60 days) VL with
soluble anti-CD3 mAb resulted in intense secretion of TGF-ß1,
compared with CD4+ T cells from control mice
(Fig. 1A). Addition of soluble anti-CTLA-4 mAb, but not
an isotype control mAb, markedly decreased TGF-ß1 secretion following
activation (Fig. 1A). Secretion of TGF-ß could be blocked
by 85% by treatment with anti-CD4 mAb GK1.5 (Fig. 1B),
indicating that most of the TGF-ß produced in this system comes from
CD4+ T cells. These results suggest that
increased TGF-ß secretion by CD4+ T cells from
VL is dependent on CTLA-4 engagement. We then investigated a role for
TGF-ß in the reciprocal regulation of IFN- production by CTLA-4
blockade (13). Following stimulation with soluble
anti-CD3, splenic CD4+ T cells from kalaazar
produced limited amounts of IFN- (Fig. 1C). However,
confirming our previous results, a marked increase in IFN-
production was observed following CTLA-4 blockade with soluble
anti-CTLA-4 mAb (Fig. 1C). Addition of exogenous
rTGF-ß1 at doses similar to the endogenous levels measured in the
cultures (100 pg/ml) completely prevented IFN- production by
CD4+ T cells (Fig. 1C). These results
suggest that TGF-ß mediates the suppressive effect of CTLA-4
engagement on IFN- production.
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). Highly purified
CD4+ T cells from either control or infected (VL)
mice were isolated from spleens and stimulated with immobilized
anti-CD3 mAb in the presence of coimmobilized anti-CTLA-4 mAb
or an isotype control mAb. TGF-ß1 secretion was measured in the
supernatants. Activation of control CD4+ T cells
with anti-CD3 plus anti-CTLA-4-immobilized Abs was required to
induce TGF-ß secretion (Fig. 2A). Activation of
CD4+ T cells from chronic VL with immobilized
anti-CD3 alone also resulted in TGF-ß secretion, but activation
with coimmobilized anti-CD3 plus anti-CTLA-4 mAbs resulted in
markedly increased TGF-ß secretion, which was nearly 6-fold higher
than that seen in control CD4+ T cells (Fig. 2A). We employed a splenic CD4+ T cell
population containing M, obtained from L.
chagasi-infected mice, to investigate the effect of direct CTLA-4
engagement in the response to soluble L. chagasi Ag Lcr1
(Fig. 2B). Again, Lcr1 stimulation alone (plus plate-bound
control hamster mAb) induced limited TGF-ß production. Engagement of
CTLA-4 induced by plate-bound anti-CTLA-4 mAb markedly increased
TGF-ß production (Fig. 2B). These results demonstrate that
CD4+ T cells from chronic VL secrete several-fold
more TGF-ß than primary T cells from naive mice, and that CTLA-4
engagement is an effective signal for TGF-ß secretion. Using highly
purified CD4+ T cells from kalaazar, we also
investigated the role of endogenous TGF-ß production in the
reciprocal regulation of IFN- production by CTLA-4 engagement (Fig. 2C). Stimulation of the purified T cells with plate-bound
anti-CD3 (plus coimmobilized control hamster mAb) induced
substantial IFN- production (Fig. 2C). This result
contrasts with deficient IFN- production by splenic
CD8- cells induced by soluble anti-CD3 (Fig. 1
C) and suggests that the T cell defect in kalaazar depends
on cell interactions with non-T cells. In fact, stimulation with
plate-bound anti-CD3 in the presence of coimmobilized
anti-CTLA-4 almost completely blocked IFN- production (Fig. 2C). More important, however, IFN- production could be
almost completely restored in these cultures following addition of
neutralizing anti-TGF-ß1 Ab (Fig. 2C). These results
demonstrate that endogenous TGF-ß production mediates the suppression
of IFN- production caused by CTLA-4 engagement.
