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Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 producing cells in both spleen and
liver, and dramatically accelerated the development of a hepatic
granulomatous response. The expression of mRNA for the CXC chemokine
IP-10 was also elevated above that seen in control Ab treated mice,
and was directly correlated with the frequency of IFN- producing
cells. In contrast, macrophage inflammatory protein-1
(MIP-1) and
monocyte chemotactic protein-1 (MCP-1) mRNA levels were unaffected by
anti-CTLA-4 treatment, suggesting that CTLA-4 blockade may exert
selective effects on chemokine expression. These changes in tissue
response and cytokine/chemokine production were accompanied by a 50 to
75% reduction of parasite load in the spleen and liver of
anti-CTLA-4-treated animals compared to controls. Furthermore,
administration of anti-CTLA-4 mAb 15 days after L.
donovani infection, when parasite burden is increasing in both
organs, also resulted in enhanced resistance. Thus, these studies
indicate a potent immunomodulatory and potentially therapeutic role for
interventions targeted at CTLA-4. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To extend these observations, a number of investigators have recently
examined the effects of CTLA-4 blockade in a variety of disease models.
For example, Leach et al. (15) demonstrated that anti-CTLA-4 mAb
treatment caused rejection of pre-established murine colon carcinoma,
and that this rejection resulted in subsequent immunity to secondary
exposure to the homologous tumor. Ab-mediated blockade of CTLA-4 also
enhanced responses in a prostate cancer model, ranging from marked
reduction in tumor growth to complete rejection (16). A role for CTLA-4
in the regulation of autoimmune disease has also been demonstrated.
Blockade of CTLA-4 promoted the onset of experimental autoimmune
encephalomyelitis and increased disease severity, associated with
enhanced production of the encephalitogenic cytokines TNF-, IFN-
and IL-2 (8, 17). More recently, McCoy et al. (18) have demonstrated
that CTLA-4 blockade promotes rapid and protective primary responses to
Nippostrongylus brasiliensis.
We have recently shown that sustained blockade of B7-2 following infection with Leishmania donovani, the causative agent of visceral leishmaniasis, resulted in enhanced Th1 and Th2 cytokine responses and a significant decrease in liver parasite burden 28 days after infection (19). The action of anti-B7-2 mAb was not dependent on interference with early T cell activation events, since delaying the start of mAb treatment until day 3 postinfection (p.i.)3 was as effective as beginning it on day 0. Hence, this data supported a model in which later B7-2/CTLA-4 interactions were critical in limiting antileishmanial responses. However, as interactions between B7-2 and CD28 may also be required for optimal T cell activation later in infection (20), we suggested that specific blockade of CTLA-4 rather than its ligand would have an even more dramatic effect on antileishmanial immunity.
Here, we demonstrate that CTLA-4 can play a significant role in
regulating host defense against an intracellular pathogen. A single
dose of anti-CTLA-4 mAb administered on day 1 p.i. with
L. donovani significantly decreased parasite burdens in the
liver and, importantly, in the spleen of infected BALB/c mice. This
enhancement of antileishmanial resistance corresponded to increased
cytokine production (IFN- and IL-4), increased expression of the CXC
chemokine IP-10, and the more rapid acquisition of a tissue
granulomatous response. In addition, a single dose of anti-CTLA-4
was also shown to limit the course of established disease. These data
confirm the potential for immunomodulation of infectious diseases using
reagents that target receptors intimately involved in T cell
activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice aged 6 to 8 wk were purchased from Tuck and Co. (Essex, U.K.), and were housed under conventional conditions. L. donovani amastigotes (LV9) were obtained from the infected spleen of a Syrian hamster and were isolated by homogenization and saponin lysis as previously described (21). BALB/c mice were infected with 2 x 107 amastigotes i.v., via the lateral tail vein in 200 µl of RPMI 1640 medium (GIBCO, Paisley, U.K.).
Abs
The hybridoma cell line, producing the hamster anti-CTLA-4 mAb 4F10, (gift from Dr. Jeffrey Bluestone, University of Chicago) was cultured in complete culture medium (RPMI 1640 medium supplemented with 5% (v/v) FCS, 2 mM sodium pyruvate, 1 mM L-glutamine, 0.5 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin; GIBCO). The Ab was affinity purified from culture supernatants using protein G. Control normal hamster IgG (HIgG) Ab was purchased from ICN (Thame, U.K.). Mice were treated with a single dose of either 100 µg of anti-CTLA-4 or HIgG, given i.p., on day 1. In some experiments, a single dose of either 100 µg of anti-CTLA-4 or HIgG, was administered on day 14 p.i.
