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The Journal of Immunology, 2002, 169: 3232-3241.
Copyright © 2002 by The American Association of Immunologists

The Early IL-4 Response to Leishmania major and the Resulting Th2 Cell Maturation Steering Progressive Disease in BALB/c Mice Are Subject to the Control of Regulatory CD4+CD25+ T Cells1

Abraham Aseffa2,*, Alain Gumy2,*, Pascal Launois*, H. Robson MacDonald{dagger}, Jacques A. Louis* and Fabienne Tacchini-Cottier3,*

* World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, and {dagger} Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by V{beta}4V{alpha}8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 107 BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 107 control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
After infection with Leishmania major, mice from the majority of inbred strains develop lesions that resolve spontaneously leaving the animals immune to reinfection. In contrast, mice from few inbred strains (e.g., BALB), referred to as genetically susceptible, develop progressive lesions that do not heal and do not become immune to reinfection. These genetically determined resistance and susceptibility to L. major have been demonstrated to result from the development of CD4+ Th1 and Th2 responses, respectively (1). Among the many stimuli possibly influencing the maturation of distinct effector cells from common CD4+ T cell precursors, cytokines play a major role (2, 3, 4, 5). The results obtained in vitro using CD4+ T cells from TCR {alpha}{beta} transgenic mice demonstrating the essential role of IL-12 and IL-4 in Th1 and Th2 cell maturation, respectively, have been validated in vivo using the murine model of infection with L. major (6, 7, 8, 9, 10). In this context, we have documented a burst of IL-4 mRNA expression in the draining lymph nodes of BALB/c mice within 16 h after infection with L. major (11). Remarkably, this rapid IL-4 response occurs during the period when neutralizing IL-4 Abs are capable of redirecting protective Th1 development in BALB/c mice (8, 9). The cognate recognition of a single epitope of the Leishmania homolog of mammalian RACK1 (LACK)4 (12) was demonstrated to drive this early IL-4 response by a restricted population of MHC class II restricted CD4+ T cells that express the V{beta}4 V{alpha}8 TCR chains (13). The causal relationship between this rapid accumulation of IL-4 transcripts and the development of aberrant Th2 responses leading to a susceptible phenotype in BALB/c mice was established (14, 15). Interestingly, the possibility of modulating this rapid IL-4 response by treatment with either exogenous IL-12 or IFN-{gamma} (11) suggests that these cells are not irreversibly committed to IL-4 production. In fact, we have recently documented the functional plasticity of these cells in terms of cytokines production (16). Together, these results suggest that these cells are sensitive to regulatory processes.

In the past few years, the concept that subpopulations of T cells were specialized in the suppression of immune responses has been revisited. Considerable attention has been given to a minor subpopulation of CD4+ T cells constitutively expressing CD25, the {alpha}-chain of the IL-2R. Both in mice and humans, these cells, named regulatory T cells, have been shown capable of suppressing the proliferation of other T cell populations (reviewed in Refs. 17 and 18).

The present study was undertaken to determine whether or not, following infection of BALB/c mice with L. major, the early production of IL-4 by LACK-reactive V{beta}4 V{alpha}8 CD4+ T cells and the resulting Th2 responses were subject to the control of regulatory CD4+CD25+ T cells. The results obtained show that CD4+CD25+ T cells negatively regulate the magnitude of the early IL-4 response to L. major in BALB/c mice as well as the importance of subsequent Th2 cell maturation. These data suggest that CD4+CD25+ T cells may also regulate harmful immune responses to infectious pathogens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female BALB/c and C57BL/6 mice were purchased from Harlan Olac (Bicester, U.K.). Female C.B.-17 SCID mice were purchased from IFFA-Credo (St. Germain sur l’Arbresle, France). All mice were used at 6–8 wk of age.

Reagents and Abs

Anti-CD3 (145-2C11), anti-FcR (2.4G2), anti-CD25 (PC61) (19), and anti-IL-4 (11B11) mAbs were affinity purified on a protein A column from hybridoma culture supernatants. Cychrome- and PE-conjugated anti-CD4 (GK1.5), PE-conjugated anti-CD45R/B220 (RA3-6B2), FITC-conjugated anti-CD8 (53-6.7), PE-conjugated anti-CD69 (H1.2F3), and anti-CD62L (MEL-14) were purchased from BD PharMingen (San Diego, CA). Purified, unlabeled, and FITC-conjugated anti-CD25 (5A2) (20), was kindly provided by Dr. M. Nabholz (Swiss Institute for Cancer Research, Epalinges, Switzerland). PE-conjugated PC61 was obtained from Dr. A. Wilson (Ludwig Institute of Cancer Research, Epalinges, Switzerland). CFSE was purchased from Molecular Probes (Eugene, OR). Flow cytometry analysis was performed on a FACScan using CellQuest software (BD Biosciences, Mountain View, CA).

Parasites, infection, and treatment of mice

L. major LV 39 (MRHO/Sv/59/P-strain) were maintained in vivo and grown in vitro as described previously (21). Groups of three to seven mice were infected s.c. in the hind footpads with 3 x 106 stationary phase L. major promastigotes in a final volume of 50 µl. Mice from designated groups received 1 mg of PC61 mAb i.p. 72 h before infection. Some mice were also treated with anti-IL-4 mAb (11B11) i.p. at indicated doses and time points. Disease progression was monitored using a Vernier caliper to measure footpad size. In designated experiments, footpad tissues were used to create limiting dilutions for quantitation of viable parasite burdens as previously described (22).

