CTLA-4 has recently been shown to act as a negative regulator of T cell activation. Here we provide evidence that blockade of CTLA-4 can result in enhanced host resistance to an intracellular pathogen. The administration of anti-CTLA-4 mAb 4F10 to BALB/c mice, 1 day following infection with Leishmania donovani, enhanced the frequency of IFN-γ 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 γIP-10 was also elevated above that seen in control Ab treated mice, and was directly correlated with the frequency of IFN-γ producing cells. In contrast, macrophage inflammatory protein-1α (MIP-1α) 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.

Cytotoxic T lymphocyte Ag-4 (CTLA-4) was identified more than 10 years ago from an activated T cell cDNA library (1). CTLA-4 (CD152) is expressed on activated CD4+ and CD8+ T cells and binds to the costimulatory ligands, B7-1 (CD80) and B7-2 (CD86), with a 20-fold higher affinity than CD28 (2, 3). Unlike CD28, expression of CTLA-4 is rarely detected on unstimulated T cells, and peak expression occurs between 24 and 72 h postactivation (2, 3, 4). CD28 had been shown previously to provide the critical second signal required for optimal T cell activation, and the structural homology between CTLA-4 and CD28, and the common use of the B7 family of ligands, initially suggested that CTLA-4 function would compliment that of CD28 (5). However, a role for CTLA-4 in the negative regulation of T cell function has now been established (6, 7, 8, 9, 10). Notably, the phenotype of mice genetically deficient for CTLA-4 indicates that this molecule is vital in regulating lymphocyte homeostasis; CTLA-4 −/− mice display a profound lymphoproliferative disorder that is usually fatal by 4 to 5 wk of age (11, 12). Physiologically, CTLA-4 mediated termination of T cell responses may facilitate the generation of memory T cells ready to respond to Ag after decay of CTLA-4 expression (reviewed in Refs. 9 and 13). Alternatively, a role for CTLA-4 in the regulation of anergy has also been recently proposed based on the observations that peptide induced anergy in DO11.10 TCR transgenic mice is prevented by the blockade of CTLA-4 (14).

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-α, IFN-γ 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-γ and IL-4), increased expression of the CXC chemokine γIP-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.

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 × 107 amastigotes i.v., via the lateral tail vein in 200 μl of RPMI 1640 medium (GIBCO, Paisley, U.K.).

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.

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 (×63) with sections derived from two mice per group.

An ELISPOT assay was used to enumerate the frequency of IFN-γ 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-γ ELISPOT were the mAb R46A2 and the rabbit polyclonal anti-IFN-γ, 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-γ) 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-γ 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).

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).

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), γIP-10, monocyte chemotactic protein-1 (MCP-1) (24), and macrophage inflammatory protein-1α (MIP-1α) (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.

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).

FIGURE 1.

CTLA-4 blockade enhances resistance to L. donovani. BALB/c mice were infected with L. donovani amastigotes and were then treated 24 h later with either 100 μg of control HIgG mAb (▪) 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).

FIGURE 1.

CTLA-4 blockade enhances resistance to L. donovani. BALB/c mice were infected with L. donovani amastigotes and were then treated 24 h later with either 100 μg of control HIgG mAb (▪) 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).

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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.

Table I.

Tissue inflammation is transiently increased following anti-CTLA-4 administrationa

Day p.i.Spleen/Body IndexLiver/Body Index
HIgGAnti-CTLA-4HIgGAnti-CTLA-4
Naive 0.62 ± 0.02 0.72 ± 0.10 5.11 ± 0.16 4.88 ± 0.06 
14 1.00 ± 0.07 1.96 ± 0.07* 5.71 ± 0.23 6.97 ± 0.16† 
28 3.06 ± 0.09 2.49 ± 0.16‡ 6.61 ± 0.57 6.52 ± 0.25 
56 5.06 ± 0.32 5.78 ± 1.10 7.88 ± 0.47 7.28 ± 0.26 
Day p.i.Spleen/Body IndexLiver/Body Index
HIgGAnti-CTLA-4HIgGAnti-CTLA-4
Naive 0.62 ± 0.02 0.72 ± 0.10 5.11 ± 0.16 4.88 ± 0.06 
14 1.00 ± 0.07 1.96 ± 0.07* 5.71 ± 0.23 6.97 ± 0.16† 
28 3.06 ± 0.09 2.49 ± 0.16‡ 6.61 ± 0.57 6.52 ± 0.25 
56 5.06 ± 0.32 5.78 ± 1.10 7.88 ± 0.47 7.28 ± 0.26 
a

BALB/c mice were infected with L. donovani and treated at day 1 p.i. with either control hamster IgG (HIgG) or anti-CTLA-4 mAb 4F10. Data represent the organ/body weight ratios at the stated times p.i., calculated for four individual mice per group. Data are representative of two individual experiments performed. *, †, ‡, Differences between control and anti-CTLA-4-treated mice at significance levels of p < 0.001, p < 0.005, and p < 0.02, respectively.

