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The Journal of Immunology, 2002, 168: 4628-4635.
Copyright © 2002 by The American Association of Immunologists

Rapid IL-4 Production by Leishmania Homolog of Mammalian RACK1-Reactive CD4+ T Cells in Resistant Mice Treated Once with Anti-IL-12 or -IFN-{gamma} Antibodies at the Onset of Infection with Leishmania major Instructs Th2 Cell Development, Resulting in Nonhealing Lesions1

Pascal Launois2,*, Alain Gumy*, Hayo Himmelrich*, Richard M. Locksley{dagger}, Martin Röcken{ddagger} and Jacques A. Louis3,*

* World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, Lausanne, Switzerland; {dagger} Departments of Medicine and Microbiology/Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and {ddagger} Department of Dermatology, Ludwig Maximilian University, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4+ T cells expressing the V{beta}4-V{alpha}8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-{gamma} at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4+ T cells also expressing the V{beta}4-V{alpha}8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-{gamma} Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-{gamma} Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Genetically determined resistance and susceptibility to Leishmania major in mice result from polarized Th1 and Th2 responses, respectively. Unlike mice from most inbred strains (e.g., C57BL/6 and CBA), which are resistant, BALB/c mice infected with L. major sustain progressive disease and develop aberrant Th2 responses (1).

Among the many stimuli influencing the differentiation of distinct CD4+ Th responses, cytokines themselves critically regulate this process. Using the murine model of infection with L. major, the validation in vivo of results obtained in vitro with naive CD4+ T cells from TCR {alpha}{beta} transgenic mice has established the crucial role of IL-12 in Th1 cell maturation (2, 3, 4) and IL-4 in Th2 cell development (5, 6). The disclosure of a rapid burst of IL-4 mRNA expression in CD4+ T cells from BALB/c mice following i.v. or s.c. injection of L. major (7) has provided grounds for the demonstrated power of anti-IL-4 neutralizing Ab in redirecting Th1 cell development in these mice (5). The CD4+ T cells responsible for this early IL-4 response to L. major express a highly restricted TCR repertoire (V{beta}4-V{alpha}8) and respond in a cognate fashion to a single dominant I-Ad-restricted epitope of the Leishmania homolog of mammalian RACK1 (LACK)4 Ag from L. major (8). The requirement for these cells and the IL-4 they produce for subsequent Th2 cell maturation and disease progression in BALB/c mice was established (9). In contrast to BALB/c mice, mice from various resistant strains (e.g., C57BL/6, CBA, and C3H) did not generate rapid IL-4 mRNA expression following infection with L. major (7). Furthermore, administration of exogenous IL-12 or IFN-{gamma} to BALB/c mice at the time of parasite inoculation readily down-regulated the early IL-4 response and, conversely, neutralization of IL-12 or IFN-{gamma} at the initiation of infection in C57BL/6 mice or inactivation of the IL-12 or IFN-{gamma} gene allowed the expression of a rapid IL-4 response to L. major in these resistant mice (Refs. 7, 10 , and 11 and our unpublished observations).

The aim of this study was to characterize the cellular source and the Ag specificity of the early IL-4 response observed in resistant mice treated with anti-IFN-{gamma} or anti-IL-12 at the onset of infection. Because such treatments reverse the natural resistance of these mice to L. major, we assessed the possibility that the IL-4 produced during the early stage of infection also plays a role in instructing aberrant Th2 cell maturation, resulting in nonhealing lesions.


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

Female BALB/c and C57BL/6 mice were purchased from Iffa Credo (L’Arbresle, France) or from Harlan Olac (Bicester, U.K.) and used at 6–10 wk of age, unless otherwise specified. I-E-transgenic C57BL/6 mice were obtained from D. Lo (The Scripps Clinic and Research Foundation, La Jolla, CA) (12).

Parasites and infection

L. major LV 39 (MRHO/Sv/59/P strain) were maintained in vivo and grown in vitro as described (13). Mice were infected s.c. in one hind footpad with 3 x 106 stationary phase L. major promastigotes in a final volume of 50 µl. In designated experiments, footpad tissues were used to create limiting dilutions for quantitation of viable parasite burdens as previously described (14).