Lymphocyte cultures and detection of cytokines in supernatants

Draining popliteal lymph node cells (5 x 106) were cultured in a final volume of 1 ml in DMEM supplemented with 5% heat inactivated FCS, 216 µg/ml L-glutamine, 5 x 10-5 M 2-ME, and 10 mM HEPES at 37°C in an atmosphere of 7% CO2 in the presence or absence of UV-irradiated L. major promastigotes (1 x 106/ml) or soluble anti-CD3 (2 µg/ml). Supernatants were collected at 48 h from cultures stimulated with soluble anti-CD3 or at 72 h for cultures stimulated with Leishmania Ags, and frozen at -20°C until use. IFN-{gamma} was measured in supernatants by ELISA as described (23). Mouse rIFN-{gamma} (supernatant of L1210 cells transfected with the murine IFN-{gamma} gene; a gift from Y. Watanabe, Kyoto University, Kyoto, Japan) was used as the standard. The limit of detection of the assay was 10 U/ml. IL-4 was measured by a bioassay using the CTLL-44 cell line (a gift from P. Erb, University of Basel, Basel, Switzerland) as described (24). Recombinant murine IL-4 secreted by X63Ag-653 cells (a gift from F. Melchers, Basel Institute of Immunology, Basel, Switzerland) was used as the standard. The limit of detection of the assay was 20 pg/ml. For proliferation assays, 5 x 105 cells were cultured with UV-irradiated stationary phase L. major promastigotes (1 x 106/ml) or anti-CD3 (0.5 µg/ml) in 200 µl DMEM/5% FCS, and were pulsed with 1 µCi of [3H]thymidine for the final 6 h of culture. Harvested cells were measured for radioactivity using a beta scintillation counter.

RNA extraction and competitive PCR

Total mRNA was extracted from popliteal lymph node cells as described (11). First strand cDNA synthesis was performed using a first strand cDNA synthesis kit according to the manufacturer’s directions (Pharmacia, Uppsala, Sweden). The polycompetitor plasmid (pQRS) was used to quantitate amounts of transcripts for IFN-{gamma}, IL-4, and the constitutively expressed hypoxanthine guanine phosphoribosyl transferase gene, using primers and PCR conditions as described (25). Briefly, the cDNA was used as a template in the presence of serial 5-fold dilutions of pQRS. After separation of the PCR products by electrophoresis in agarose gel containing ethidium bromide, the ratio of IL-4 or IFN-{gamma} transcripts to hypoxanthine guanine phosphoribosyl transferase transcripts was calculated. The results are expressed as the fold increase in cytokine mRNA in mice infected with L. major compared with that in noninfected, but otherwise similarly treated, control mice.

Cell sorting and reconstitution of SCID mice

CD4+CD25+ cells were depleted from total spleen cells of naive wild-type BALB/c mice using magnetic cell sorting columns (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s directions. In brief, total spleen cells depleted of RBC by lysis in Tris-buffered NH4Cl solution were first stained with anti-CD25 mAb 5A2 for 20 min on ice. Following washing, cells were resuspended with magnetic microbeads that had been conjugated with goat anti-rat IgG Ab and the CD25-depleted population was negatively selected after immobilization with a magnet. This procedure generally resulted in the removal of CD4+CD25+ cells from the spleen cell suspension to background levels of <1.5% of total CD4+ cells, as detected by flow cytometry with subsequent staining using PC61, a second anti-CD25 mAb that binds to a different epitope on the IL-2R{alpha} molecule (19, 20). In some experiments, depletion was performed with a combination of FITC-labeled 5A2 and anti-FITC microbeads. The efficiency of depletion was similar.

C.B.-17 SCID mice were bled in the tail vein and PBMC were obtained by Ficoll-Hypaque centrifugation. The PBMC were stained for CD4, CD8, and B220 and screened by flow cytometry. SCID mice that had <1% peripheral B or T cells were reconstituted i.v. with designated numbers of spleen cells from wild-type BALB/c mice. In some experiments, SCID mice were reconstituted with CFSE-labeled spleen cells as above.

Cell counting

Cells were either counted using a Neubauer chamber or using the FACS; cell suspensions were resuspended in a volume of 300 µl, and 105 bacterial count microbeads (Molecular Probes) were added to each tube and cells were then analyzed by FACS. Gates were assigned to the microbeads and lymphocytes by forward and size scatter characteristics. The ratio of beads to lymphocytes was determined for each specimen from the electronic counts on FACScan.

Statistics

Statistical analysis was done using the two-tailed t test for unpaired data. The estimation of the frequency of parasites by limiting dilution was calculated by the Taswell method using the program Estimfree (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Depletion of CD4+CD25+ regulatory T cells before infection with L. major exacerbates the development of lesions in BALB/c mice

To study the role of the CD4+CD25+ regulatory T cells in vivo, BALB/c mice were treated once with 1 mg of {alpha}CD25 mAb PC61 i.p. (19). Seventy-two hours following such treatment, the proportion of CD4+CD25+ T cells dropped from around 10% to below 2% of the CD4+ T cell population in lymph nodes (data not shown). The percentage of CD4+CD25+ T cells within the lymph nodes remained low for over 10 days. The percentage of the CD4+CD25+ subpopulation in peripheral blood was also reduced to around 2% of the circulating CD4+ T cells (data not shown). This depletion was also long-lasting as reported by others (27).