Determination of parasite burden and histologic responses
Parasite burdens are expressed as Leishman-Donovan units (where LDU represents the number of parasites per 1000 host cell nuclei multiplied by the organ weight) and were determined from Giemsa-stained tissue impression smears, as previously described (21). For histologic analysis, liver samples were embedded in OCT compound (Raymond Lamb, London, U.K.) and then snap frozen in isopentane/liquid nitrogen. Sections (6 µm) were cut with a cryostat, fixed for 10 min in acetone, and then stained by conventional methods with hematoxylin. To determine the degree of cellular response, each infected foci was scored as 1) infected Kupffer cell (KC) with no cellular reaction, 2) fused KCs with few or no associated inflammatory cells, 3) immature granuloma, comprising usually fused infected KCs with limited cellular infiltrate, and 4) mature granuloma, showing extensive epitheliod cell development, fused KCs, and organized cellular infiltrate. The data represent the mean ± SEM derived from counting 100 infected KCs per mouse (n = 4 mice per treatment group from two experiments). In addition, to determine granuloma density in the tissue, the total number of granulomas (immature and mature) were scored in 50 consecutive microscopic fields (x63) with sections derived from two mice per group.
Enzyme-linked immunospot (ELISPOT) assays
An ELISPOT assay was used to enumerate the frequency of IFN-
and IL-4 producing cells from the liver and spleen from each treatment
group, as described elsewhere (19). Briefly, hepatic mononuclear cells
were isolated following perfusion of the portal vein with ice-cold
perfusion buffer (PBS, 0.5 mM EDTA, and 5 mM glucose) and removal of
the gall bladder. After collagenase treatment, viable cells were
isolated over Histopaque 1083 (Sigma, Poole, U.K.) washed, and
resuspended in complete culture medium. Spleen cell suspensions were
prepared using a 20-µm sieve, and the erythrocytes were lysed using
Tris-buffered ammonium chloride (140 mM NH4Cl, 17 mM Tris,
pH,7.5). After washing, the cells were resuspended in complete medium.
Ab pairs for the IFN- ELISPOT were the mAb R46A2 and the
rabbit polyclonal anti-IFN-, and for the IL-4 ELISPOT, mAb
11B11 and biotinylated-polyclonal goat anti-IL-4 (Genzyme,
Cambridge, MA). Millititer HA plates (Millipore, Watford, U.K.)
were coated overnight with Ab at 4°C and then blocked with complete
medium. After washing, freshly explanted cells were added in triplicate
in serial dilutions and incubated for 20 h at 37°C in a 5%
CO2 incubator. After removal of cells, secondary Abs were
added at 1:1000 (v/v; for anti-IFN-) and 1:250 (v/v; for
anti-IL-4) in PBS and 0.05% Tween-20 (Sigma, Poole, U.K.;
overnight at 4°C). After washing, alkaline phosphatase-conjugated
goat anti-rabbit IgG or avidin (1:20,000 (v/v) in PBS and Tween
containing 1% BSA) was added overnight at 4°C. Spots representing
single IFN- and IL-4-producing cells were detected using the
substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
(Sigma Fast, BCIP/NBT), and dissolved in 10 ml of deionized water. Data
represent total cytokine producing cells/106 splenocytes or
hepatic mononuclear cells, calculated from serially diluted
samples. Data represent results from individual mice (n
= 3/group).
Determination of Ab responses
Specific anti-Leishmania responses were determined by ELISA. Nunc Maxisorp plates (Life Technologies, Paisley, U.K.) were coated overnight with 100 µl of soluble leishmanial Ag at a concentration of 10 µg/ml in sodium carbonate-bicarbonate buffer (pH 9.6). After washing with PBS/Tween, plates were blocked with PBS containing 2% BSA, and then incubated with test serum samples (1 h at 37°C). After washing, biotinylated anti-IgG1 and IgG2a (Serotec, Oxford, U.K.) were added (1:1000 (v/v) in wash buffer), washed and then incubated with streptavidin-horseradish peroxidase (Serotec, Oxford, U.K.). Bound enzyme was detected using the ABTS substrate (2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid; Sigma) and optical density read at 405 nm using an ELISA reader (Molecular Devices, Menlo Park, CA).