Reagents, Abs, and treatment of mice

Recombinant LACK protein from L. major was produced in Escherichia coli from the expression plasmid pET3a-{delta}9-rLACK and purified on Ni-nitrilotriacetic acid resin, as described (8). Mice were injected with 5 µg of LACK into one hind footpad. The following mAbs were used in this study: KT4–10 (anti-V{beta}4) and KT50.1 (anti-V{alpha}8). FITC-conjugated goat anti-rat (Caltag Laboratories, San Francisco, CA) IgG antisera were used to stain cells incubated with anti-V{beta}- or V{alpha}-specific hybridoma supernatants. Anti-murine IFN-{gamma} and anti-IL-4 mAbs were produced from the XMG 1.2 rat hybridoma (15) and from the 11B11 rat hybridoma (16), respectively. Anti-murine IL-12 mAbs were produced from the C17.15 and the C17.8 rat hybridoma was kindly provided by G. Trinchieri (Wistar Institute of Anatomy and Biology, Philadelphia, PA). Anti-DNP rat mAbs LO-DNP-57 (17), kindly provided by H. Bazin (University of Louvain, Louvain, Belgium), were used as control Abs. Mice were given 1 mg of anti-IFN-{gamma}, anti-IL-12, or anti-DNP mAbs 18 h before infection with L. major or injection of LACK.

Infection of adult mice with MMTV

Exogenous mouse mammary tumor viruses (MMTV)(SIM) and MMTV(SW), encoding a V{beta}4- or V{beta}6-specific superantigen (18, 19), respectively, were used in this study. C57BL/6 mice expressing an I-E transgene in B cells (12) were infected with MMTV as adults through the s.c. injection of a milk-derived virus preparation into one hind footpad, as described (8). Various times after, mice were bled and the percentage of V{beta}4 and V{beta}6 cells among CD4+ T cells was followed by flow cytometry in peripheral blood lymphocytes.

Fluorescent cell sorting

CD4+ T cells were purified from the draining lymph nodes using MACS (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer’s conditions. Briefly, cells were suspended with magnetic microbeads that had been conjugated with anti-CD4 mAb (GK1.5) and isolated after immobilization with a magnet. The enriched (85%) CD4+ T cell populations were stained with anti-V{beta}4- or -V{alpha}8-specific mAbs followed by a FITC-conjugated anti-rat IgG antisera. Cells were sorted into V{beta}4- or V{alpha}8-positive and -negative populations using a FACStarPlus flow cytometer (BD Biosciences, Mountain View, CA). The purity of the sorted populations that express either the V{beta}4 or V{alpha}8 TCR chain was 99%.

RNA extraction and competitive PCR analysis

Total RNA was extracted from cells of draining popliteal lymph nodes as described (7). First-strand cDNA synthesis was performed using a first-strand cDNA synthesis kit according to the manufacturer’s directions (Pharmacia Biotech, Uppsala, Sweden). The polycompetitor plasmid (pQRS) was used to quantitate amounts of transcripts for IFN-{gamma}, IL-4, and the constitutively expressed HPRT gene, using primers and PCR conditions as described (20). IL-12 p35 and IL-12 p40 mRNA were monitored with the pMus3 competitor (Sanofi, Labège, France) as described (21). The first-strand cDNA was used directly as a template in the presence of serial 5-fold dilutions of pQRS or pMus3/pHos3 competitor. After separation of the PCR products by agarose gel electrophoresis, the ratio of IFN-{gamma}, IL-4, IL-12 p35, and IL-12 p40 to HPRT transcripts was calculated. The results are shown as the fold increases in cytokine mRNA in mice infected with L. major or injected with the LACK protein, as compared with control mice.