We have previously reported that when using an ELISPOT assay, both IFN-γ and IL-4-producing cells can be detected following L. donovani infection (19, 27). The frequency of IFN-γ 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-γ-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-γ 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-γ 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-γ 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-γ 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.

FIGURE 2.

CTLA-4 blockade increases the frequency of IFN-γ and IL-4-producing cells. Spleen and liver mononuclear cell preparations from HIgG and anti-CTLA-4 treated L. donovani-infected mice were assayed for the frequency of IFN-γ (▵) 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-γ-producing cells per 106 cells in the spleen and liver of naive mice was 933 ± 140 and 1113 ± 186, respectively. For IL-4-producing cells, the frequencies were 520 ± 83 and 667 ± 94, respectively. Treatment of naive mice with anti-CTLA-4 had no effect on these frequencies (data not shown). Treatment with anti-CTLA-4 enhanced the frequency of cells producing both cytokines relative to infected HIgG treated controls in both organs (p < 0.01 in each case).

FIGURE 2.

CTLA-4 blockade increases the frequency of IFN-γ and IL-4-producing cells. Spleen and liver mononuclear cell preparations from HIgG and anti-CTLA-4 treated L. donovani-infected mice were assayed for the frequency of IFN-γ (▵) 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-γ-producing cells per 106 cells in the spleen and liver of naive mice was 933 ± 140 and 1113 ± 186, respectively. For IL-4-producing cells, the frequencies were 520 ± 83 and 667 ± 94, respectively. Treatment of naive mice with anti-CTLA-4 had no effect on these frequencies (data not shown). Treatment with anti-CTLA-4 enhanced the frequency of cells producing both cytokines relative to infected HIgG treated controls in both organs (p < 0.01 in each case).

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In addition, as observed following anti-B7-2 treatment (19), treatment with anti-CTLA-4 mAb did not appreciably alter the ratio of IFN-γ 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).

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 3,A, 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. 3,B 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. 3,B 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. 4 C). 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.

FIGURE 3.

Rapid granuloma formation follows treatment with anti-CTLA-4 mAb. The development of hepatic granulomas was determined from hematoxylin-stained liver cryosections. A, Granuloma density was determined from 50 consecutive microscopic fields (×63). Data are representative of two independent experiments (n = 2 mice for each experiment performed) and represent the mean number of granulomas per 50 fields ± SE. B, Histologic response around individual infected KC in control (open bars) and anti-CTLA-4 treated (hatched bars) mice was scored, as described in Materials and Methods. Data represent the frequency of cells showing no response (KC), KC fusion (FKC), immature granulomas (IG), and mature granulomas (MG). Data were obtained from 100 infected KC per mouse (n = 4 mice per group from two independent experiments).

FIGURE 3.

Rapid granuloma formation follows treatment with anti-CTLA-4 mAb. The development of hepatic granulomas was determined from hematoxylin-stained liver cryosections. A, Granuloma density was determined from 50 consecutive microscopic fields (×63). Data are representative of two independent experiments (n = 2 mice for each experiment performed) and represent the mean number of granulomas per 50 fields ± SE. B, Histologic response around individual infected KC in control (open bars) and anti-CTLA-4 treated (hatched bars) mice was scored, as described in Materials and Methods. Data represent the frequency of cells showing no response (KC), KC fusion (FKC), immature granulomas (IG), and mature granulomas (MG). Data were obtained from 100 infected KC per mouse (n = 4 mice per group from two independent experiments).

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FIGURE 4.

Granuloma development following anti-CTLA-4 mAb treatment. Photomicrographs of tissue responses 14 days p.i. with L. donovani. A, Immature granuloma from control mice. B and C, Mature granulomas from mice treated with anti-CTLA-4. Note that in C extensive epitheliod cell generation has occurred even in though the degree of mononuclear cell cuffing is reduced compared with that seen in B. Parasitized cells are indicated by arrowheads. (Hematoxylin stained, ×100 magnification.)

FIGURE 4.

Granuloma development following anti-CTLA-4 mAb treatment. Photomicrographs of tissue responses 14 days p.i. with L. donovani. A, Immature granuloma from control mice. B and C, Mature granulomas from mice treated with anti-CTLA-4. Note that in C extensive epitheliod cell generation has occurred even in though the degree of mononuclear cell cuffing is reduced compared with that seen in B. Parasitized cells are indicated by arrowheads. (Hematoxylin stained, ×100 magnification.)