Measurement of chemokine mRNA levels
mRNA was extracted from the liver tissue samples using
Tri-reagent (Sigma), according to the manufacturers protocol.
Measurement of chemokine mRNA accumulation was determined using a
semiquantitative RT-PCR, as previously described elsewhere (22). PCR
products were vacuum blotted onto nylon membrane (Amersham, Bucks,
U.K.) and then hybridized with specific oligonucleotide probes
conjugated to horseradish peroxidase. Primers and probes for
hypoxanthine-guanine phosphoribosyltransferase (HPRT) (23), IP-10,
monocyte chemotactic protein-1 (MCP-1) (24), and macrophage
inflammatory protein-1 (MIP-1) (25) have been described
elsewhere. Specific products were revealed using the ECL
chemiluminesence system (Amersham) according to manufacturers protocol,
and recorded on x-ray film (Kodak, Rochester, NY). Autoradiographs were
analyzed using Phoretix 1D software (Phoretix, Newcastle, U.K.); then
chemokine mRNA levels are expressed as arbitrary densitometric units
(mean ± SD for three individual mice), calculated relative to the
intensity of the signal generated by the noninducible, constitutively
expressed housekeeping gene HPRT.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Maximal surface expression of CTLA-4 on murine and human T cells
can be detected between 24 and 72 h postactivation (4, 26).
Therefore, to address the role of CTLA-4 in antileishmanial immunity,
we treated BALB/c mice with hamster anti-CTLA-4 mAb 1 day p.i. with
L. donovani. Previous studies have shown that this dose of
anti-CTLA-4 mAb and the kinetics of administration will antagonize
CTLA-4 function (17). A representative course of infection (one of
three independent experiments performed) is shown in Figure 1
. Infection in mice treated with the
control hamster IgG followed an expected course, with parasite burdens
in the liver reaching their peak at day 28 p.i., and subsequently
declining. In contrast, parasite burdens in the spleen of control
animals failed to come under immunologic control during the study
period. Mice treated with anti-CTLA-4 mAb showed significantly
enhanced resistance to infection, with peak parasite burden in the
liver reduced by 59% (p < 0.01). At day
56 p.i., even though control BALB/c mice have by this time
significantly reduced their liver parasite burden, those animals
receiving anti-CTLA-4 mAb had a 75% reduction in liver parasite
burden relative to controls (p < 0.01). We
have previously shown that parasite burden in the spleen of infected
BALB/c mice is often refractory to therapeutic regimes which
nonetheless enhance resistance in the liver (19, 27). Thus, it is of
considerable interest that treatment with anti-CTLA-4 mAb also
significantly decreased parasite burden in the spleen (by 48% at day
28, and 69% at day 56; p < 0.02).
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In naive mice, the administration of anti-CTLA-4 mAb did not
result in nonspecific inflammation in either spleen or liver (as
assessed by measurement of organ/body weight indices; Table I). However, infection with L.
donovani is associated with progressive hepato-splenomegaly (21).
In mice treated with anti-CTLA-4 mAb, the degree of hepatomegaly
was increased above that of control infected mice at day 14 post
infection (liver/body weight index of 5.713 ± 0.230 vs 6.968
± 0.158 in control and anti-CTLA-4 mAb treated mice,
respectively). However, hepatomegaly returned to the level seen in
control infected mice by day 28 p.i. and thereafter (Table I). The
transient increase in splenomegaly was even more pronounced, with an
approximate doubling of spleen/body weight index in
anti-CTLA-4-treated mice (1.003 ± 0.07 vs 1.955 ± 0.073
in control and anti-CTLA-4 mAb treated mice, respectively). At day
28 p.i. the spleens of anti-CTLA-4-treated mice were slightly
but significantly reduced in size compared with control infected mice,
but there were no significant differences by day 56 p.i. Hence,
the most marked effect of this treatment in both organs is a transient
elevation of the inflammatory response.
