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The Journal of Immunology, 2006, 177: 4636-4643.
Copyright © 2006 by The American Association of Immunologists, Inc.

Inhibition of IL-2 Induced IL-10 Production as a Principle of Phase-Specific Immunotherapy1

Manish Bodas2, Nitya Jain2, Amit Awasthi, Sunil Martin, Raghu Kumar Penke Loka, Dineshkumar Dandekar, Debashis Mitra and Bhaskar Saha3

National Centre for Cell Science, Ganeshkhind, Maharashtra, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leishmania donovani, a protozoan parasite, inflicts a fatal disease, visceral leishmaniasis. The suppression of antileishmanial T cell responses that characterizes the disease was proposed to be due to deficiency of a T cell growth factor, IL-2. We demonstrate that during the first week after L. donovani infection, IL-2 induces IL-10 that suppresses the host-protective functions of T cells 14 days after infection. The observed suppression is concurrent with increased CD4+glucocorticoid-induced TNF receptor+ T cells and Foxp3 expression in BALB/c mice, implicating IL-2-dependent regulatory T cell control of antileishmanial immune responses. Indeed, IL-2 and IL-10 neutralization at different time points after the infection demonstrates their distinct roles at the priming and effector phases, respectively, and establishes kinetic modulation of ongoing immune responses as a principle of a rational, phase-specific immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
One of the basic selection pressures that guided the coevolution of the parasites and the hosts was the need to evade or eliminate each other, respectively (1). Although Leishmania replicates as amastigotes within the macrophages and evades their antileishmanial functions (2), macrophages present leishmanial Ags to antiparasite T cells (3). Although immune evasion promotes, host-protective T cells ameliorate the infection. Thus, the Leishmania-macrophage interactions are poised to be a dynamic one, regulating immune response parameters differently at different phases of the antileishmanial immune response. Indeed, during the first week of Leishmania major infection, the priming phase when the infection is established, CD4+ T cells release a wide array of cytokines, including IL-2 and IFN-{gamma} (3). These two cytokines were proposed to execute a transient host-protective immunity (3), but during the ensuing effector phase, the host-protective T cell functions are suppressed (3, 4). The observations imply that the immune responses during the priming phase, the first week after infection, may critically influence the onset of the T cell suppression, the characteristic feature of chronic Leishmania donovani infection. However, the mechanism of the T cell suppression remains unknown. Because IL-2 was originally identified as a potent T cell growth factor (5), the impaired T cell response in experimental L. donovani infection was proposed to be due to suppressed IL-2 production (6, 7). According to some reports, visceral leishmaniasis patients have decreased IL-2 production (8, 9). By contrast, according to a report on visceral leishmaniasis patients, IL-2 production by nonadherent cells in response to leishmanial Ag was not reduced (10); and in susceptible mice, IL-2 administration during the first week after the infection had no antileishmanial effects (M. Bodas, N. Jain, and B. Saha, unpublished observation). Thus, the role of IL-2 in the induction of host-protective T cells in the first week and in the later period after L. donovani infection remains unknown. Therefore, the effects of anti-IL-2 plus anti-IL-2R Ab treatment of BALB/c mice, a susceptible host, on the course of L. donovani infection were used to probe the dynamicity and evolution of antileishmanial T cell responses during the progressive infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Leishmania parasite and animals

BALB/c mice were from The Jackson Laboratory and were maintained in the National Centre for Cell Science’s Experimental Animal Facility. Mice were infected (i.v.) with 2 x 107 L. donovani stationary-phase promastigotes (strain LV9). At the end of infection, as indicated, the mice were euthanized and the spleens were collected and weighed. The cut surface of a transverse section of the spleens was stamped onto grease-free microscope glass slides. The stamps were dried, methanol fixed, and Giemsa stained. The slides were examined under a light microscope (E600; Nikon) for enumerating the number of amastigotes per 1000 host cell nuclei. The parasite load was expressed as Leishman-Donovan unit, which is calculated by multiplying the spleen weight (in grams) by the number of amastigotes per 1000 host cell nuclei (11). The experiments accorded with the Committee for the Purpose of Control and Supervision of Experiments on Animals-approved protocols.

Reagents

The Abs and the cytokines were procured from BD Pharmingen and Santa Cruz Biotechnology. Nylon wool was from Robbins Scientific, and the CD4+ T cell purification mixture was from StemCell Technologies. Anti-IL-2 (clone HB11674; DNAX), anti-IL-10 (clone HB 10739; DNAX), and anti-CD25 hybridomas were from American Type Culture Collection. The Abs were purified using protein G columns. The isotype-specific Abs were procured from BD Pharmingen.