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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-1α, MCP-1, and γIP-10 mRNA was determined relative to the housekeeping gene HPRT. Although the accumulation of MIP-1α 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-γ-producing cells determined in the liver by ELISPOT analysis (Fig. 5, A and B). In contrast, the accumulation of mRNA encoding the CXC chemokine γIP-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 γIP-10 mRNA were directly correlated with the number of hepatic IFN-γ-producing cells (Fig. 5 C). Thus, anti-CTLA-4 mAb appears to have a selective effect on chemokine responses within the L. donovani-infected liver.

FIGURE 5.

Elevated levels of γIP-10 mRNA accumulation following anti-CTLA-4 mAb treatment correlates with an increased frequency of IFN-γ-producing cells. Hepatic MCP-1 (A), MIP-1α (B), and γIP-10 (C) mRNA levels were determined by RT-PCR from control (□, ▪) and anti-CTLA-4-treated (▵, ▴) mice at day 3 p.i. (open symbols) and day 7 p.i. (closed symbols). Data are plotted against the frequency of hepatic IFN-γ-producing cells, determined by ELISPOT assay, for each individual mouse studied (n = 3 per group at each time point). A significant degree of correlation between IFN-γ and γIP-10 was determined by linear regression (r = 0.53, p < 0.006).

FIGURE 5.

Elevated levels of γIP-10 mRNA accumulation following anti-CTLA-4 mAb treatment correlates with an increased frequency of IFN-γ-producing cells. Hepatic MCP-1 (A), MIP-1α (B), and γIP-10 (C) mRNA levels were determined by RT-PCR from control (□, ▪) and anti-CTLA-4-treated (▵, ▴) mice at day 3 p.i. (open symbols) and day 7 p.i. (closed symbols). Data are plotted against the frequency of hepatic IFN-γ-producing cells, determined by ELISPOT assay, for each individual mouse studied (n = 3 per group at each time point). A significant degree of correlation between IFN-γ and γIP-10 was determined by linear regression (r = 0.53, p < 0.006).

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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.

FIGURE 6.

CTLA-4 blockade enhances disease resolution in established infection. BALB/c mice were infected with L. donovani and at day 14 p.i. (arrow) were treated with 100 μg HIgG (▪) or anti-CTLA-4 (▴). Parasite burden in liver (A) and spleen (B) was determined at day 28 p.i. Control and treated groups were significantly different at p < 0.005 (in the liver) and p < 0.02 (in the spleen) using Student’s t test. Data represent one of three independent experiments.

FIGURE 6.

CTLA-4 blockade enhances disease resolution in established infection. BALB/c mice were infected with L. donovani and at day 14 p.i. (arrow) were treated with 100 μg HIgG (▪) or anti-CTLA-4 (▴). Parasite burden in liver (A) and spleen (B) was determined at day 28 p.i. Control and treated groups were significantly different at p < 0.005 (in the liver) and p < 0.02 (in the spleen) using Student’s t test. Data represent one of three independent experiments.

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It has recently become clear that ligation of CD28 and CTLA-4 by B7-1 and B7-2 provides an exquisite degree of fine tuning on TCR-mediated T cell activation. This finding has led to the suggestion that these receptor-ligand interactions may provide targets for the manipulation of immune responses during autoimmunity, in tumor immunotherapy (15, 16, 29, 30, 31), or in helminth infection (18). We now demonstrate that blockade of CTLA-4 in vivo leads to increased host resistance to an intracellular pathogen, L. donovani. These data strengthen the case for interventions aimed at this receptor in combating infectious diseases.

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-γ 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-1α, MCP-1, and γIP-10 are all initially induced in a T cell-independent fashion, with peak mRNA accumulation detected at 5 h p.i. Subsequently, MIP-1α 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-1α nor MCP-1 is produced by CD4+ or CD8+ T cells. In contrast, γIP-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 γIP-10 mRNA. Based on these data, we have suggested that γIP-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-1α, 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 γIP-10 mRNA accumulation, but not that for MIP-1α or MCP-1, is increased following blockade of CTLA-4 in vivo, and that this directly correlates with an increased frequency of hepatic IFN-γ-producing cells. However, a causal link between CTLA-4 ligation, γIP-10 production by T cells, and the observed tissue response occurring in L. donovani-infected mice will require future availability of neutralizing Abs to γIP-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.

We thank Dr. J. Bluestone for providing the 4F10 hybridoma cell line, Dr. P. Perrin for purified Abs, Helen Counihan and David Little for assistance with cryotomy, and Dan Salaman for help in preparation of the figures.

1

This work was supported by grants from the British Medical Research Council and the Wellcome Trust.

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-1α, macrophage inflammatory protein-1α.

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.

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