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We have previously reported that when using an ELISPOT assay, both
IFN- and IL-4-producing cells can be detected following L.
donovani infection (19, 27). The frequency of IFN- and
IL-4-producing cells isolated from the spleen of control infected mice
at day 7 p.i. was not significantly different from that in naive
mice (Fig. 2). In contrast,
IFN--producing cells, and to a lesser extent IL-4-producing cells,
were increased in frequency in the liver of these infected control mice
at day 7 p.i. In mice treated with anti-CTLA-4 mAb, the
frequency of both IFN- and IL-4-producing cells was significantly
increased compared with control infected mice in both spleen and liver
(Fig. 2). Despite these increases in frequency of IFN- and
IL-4-producing cells, however, there were no significant differences in
day 7 parasite burden in either organ (327 ± 54 LDU and 267
± 6 LDU in the liver, and 3 ± 1 LDU and 3 ± 1 LDU in the
spleen of control vs anti-CTLA-4, respectively). Furthermore, flow
cytometry indicated that there was no appreciable change in
CD4:CD8:B220+ ratio in either organ as a result of
anti-CTLA-4 treatment (data not shown). In contrast to the
differences observed in frequency of IFN- and IL-4-producing cells
at day 7 p.i., the frequency of cells producing these cytokines
was not significantly different between control and treated mice at day
14 p.i (data not shown). Thus, the increased frequency of IFN-
and IL-4-producing cells resulting from anti-CTLA-4 treatment was
only observed early in infection and preceded changes in parasite
burden in the tissues.
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and IL-4-producing cells, suggesting that no bias toward
either a Th1 or Th2 response occurred as a result of this treatment.
This suggestion was strengthened by an analysis of parasite-specific
IgG1 and IgG2a isotype responses over the entire course of infection
(until day 56 p.i.), which failed to show any significant
difference at any time point between control infected mice and those in
which CTLA-4 had been blocked (data not shown). Rapid granuloma formation accompanies anti-CTLA-4 mAb administration
Granuloma formation is a key process for the control of L.
donovani infection and precedes parasite clearance (22, 28).
Therefore, it was possible that the changes in the frequency of
cytokine producing cells, noted above, could also be translated into
later changes in the local tissue response. Therefore, to test this
hypothesis, we made a quantitative analysis of granuloma formation at
day 14 p.i., when parasite burden was still similar in both
anti-CTLA-4 mAb-treated and control mice (Fig. 1
). Initially, we
determined the density of granulomas in the liver of these mice. As
shown in Figure 3A, the
density of hepatic granulomas was 
5-fold greater in mice
treated with anti-CTLA-4 mAb than in control Ab-treated mice. It
was immediately apparent, however, that the tissue response was also
qualitatively different. Therefore, we examined the effect of
anti-CTLA-4 treatment on the extent of granuloma maturation. In
control infected mice at day 14 p.i., 60% of infected KC have
attracted a focused cellular response, and those granulomas present are
at an immature stage of development (Figs. 3B and
4A). Mature granulomas are
normally rare at this time point. In marked contrast, 90% of
infected KC in anti-CTLA-4 mAb-treated mice have a focal cellular
response, and >50% of the granulomas formed had progressed to full
maturity (Figs. 3B and 4B). However, it should be
noted that mature granulomas in anti-CTLA-4 mAb-treated mice did
not always contain an extensive mononuclear cell cuff, though the
degree of epitheliod cell development clearly differentiated these from
immature granulomas seen in control mice (Fig. 4C). By day
28 p.i., there was little to differentiate the hepatic tissue
response in the two groups of mice, either by density or maturity, with
the exception that those from anti-CTLA-4 mAb-treated mice
contained fewer parasites (data not shown). Thus, blockade of CTLA-4
enhances granuloma formation, both qualitatively and quantitatively,
after infection with L. donovani.
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IP-10
expression.