Lymphocyte cultures and detection of cytokines in supernatants

Popliteal lymph node cells (5 x 106) were stimulated with UV irradiated L. major promastigotes (1 x 106) in a final volume of 1 ml. Cells were cultured in DMEM supplemented with 5% heat inactivated FCS, L-glutamine (216 µg/ml), 5 x 10-5 M 2-ME, and 10 mM HEPES in an atmosphere of 7% CO2 at 37°C. Culture supernatants were collected after 72 h and stored at -20°C until use. IFN-{gamma} was measured in supernatants by ELISA as described (22). Mouse recombinant IFN-{gamma} (supernatant of L1210 cells transfected with the murine IFN-{gamma} gene; a gift of Y. Watanabe, Kyoto University, Kyoto, Japan) was used as standard. IL-4 was measured by a bioassay using the CTLL-44 cell line (gift of P. Erb, University of Basel, Basel, Switzerland) as described (23). Recombinant murine IL-4 expressed in X63Ag-653 cells (gift of F. Melchers, Basel Institute of Immunology, Basel, Switzerland) was used as standard. The limits of detection of these assays were 10 U/ml for IFN-{gamma} and 20 pg/ml for IL-4.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Infection with L. major rapidly induces increased IL-4 mRNA levels in C57BL/6 mice treated with anti-IL-12 or anti-IFN-{gamma} 1 day before parasite inoculation

Groups of BALB/c and C57BL/6 mice treated with anti-DNP, anti-IL-12, or anti-IFN-{gamma} mAbs were injected s.c. with L. major in the hind footpads. At various times after infection the draining popliteal lymph nodes were removed for RNA extraction and IL-4 mRNA levels were determined by semiquantitative RT-PCR. Results depicted in Fig. 1Go confirm that susceptible BALB/c mice, in contrast to resistant C57BL/6 mice, exhibit a burst of IL-4 mRNA expression in draining lymph nodes 16 h after infection with L. major (7). Treatment of C57BL/6 mice with either anti-IFN-{gamma} or anti-IL-12 clearly allowed these mice to also generate IL-4 transcripts as soon as 16 h after parasite inoculation. At this time, the levels of IL-4 mRNA expression were similar to those found in BALB/c mice. As reported, kinetics analysis of IL-4 mRNA expression in response to L. major in BALB/c mice showed a burst peaking at 16 h with IL-4 mRNA levels returning to baseline values at 48 h before reaching again (5 days later) elevated levels that remained stable during the course of infection (7). Interestingly, the kinetics of IL-4 mRNA expression in both C57BL/6 and BALB/c mice treated with anti-IFN-{gamma} or anti-IL-12 were somewhat different, with a burst at 16 h that remained stable at least during the first 10 days of infection (Fig. 1Go).



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  		FIGURE 1. Kinetics of IL-4 mRNA expression in popliteal lymph nodes following the s.c. injection of 3 x 106 stationary phase L. major into the hind footpad. BALB/c mice and C57BL/6 mice (three per group), treated or not with anti-IL-12 or -IFN-{gamma} Abs, were infected with 3 x 106 L. major. At different times after infection cells were isolated from popliteal lymph nodes, RNA was extracted, and the levels of IL-4 mRNA were determined by semiquantitative RT-PCR. For mice not treated with Abs, the results are expressed as the fold increase in IL-4 mRNA in infected mice compared with that in noninfected mice. For mice treated with Abs, the results are expressed as the fold increase in IL-4 mRNA compared with that in mice noninfected with L. major but treated with the same Abs. Bars represent the mean ± SD of triplicate determinations. The results are from one of two experiments that gave similar results.