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To investigate the role of TGF-ß secretion in the
anti-parasite response conducted by CD4+ T
cells from VL, we measured the endogenous parasite load in cultures
following CD4+ T cell activation with recombinant
L. chagasi Ag Lcr1 under different experimental conditions
(Fig. 3). Splenic
CD8- cells, containing infected M, were
activated in vitro, nonadherent cells were removed, and intracellular
parasite load was measured in Schneider medium. Stimulation with
L. chagasi Ag Lcr1 failed to reduce intracellular parasite
load in the presence of an isotype control mAb, but almost completely
eliminated endogenous parasites in the presence of CTLA-4 blockade with
soluble anti-CTLA-4 mAb (Fig. 3A). Stimulation with Lcr1
also failed to eliminate parasites in the presence of control chicken
Ig, but almost completely eliminated intracellular parasites in the
presence of neutralizing anti-TGF-ß1 Ig (Fig. 3A).
These results suggest that endogenous TGF-ß production is sufficient
to explain the dependence of parasite growth on CTLA-4 function. In
fact, leishmanial Ag Lcr1 also induced intense TGF-ß1 secretion in
these cultures and, again, increased TGF-ß secretion was prevented if
CTLA-4 blockade was introduced concomitant to activation (Fig. 3B).
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production. The autocrine effect of TGF-ß1 secretion on both IL-2 and
IFN- production by CD4+ T cells was
investigated by neutralizing TGF-ß (Fig. 4). Addition of anti-TGF-ß1 Ab
failed to increase IL-2 production, but markedly increased IFN-
production by CD4+ T cells activated by
leishmanial Ag Lcr1 (Fig. 4). These results suggest that deficient IL-2
secretion does not result from TGF-ß production. Taken together,
these results indicate that TGF-ß plays a critical role in
suppression of the anti-parasite response of
CD4+ T cells from VL, specially in the case of
IFN- secretion.
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To demonstrate the role of TGF-ß in CTLA-4-mediated suppression
of anti-parasite responses, we established cocultures of
CD4+ T cells plus infected M from spleens of
L. chagasi-infected mice. Cells were stimulated with either
soluble Lcr1 or immobilized anti-CD3, both in the presence of
coimmobilized anti-CTLA-4 mAbs. Endogenous parasite load was
assessed at the end of 4-day culture, which increases the ability to
detect the parasite load, compared with 3-day cultures. Different from
CD8- spleen cells (Fig. 3), the
CD4+ T plus M cell population gave increased
parasite replication values upon stimulation with both Lcr1 and bound
anti-CD3 (Fig. 5, A and
B). This enhanced parasite growth was reproducible and
presumably resulted from enrichment of fully functional APCs in these
cultures. In addition, costimulation with immobilized anti-CTLA-4,
but not with an isotype control mAb, markedly increased parasite
replication in cultures activated with either Lcr1 (Fig. 5A)
or plate-bound anti-CD3 (Fig. 5B) in the presence of
control chicken Ig. Addition of neutralizing anti-TGF-ß1 Ig
essentially eliminated parasite replication in cultures costimulated
with plate-bound anti-CTLA-4 (Fig. 5, A and
B). Interestingly, addition of anti-TGF-ß1
neutralizing Ab to cultures stimulated with Lcr1 or anti-CD3 alone
resulted in only partial reduction of the intracellular parasite burden
(Fig. 5, A and B). This latter result suggests
that other cytokines produced by T cells also increase parasite growth,
but that extensive CTLA-4 engagement renders TGF-ß the dominant T
cell-derived cytokine promoting parasite replication. As a counterproof
for the role of TGF-ß, we investigated the effect of exogenous
TGF-ß addition on the parasite load of cultures subjected to CTLA-4
blockade (Fig. 6). CTLA-4 blockade almost
completely eliminated the intracellular load of L. chagasi
following activation of CD8- splenocytes with
L. chagasi Ag Lcr1 (Fig. 6A) or soluble
anti-CD3 (Fig. 6B). Addition of rTGF-ß1 to cultures
subjected to CTLA-4 blockade restored growth of L. chagasi
in a dose-dependent fashion and for both kinds of T cell stimulation
(Fig. 6, A and B). Restoration of parasite growth
with 50 pg/ml or more of extra exogenous TGF-ß is compatible with the
endogenous levels of TGF-ß (90–220 pg/ml) produced by
CD8- splenocytes following activation with
either anti-CD3 or Lcr1. Interestingly, addition of an excess (20
ng/ml) of murine rIL-10 also restored growth of L. chagasi,
although less efficiently than TGF-ß (Fig. 6A). Taken
together, the results demonstrate that increased TGF-ß1 secretion
mediates enhanced parasite replication effected by CTLA-4
engagement.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to increase intracellular parasite replication.