Compared with similarly infected control BALB/c mice, mice treated with 1 mg of PC61 mAb 72 h before infection with 3 x 106 L. major promastigotes developed significantly larger lesions (Fig. 1GoA) that contained higher numbers of parasites (Fig. 1GoB). The number of parasites measured in lesions of PC61-treated BALB/c mice was consistently significantly higher than that measured in lesions of BALB/c mice in five different experiments (p = 0.019). The development of severe lesions in BALB/c mice depleted of CD4+CD25+ T cells was correlated with an enhanced IL-4-producing Th2 response. Results in Fig. 1GoC show that 15 days after infection with L. major the amounts of IL-4 transcripts in draining lymph nodes are already five times higher in BALB/c mice depleted of CD4+CD25+ cells than in similarly infected control BALB/c mice. Comparable results were obtained when supernatants of L. major-activated lymph node cells were analyzed for the accumulation of IL-4 (Fig. 1GoD). This difference in IL-4 production between CD4+CD25+ cell-depleted and normal BALB/c mice was also observed at later times after infection (data not shown). In most experiments, the level of IFN-{gamma} in supernatants of cultures from draining lymph nodes of mice depleted of CD4+CD25+ cells was equivalent to, or only slightly higher, than that measured in cultures of lymph nodes from nondepleted BALB/c mice (Fig. 1GoD).



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  		FIGURE 1. Infection of BALB/c mice depleted of CD4+CD25+ regulatory cells with L. major leads to an exacerbated course of disease and an enhanced Th2 response. A, BALB/c, C57BL/6, and BALB/c mice depleted of CD4+CD25+ cells were infected with 3 x 106 stationary phase L. major promastigotes s.c. in the footpads. The course of infection was monitored by weekly measurement of the diameter of footpads with a metric caliper. The mean size of lesions (increase in diameter of footpads due to infection) and SD are shown (four mice per group). Similar results were obtained in three other experiments. *, p < 0.001 between CD4+CD25+ depleted vs nondepleted BALB/c mice. B, Parasite load in the footpads of mice sacrificed 15 and 42 days after infection with L. major was determined by limiting dilution assay. The mean counts of two footpads per group is shown. Data are representative of two experiments. C, IL-4 mRNA levels in popliteal lymph nodes of BALB/c mice depleted or not of CD4+CD25+ T cells. BALB/c mice and BALB/c mice depleted of CD4+CD25+ T cells were infected with 3 x 106 stationary phase L. major promastigotes s.c. in the hind footpads. Control groups were untreated or noninfected BALB/c mice treated with PC61. Fifteen days later, mice were sacrificed, mRNA was extracted from their popliteal lymph nodes and the relative level of IL-4 mRNA was determined by semiquantitative RT-PCR as described in Materials and Methods (n = 4 mice per group). Results are expressed as fold increase relative to similarly treated, noninfected control mice. The results represent one of three independent experiments. D, IL-4 and IFN-{gamma} production by lymph node cells of mice 15 days after infection with L. major. Popliteal lymph node cells were isolated 15 days after infection with L. major and 5 x 106 cells from mice of each group (three mice per group) were stimulated in vitro with 106 UV-irradiated L. major promastigotes for 72 h. IFN-{gamma} and IL-4 production was evaluated in the supernatant as described in Materials and Methods. Results are expressed as mean and SD of triplicate measurements. The data shown are representative of two independent experiments.

 
Depletion of CD4+CD25+ T cells in C57BL/6 mice before infection with L. major did not alter the course of infection (data not shown). In three independent experiments, no significant difference in the size of lesions between PC61-treated and control C57BL/6 mice was observed, and both groups resolved their lesions. Furthermore, the level of cytokines produced (no IL-4, high level of IFN-{gamma}) in the draining lymph nodes of both groups of mice did not differ. However, parasite load in the footpads of CD25+ T cell-depleted C57BL/6 mice 5 wk after infection was 10 times lower than in control C57BL/6 mice infected with L. major simultaneously; 8 wk after infection parasites in the lesions of PC61 treated mice disappeared almost completely (75% of mice with no parasites detectable and 25% of mice with 3–10 parasites/footpad) while C57BL/6 mice not depleted of CD25+ T cells still had 0.5- 6.7 x 103 parasites per lesion.

The early IL-4 response to L. major is significantly enhanced in BALB/c mice depleted of CD4+CD25+ cells

We have previously documented a burst of IL-4 mRNA expression in draining lymph nodes of BALB/c mice within 1 day of infection with L. major (11). This early IL-4 burst occurs in a restricted population of LACK-reactive CD4+ T cells expressing the V{beta}4V{alpha}8 TCR chains (28). The causal relationship between this early IL-4 response and subsequent Th2 cell maturation in BALB/c mice was demonstrated (15).

To investigate whether or not CD4+CD25+ regulatory T cells control the early IL-4 mRNA response to L. major, BALB/c mice treated or not with 1 mg of anti-CD25 PC61 mAb were inoculated, 3 days later, with L. major in one hind footpad. Sixteen hours after infection, total mRNA from draining lymph nodes was analyzed for IL-4 mRNA expression using a semiquantitative RT-PCR. Compared with infected mice not treated with PC61 mAb, higher levels (6–10 times) of IL-4 mRNA transcripts were consistently observed in lymph nodes of CD4+CD25+ T cell-depleted mice (Fig. 2Go). This increase in IL-4 mRNA was confirmed in five independent experiments where, 16 hr after infection with L. major, the level of IL-4 mRNA measured in PC61-treated mice was significantly higher (p = 0.0001) compared with that measured in BALB/c mice that were not injected with the mAb. The IL-4 mRNA was detected only in the V{beta}4V{alpha}8 CD4+ T cell population (data not shown). Comparable results were obtained by real-time PCR, 16 h after infection with L. major (data not shown). It is noteworthy that in the absence of infection with L. major, treatment of BALB/c mice with the mAb PC61 did not affect the basal level of IL-4 mRNA transcripts. In five independent experiments, no statistically significant difference was observed between the level of IFN-{gamma} transcripts measured in draining lymph nodes of BALB/c mice depleted or not of CD25+ cells 16 h after infection with L. major (data not shown).