 
The LACK Ag from L. major induces rapid IL-4 mRNA expression in C57BL/6 mice treated with anti-IFN-{gamma} or anti-IL-12 mAb 1 day before injection

The early IL-4 response to L. major in BALB/c mice results from the cognate recognition of an immunodominant T cell epitope in the LACK Ag by specific CD4+ T cells (8). To determine whether the LACK Ag was also responsible for the rapid induction of IL-4 transcripts in response to L. major in C57BL/6 mice treated with anti-IFN-{gamma} or anti-IL-12 mAb, C57BL/6 mice were treated with 1 mg of anti-IFN-{gamma} mAb and 18 h later injected in the footpads with either 5 µg of recombinant LACK or L. major promastigotes. The lymph node cells were collected at designated periods for analysis of IL-4 mRNA expression using RT-PCR. Transcripts for IL-4 were readily detected, with a peak at 16 h that only slowly decayed over the ensuing 10 days (Fig. 2Go). Similar results were obtained in C57BL/6 mice treated with anti-IL-12, and no IL-4 mRNA was detected in control C57BL/6 mice receiving anti-DNP mAb before injection of LACK or L. major (data not shown). It is noteworthy that the I-Ab-restricted epitope of LACK triggering this early IL-4 response in anti-IFN-{gamma}-treated C57BL/6 mice has recently been mapped and found clearly distinct from the I-Ad-restricted epitope (aa 156–173) eliciting this IL-4 response in BALB/c mice (Ref. 8 and P. Launois, S. Pingel, R. M. Locksley, and J. A. Louis, manuscript in preparation).



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  		FIGURE 2. The LACK protein from L. major rapidly induces IL-4 mRNA expression in draining lymph node cells from C57BL/6 mice treated with anti-IFN-{gamma} Abs. C57BL/6 mice (five mice per group) were treated with 1 mg of anti-IFN-{gamma} Abs 1 day before inoculation with 3 x 106 L. major promastigotes or injection of 5 µg recombinant LACK in one hind footpad. At various times thereafter cells were isolated from popliteal lymph nodes, RNA was extracted, and the levels of IL-4 mRNA were determined by semiquantitative RT-PCR. Results are expressed as the fold increase in IL-4 mRNA as compared with mice given only anti-IFN-{gamma} Abs. Bars represent the mean ± SD of triplicate determinations. Similar results were obtained in two separate experiments.

 
TCR V{beta} and V{alpha} chains used by CD4+ T cells that express IL-4 mRNA in response to L. major or LACK in C57BL/6 mice treated with anti-IFN-{gamma} or -IL-12 mAbs

We have previously demonstrated that the burst of IL-4 mRNA expression seen in draining lymph nodes of BALB/c mice 16 h after infection with L. major or injection of LACK occurs within CD4+ T cells that express V{beta}4-V{alpha}8 TCRs (8). Because the rapid induction of IL-4 transcripts in anti-IFN-{gamma}- or anti-IL-12-treated C57BL/6 mice also occurs in response to the LACK Ag from L. major, experiments were conducted to determine the CD4+ T cell origin of this early IL-4 response and their TCR V{beta} and V{alpha} usage. CD4+ T cells purified from the draining lymph nodes of anti-IFN-{gamma}- or anti-IL-12-treated C57BL/6 mice 16 h after infection with L. major or injection of LACK were first demonstrated to be the only source of IL-4 transcripts (data not shown). Then, lymph node CD4+ T cells were stained with anti-V{beta}4- or anti-V{alpha}8-specific mAb, and after purification by FACS sorting total RNA was isolated from the V{beta}4-positive and -negative or V{alpha}8-positive and -negative populations and analyzed for the presence of IL-4 mRNA using RT-PCR. Results in Fig. 3Go clearly show that 16 h after infection with L. major (Fig. 3GoA) or injection of LACK (Fig. 3GoB) the burst of IL-4 mRNA expression in C57BL/6 mice treated previously with anti-IFN-{gamma} mAb occurs in CD4+ T cells expressing the V{beta}4-V{alpha}8 TCR chains.