Kinetics of CD4+ T cell proliferation during progressive L. donovani infection

CD4+ T cells were purified from the naive and L. donovani-infected mice on various days after infection. CD4+ T cells were purified (>98%) using the murine CD4+ T cell enrichment mixture, as per the manufacturer’s protocol. CD4+ T cells were incubated in 96-well plate at 2 x 105 cells/well with anti-CD3 (0.5 µg/ml) plus anti-CD28 (2 µg/ml) for 60 h. Cells were pulsed with 1 µCi of [3H]thymidine (BRIT) for 12 h, and proliferation was assessed using liquid scintillation counter (TopCount; Packard Life Sciences).

T cell suppression assay

A total of 5 x 104 CD4+ T cells was cocultured with 2.5 x 104 CD4+ T cells isolated from the spleen of mice after different days of infection, as indicated (Fig. 1C), and anti-CD3 (0.5 µg/ml) plus anti-CD28 (2 µg/ml) Abs. In other experiments, the indicated numbers of splenic CD4+ T cells were added from the naive or L. donovani-infected mice 3 wk after infection (Figs. 2E and 3D). The suppression was expressed as the percentage of decrease of the [3H]TdR incorporation observed in control cultures.


Figure 1
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  		FIGURE 1. L. donovani infection progresses in two phases in BALB/c mice, a susceptible host. A, Parasite load and DTH response in L. donovani-infected BALB/c mice. BALB/c mice infected i.v. with 2 x 107 L. donovani promastigotes were sacrificed every week. Splenic parasite load was determined by stamp-smear method and expressed as Leishman-Donovan unit (LDU; circle). Some mice were also injected with CSA (40 µg/mouse) in their left hind footpad, and the net swelling (triangle) was recorded in comparison with the uninjected footpad thickness 24 h after the Ag injection. B, Splenic CD4+ T cells were isolated from naive and L. donovani-infected mice every week after the infection, as indicated, and the proliferation of the T cells in response to anti-CD3 (0.5 µg/ml) plus anti-CD28 (2 µg/ml) was assessed by a standard [3H]thymidine incorporation assay. C, The splenic CD4+ T cells (5 x 104) from naive mice were cocultured with splenic CD4+ T cells (2.5 x 104) isolated from the L. donovani-infected mice on the indicated days of infection, and the proliferation assay was performed, as described in Materials and Methods. D, CD4+ T cells were purified by negative selection from the spleens of naive and 21-day infected BALB/c mice. The cells were stained with anti-CD3 PE and anti-CD28 FITC. The cells were analyzed by a FACSVantage flow cytometer. The results are presented as histograms. The error bars represent mean ± SD. The experiments were performed at least three times, and representative data are shown.

 

Figure 2
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  		FIGURE 2. In L. donovani infection, t-reg cells contribute to the suppression. A, CD25 expression on CD4+ T cells from naive and 2-wk infected BALB/c mice. B, GITR expression on splenic CD4+ T cells from naive and 2-wk infected BALB/c mice. C, Foxp3 expression in anti-CD3 plus anti-CD28-stimulated CD4+T cells from naive and 2-wk infected BALB/c mice. The CD4+ T cells from the spleens of naive and infected BALB/c mice were cultured in medium or with anti-CD3 (0.5 mg/ml) plus anti-CD28 (2 mg/ml) for 12 h. The cells were then washed with PBS, and RNA was extracted for the RT-PCR using Foxp3 primers, as described in Materials and Methods. D, The anti-CD3 plus anti-CD28-induced proliferation of Leishmania-infected BALB/c-derived CD4+ T cells was less than those from the naive mice (*, p < 0.001). CD4+CD25+ T cell removal increased the proliferation of CD4+ T cells significantly (p < 0.001). E, The negatively selected CD4+CD25– T cells (5 x 104) were stimulated with anti-CD3 plus anti-CD28 Abs. The indicated numbers of naive or Leishmania-infected BALB/c-derived CD4+CD25+ T cells were added. The cells from only the infected mice inhibited the proliferation of the CD4+CD25– T cells in a dose-dependent manner, as assessed by a standard 3-day proliferation assay. The error bars represent mean ± SD (*, p < 0.001). The experiments were performed at least three times, and representative data are shown.

 

Figure 3
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  		FIGURE 3. CD4+ T cell-expressed IL-10 mediates the suppressor function. A, Cytokine profile of splenic CD4+ T cells from naive and infected BALB/c mice. The cells (105/well in a 96-well plate) were stimulated with anti-CD3 (0.5 µg/ml) plus anti-CD28 (2 µg/ml) for 36 h. The cell culture supernatants from anti-CD3 plus anti-CD28-stimulated CD4+ T cell cultures were assayed for the cytokines by ELISA. B, rIL-2 (10 ng/ml; tested till 100 ng/ml, but without any enhancement in proliferation (data not shown)) failed to restore the proliferation defect in CD4+ T cells from Leishmania-infected BALB/c mice (*, p < 0.001). C, Proliferation of infected BALB/c-derived CD4+ T cells was restored by anti-IL-10 Ab (*, p < 0.001; 10 µg/ml). D, IL-10 neutralization abrogates the suppressive effect of the CD4+ T cells (the number of cells added is indicated on the x-axis) from Leishmania-infected mice on naive CD4+ T cell (5 x 104 cells/well) proliferation (*, p < 0.001).