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  		FIGURE 2. Depletion of CD4+CD25+ T cells in BALB/c mice enhances the early IL-4 burst observed 1 day after infection with L. major. BALB/c mice were treated with 1 mg PC61 i.p. and infected s.c. with 3 x 106 stationary phase L. major promastigotes 72 h later. Control groups consisted of noninfected mice treated with PC61, and mice infected without any prior treatment. Mice (four to five per group) were sacrificed 16 h after infection and mRNA was extracted from popliteal lymph nodes. The relative level of IL-4 mRNA was determined by semiquantitative RT-PCR (as described in Materials and Methods). For mice injected with the PC61 mAb, results are expressed as the increase in IL-4 mRNA in popliteal lymph nodes of mice treated with PC61 and infected with L. major compared with that measured in noninfected mice similarly treated with the mAb. For untreated mice, the results are expressed as fold increase in IL-4 mRNA in mice infected with L. major compared with that in noninfected mice. Results are representative of three independent experiments.

 
Demonstration of the inhibitory role of CD4+CD25+ regulatory cells on disease progression and Th2 cell development using an adoptive cell transfer system

In addition to depleting cells from the minor CD4+CD25+ regulatory subset, treatment of mice with the anti-CD25 mAb PC61 could also lead to depletion of other CD4+ T cells induced to express CD25 following activation by L. major Ags. To circumvent this potential problem, we directly tested the regulatory potential of CD4+CD25+ cells using an adoptive cell transfer system originally described by Mitchell et al. (29) and subsequently by others (30, 31, 32). Reconstitution of syngeneic nu/nu or SCID mice with 107 spleen cells from naive BALB/c mice was demonstrated to render these otherwise highly susceptible immunocompromised mice resistant to infection with L. major. These reconstituted SCID mice were shown to develop polarized Th1 response to infection. Conversely, reconstitution of SCID mice with 108 spleen cells from naive BALB/c mice was shown to restore the susceptible phenotype characterized by the development of unhealing lesions. These reconstituted SCID mice developed polarized Th2 differentiation following infection with L. major.

Therefore, to study the role of CD4+CD25+ regulatory cells in Th cell maturation following infection with L. major, SCID mice were adoptively transferred with either 107 spleen cells obtained from normal naive BALB/c mice, or 107 spleen cells depleted in CD25+ cells by MACS sorting. Five days later, all mice were infected with 3 x 106 L. major into the hind footpad and the development of lesions was monitored. Results in Fig. 3GoA confirm that SCID mice reconstituted with 107 naive BALB/c spleen cells are capable of controlling infection. In contrast, mice reconstituted with spleen cells depleted in CD25+ cells developed progressive lesions (Fig. 3GoA). Estimation of the numbers of viable parasites in lesions by limiting dilution analysis substantiated these findings because 10 wk after infection, the parasite burden was 3–5 log higher in lesions of SCID mice reconstituted with CD25+ cell-depleted spleen cells compared with mice reconstituted with unseparated spleen cells (Fig. 3GoB). Susceptibility of these mice to L. major was correlated with ultimate Th2 cell development because after stimulation with L. major in vitro their lymph node cells produced elevated amounts of IL-4 and reduced amounts of IFN-{gamma} (Fig. 3GoC).



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  		FIGURE 3. SCID mice reconstituted with 107 spleen cells from syngeneic BALB/c mice depleted of CD4+CD25+ T cells mount a polarized Th2 response to L. major infection and develop a progressive disease. A, Evolution of lesion in SCID mice reconstituted with BALB/c spleen cells depleted of CD4+CD25+ T cells. SCID mice were reconstituted with 107 total spleen cells or 107 spleen cells depleted of CD4+CD25+ T cells as described in Materials and Methods. Nonreconstituted SCID and BALB/c mice were used as controls. Each group of mice was infected with 3 x 106 stationary phase L. major promastigotes and the course of infection was monitored by measuring the diameter of the footpads weekly with a metric caliper. The mean size of lesions (increase in diameter of footpads due to infection) and SD for three to five mice per group is shown. Similar results were obtained in three other experiments including three to seven mice per group. *, p < 0.001 between the size of lesions in SCID mice that received spleen cells depleted vs not depleted of CD4+CD25+ regulatory T cells. B, Parasite burden in the footpads of SCID mice reconstituted with BALB/c spleen cells depleted or not of CD4+CD25+ T cells 10 wk after infection with L. major. SCID mice were reconstituted with 107 total spleen cells or BALB/c splenocytes depleted of CD4+CD25+ regulatory T cells and infected 5 days later with 3 x 106 stationary phase L. major promastigotes in the footpads. Nonreconstituted SCID mice were similarly infected as control. Ten weeks later, mice were sacrificed and the parasite burden in their footpads was determined by limiting dilution analysis as described in Materials and Methods. The results are the mean counts from two footpads per group in two separate experiments. C, Cytokine production in lymph node cells from SCID mice reconstituted with spleen cells depleted of CD4+CD25+ T cells and infected with L. major. Popliteal lymph node cells were isolated 10 wk after infection and 5 x 106 cells from each group of reconstituted mice (three mice per group) were stimulated in vitro with 106 UV-irradiated L. major promastigotes for 72 h. IFN-{gamma} and IL-4 production was evaluated in the supernatants as described in Materials and Methods. Results are expressed as mean of three measurements. The data shown are representative of three independent experiments. n.d., not detectable.