The striking difference in granuloma development seen in mice
treated with anti-CTLA-4 mAb led us to examine whether chemokines
involved in this process4
were differentially regulated in treated vs control mice. Total liver
RNA from mice at day 3 and day 7 p.i. was subjected to RT-PCR, and
the accumulation of MIP-1, MCP-1, and IP-10 mRNA was determined
relative to the housekeeping gene HPRT. Although the accumulation of
MIP-1 and MCP-1 mRNA increased slightly from day 3 to day 7
p.i., there was no significant difference between control mice and
those receiving anti-CTLA-4. The levels of these two chemokines was
also independent of the frequency of IFN--producing cells determined
in the liver by ELISPOT analysis (Fig. 5,
A and B). In contrast, the accumulation of mRNA
encoding the CXC chemokine IP-10 increased from day 3 p.i. to
day 7 p.i. and was elevated in mice receiving anti-CTLA-4
compared with control infected mice. Notably, increases in IP-10
mRNA were directly correlated with the number of hepatic
IFN--producing cells (Fig. 5C). Thus, anti-CTLA-4 mAb
appears to have a selective effect on chemokine responses within the
L. donovani-infected liver.
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The data presented above illustrate that a single injection of
anti-CTLA-4 mAb early after infection has dramatic effects on host
resistance. To determine whether this mAb was also capable of altering
the course of established disease, L. donovani infected mice
were treated with a single dose of 100 µg of anti-CTLA-4 mAb or
hamster IgG 14 days p.i., a time at which parasite burden in both
organs is increasing. Dramatically, the administration of
anti-CTLA-4 mAb significantly enhanced resistance in both the liver
(p < 0.005) and spleen
(p < 0.02), even after initial T cell
responses have been established (Fig. 6).
Hence, blockade of CTLA-4 has the capacity to act in a therapeutic
manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies in experimental visceral leishmaniasis have
illustrated that the degree of host resistance to L.
donovani is under both genetic and organ-specific control (27, 32, 33, 34). Modifying the genetically determined level of hepatic
resistance has been readily achievable by the use of cytokine therapy
(35, 36), the administration of neutralizing anticytokine Abs (27, 37, 38, 39), and also in a more limited way by vaccination (40, 41). In
contrast, the genetically determined course of disease in the spleen
has been more resistant to immunomodulation. For example,
neutralization of IL-12 (27) or IL-10 (S. C. Smelt and P. M. Kaye,
unpublished data) fails to affect early parasite growth in this
organ compared with the dramatic (and opposing) effects of these
treatments on liver parasite burden. These data have collectively
suggested that, particularly early during infection, distinct
antiparasite effector mechanisms are operating in these two organs
(27). Therefore, a notable feature of the present study is that
blockade of CTLA-4 had a similar and dramatic effect on parasite burden
in both organs. The effect of blockade of CTLA-4 contrasts
significantly with the reported effect of anti-B7-2 mAb in this
model, in which the spleen was again refractory to the beneficial
effects of this treatment (19). We are currently addressing possible
mechanisms to explain the difference in efficacy of these mAbs in
mediating parasite clearance from the spleen. In our studies with
anti-B7-2 mAb, we employed a continual dosage schedule over the
first 14 days p.i., and as a consequence, partial blockade of CD28
function would also have occurred over this time period. It has been
noted previously that although CD28 may not be essential for primary T
cell activation (19, 42), ligation of CD28 subsequently enhances and
sustains T cell responses (20). If CD28 blockade is the correct
explanation for these contrasting results, T cells in the spleen and
liver might be expected to have different requirements for continued
CD28-mediated signals. Alternatively, the expression of effective
immunity against L. donovani in the spleen may require
additional cell types, which are CD28-dependent in their activation
requirements. In this respect, we have suggested that NK cells may play
a more prominent role in antileishmanial immunity in the spleen
compared with the liver (27), and NK cells have been shown to utilize
CD28/B-7 mediated pathways during their activation for IFN-
production (43, 44). NK cell activation may be regulated directly by
CTLA-4, or indirectly as a consequence of their requirement for T
cell-derived IL-2 (45, 46). Although the precise mechanisms remain to
be elucidated, our data nevertheless suggest a critical role for CTLA-4
in regulating the efficacy of antileishmanial mechanisms operating in
both tissues.
The altered tissue responses that result from anti-CTLA-4 mAb treatment are striking. The kinetics of granuloma development following L. donovani infection in BALB/c mice has been well documented (22, 28, 47, 48). A predominantly mononuclear cell infiltrate is detectable from day 3 p.i., and by day 7 p.i. small numbers of inflammatory cells accumulate around infected KC. By day 14, readily observable accumulations of predominantly mononuclear cells can be seen associated with 40 to 60% of the infected KC in the tissue. However, an organized mantle of lymphocytes and monocytes, of variable cellularity, as well as extensive epitheliod cell development is usually not observed until day 28 p.i., and by this stage a greater proportion of infected KC are also associated with a tissue response. In anti-CTLA-4-treated mice, all facets of the tissue response are accelerated, with >90% of infected KCs becoming the focus of a rapidly maturing granulomatous response by day 14 p.i.
To understand the process of granuloma formation and maturation in more
detail, we have recently examined the role of chemokines in this
process.4 Our initial studies indicate that the regulation
of chemokine synthesis is biphasic following L. donovani
infection. The CC and CXC chemokines MIP-1, MCP-1, and IP-10 are
all initially induced in a T cell-independent fashion, with peak mRNA
accumulation detected at 5 h p.i. Subsequently, MIP-1 and MCP-1
decline to baseline levels at 24 h and remain so until T
cell-dependent mechanisms induce re-expression from day 7 to 14 onward.