 
Cytokine ELISA

IL-2, IL-4, IFN-{gamma}, and IL-10 in culture supernatants were assayed by ELISA using paired mAbs (BD Pharmingen).

RT-PCR IL-10 and FoxP3

RT-PCR was performed, as described (12). The primers used are as follows: IL-10 forward, 5'-TCA CTC TTC ACC TGC TCC AC-3' and reverse, 5'-TCA CTC TTC ACC TGC TCC AC-3'; FoxP3 forward, 5'-CAG CTG CCT ACA GTG CCC CTA G-3' and reverse, 5'-CAT TTG CCA GCA GTG GGT AG-3'; for equal loading control, beta-actin primers forward, 5'-TAC CAC TGG CAT CGT GAT GGA CT-3' and reverse, 5'-TTT CTG CAT CCT GTC GGC AAT-3' were used.

Flow cytometry

Splenocytes from the uninfected and infected BALB/c mice were stained with FITC- or PE-conjugated mAb to glucocorticoid-induced TNF receptor (GITR),4 CD25, and CD4, as indicated, and were analyzed by a FACSVantage (BD Biosciences). The Abs were procured from BD Pharmingen.

IL-2 is required for the generation of the T cells that suppress naive T cell proliferation

Naive BALB/c mice-derived CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 in presence or absence of IL-2- or anti-IL-10-neutralizing Ab, followed by a rest for 3 days and restimulations with anti-CD3 plus anti-CD28 for [3H]thymidine incorporation assay.

Macrophage-T cell coculture for parasite-killing assay

Thioglycolate-elicited peritoneal macrophages (5 x 104/well) were cultured in 16-well tissue culture slides (Nunc). The macrophages were infected with L. donovani promastigotes at a 1 macrophage:10 parasite ratio for 12 h. The extracellular parasites were washed out, and the infection continued for another 36 h, followed by addition of 1.5 x 105 CD4+ T cells isolated from the spleen of the mice, as indicated. After 24-h macrophage-T cell coculture, the T cells were washed out, wells were removed, and the slides were then fixed, Giemsa stained, and examined under a light microscope (magnification x1500), for enumerating the number of amastigotes per 100 macrophages.

In some experiments, in which the inhibitory effect of IL-10 on the antileishmanial functions of IFN-{gamma} was assessed, the macrophages were preincubated with IL-10 (20 ng/ml) for 12 h before infection. After 12-h infection with the parasite and washing out the extracellular parasites, the macrophages were treated with IFN-{gamma} (50 ng/ml). The dose of IFN-{gamma} was selected after a titration of the IFN-{gamma} doses against its antileishmanial effects.

Therapeutic effect of IL-2 and IL-10 on Leishmania infection in vivo

The L. donovani-infected BALB/c mice were treated with anti-IL-2 plus anti-IL-2R (each 50 µg/mouse) i.p. for 5 days, either from the day of infection or beginning on different days after the infection, as indicated. The mice were also treated with the anti-IL-10 Ab (50 µg/mouse), as indicated. The Abs, whenever used in vitro, were used at a concentration of 10 µg/ml. Isotype-matched control Abs were procured from BD Pharmingen and used at the same concentration as that of the other Abs.

Preparation of crude soluble Ag (CSA) and delayed-type hypersensitivity (DTH) response

The stationary-phase promastigotes were washed in PBS and were subjected to freeze-thaw cycles for six times, followed by sonication, as described earlier (13). The suspension was clarified by microfuging (Brinkman Instruments) at 40°C for 10 min. The supernatants were filtered through a 0.22-µm filter, and the proteins were estimated. The supernatant was used as the leishmanial CSA.

The DTH response was measured 24 h after the s.c. injection of CSA (40 µg) in one of the hind footpads of naive or L. donovani-infected mice. The difference in the thickness between the hind footpad that received the Ag and the other one that received saline was the net swelling providing the measure of DTH response.

For adoptive transfer of DTH by CD4+ T cells, the cells from the naive or infected BALB/c mice on different days of infection, as indicated, were transferred s.c. into the left hind footpad of naive BALB/c mice with 40 µg of CSA (2 x 106 cells/mouse). Swelling was measured 24 h after the Ag injection. In some experiments, to test the suppressive effects of the day 21 postinfection CD4+ T cells, as described, the indicated number of the T cells was transferred (Fig. 4B).