 
It is noteworthy that reconstituted SCID mice were infected with L. major 5 days after adoptive cell transfer, a time when significant repopulation of lymph nodes with donor cells had occurred. Using flow cytometry and establishing a ratio of 105 bacterial count microbeads to lymphocytes, an equivalent number of cells was measured in the lymph nodes and spleens of SCID mice reconstituted with CD25+-depleted or undepleted spleen cells 5 days after reconstitution (data not shown).

Demonstration of the inhibitory role of CD4+CD25+ regulatory cells on the early IL-4 response to L. major in BALB/c mice using an adoptive cell transfer system

The requirement for LACK-reactive V{beta}4 V{alpha}8 CD4+ T cells and the IL-4 they produce, within one day of infection with L. major for subsequent Th2 cell development and expression of a susceptible phenotype in BALB/c mice, has been demonstrated (13, 14). Therefore, experiments were designed to determine whether the susceptible phenotype of mice reconstituted with CD25+ cell-depleted spleen cells and the resistant phenotype of SCID mice reconstituted with 107 BALB/c spleen cells were correlated with the expression of an early IL-4 mRNA burst in response to L. major or lack thereof, respectively. Five days after reconstitution with either 107 total or CD25-depleted spleen cells, SCID mice were infected with 3 x 106 L. major and 16 h later IL-4 transcripts were quantitated in their draining lymph node cells by RT-PCR. The lymph nodes of SCID mice that received CD25-depleted BALB/c spleen cells showed a rapid increase in IL-4 mRNA similar in magnitude to that observed in BALB/c mice simultaneously infected with L. major. In contrast, no increase in IL-4 mRNA expression was observed in lymph node cells of mice that received an equivalent number of BALB/c spleen cells not depleted in CD4+CD25+ regulatory T cells (Fig. 4Go). These results show that in this model system, as in BALB/c mice, an early IL-4 burst precedes Th2 cell maturation. Experiments aimed at identifying the cellular origin of the rapid IL-4 mRNA burst in SCID mice reconstituted with CD25+ cell-depleted spleen cells have revealed that it occurred in CD4+ T cells that express the V{beta}4 TCR chain (A. Gumy, unpublished data).



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  		FIGURE 4. Draining lymph node cells from SCID mice reconstituted with BALB/c spleen cells depleted of CD4+CD25+ T cell and infected with L. major show an early IL-4 burst similar to that observed in BALB/c mice 16 h after infection. SCID mice were reconstituted with 107 BALB/c splenocytes depleted or not of CD4+CD25+ T cells in vitro and infected or not with 3 x 106 stationary phase L. major promastigotes in the footpad. Four mice per group were sacrificed 16 h after infection and mRNA was extracted from their popliteal lymph nodes. The relative levels of IL-4 mRNA were determined by semiquantitative RT-PCR (Materials and Methods). Results are expressed as fold increase relative to the noninfected similarly treated control mice. This is a representative experiment of five.

 
Because use of anti-CD25 mAb is likely depleting all CD25+ T cells including the small percentage (<2%) of CD4-CD25+ T cells, experiments were performed to directly assess the suppressive role of CD4+CD25+ T cells on the early IL-4 mRNA burst. FACS-sorted CD4+CD25+ T cells (2.8 x 105) isolated from BALB/c spleen were added to spleen cells depleted of CD25+ T cells by MACS and 107 cells were injected i.v. into SCID mice. Five days later, mice were injected with 3 x 106 L. major promastigotes and IL-4 mRNA was measured in their draining lymph nodes 16 h later. No significant increase in IL-4 mRNA was detectable in the draining lymph nodes of these reconstituted SCID mice when compared with similarly reconstituted uninfected SCID mice. However, SCID mice that received 107 CD25-depleted spleen cells with no inclusion of CD4+CD25+ cells showed an increase of 38 times in IL-4 mRNA as compared with similarly reconstituted SCID mice that were not infected (data not shown). These results show that CD4+CD25+ T cells are indeed regulating the early IL-4 mRNA burst observed 16 h after parasite inoculation.

Depletion of CD4+CD25+ cells leads to enhanced proliferation of CD4+ T cells following infection of mice with L. major

Once activated, CD4+CD25+ cells are capable of inducing cell cycle arrest of activated CD4+ T cells in an Ag-nonspecific manner (33, 34). In most of our experiments with BALB/c mice, the total lymphocyte count from the popliteal lymph nodes of CD25+ depleted mice was higher than that from nondepleted mice for the same duration of infection (data not shown). Therefore, we tested whether draining lymph node cells from CD25-depleted and nondepleted BALB/c mice infected with L. major differed in their proliferative capacity to specific stimulation in vitro. As shown in Fig. 5Go, cells from CD25-depleted mice showed a higher rate of thymidine incorporation in response to L. major or anti-CD3 in culture. This was also observed in nonrestimulated cells as a higher background. Surprisingly, this difference was still apparent 5 wk after infection, at least for lymph node cells stimulated with anti-CD3, suggesting that the state of activation of CD4+ cells in the periphery has been altered by the absence of the CD4+CD25+ T cells in vivo during the initial infection with L. major.



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  		FIGURE 5. Absence of CD4+CD25+ regulatory T cells leads to enhanced proliferation of BALB/c lymph node cells in response to antigenic restimulation in vitro. BALB/c and BALB/c mice depleted of CD4+CD25+ T cells by i.p. administration of 1 mg of PC61 mAb 72 h before infection were injected with 3 x 106 stationary phase L. major promastigotes s.c. in the hind footpads (four mice per group). Draining lymph node cells were removed at 15 days after infection and 5 x 105 cells were stimulated with 0.5 µg anti-CD3 or 2 x 105 UV-irradiated L. major for 48 or 72 h, respectively. [3H]Thymidine was added for the last 6 h of culture. Shown are the mean and SD of scintillation cpm for triplicate cultures. Data are representative of two separate experiments.