Importantly, at day 14 p.i., neither MIP-1 nor MCP-1 is
produced by CD4+ or CD8+ T cells. In contrast,
IP-10 mRNA accumulation is maintained over the first 3 days p.i. in
a T cell-dependent manner, and by day 14 p.i., T cells themselves
contribute 50% of the accumulated tissue IP-10 mRNA. Based on
these data, we have suggested that IP-10 is a major physiologic
mediator of inflammation and granuloma formation following L.
donovani infection.4 It is therefore of interest that
chemokine production by T cells has recently been shown to be
selectively influenced by costimulation. Herold et.al. (49) have
demonstrated the critical role of CD28 in the induction of MIP-1,
but not RANTES, in murine T cells, whereas in human
lymphomas, RANTES promotor activity is linked to CD28
costimulation (50). We have now shown that hepatic IP-10 mRNA
accumulation, but not that for MIP-1 or MCP-1, is increased
following blockade of CTLA-4 in vivo, and that this directly correlates
with an increased frequency of hepatic IFN--producing cells.
However, a causal link between CTLA-4 ligation, IP-10 production by
T cells, and the observed tissue response occurring in L.
donovani-infected mice will require future availability of
neutralizing Abs to IP-10.
In the absence of a CTLA-4 signal, there are at least three possible fates for individual T cells. First, T cells may fail to be rendered anergic. Perez et al. (14) have suggested that CTLA-4 ligation is a critical step in anergy induction rather than a default pathway induced through the absence of CD28-mediated costimulation. We have previously shown that infection with L. donovani results in a reduction in macrophage expression of B7-1 in vitro and in vivo (51). Immediately following L. donovani infection, marginal zone macrophages and marginal metallophils contain the majority of intracellular parasites (46), and as a consequence many T cells entering the periarteriolar lymphocytic sheath may first encounter Ag presented by these macrophages (52). Furthermore, as granulomas develop in the liver, the surface of the infected KC may become a major site of TCR engagement. Under these conditions of locally reduced costimulation, resulting from infection with L. donovani, the higher affinity of CTLA-4 compared with CD28 may normally favor signaling through this receptor and the preferential induction of anergy (13, 14). Blockade of CTLA-4 may therefore facilitate B7-CD28 interactions and prolong activation and cytokine production. Similar "competition" models have been applied to account for the beneficial effects of anti-CTLA-4 mAb in tumor models (16, 31). Second, unrestrained activation mediated by TCR and CD28 signaling may lead these cells to undergo extensive proliferation, similar to that seen in mice genetically deficient in CTLA-4 (11, 12). The hepato-splenomegaly seen early after anti-CTLA-4 mAb treatment in infected, but not naive, mice and the enhanced frequency of cytokine producing cells in both organs would tend to support this. Finally, in the absence of CTLA-4 signaling there may be a more rapid progression to activation induced cell death (31). A rapid loss of cells following delivery of their effector function might account for the transient nature of both the hepato-splenomegaly and increases in frequency of cytokine producing cells that we observe. Experiments are now underway to differentiate between these possibilities.
Finally, we have shown that late administration of anti-CTLA-4 mAb is able to exert beneficial effects on the course of established infection. Unlike L. major infections in mice, where immunologic control of the course of disease is established within the first few days after infection (53), the control of L. donovani infection has been shown to be continually amenable to experimental manipulation. Thus, anticytokine (27) and cytokine (35, 54) therapy given late in infection has been shown to influence disease outcome and has provided the impetus for the recent clinical evaluation of immunomodulatory therapies (41, 55, 56, 57). Data presented in this report indicates that blockade of CTLA-4 is a potent therapeutic strategy, but it will remain to be seen whether interventions targeted at CTLA-4 have any future in the treatment of visceral leishmaniasis in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul M. Kaye, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom. E-mail address:
3 Abbreviations used in this paper: p.i., postinfection; LDU, Leishman-Donovan units; KC, Kupffer cell; ELISPOT, enzyme-linked immunospot; HPRT, hypoxanthine-guanine phosphoribosyltransferase; MCP-1, monocyte chemotactic protein-1; MIP-1, macrophage inflammatory protein-1.