Figure 4
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  		FIGURE 4. CD25+ T cells in the day 21 infected spleen exert the suppressive effect. A, As described in Materials and Methods, 5 x 104 naive CD4+ T cells were cocultured with 2.5 x 104 CD4+ or CD4+CD25– or CD4+CD25+ cells isolated from the spleen of mice 21 days after infection (D21), and were stimulated with anti-CD3 (0.5 µg/ml) plus anti-CD28 (2 µg/ml) Abs in presence or absence of anti-IL-10 Abs, as indicated. The proliferation assay was performed by a standard [3H]TdR incorporation assay (*, p < 0.001). B, CD4+CD25+ T cells from 21-day infected BALB/c mice suppress the adoptively transferable DTH response. The CD4+ T cells, unfractionated or fractionated into CD25+ or CD25– cells, as indicated, from the 21-day infected BALB/c mice, were transferred s.c. into the left hind footpad of naive BALB/c mice with 40 µg of CSA (2 x 106 cells/mouse) and in presence or absence of anti-IL-10 Ab (100 µg/mouse). Swelling was measured 24 h after the Ag injection (*, p < 0.001). C, Twenty-one-day infected mice suppress the parasite control. As described in Materials and Methods, the BALB/c-derived peritoneal macrophages were cocultured with the CD4+ T cells, either as such or depleted of CD25+ T cells or with the addition of the indicated numbers of CD4+CD25+ T cells, in presence of anti-IL-10 Ab (*, p < 0.002; 10 µg/ml). The control Ab that matched the isotype of the anti-IL-10 Ab did not have any effect on the CD4+CD25+ T cell-regulated parasite growth in macrophages. The error bars represent mean ± SD. The experiments were performed at least three times, and representative data are shown.

 
Statistical analyses

The in vitro experiments were performed at least in triplicates. A minimum of five mice per group was used for any in vivo experiment. The results are described as mean ± SD. The significance of difference between the means was determined by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
L. donovani infection progresses in two phases

We infected BALB/c mice with L. donovani (11) and assessed the weekly progression of infection and T cell functions. We observed that the infection progressed in two phases: during the first 2 wk after the infection, the increase in splenic parasite burden was slow, but during the late phase starting from 14 days after infection, the parasite burden increased rapidly (Fig. 1A). The DTH response to leishmanial Ags was high during the first 2 wk, but died down from 14 days after the infection (Fig. 1A). Similarly, the CD4+ T cells proliferated well in response to TCR stimulation during the first 2 wk after infection, but were suppressed in the effector phase (Fig. 1B). In fact, the CD4+ T cells isolated from the infected mice were cocultured with the naive CD4+ T cells in presence of anti-CD3 and anti-CD28 Ab. It was observed that the CD4+ T cells from 14 days postinfection onward suppressed the proliferation of naive CD4+ T cells (Fig. 1C). The observed suppression of CD4+ T cell proliferation was not due to decreased expressions of CD3 and CD28, as the expressions of CD3 and CD28 on CD4+ T cells from both naive and L. donovani-infected mice were comparable (Fig. 1D). These observations suggest that during the first week of L. donovani infection, when the Ag-specific T cells are primed, the host-protective antileishmanial response was intact, resulting in slow parasite growth. However, 2 wk after infection, the host-protective effector functions of these T cells were suppressed by the CD4+ T cells with suppressor activities, resulting in the rapid rise in splenic parasite load.

The suppressor activities are contributed by regulatory T (t-reg) cells during L. donovani infection

Because the t-reg cells are shown to have suppressor activity in a variety of settings, including L. major infection (12, 14, 15, 16), we examined whether t-regs are phenotypically identifiable in spleen 21 days after the infection. We observed that the numbers of CD4+ T cells expressing CD25 (IL-2R-{alpha} that binds IL-2), a marker of t-reg cells (12), were comparable between the uninfected and L. donovani-infected mice (Fig. 2A). A previous report also suggested that in L. donovani infection, IL-2 binding by T cells is not impaired (17). However, the number of GITR+CD4+ T cells increased (Fig. 2B), but the Foxp3 expression remained unchanged (Fig. 2C). In the proliferation assay, depletion of CD25+ T cells increased the proliferation of CD4+ T cells (Fig. 2D) and addition of the CD25+ T cells from infected, but not naive BALB/c mice reinstated the suppression in a dose-dependent manner (Fig. 2E). The lack of suppression by the addition of naive CD4+CD25+ T cells in our system could be due to the absence of the accessory cells that were used in the original assay system developed by Thornton et al. (18, 19). In contrast to the contact-dependent suppression in the system developed by Thornton et al. (18, 19), it is possible that in our system, the suppression was mediated by IL-10, as shown later in several assays. Although the increased GITR staining and the functional assays indicate a t-reg-like suppressor activity in L. donovani infection, our observations suggest that the expression of the suggested t-reg markers such as CD25, GITR, and Foxp3 in CD4+ T cells may not necessarily correlate with their suppressor activity.