 
No preferential expansion of V{beta}4+ cells was found when cultured cells were stained and tested by flow cytometry (data not shown), suggesting that the state of activation rather than the preferential expansion of V{beta} subsets leads to the increased proliferation observed in lymph node cell cultures from mice depleted of CD4+CD25+ T cells.

Upon stimulation with L. major in vitro, the proliferative response of lymph node cells obtained 10 wk after infection with L. major from SCID mice reconstituted with 107 CD25+ T cell-depleted splenocytes was clearly higher than that observed in lymph node cells of similarly infected SCID mice reconstituted with the same number of total spleen cells (Fig. 6Go).



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  		FIGURE 6. Increased in vitro proliferative response to L. major stimulated lymph node cells isolated from infected SCID mice reconstituted with CD4+CD25+-depleted spleen cells. Thymidine incorporation of cells from SCID mice reconstituted with spleen cells depleted or not of CD4+CD25+ T cells and subsequently infected with L. major. Draining lymph node cells were removed 10 wk after infection and 5 x 105 cells were restimulated in vitro with 2 x 105 UV-irradiated L. major for 72 h. [3H]Thymidine was added for the last 6 h of culture. Shown are the mean and SD of scintillation cpm for triplicate cultures. Data are representative of two separate experiments.

 
Neutralization of IL-4 prevents Th2 cell maturation and susceptibility to L. major in SCID mice reconstituted with 107 BALB/c spleen cells depleted of CD4+CD25+ T cells

Treatment of BALB/c mice with anti-IL-4 mAb at the initiation of infection is capable of redirecting protective Th1 cell development resulting in resistance to L. major (8, 9, 28). Similarly to BALB/c mice, infection with L. major of SCID mice reconstituted with 107 BALB/c spleen depleted of the CD4+CD25+ regulatory T cell population results in a rapid burst of IL-4 mRNA expression in draining lymph node cells (Fig. 4Go). Experiments were then designed to determine whether the Th2 response developing in these mice was also instructed by the IL-4 produced as a result of this early IL-4 mRNA burst. SCID mice reconstituted with 107 BALB/c spleen cells depleted of CD25+ cells were infected with 3 x 106 L. major and treated or not with 1 mg of anti-IL-4 mAb 11B11 at the onset of infection. Control groups included similarly infected nonreconstituted SCID mice, SCID mice reconstituted with 107 unseparated spleen cells, and BALB/c mice treated or not with anti-IL-4 mAb. Monitoring the development of lesions in mice from these various groups clearly showed that, similarly to BALB/c mice, treatment with anti-IL-4 renders SCID mice reconstituted with 107 spleen cells devoid of CD25+ T cells fully resistant to L. major (Fig. 7GoA). Furthermore, the numbers of parasites recovered after culture in vitro of footpad tissues, removed 12 wk after infection, confirmed that parasite growth was controlled in these mice (2 x 103 L. major/footpad lesion vs 3 x 108 in control mice not treated with anti-IL-4 mAb). Resistance to infection was correlated with the ultimate development of Th1 responses 12 wk after infection. Results in Fig. 7GoB show that SCID mice reconstituted with spleen cells free of CD25+ cells and treated with anti-IL-4 mAb exhibited a >10-fold decrease in the amounts of IL-4 transcripts in their draining lymph node lymphocytes compared with similarly infected SCID mice not treated with anti-IL-4 mAb. As previously observed, it is noteworthy that the responses of the mice from the various groups could not be discriminated on the basis of the amounts of IFN-{gamma} transcripts. Comparable results were obtained when supernatants of specifically activated lymph node cells were analyzed for the accumulation of IL-4 or IFN-{gamma} (data not shown).



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  		FIGURE 7. Neutralization of IL-4 abrogates the Th2 response of SCID mice reconstituted with CD4+CD25+ T cell-depleted splenocytes following L. major infection and leads to control of lesions. A, SCID mice were reconstituted with 107 spleen cells depleted or not of CD4+CD25 regulatory T cells and infected with 3 x 106 L. major promastigotes 5 days later. A group of mice reconstituted with depleted cells was treated with 1 mg of anti-IL-4 mAb i.p. at the time of infection. Lesion size was monitored with a metric caliper. Data are from five mice per group. n.r., SCID mice nonreconstituted. *, p < 0.00001 between SCID + 107 depleted and SCID + 107 depleted anti IL-4. B, mRNA was isolated from the lymph nodes of mice 12 wk after infection with L. major. cDNA was prepared from each group, and semiquantitative PCR was performed for IL-4 and IFN-{gamma} as described in Material and Methods. Results are expressed as fold increase in mRNA of mice infected with L. major compared with mice similarly treated but not infected. n.r., SCID mice nonreconstituted.

 
Importantly, the anti-IL-4 treatment did not affect the proliferation rate of transferred CD4+ T cells in reconstituted SCID mice as assessed by determining the CFSE profile in draining lymph node cells 15 days after transfer of CFSE-labeled spleen cells, i.e., 10 days after infection with L. major (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that compared with normal BALB/c mice, BALB/c mice depleted of CD4+CD25+ regulatory T cells three days before infection with L. major develop significantly more severe lesions that contain higher numbers of parasites. This enhanced disease progression was correlated with the faster development of robust IL-4-producing CD4+ T cell responses. In the absence of CD4+CD25+ regulatory T cells, the early IL-4 transcriptional burst seen in BALB/c mice within 1 day of infection with L. major was significantly enhanced. Inhibition of the activation of MHC class II-restricted parasite-specific CD4+ T cells due to the persistence of the anti-CD25 PC61 depleting mAb is unlikely to account for the observed effects. Indeed, SCID mice reconstituted with BALB/c spleen cell populations, depleted of CD25+ regulatory T cells in vitro, also exhibited increased levels of IL-4 transcripts in the draining lymph nodes 1 day following infection with L. major, enhanced disease progression and Th2 cell development.