To our knowledge, this is the first report of a direct link between
TGF-ß production and CTLA-4-mediated suppression of cellular immunity
in an experimental disease model.
Although evidence accumulated that CTLA-4 plays a dominant negative
regulatory role in T cell activation (1, 2, 3, 4), the mechanism
of CTLA-4 down-regulation remains undefined (1, 2).
Recently, a new picture emerged with the demonstration that CTLA-4
engagement induces production of TGF-ß by CD4+
T cells, and that TGF-ß accounts, at least in part, for the
suppressive effects of CTLA-4 engagement (5). The
hypothesis of a functional link between TGF-ß secretion and the
B7/CTLA-4 pathway is intriguing, given their overlapping roles in
immune tolerance and in suppression of autoimmunity and antitumor
immunity (5). TGF-ß is a multifunctional cytokine with
potent immunosuppressive effects on proliferation, cytokine production,
and cytotoxic activity of T cells (6, 7, 25, 26, 27). TGF-ß
has been reported to inhibit Th1 T cell development driven by IL-12 and
IFN- (26) and to block IL-12-induced Jak-Stat signaling
and IFN- production by activated T cells (27). These
activities strongly suggest a deleterious role for TGF-ß on host
protective responses mounted by anti-parasite Th1 T cells. In
addition, TGF-ß induces down-regulation of some microbicidal M
functions, increases intracellular replication of the cutaneous
leishmaniasis agent Leishmania braziliensis, and reactivates
latent L. braziliensis infection in vivo (8, 14). Recently, an immunopathogenic role for TGF-ß has also
been suggested in experimental VL (16, 17). TGF-ß has
been identified as the soluble mediator suppressing IFN- production
by hepatic granuloma cells from susceptible mice infected with L.
chagasi (16). Moreover, treatment of infected mice
with an adenoviral vector expressing TGF-ß exacerbated the infection
(16).
We have previously demonstrated that splenic T cells from mice infected
with L. chagasi are unresponsive to either Ag or
anti-CD3 stimulation and that parasites could grow unchecked in
spleen cell cultures (13). Blockade of CTLA-4 restored T
cell responsiveness, increased IL-2, IFN-, and IL-4 production, and
induced rapid parasite clearance following T cell activation
(13), suggesting a major role for CTLA-4 in the
down-regulation of T cell responses in VL. In the present study, we
found that splenic T cells from murine VL secrete large amounts of
TGF-ß upon stimulation with L. chagasi recombinant Ag Lcr1
or anti-CD3. Increased TGF-ß secretion could be either prevented
by CTLA-4 blockade or induced by CTLA-4 engagement. In addition, we
demonstrated that TGF-ß mediates the reciprocal regulation of IFN-
production by CTLA-4 engagement. We have also established cocultures of
splenic CD4+ T cells and infected M from VL to
investigate the interplay between CTLA-4 engagement, TGF-ß
production, and the control of intracellular replication of L.
chagasi. In unmanipulated cultures, activation of T cells resulted
in TGF-ß secretion and increased parasite growth. Cross-linkage of
CTLA-4 by immobilized anti-CTLA-4 mAb markedly increased both
TGF-ß production by CD4+ T cells and parasite
replication in M. Paracrine exacerbation of parasite growth in M
was strictly dependent on secreted TGF-ß, because it could be
completely prevented by TGF-ß neutralization. Interestingly, the
deleterious role of TGF-ß was less critical in the absence of
extensive CTLA-4 cross-linkage, as the neutralizing anti-TGF-ß Ig
only partially reduced parasite replication. These results suggest that
other unidentified lymphokines can cooperate with TGF-ß. One
candidate is IL-10, because both rIL-10 and rTGF-ß1 are able to
restore parasite replication in cultures subjected to CTLA-4 blockade.