4 S. E. Cotterell, C. R. Engwerda, and P. M. Kaye. Leishmania donovani infection initiates T cell-independent chemokine responses, which are subsequently amplified in a T cell-dependent manner. Submitted for publication.
Received for publication January 5, 1998. Accepted for publication June 17, 1998.
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 M. F. Hassan, Y. Zhang, C. R. Engwerda, P. M. Kaye, H. Sharp, and Q. D. Bickle The Schistosoma mansoni Hepatic Egg Granuloma Provides a Favorable Microenvironment for Sustained Growth of Leishmania donovani Am. J. Pathol., September 1, 2006; 169(3): 943 - 953. [Abstract] [Full Text] [PDF]
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 D. Homann, W. Dummer, T. Wolfe, E. Rodrigo, A. N. Theofilopoulos, M. B. A. Oldstone, and M. G. von Herrath Lack of Intrinsic CTLA-4 Expression Has Minimal Effect on Regulation of Antiviral T-Cell Immunity J. Virol., January 1, 2006; 80(1): 270 - 280. [Abstract] [Full Text] [PDF]
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 M. Boudewijns, A. Jeurissen, M. Wuyts, L. Moens, L. Boon, J. J. Van Neerven, A. Kasran, L. Overbergh, C. Lenaerts, M. Waer, et al. Blockade of CTLA-4 (CD152) enhances the murine antibody response to pneumococcal capsular polysaccharides J. Leukoc. Biol., November 1, 2005; 78(5): 1060 - 1069. [Abstract] [Full Text] [PDF]
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H. W. Murray Prevention of Relapse after Chemotherapy in a Chronic Intracellular Infection: Mechanisms in Experimental Visceral Leishmaniasis J. Immunol., April 15, 2005; 174(8): 4916 - 4923. [Abstract] [Full Text] [PDF]
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C. R. Engwerda, M. Ato, S. Stager, C. E. Alexander, A. C. Stanley, and P. M. Kaye Distinct Roles for Lymphotoxin-{alpha} and Tumor Necrosis Factor in the Control of Leishmania donovani Infection Am. J. Pathol., December 1, 2004; 165(6): 2123 - 2133. [Abstract] [Full Text] [PDF]
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G. A. Martins, C. E. Tadokoro, R. B. Silva, J. S. Silva, and L. V. Rizzo CTLA-4 Blockage Increases Resistance to Infection with the Intracellular Protozoan Trypanosoma cruzi J. Immunol., April 15, 2004; 172(8): 4893 - 4901. [Abstract] [Full Text] [PDF]
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C. L. Meier, M. Svensson, and P. M. Kaye Leishmania-Induced Inhibition of Macrophage Antigen Presentation Analyzed at the Single-Cell Level J. Immunol., December 15, 2003; 171(12): 6706 - 6713. [Abstract] [Full Text] [PDF]
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H. W. Murray, C. M. Lu, E. B. Brooks, R. E. Fichtl, J. L. DeVecchio, and F. P. Heinzel Modulation of T-Cell Costimulation as Immunotherapy or Immunochemotherapy in Experimental Visceral Leishmaniasis Infect. Immun., November 1, 2003; 71(11): 6453 - 6462. [Abstract] [Full Text] [PDF]
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S. Stager, J. Alexander, K. C. Carter, F. Brombacher, and P. M. Kaye Both Interleukin-4 (IL-4) and IL-4 Receptor {alpha} Signaling Contribute to the Development of Hepatic Granulomas with Optimal Antileishmanial Activity Infect. Immun., August 1, 2003; 71(8): 4804 - 4807. [Abstract] [Full Text] [PDF]
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S. Ahmed, M. Colmenares, L. Soong, K. Goldsmith-Pestana, L. Munstermann, R. Molina, and D. McMahon-Pratt Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis Infect. Immun., January 1, 2003; 71(1): 401 - 410. [Abstract] [Full Text] [PDF]
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R. J. DiPaolo and E. R. Unanue Cutting Edge: The Relative Distribution of T Cells Responding to Chemically Dominant or Minor Epitopes of Lysozyme Is Not Affected by CD40-CD40 Ligand and B7-CD28-CTLA-4 Costimulatory Pathways J. Immunol., September 15, 2002; 169(6): 2832 - 2836. [Abstract] [Full Text] [PDF]
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T. Jacobs, S. E. B. Graefe, S. Niknafs, I. Gaworski, and B. Fleischer Murine Malaria Is Exacerbated by CTLA-4 Blockade J. Immunol., September 1, 2002; 169(5): 2323 - 2329. [Abstract] [Full Text] [PDF]