Studying the requirement for IL-2 signaling on the progression of lesions in BALB/c mice infected with L. major, others have reported that the biweekly administration of anti-IL-2R{alpha} (CD25) mAb PC61 during the first 4 wk of infection renders BALB/c mice resistant (35). In these experiments, it is likely that persistent blocking of the IL-2R and/or depletion of CD4+ T cells, that are induced to express the IL-2R{alpha} chain following specific activation, preferentially interfered with the expansion of recently activated CD4+ Th2 cells because IL-2 signaling has been reported to be required for the establishment and maintenance of Th2 responses (35, 36, 37). Interestingly, in this study restricting the administration of the anti-IL-2R{alpha} PC61 mAb to the first day of infection with L. major either did not modify or sometimes exacerbated disease progression (35). Because we show in this study that an interval of 3 days after the injection of PC61 mAb is required for the maximal depletion of CD25+ T cells, it is likely that when the anti-CD25 mAb is administered the day of infection, the exacerbating effect on disease progression varies depending upon the numbers of CD25+ regulatory T cells available. In this context, at least in vitro, the degree of suppression mediated by CD4+CD25+ regulatory T cells has been demonstrated to be proportional to the numbers of regulatory T cells (34).

Even in the absence of antigenic stimulation, the results in this report show an increased proliferation and repopulation of lymph nodes by adoptively transferred CD4+ T cells in syngeneic SCID recipients when the CD4+CD25+ population was removed from the spleen cell inoculum before transfer. That CD25+ regulatory cells regulate the size of the peripheral lymphoid compartment is also supported by several other observations. Thus, a shortage of CD25+ regulatory T cells has been reported in IL-2- or IL-2R{alpha}-deficient mice that exhibit a dysregulation of both the size and the content of their peripheral lymphoid compartment resulting in autoimmunity (38, 39). Similarly, the activated/memory CD45RBlowCD4+ T cell population, containing natural regulatory T cells, was clearly shown to limit the peripheral expansion of naive CD45RBhighCD4+ T cells when both CD4+ T cell subpopulations were transferred into syngeneic Rag-20/0 recipients (40). Furthermore, depletion of CD25+ cells in vivo with the PC61 mAb led to an increased expansion of adoptively transferred C57BL/6 spleen T cells in syngeneic nude mice (39).

In contrast to naive classical CD4+ T cells that need to be activated to express CTLA-4, CD4+CD25+ regulatory cells have been shown to express CTLA-4 constitutively (41, 42, 43). Blockade of CTLA-4 using anti-CTLA-4 mAb has been reported to decrease the suppressive capacity of CD25+ regulatory T cells in vitro (43) and to interfere in vivo with the ability of these cells to control intestinal inflammation (42, 43). It is presently not known whether the CTLA-4 molecules on CD25+ regulatory T cells prevent interaction between the CD28 molecules on target cells with the B7 (CD80/CD86) molecules on APC (43) or, alternatively, cross-linking the CTLA-4 molecules on CD25+ cells results in TGF-{beta} production (44). In this context, treatment with anti-CTLA-4 mAb has been shown to exacerbate disease progression and to lead to enhanced Th2 responses in BALB/c mice infected with L. major (45) effects similar to those reported in this study following depletion of CD4+CD25+ regulatory T cells. Using different antigenic systems, other studies have shown that mice expressing a transgenic TCR on a CTLA-4-/- background preferentially develop Th2 responses and conversely that signaling through CTLA-4 inhibits Th2 maturation (46). Thus CTLA-4 would limit the magnitude of Th2 differentiation. Therefore, the similarities between the effects observed either in the absence of CD4+CD25+ regulatory T cells or following the blockade of CTLA-4 support the hypothesis that CTLA-4 may play a role in the suppressive activity of CD4+CD25+ regulatory T cells.

The suppressive capacity of the CD4+CD25+ population likely results, at least in part, from its ability to inhibit IL-2 transcription and IL-2 production in the target T cell population (47). As a result, these responder cells fail to proliferate and undergo cell cycle arrest at the G0/G1 phase (47). Whether this inhibition, dependent on cell contact between the regulatory and the responder cells, is mediated by other cytokines, in soluble or membrane-bound forms, is still a matter of debate (reviewed in Refs. 18 and 48). In this context, we show in this study that up to 5 wk after infection with L. major, the draining lymph node cells of either BALB/c mice depleted of CD4+CD25+ regulatory T cells or SCID mice reconstituted with spleen cells depleted in regulatory T cells in vitro exhibited significantly enhanced proliferative responses upon specific restimulation in vitro. Likewise, following infection with L. major, enhanced CD4+ T cell proliferation was also observed in vivo in SCID recipients of CSFE-labeled CD4+CD25+-depleted BALB/c spleen cell populations. This increased proliferative capacity likely results from enhanced IL-2 production in mice lacking CD25+ regulatory cells because we have detected, soon after infection with L. major, higher levels of IL-2 transcripts in draining lymph node cells of BALB/c mice depleted of CD25+ T cells as compared with normal BALB/c mice (A. Gumy, unpublished data).