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  		FIGURE 3. The CD4+ T cells from anti-IFN-{gamma}-treated C57BL/6 mice that express rapidly IL-4 mRNA transcripts in response to infection with L. major or injection of LACK express the V{beta}4-V{alpha}8 TCR chains. Sixteen hours after infection with L. major (A) or injection of 5 µg of LACK (B), CD4+ cells from draining lymph nodes of anti-IFN-{gamma}-treated C57BL/6 mice (five mice per group) were enriched by MACS sorting. After staining with either anti-V{beta}4 or anti-V{alpha}8 Abs CD4+ T cells were sorted by flow cytometry into positive or negative V{beta}4 or V{alpha}8 subpopulations, respectively. RNA was extracted from these cells and the relative levels of IL-4 mRNA were determined by semiquantitative RT-PCR. Results are expressed as the fold increase in IL-4 mRNA in mice infected with L. major as compared with noninfected mice. Bars represent the mean ± SD of triplicate determinations. Similar results were obtained in two individual experiments.

 
Treatment of V{beta}4-deficient C57BL/6 mice with anti-IFN-{gamma} mAb does not allow the expression of a rapid IL-4 response to L. major

The 3' long terminal repeats of the mouse mammary tumor viruses (MMTV)(SIM) and MMTV(SW) encode a superantigen that leads initially to local stimulation and subsequently to systemic deletion of CD4+ T cells expressing the V{beta}4 or V{beta}6 TCR chains, respectively (18, 19). Because the superantigenic property of MMTV(SIM) requires the presence of MHC class II I-E molecules (24), C57BL/6 mice transgenic for the I-E molecules were used to assess in vivo the role of V{beta}4-V{alpha}8 CD4+ T cells in the early IL-4 response to L. major in anti-IFN-{gamma} mAb-treated C57BL/6 mice.

Groups of I-E transgenic C57BL/6 mice treated 20 wk previously with either MMTV(SIM) or MMTV(SW) and uninfected age-matched I-E transgenic and normal C57BL/6 mice were treated with anti-IFN-{gamma} mAb 18 h before infection with L. major. MMTV(SIM)- and MMTV(SW)-infected I-E transgenic C57BL/6 mice had drastically reduced numbers (<2%) of V{beta}4 or V{beta}6 CD4+ T cells in their peripheral blood lymphocytes, respectively. The draining lymph node cells were harvested after 16 h of infection for analysis of total mRNA for IL-4 transcripts. Similarly infected BALB/c mice were also used as controls. In contrast to MMTV(SW)-infected or normal I-E transgenic C57BL/6 mice, treatment with anti-IFN-{gamma} did not allow the generation of IL-4 transcripts in V{beta}4-deficient I-E transgenic C57BL/6 mice (Fig. 4Go). Further kinetic analysis of the IL-4 response in these mice showed that this did not result from a shifted kinetic response to L. major (data not shown) but rather from the absence of V{beta}4 CD4+ T cells.



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  		FIGURE 4. Early IL-4 mRNA expression in response to L. major does not occur in draining lymph nodes of V{beta}4-deficient C57BL/6 mice treated with anti-IFN-{gamma} 1 day before infection. I-E transgenic C57BL/6 mice deficient in either V{beta}4-positive or V{beta}6-positive CD4+ cells as a result of exposure to MMTV(SIM) or MMTV(SW) (five mice per group) were treated with 1 mg of anti-IFN-{gamma} Abs 18 h before inoculation of 3 x 106 L. major into one hind footpad. Similarly infected BALB/c and anti-IFN-{gamma}-treated I-E transgenic and normal C57BL/6 mice were used as control (five mice per group). Sixteen hours later RNA was extracted from their draining lymph node cells and IL-4 mRNA levels were determined by semiquantitative RT-PCR. Results are expressed as the fold increase in IL-4 mRNA as compared with noninfected mice. Bars represent the mean ± SD of triplicate determinations. Results are from one of two experiments that gave similar results.