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 A. M. Doyle, A. C. Mullen, A. V. Villarino, A. S. Hutchins, F. A. High, H. W. Lee, C. B. Thompson, and S. L. Reiner Induction of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) Restricts Clonal Expansion of Helper T Cells J. Exp. Med., September 24, 2001; 194(7): 893 - 902. [Abstract] [Full Text] [PDF]
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 H. W. Murray Clinical and Experimental Advances in Treatment of Visceral Leishmaniasis Antimicrob. Agents Chemother., August 1, 2001; 45(8): 2185 - 2197. [Full Text] [PDF]
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D. Pietrella, S. Perito, F. Bistoni, and A. Vecchiarelli Cytotoxic T Lymphocyte Antigen Costimulation Influences T-Cell Activation in Response to Cryptococcus neoformans Infect. Immun., March 1, 2001; 69(3): 1508 - 1514. [Abstract] [Full Text] [PDF]
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S. Stager, D. F. Smith, and P. M. Kaye Immunization with a Recombinant Stage-Regulated Surface Protein from Leishmania donovani Induces Protection Against Visceral Leishmaniasis J. Immunol., December 15, 2000; 165(12): 7064 - 7071. [Abstract] [Full Text] [PDF]
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 N. A. Gomes, V. Barreto-de-Souza, and G. A. DosReis Early in vitro priming of distinct Th cell subsets determines polarized growth of visceralizing Leishmania in macrophages Int. Immunol., September 1, 2000; 12(9): 1227 - 1233. [Abstract] [Full Text] [PDF]
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T. McGaha and J. W. Murphy CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response Infect. Immun., August 1, 2000; 68(8): 4624 - 4630. [Abstract] [Full Text] [PDF]
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E. L. Masteller, E. Chuang, A. C. Mullen, S. L. Reiner, and C. B. Thompson Structural Analysis of CTLA-4 Function In Vivo J. Immunol., May 15, 2000; 164(10): 5319 - 5327. [Abstract] [Full Text] [PDF]
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S. C. Smelt, S. E. J. Cotterell, C. R. Engwerda, and P. M. Kaye B Cell-Deficient Mice Are Highly Resistant to Leishmania donovani Infection, but Develop Neutrophil-Mediated Tissue Pathology J. Immunol., April 1, 2000; 164(7): 3681 - 3688. [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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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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J. Kirman, K. McCoy, S. Hook, M. Prout, B. Delahunt, I. Orme, A. Frank, and G. Le Gros CTLA-4 Blockade Enhances the Immune Response Induced by Mycobacterial Infection but Does Not Lead to Increased Protection Infect. Immun., August 1, 1999; 67(8): 3786 - 3792. [Abstract] [Full Text] [PDF]
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B. Vester, K. Muller, W. Solbach, and T. Laskay Early Gene Expression of NK Cell-Activating Chemokines in Mice Resistant to Leishmania major Infect. Immun., June 1, 1999; 67(6): 3155 - 3159. [Abstract] [Full Text] [PDF]
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M. M. Elloso and P. Scott Expression and Contribution of B7-1 (CD80) and B7-2 (CD86) in the Early Immune Response to Leishmania major Infection J. Immunol., June 1, 1999; 162(11): 6708 - 6715. [Abstract] [Full Text] [PDF]
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 J Alexander, A. Satoskar, and D. Russell Leishmania species: models of intracellular parasitism J. Cell Sci., January 9, 1999; 112(18): 2993 - 3002. [Abstract] [PDF]
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 C.A. CHAMBERS and J.P. ALLISON CTLA-4 -- The Costimulatory Molecule That Doesn't: Regulation of T-cell Responses by Inhibition Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 303 - 312. [Abstract] [PDF]
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) or anti-CTLA-4 (
). Liver (A) and
spleen (B) parasite burdens were determined from tissue
smears and are represented as mean LDU ± SE for four mice per
group. The data is representative of three individual experiments
performed (*) and (**) denotes significance of
p < 0.01 and p < 0.02,
respectively (Student’s t test).
) and IL-4 (
)-producing
cells using ELISPOT at day 7 p.i. Each point represents an
individual mouse, and horizontal bars represent mean values. The
frequency of IFN-
, 