Cell cycling favors cytokine gene expression. Although entry into the S phase is necessary for the expression of the IFN-{gamma} and IL-4 genes (49, 50), there is a controversy regarding possible differences in the number of cell divisions required for naive CD4+ T cells to differentiate toward either IFN-{gamma}-producing Th1 cells or IL-4-producing Th2 cells (51). In the absence of CD4+CD25+ regulatory T cells, results presented in this study show that donor CD4+ T cells proliferate more intensively in response to L. major in vitro. Combined with the present demonstration of the inhibitory role of CD4+CD25+ regulatory T cells on Th2 cell maturation following infection with L. major, these results could indicate that the magnitude of the Th2 response is dependent upon the proliferation rate. However, more likely is that the enhanced IL-4 production by V{beta}4V{alpha}8 CD4+ T cells during the first day of infection with L. major observed in the absence of CD4+CD25+ regulatory T cells could account for the subsequent development of magnified Th2 responses. Such a hypothesis is strongly supported by the results in this report showing that neutralization of the IL-4 produced during the early stage of infection with L. major led to a significant inhibition of Th2 cell development in SCID mice reconstituted with syngeneic spleen cells depleted of CD4+CD25+ regulatory T cells without affecting the proliferation rate of the transferred CD4+ T cells. Therefore, we believe that an excessive production early after infection of the IL-4, necessary for instructing further Th2 cell development, by LACK-reactive V{beta}4V{alpha}8 CD4+ T cells is a major consequence of the depletion of CD25+ regulatory T cells in this experimental system. The requirement for LACK-reactive V{beta}4V{alpha}8 CD4+ T cells and the IL-4 they produce during the first day of infection with L. major for subsequent Th2 cell maturation and disease progression in susceptible BALB/c mice has been firmly established (13, 15, 28). The mechanism by which CD25+ regulatory T cells down-regulate IL-4 production by LACK-specific V{beta}4V{alpha}8 CD4+ T cells is not known. However, given our recent findings that this early IL-4 response to L. major is regulated by IL-2 (A. Gumy, unpublished data), it is possible that a CD25+ regulatory T cell-mediated inhibition of IL-2 gene transcription could affect IL-4 production.

Although anergic in terms of proliferation, CD4+CD25+ regulatory T cells have been reported to express their suppressive activity at concentrations of Ags significantly lower than those necessary for activation of other (naive) T cells (34). In this context, results from elegant experiments recently indicated that the development of CD4+CD25+ regulatory T cells requires higher avidity of their TCR for MHC class II/self-peptides than other (naive) T cells (52). The LACK Ag of L. major is the Leishmania homolog of mammalian RACK1 and there is some degree of homology between the two proteins, particularly within the region of the immunodominant I-Ad epitope of LACK eliciting the rapid IL-4 response by V{beta}4V{alpha}8 CD4+ T cells in BALB/c mice (12, 14). It is thus tempting to speculate that some CD4+CD25+ regulatory T cells expressing TCR with high avidity for a peptide in mammalian RACK1 are positively selected in the thymus and readily activated in the periphery to exert suppression following injection of LACK. The mechanism by which regulatory CD4+CD25+ T cells suppress IL-4 production by LACK-reactive cells remains elusive. However, if regulatory CD25+ T cells and V{beta}4V{alpha}8 CD4+ T cells recognize their specific epitope on the same APC, the possibility that activated regulatory CD25+ T cells renders this APC unable to provide costimulatory signals necessary for IL-2 transcription in V{beta}4V{alpha}8 CD4+ T cells is attractive. In this context, some observations already strongly suggest that the CD25+ suppressor T cell population acts on APC (17).

Noteworthy, depletion of CD25+ T cells in C57BL/6 mice before infection with L. major had no significant effect on the resolution of lesion; however, the transient absence of CD25+ regulatory T cells resulted in reduced parasite numbers within lesions and suppressed almost totally the parasite reservoir remaining normally in clinically cured C57BL/6 mice. CD4+CD25+ regulatory T cells have been reported to produce IL-10 in vivo (53, 54). Because IL-10 is a cytokine important in controlling the residual parasites in clinically cured C57BL/6 mice (55), depletion of regulatory T cells which last for over a month, could result in loss of IL-10 and thus reduce the number of residual parasites within the lesions. In BALB/c mice, depletion of CD4+CD25+ T cells leads to the opposite phenomenon, i.e., increased parasite growth. Thus in strains of mice susceptible to infection with L. major, CD4+CD25+ T cells act very early on cells responsible for driving Th2 differentiation, regulating early IL-4 secretion, but in resistant strains developing Th1 responses following infection, CD4+CD25+ may produce the IL-10 recently found necessary for the persistence of parasites in clinically cured animals (55). Additional experiments are needed to further decipher the mechanism of action for CD4+CD25+ in strains of mice resistant to infection with L. major.

In summary, in this study we have shown that regulatory T cells, highly efficient in controlling self-reactive effector T cells and preventing autoimmunity, are also able to restrain the development of detrimental Th2 responses to an intracellular parasite in genetically susceptible hosts. Defining the fine specificities of CD25+ regulatory T cells, understanding the cellular and molecular mechanisms involved in suppression and their role in controlling pathologies induced by specific Th subsets remain important issues that deserve further studies. This knowledge could ultimately lead to the design of new strategies for manipulating the development of effector responses to the host’s benefit.


   Acknowledgments
 
We thank Consolée Aletti for technical assistance.


   Footnotes
 
1 This work was supported by a grant from the Swiss National Science Foundation (to J.A.L.). Back

2 A.A. and A.G. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Fabienne Tacchini-Cottier, World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, 155, Chemin des Boveresses, CH-1066 Epalinges, Switzerland. E-mail address: Fabienne.Tacchini-Cottier{at}ib.unil.ch Back

4 Abbreviation used in this paper: LACK, Leishmania homolog of mammalian RACK1. Back

Received for publication May 3, 2002. Accepted for publication July 9, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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