 
Treatment of V{beta}4-deficient C57BL/6 mice with anti-IFN-{gamma} has no effect on disease progression and does not redirect Th2 cell maturation

Results in Fig. 5GoA confirm that treatment of C57BL/6 mice with either anti-IL-12 or -IFN-{gamma} mAb at the initiation of infection with L. major significantly alters their resistant phenotype (2, 3, 4, 25). Estimation of the numbers of viable parasites recovered after culture in vitro of footpad tissue under limiting culture conditions (14) confirmed that although parasite growth was controlled in anti-DNP-treated control C57BL/6 mice 65 days after infection, footpad lesions of C57BL/6 mice treated once with anti-IL-12 or anti-IFN-{gamma} mAb contained significantly elevated numbers of parasites (data not shown). However, the size of lesions in these mice did not reach the magnitude of lesions developing in genetically susceptible BALB/c mice. Compared with anti-DNP-treated control mice, C57BL/6 mice treated once with anti-IL-12 or -IFN-{gamma} mAb at the onset of infection exhibited, 45 days later, 10-fold increases in the amounts of IL-4 transcripts in their draining lymph node lymphocytes, with a decrease in the amounts of IFN-{gamma} transcripts (Fig. 5GoB). Comparable results were obtained when supernatants of cultures of specifically stimulated designated lymph node cell populations were analyzed for the accumulation of IL-4 and IFN-{gamma} (data not shown). Thus, the effect of treatment of resistant C57BL/6 mice with a single dose of either anti-IL-12 or anti-IFN-{gamma} mAb on disease progression correlates with the ultimate development of IL-4-producing T cell responses.



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  		FIGURE 5. Course of infection with L. major in C57BL/6 mice treated with anti-IL-12 or -IFN-{gamma} Abs at the initiation of infection and cytokine transcripts in draining lymph node cells 45 days after infection. C57BL/6 mice (six per group) were treated with 1 mg of control anti-DNP, anti-IL-12, anti-IFN-{gamma}, or anti-IFN-{gamma} and anti-IL-4 Abs 1 day before inoculation with 3 x 106 L. major promastigotes in one hind footpad. Similarly infected BALB/c mice were used as control. The size of footpad lesions was monitored using a Vernier caliper. The mean size of lesions ± SD is shown in A. Cells were isolated from popliteal lymph nodes of two mice 45 days after infection, RNA was extracted, and the levels of IFN-{gamma} and IL-4 mRNA were determined by semiquantitative RT-PCR. Results are expressed as the fold increase in IL-4 or IFN-{gamma} mRNA as compared with noninfected mice (B). Courses of infection in C57BL/6 mice (three mice per group) treated with anti-IFN-{gamma} alone or with anti-IFN-{gamma} and anti-IL-4 are shown in C. Comparable results were obtained in another separate experiment.

 
The early IL-4 response to L. major/LACK has been demonstrated to be responsible for the aberrant Th2 differentiation that occurs with progressive disease in susceptible BALB/c mice (8). Similarly, administration of neutralizing anti-IL-4 mAb to C57BL/6 mice, at the time of parasite inoculation, abrogated the effect of treatment with a single dose of anti-IFN-{gamma} mAb on the progression of lesions (Fig. 5GoC) and the subsequent development of IL-4-producing T cell responses (data not shown). Experiments were then designed to further determine whether the ability of treatment with anti-IFN-{gamma} to allow the expression of a rapid IL-4 response to L. major in resistant C57BL/6 mice explains its effects on the subsequent development of IL-4-producing T cells, resulting in nonhealing lesions. Groups of C57BL/6, I-E transgenic C57BL/6, and V{beta}4- or V{beta}6-deficient I-E transgenic C57BL/6 mice, as a result of long-term infection with MMTV(SIM) or MMTV(SW), respectively, were treated with anti-IFN-{gamma} mAb 1 day before infection with L. major. As assessed by measurements of the footpad lesions over time, V{beta}4 CD4-deficient I-E transgenic C57BL/6 mice were able to resolve their lesions (Fig. 6Go) in a manner not different from concurrently infected C57BL/6 or I-E transgenic C57BL/6 mice (Fig. 6Go). Mice from all other groups treated with anti-IFN-{gamma} 1 day before infection with L. major developed progressive lesions that again reached a plateau at 8 wk after infection (Fig. 6Go). Similar results were obtained when disease progression was assessed by estimating the numbers of parasites recovered after culture in vitro of footpad tissues removed 6.5 wk after infection (Fig. 7Go). The effect of treatment with anti-IFN-{gamma} on the course of lesions was correlated with the ultimate development of polarized Th responses. Forty-two days after infection, mice from the various groups were sacrificed and their draining lymph nodes were harvested for cytokine assays. Results in Fig. 8Go show that supernatants from lymph node cell populations of anti-IFN-{gamma}-treated V{beta}4-deficient C57BL/6 mice contain, after specific stimulation with L. major, 5–15 times less IL-4 than supernatants of similarly stimulated lymph node cells of anti-IFN-{gamma} treated C57BL/6, I-E transgenic C57BL/6, or V{beta}6-deficient I-E transgenic C57BL/6 mice. Comparable results were obtained when total mRNA from the lymph nodes of mice from the designated groups was analyzed for IL-4 transcripts by semiquantitative RT-PCR (data not shown). It is noteworthy that the responses of the various groups could not be discriminated on the basis of either the IFN-{gamma} produced in vitro (Fig. 8Go) or amounts of IFN-{gamma} transcripts (data not shown).