IL-10 produced by the CD4+ T cells mediates the suppressor function

The lower T cell proliferation and DTH response in the effector phase of the antileishmanial immune response could result from three possible mechanisms. First, because IL-2 was originally identified as a potent T cell growth factor (5), the suppression of T cell proliferation could be due to IL-2 deficiency (6, 7). Second, because the host protection against Leishmania infection was proposed to be mediated by IFN-{gamma} (20), the observed rapid parasite growth could be due to less IFN-{gamma} production in that phase of antileishmanial T cell response. Finally, there could be active suppression of the host-protective antileishmanial T cell response due to exaggerated production of counteractive disease-promoting cytokines such as IL-4 and IL-10 (21, 22). Therefore, we examined the kinetics of the production of these cytokines by anti-CD3 plus anti-CD28-stimulated CD4+ T cells during the course of L. donovani infection. First, the production of IL-2 was increased till day 14 after infection, after which its level was maintained (Fig. 3A), suggesting that the observed suppression was not due to deficiency in IL-2 production. In fact, IL-2 supplementation failed to restore CD4+ T cell proliferation in response to anti-CD3 plus anti-CD28 (Fig. 3B). In contrast, the CD4+ T cells from 7 days postinfection produced a higher IFN-{gamma} as compared with that observed during the late phase of infection (Fig. 3A). In contrast, IL-4 production increased till day 14 postinfection and remained steady during the later course of infection (Fig. 3A), whereas the production of IL-10 continued to increase during the course of infection (Fig. 3A). These observations suggested that the observed suppression of CD4+ T cell proliferation was due to exaggerated IL-4 and IL-10 production. Therefore, we assessed the relative importance of these two cytokines in the T cell proliferation assays. It was observed that IL-10, but not IL-4, neutralization restored the CD4+ T cell proliferation significantly (Fig. 3C). In fact, IL-4 appeared to exert antiproliferative effect on naive CD4+ T cells (Fig. 3C). Similarly, the suppression of naive CD4+ T cell proliferation by the infected CD4+ T cells was prevented by IL-10 neutralization, but not by IL-2 supplementation (Fig. 3D). These observations suggested that IL-10 had an antiproliferative effect and that the suppression of CD4+ T cell proliferation was affected by IL-10, but the significance of this antiproliferative effect of IL-10 on T cells remains to be examined. However, it is possible that IL-10-rich splenic microenvironment in the effector phase of the antileishmanial immune response inhibits the proliferation of the freshly infiltrating naive T cells and thereby debilitates the host-protective response further.

Indeed, depletion of CD4+CD25+ T cells restored the T cell proliferation significantly in response to anti-CD3 plus anti-CD28 stimulation, and addition of CD4+CD25+ T cells decreased the proliferation; the suppression was preventable with the anti-IL-10 Ab (Fig. 4A). Similar IL-10-dependent suppressor effects of CD4+CD25+ T cells were observed in the adoptive DTH transfer experiments as well (Fig. 4B). The depletion of CD25+ T cells restored the antileishmanial effect of CD4+ T cells isolated from the 21-day infected mice, and addition of the titrated numbers of CD4+CD25+ T cells back to the macrophage-T cell coculture reinstated the suppression of the antiparasitic effect of those CD4+CD25– T cells (Fig. 4C). In addition, these CD25+ T cells failed to reinstate the suppression in presence of the anti-IL-10 Ab, suggesting that the CD25+ T cell-secreted IL-10 was responsible for the suppression of the host-protective effect of the CD4+ T cells.

IL-10 abrogates host-protective function of T cells

To test the antiproliferative effect of IL-10, CD4+ T cells were incubated with the indicated amount of IL-10 for 12 h, followed by stimulation with anti-CD3 plus anti-CD28. It was observed that the IL-10-pretreated CD4+ T cells had a significantly less proliferation than the untreated cells (Fig. 5A), indicating an antiproliferative effect of the cytokine. Thus, it is possible that IL-10 prevents the expansion of the host-protective T cells inhibiting the host-protective functions.


Figure 5
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  		FIGURE 5. lL-10 abrogates the host-protective functions of T cells. A, IL-10-preincubated CD4+ T cells suppress the proliferation of naive CD4+ T cells (*, p < 0.001). Naive CD4+ T cells (105 cells/well in a 96-well plate) were cultured with anti-CD3 (0.5 µg/ml) + anti-CD28 (2 µg/ml) in presence or absence of the indicated number of IL-10-treated CD4+ T cells for 60 h, followed by [3H]thymidine pulsing for 12 h. The proliferation was assessed by [3H]thymidine incorporation. B, DTH response to leishmanial Ag during progressive L. donovani infection in BALB/c mice was assessed by adoptive transfer of CD4+ T cells in the footpads of naive BALB/c mice. IL-10 neutralization relieves the 21 days (D-21) postinfection CD4+ T cells’ suppression on 10 days (D-10) postinfection CD4+ T cell-mediated antileishmanial DTH response (*, p < 0.001). C, Amastigote burden in macrophages is controlled better by the 10 days (D-10) postinfection CD4+ T cells than those from day 21 postinfection CD4+ T cells (*, p < 0.001). IL-10 prevents the suppression mediated by the 21-day postinfection CD4+ T cells (D-21). D, IL-10 reduces the IFN-{gamma} responsiveness measured in terms of IFN-{gamma}-induced decrease in amastigote number per 100 macrophages (*, p < 0.001). The experiments were performed, as described in Materials and Methods. The data, mean ± SD, presented here are representatives of three individual experiments.