However, our results suggest that increasing the extent of CTLA-4
engagement in T cells leaves TGF-ß as the dominant deleterious
cytokine produced. This effect can be explained, because TGF-ß is the
only known cytokine whose secretion is increased by CTLA-4 engagement,
and the effect can be seen in Th0, Th1, and Th2 T cell clones
(5). In contrast, CTLA-4 engagement suppresses production
of IL-2, IFN-, and IL-3 by Th1 and IL-3, IL-4, IL-5, and IL-10 by
Th2 T cell clones and cell lines (24). Therefore, it is
likely that increasing the level of CTLA-4 engagement will suppress
production of potentially cooperative cytokines, such as IL-4 and
IL-10, while, at the same time, will increase TGF-ß production. It
should be noted that CTLA-4 is already engaged in unseparated splenic
CD8- cell cultures stimulated by cell
interaction-dependent soluble stimuli, such as Lcr1 Ag or anti-CD3,
and that endogenous parasite replication is entirely dependent on the
levels of TGF-ß produced (Fig. 3). As a counter-proof for the role of
TGF-ß, we found that exogenous addition of rTGF-ß1 increased
replication of L. chagasi in a dose-dependent fashion in
cultures subjected to CTLA-4 blockade. Considering that the levels of
endogenous TGF-ß varied between 25–40 pg/ml in cultures of
CD8- splenocytes blocked with anti-CTLA-4,
the increase in parasite replication attained with extra addition of
50–100 pg/ml TGF-ß agrees well with the endogenous levels of 90–220
pg/ml TGF-ß produced by T cell activation and indicates that these
levels are immunosuppressive for T cells. Blockade of IFN-
production by rTGF-ß, and restoration of IFN- production by a
neutralizing anti-TGF-ß1 Ab, confirmed that the endogenous
TGF-ß produced was suppressive for T cells.
Our findings indicate that the negative B7/CTLA-4 costimulatory pathway prevents L. chagasi elimination in the spleens of susceptible BALB mice. These results agree with a recent study showing that CTLA-4 blockade enhances protective immunity in vivo to visceral L. donovani infection (12). However, the dominant CTLA-4 ligand appears to be B7-2 in the livers of L. donovani-infected mice (28), while we found a major role for B7-1 in the spleens of L. chagasi-infected mice (13). No relationship between the type of B7 molecule and Th cell differentiation was found, because blockade of both B7-2 in liver (28) and B7-1 in spleen (13) enhanced both Th1 and Th2 responses in mice with VL. In agreement, both studies found that CTLA-4 blockade also enhanced production of both Th1 and Th2 cytokines in liver and spleen. Regulation of T cell responses by B7-1 and B7-2 is complex and differs according to the kinetics and location of the ongoing immune response (29). The role of B7 costimulation on protective immunity against cutaneous L. major infection in mice has been investigated. B7-2 is required for a Th2 response to L. major in susceptible BALB mice (30, 31), confirming earlier studies that employed injection of CTLA-4 Ig to block B7 molecules in vivo (32). However, in the latter study, continued injection of CTLA-4 Ig into BALB mice also abrogated the acquired resistance, suggesting that a B7 ligand is necessary for late protective anti-Leishmania responses (32). In addition, B7-2 is required for an early protective Th1 response in resistant mice (30, 31). The dominant role of B7-2 in early responses mounted by both susceptible and resistant mice has been ascribed to its majoritary constitutive expression in lymph nodes draining the site of infection (31). In contrast, B7-1 could be up-regulated in the course of L. major infection and was also able to costimulate both Th1 and Th2 responses if expressed at high levels (31). In a murine model of autoimmune disease induction, B7-2 dominated the T cell response in draining lymph nodes, but B7-1 dominated the response of both splenic and tissue-infiltrating autoimmune T cells (29). Differences in kinetics and regulation of B7-1 and B7-2 expression at different sites could explain why B7-2 is the major CTLA-4 ligand in the liver in L. donovani infection (28), but B7-1 is the major CTLA-4 ligand in the later splenic response in L. chagasi infection (13).
The immunopathogenic mechanism of L. chagasi infection
leading to increased CTLA-4 modulation is currently unknown, but our
results suggest that TGF-ß production is involved. There is a marked
increase in the memory/activated CD4+ T cell
subset in the spleens of mice infected with L. chagasi (data
not shown). However, most of these cells appear to be resting memory
cells, because they require both Ag restimulation and CTLA-4 blockade
to secrete cytokines and kill the parasites (13).