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  		FIGURE 6. Course of infection with L. major in anti-IFN-{gamma}-treated I-E transgenic C57BL/6 mice deficient in V{beta}4-positive CD4+ T cells. I-E transgenic C57BL/6 mice deficient in either V{beta}4-positive or V{beta}6-positive CD4+ cells as a result of exposure to MMTV(SIM) or MMTV(SW) (four mice per group) were treated with 1 mg of anti-IFN-{gamma} Abs 18 h before inoculation with 3 x 106 L. major into one hind footpad. Similarly infected anti-IFN-{gamma}-treated- and anti-DNP-treated I-E transgenic and normal C57BL/6 mice not exposed to MMTV were used as control (five mice per group). The mean size of lesions ± SD is shown. Similar results were obtained in another separate experiment.

 


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  		FIGURE 7. Parasite load in lesions of mice infected with L. major. Data represent the number of viable parasites recovered, 45 days after infection with L. major, from the footpads of mice from the groups in Fig. 6Go, as estimated by limiting dilution assays as described in Materials and Methods. Bars represent the mean ± SD of the five individual mice of each group.

 


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  		FIGURE 8. Treatment of V{beta}4-deficient I-E transgenic mice with anti-IFN-{gamma} before infection with L. major does not interfere with Th1 cell development. IL-4 and IFN-{gamma} production by lymph node T cells, obtained 42 days after infection, from the mice of selected groups in Fig. 6Go after stimulation in vitro with L. major were measured by bioassay and ELISA, respectively, as described in Materials and Methods. Bars represent the mean ± SD of triplicate determinations. Results are from one of two experiments that gave similar results.

 
These results correlate the absence of early IL-4 transcripts in response to L. major/LACK in V{beta}4-deficient C57BL/6 mice treated with anti-IFN-{gamma} with the inability of this treatment to inhibit resolution of lesions and redirect Th2 cell maturation in these mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several experimental results, obtained a decade ago, already supported the requisite role of IL-4 in mediating both Th2 cell differentiation and susceptibility to L. major in BALB/c mice (5, 6). With this background, we have documented a burst of IL-4 mRNA expression in draining lymph nodes CD4+ T cells of susceptible BALB/c mice within 1 day of infection (7). The cognate recognition of a single epitope of the LACK (26) was demonstrated to drive this early IL-4 response by a restricted population of MHC class II-restricted CD4+ T cells that expressed the V{beta}4-V{alpha}8 TCR chains (8). The requirement of these cells and the IL-4 they produce, during the early period of T cell activation, for Th2 cell development and susceptibility to L. major was established (8, 9). Remarkably, this rapid IL-4 response to L. major was not observed in any of the mice from the resistant strains tested (7).