 
To test the role of IL-10 in the prevention of host-protective T cell functions, we used the adoptive transfer of DTH response against leishmanial Ags (13) and parasite-killing assay using macrophage-T cell coculture system. It was observed that the CD4+ T cells from the 21 days postinfection mice inhibited the antileishmanial DTH transferred by CD4+ T cells from 10 days postinfection mice and that anti-IL-10 Ab administration rescued the antileishmanial response significantly (Fig. 5B). Similarly, the 24-h infected macrophages were cocultured for additional 48 h with the 10 days postinfection or 21 days postinfection CD4+ T cells or in combination, as indicated, and in presence or absence of anti-IL-10 Ab, followed by the enumeration of amastigotes. It was observed that the macrophages cocultured with 10 days postinfection CD4+ T cells had a significantly less number of amastigotes than the macrophages cocultured with the 21 days postinfection CD4+ T cells (Fig. 5C). Neutralization of IL-10 in these cultures significantly rescued the antileishmanial functions of these T cells (Fig. 5C). However, IFN-{gamma} neutralization in these anti-IL-10-treated macrophage-T cell cocultures increased the parasite load, suggesting that IL-10 interferes with IFN-{gamma} responsiveness (data not shown). The ability of IL-10 to interfere with IFN-{gamma} responsiveness was further proved by the fact that preincubation of macrophages with IL-10 inhibited the IFN-{gamma}-induced amastigote control in macrophages (Fig. 5D). These observations indicated that IL-10 inhibited the antileishmanial functions of the host-protective CD4+ T cells.

Taken together, these observations suggest several mechanisms of the suppression observed during L. donovani infection. First, because IFN-{gamma} activates macrophages to eliminate the intracellular amastigotes (20), less IFN-{gamma} production is one possible mechanism of the observed suppression of the host-protective antileishmanial function. Second, IL-10 plays a definitive disease-exacerbative role by suppressing the host-protective T cell functions by inhibiting IFN-{gamma} production and by impairing IFN-{gamma} responsiveness. Third, because neither the IL-2 production nor the IL-2R (CD25) expression was impaired, the observed suppression was not due to IL-2 deficiency as proposed (6, 7). On the contrary, because the IL-2 production is maintained throughout the infection, it is possible that IL-2 may play a role in the generation of the T cells with suppressor activity (13, 23, 24).

IL-2 is required for IL-10-dependent suppressor activity

We tested whether the presence of IL-2 during primary stimulation resulted in decreased proliferative response upon restimulation. It was observed that the presence of IL-2 during primary stimulation decreased the proliferation upon second stimulation (Fig. 6A). The inhibition was prevented by IL-10 neutralization during primary stimulation (Fig. 6A), suggesting that IL-10 induced during primary stimulation might affect the T cells, as shown earlier (Fig. 5A). Indeed, IL-2 induced IL-10 in T cells in a dose-dependent manner (Fig. 6B).


Figure 6
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  		FIGURE 6. IL-2 is required for IL-10-dependent suppressor activity. A, CD4+ T cells were cultured with the indicated stimuli for 3 days. The cells were given 3 days of rest, followed by anti-CD3 (0.5 µg/ml) plus anti-CD28 (5 µg/ml) stimulation for 48 h. Primary culture of these T cells in absence of IL-2 induced higher proliferation during restimulation. B, IL-2-induced IL-10 production from anti-CD3 plus anti-CD28-stimulated CD4+ T cells from BALB/c mice, as assessed by RT-PCR (left panel) and ELISA (right panel). The splenic CD4+ T cells from naive BALB/c mice were stimulated with anti-CD3 plus anti-CD28 and different doses of IL-2, as indicated, for 8 h (for RT-PCR) or 36 h (for ELISA). After 8-h stimulation, the cells were processed for RNA isolation, and the RT-PCR for IL-10 was performed using IL-10-specific primers. The supernatants were collected from the 36-h-old culture and assessed for IL-10 content by ELISA. C, Anti-IL-2 plus anti-IL-2R Ab or anti-IL-10 Ab administration from the day of infection imparted resistance to the BALB/c mice (*, p < 0.001). BALB/c mice were infected with the stationary-phase promastigotes (2 x 107/mouse) and were treated with anti-IL-2 plus anti-IL-2R and/or anti-IL-10 Ab (each Ab 50 µg/mouse, i.p.) for 5 days. The control mice received only infection and isotype-matched control Abs (50 or 100 or 150 µg/mouse, depending on how much the experimental group received). D, Thirty-five days after infection, the mice were sacrificed and splenic CD4+ T cells were isolated and stimulated with anti-CD3 plus anti-CD28, as described above. After 36-h culture, the supernatants were harvested and the cytokine contents were estimated by ELISA. The treatment resulted in less IL-10, but higher IFN-{gamma} production by the CD4+ T cells from the treated mice, as compared with the infected, untreated controls.