Different from naive T cells, resting memory T cells contain
intracellular stores of CTLA-4 that are continuously recycled between
the cell surface and cytoplasm (33), suggesting more
efficient CTLA-4 signaling in this cell subset. In the present study,
we found that activated CD4+ T cells from chronic
VL secrete several-fold more TGF-ß than naive
CD4+ T cells following CTLA-4 engagement.
Moreover, the levels of TGF-ß produced by cells from chronic VL are
suppressive for IFN- production and stimulatory for parasite growth
in M. It is presently unknown how high TGF-ß-producing cells
accumulate in murine kalaazar, but it is noteworthy that TGF-ß
positively regulates its own production by CD4+ T
cells (34). It is possible that T cell interaction with
Leishmania-infected APCs favors CTLA-4 engagement and
positive regulation of TGF-ß production. First, infected splenic M
from VL down-regulate surface expression of B7 (35), which
might favor CTLA-4 engagement because of its higher affinity for B7
molecules, compared with CD28 (36). Second, CTLA-4
engagement might also favor preferential T cell survival, because
CTLA-4 engagement does not result in T cell apoptosis (5)
and TGF-ß blocks Fas ligand expression and activation-induced cell
death in T cells (37). In addition, TGF-ß production by
infected target cells (14) and secretion of type 2
cytokines (34) could cooperate in this process. Recursive
up-regulation of TGF-ß could progress until the chronic stage of
infection, when suppressive levels of autocrine TGF-ß production
would be attained following CTLA-4 engagement, leading to memory T cell
arrest. Noteworthy, we found that TGF-ß down-regulates IFN-, but
not IL-2 production by T cells from chronic VL. In contrast, we have
previously demonstrated that both IL-2 and IFN- production by these
same T cells are down-regulated by CTLA-4 (13). These
results indicate that CTLA-4 suppression cannot be solely attributed to
TGF-ß secretion and that blockade of IL-2 production is independent
on this mechanism. The latter results agree with studies showing that
TGF-ß blocks IL-12-mediated production of IFN- (27),
but not IL-2 production (6) by activated murine T cells.
Moreover, anti-TGF-ß neutralizing Ab only partially reverted the
inhibitory effect of CTLA-4 engagement on murine T cell proliferation
(5). Thus, it seems that CTLA-4 engagement blocks T cell
activation through multiple mechanisms, TGF-ß secretion being one
of them.
Our findings imply that impairment of CD4+ T cell function in chronic VL is fully reversible and does not lead to peripheral clonal deletion. It will be important to investigate to what extent our present ex vivo findings can be extended to the observed impairment of cellular responses in patients with kalaazar. In the in vivo disease process, one must consider that additional regulatory factors may be present, besides B7/CTLA-4 signaling and TGF-ß production. If this regulatory pathway proves relevant in human disease, it could be considered in the future as a target for improvement of cellular immunity and to accelerate the cure of patients with kalaazar.
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Acknowledgments |
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Footnotes |
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ncia e Technologica, Conselho Nacional de Pesquisas, Comissao de Aperfeiçoamento de Pessoal de Nival Superior, and Fundeão de Apoio à Pesquisa de Estado do Rio de Janeiro. 
2 Address correspondence and reprint requests to Dr. George A. DosReis, Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS Bloco G, Ilha do Fundão, Rio de Janeiro, RJ 21944-970, Brazil. E-mail address:
3 Abbreviations used in this paper: VL, visceral leishmaniasis; M, macrophage(s).
Received for publication July 8, 1999. Accepted for publication November 30, 1999.
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) or
anti-CTLA-4 mAbs (•) in 96-well plates (4 x 105
cells). Cultures treated with anti-CTLA-4 also received the
indicated doses of human recombinant TGF-ß1 at the start of the
experiment (•). A, Some cultures treated with soluble
anti-CTLA-4 received 20,000 pg/ml mouse rIL-10 (
) at the start
of the experiment. After 4 days in culture, nonadherent cells were
removed and the resulting parasite load was measured in adherent cells
after 4 days (A) or 7 days (B) in
Schneider medium. Results represent the mean ± SE of triplicate
cultures. A and B, The only treatment
that gave a nonsignificant difference in parasite replication was 10.0
pg/ml TGF-ß1. All other treatments gave significant growth
(p < 0.01 or p < 0.05)
compared with CTLA-4 blockade alone.