 
Because IL-2 induced IL-10 that suppressed the host-protective T cell responses, we examined the therapeutic potential of anti-IL-2 plus anti-IL-2R Ab or anti-IL-10 Ab administration in L. donovani infection in BALB/c mice. We observed that administration of the anti-IL-2 plus anti-IL-2R Ab or anti-IL-10 Ab had significant ameliorating effects (Fig. 6C). However, the protective effect of anti-IL-2 plus anti-IL-2R Ab was significantly lost at the late phase of infection. In contrast, anti-IL-10 Ab or anti-IL-10 and anti-IL-2 + anti-IL-2R together had a lasting protective effect (Fig. 6C). To find out the mechanism of such protective effect, we assayed IFN-{gamma} and IL-10 production by CD4+ T cells in these mice. It was observed that compared with those from the control mice, the Ab-treated mice had higher IFN-{gamma}, but less IL-10 (Fig. 6D). Like the parasite load, the effect of IL-10 neutralization had a longer lasting effect on the cytokine production by T cells. Therefore, taken together, these data suggested that IL-2 was crucial at the initial priming phase for the induction of IL-10-producing T cells. Because IL-10 works primarily at the effector phase establishing the observed immunosuppression 14 days after infection, it is possible that such treatments with anti-IL-2 plus anti-IL-2R or with anti-IL-10 may have phase-specific effects.

IL-2 works at the priming phase, whereas IL-10 works at the effector phase

To decipher whether IL-2 and IL-10 had any phase-specific effects, we administered the Abs at different time points after L. donovani infection, as indicated. It was observed that the early treatment with anti-IL-2 plus anti-IL-2R had a significant disease-ameliorating effect, but the delay till the second week of infection significantly diminished the protective effect (Fig. 7A). In contrast, even a delayed administration of anti-IL-10 Ab alone or in combination with anti-IL-2 plus anti-IL-2R Ab offered a significant host protection (Fig. 7A). Therefore, the result suggests that IL-2 works primarily at the priming phase, while IL-10 works in the effector phase. However, the protection in both the cases was associated with low IL-10, but high IFN-{gamma} production by the CD4+ T cells (Fig. 7B), suggesting that the suppressive functions were significantly deterred and the host-protective functions of these T cells might have recovered. Next, we isolated CD4+ T cells from the infected mice (control) or from the infected mice that were treated with the indicated Abs immediately after infection or delayed till 14 days after infection and assessed their ability to transfer DTH to syngenic naive recipients, control amastigote number in macrophages, and suppress the naive CD4+ T cell proliferation. It was observed that the delay in beginning the treatment with anti-IL-2 plus anti-IL-2R Ab significantly reduced the ability of these CD4+ T cells to adoptively transfer the antileishmanial DTH (Fig. 7C) and to control amastigote number in macrophages (Fig. 7D). These T cells, however, suppressed the proliferation of naive CD4+ T cells (Fig. 7E).


Figure 7
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  		FIGURE 7. IL-2 works at the priming phase, whereas IL-10 works at the effector phase. A, A delay in anti-IL-2 plus anti-IL-2R treatment lost its efficiency to reduce splenic parasite burden, but cotreatment with anti-IL-10 Ab restored the efficacy (*, p < 0.001). The mice were infected on day 0. Some mice were treated i.p. with the indicated Abs (at doses as described above) from days 0 to 7; the others received the indicated Abs from days 8 to 14, and days 15 to 21. The mice were sacrificed 28 days after infection. The splenic parasite load was determined by the stamp-smear method, as described in Materials and Methods. B, A delayed administration of anti-IL-2 plus anti-IL-2R Ab resulted in the loss of IFN-{gamma}-inducing ability. The splenic CD4+ T cells from the mice described in A were isolated and were stimulated with anti-CD3 plus anti-CD28 for 36 h, as described in Materials and Methods. The cell culture supernatants were assayed for IL-10 and IFN-{gamma} contents by ELISA. C, A delayed administration of anti-IL-2 plus anti-IL-2R Ab resulted in the loss of adoptively transferable antileishmanial DTH function. The mice from the experiment described in A were sacrificed 28 days after infection. The splenic CD4+ T cells were isolated and adoptively transferred to a hind footpad of naive syngenic recipients with CSA (40 µg/mouse) in a total volume of 50 µl. The swelling of the injected footpad was compared with that of uninjected footpad. The net swelling was taken as the DTH response. Five mice were used per group of recipients. D, A delayed administration of anti-IL-2 plus anti-IL-2R Ab resulted in the loss of parasite-killing functions. Anti-IL-10 rescues the function. The CD4+ T cells from the experiment described in A were cocultured with the L. donovani-infected macrophages for 24 h, as described in Materials and Methods. The macrophages were then stained with Giemsa and enumerated under a light microscope for amastigote numbers per 100 macrophages. E, A delayed administration of anti-IL-2 plus anti-IL-2R Ab resulted in the reinstatement of the suppression. The CD4+ T cells were isolated from the mice groups, which were treated during the first or third week after infection, as shown, with the indicated Abs, as described above. The CD4+ T cells (2.5 x 104) were cocultured with the CD4+ T cells (5 x 104) from naive mice, as described in Materials and Methods. The data, mean ± SD, presented here are representatives of three individual experiments.

 
Thus, the results altogether suggest that during the L. donovani infection, IL-2 significantly controls antileishmanial activity of CD4+ T cells. During the first week of infection, when the priming of these cells takes place, IFN-{gamma} is produced that executes the host-protective functions. In contrast, IL-2 induces the T cells with the regulatory functions that are mediated by IL-10. In corroboration with these findings, treatment with anti-IL-2 plus anti-IL-2R works early after infection, whereas the treatment with anti-IL-10 Ab works even late after infection. The ameliorating effects of these phase-specific treatments were associated with the recovery of the host-protective antileishmanial T cell functions. Therefore, our observations suggest kinetic modulation of an ongoing immune response as a principle of the phase-specific immunotherapy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The host’s immune response to Leishmania, a protozoan parasite, is poised to be a dynamic one just as their history of coevolution appears to be. Although immediately after injection into the host the parasite endeavors to establish the infection, the host mobilizes the T cells against the parasite, resulting in the initial control of the parasite growth. Later, the parasite uses the host’s immune control mechanisms to its own benefit, resulting in its rapid growth. One such immune control mechanism is IL-2-mediated regulation of the CD4+ T cell response. Contrary to the proposition that IL-2 might play a host-protective role in Leishmania infection (7), we demonstrate the disease-promoting role of IL-2 in L. donovani infection. IL-2 may play a similar role in L. major infection as well (25). One possible mechanism of such discrepancy could arise from the form of the parasite used for infection; although Murray et al. (7), used amastigotes, we and Heinzel et al. (25) used promastigotes. However, in our studies, when we compared the effects of promastigotes and amastigotes, we did not find any significant differences in terms of the T cell subset modulation and DTH response (M. Bodas and B. Saha, data not shown).

As far as the role of IL-2 in experimental visceral leishmaniasis is concerned, during the early phase of infection, IL-2 induces both IFN-{gamma}- and IL-10-secreting T cells, perhaps as a function of the available IL-2 concentration, but IL-10 suppresses the IL-12 production by the APCs by differential regulation of MAPKs (26), reduces IL-12R expression on T cells (B. Saha, unpublished observation), and impairs IFN-{gamma} responsiveness of macrophages, resulting in the suppression of IFN-{gamma}-mediated amastigote elimination. Being residents of the IL-10-rich splenic microenvironment, these T cells do not proliferate and suppress the activation of the infiltrating naive T cells, a phenomenon reminiscent of infectious tolerance (27). Thus, Leishmania exaggerates and exploits the host’s IL-10-dependent autoregulatory or a feedback servo-mechanism that prevents excessive inflammation-mediated host-tissue pathology, but supports unhindered parasite growth (25, 26). Such a mechanism may operate in those viral infections in which the viral IL-10 (28, 29) may skew the immune response in a similar way. It is possible that our proposed phase-specific immunotherapy interferes with the IL-2-dependent expansion of the IL-10-secreting cells early after infection and with their effector functions at a later phase. The previously reported experiments with IL-10 blockade, albeit without any kinetic analyses of the evolution and function of such CD4+ T cells, might also work the same way, resulting in the observed host-protective effect against the parasite (30, 31).

Because parasites coevolved with their hosts to evade the host’s immune responses, the immune system coevolved to adapt to the pressure exerted by these persistent pathogens. Therefore, during an ongoing immune response in an infection, particularly where the constantly changing parasite load modulates the immune response, the host-parasite interaction is rather dynamic than being static and steady state. Based on the kinetics of evolution of the T cells with suppressor functions and their mechanism of action, we demonstrate a novel stage-specific immunotherapy of leishmaniasis that may be applicable to other diseases, such as allograft reactions and autoimmune diseases, particularly where the Ags persist.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Council for Scientific and Industrial Research and Department of Biotechnology grants (to M.B., A.A., S.M., and R.K.). Back

2 M.B. and N.J. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Bhaskar Saha, National Centre for Cell Science, Ganeshkhind, Pune 411 007, India. E-mail address: sahab{at}nccs.res.in Back

4 Abbreviations used in this paper: GITR, glucocorticoid-induced TNF receptor; CSA, crude soluble Ag; DTH, delayed-type hypersensitivity; t-reg, regulatory T. Back

Received for publication March 27, 2006. Accepted for publication July 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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