From these results, it has been speculated that the LACK-specific V{beta}4-V{alpha}8 CD4+ T cell population could represent a unique lineage in BALB/c mice that releases great amounts of IL-4 under conditions of neutral priming. Alternatively, a greater frequency of LACK-specific V{beta}4-V{alpha}8 CD4+ T cell precursors in BALB/c mice could account for the capacity of the initial IL-4 production in response to LACK to exceed the threshold required for Th2 lineage commitment (8). Neither of these hypotheses is supported by the results in this report confirming that neutralization of IL-12 or IFN-{gamma} 1 day before parasite inoculation allows the expression of an early IL-4 response in resistant C57BL/6 mice, and showing that this response is not only similar in magnitude to that of BALB/c mice but also occurs among V{beta}4-V{alpha}8 CD4+ T cells in response to the cognate recognition of a LACK epitope. This contention is also supported by prior studies demonstrating that a biased expansion of CD4+ T cells that expressed the V{beta}4-V{alpha}8 TCR occurred in the lymph node cell population, draining the site of inoculation with L. major in both susceptible and resistant mice (27). Furthermore, recent observations revealed similar frequencies of I-Ad/LACK+CD4+ T cells in lymph nodes of susceptible and resistant mice (28). However, these results were obtained using BALB/c and B10.D2 (resistant to L. major) mice transgenic for the {beta}-chain of the LACK-specific TCR to facilitate the detection of LACK-specific T cells with multivalent I-Ad immunodominant LACK peptide/MHC molecules. More likely, then, a deficiency in mechanism(s) capable of down-regulating the early IL-4 response by V{beta}4-V{alpha}8 CD4+ T cells in BALB/c mice might underlie the exquisite susceptibility of these mice to L. major. This hypothesis is strengthened by recent results, which have revealed the functional plasticity, in terms of IL-4 or IFN-{gamma} production, of LACK-reactive V{beta}4-V{alpha}8 CD4+ T cells in BALB/c mice (29). Furthermore, treatment of BALB/c mice with exogenous IL-12 was demonstrated to suppress the L. major-induced early IL-4 gene expression (7) and render these mice resistant to L. major (2, 3). Others have reported that, although IL-12 p40 is produced in BALB/c mice soon after infection, its function is inhibited by other cytokines produced simultaneously (25). In this context, the production of TGF-{beta} by macrophages from mice infected with Leishmania amazonensis has been found important for determining susceptibility to infection with this parasite (30). It is noteworthy that TGF-{beta} impairs IL-12 production and CD40 expression by macrophages (31) and inhibits T cell responsiveness to IL-12 (32).

The results presented in this work clearly indicate that C57BL/6 mice are fully capable of mounting an early IL-4 response to L. major/LACK but are possibly prevented from doing so by an even earlier IL-12 or IFN-{gamma} response. In this context we have recently observed that C57BL/6 mice exhibited a burst of IFN-{gamma} and IL-12 mRNA expression within 12 h after the s.c. injection of L. major (fold increase compared with uninfected mice: 50 for IFN-{gamma}, 9 for IL-12 p35, and 10 for IL-12 p40). Thus, the presently reported effect of a single dose of anti-IL-12 or -IFN-{gamma} mAb, at the onset of infection, on the expression of an early IL-4 response in resistant mice likely results from neutralization of the IL-12 or IFN-{gamma} rapidly produced after infection. The fact that previous results showed that treatment with anti-IL-12 can be delayed for up to 2 wk after infection and still promote susceptibility in resistant mice (4) clearly indicates that IL-12 also affects Th development in this system independently from down-regulating early IL-4 production by V{beta}4-V{alpha}8 CD4+ T cells. Thus, it is expected that, even in the absence of LACK-reactive V{beta}4-V{alpha}8 CD4+ T cells, IL-12 is still required for Th1 cell differentiation, maintenance, and resistance to infection.

A role for IL-10 in promoting susceptibility to L. major in mice is supported by recent results (33). However, our inability to reveal early IL-10 production following infection with L. major supports the notion that the role of IL-10 is mainly to render host cells less responsive to IFN-{gamma} for intracellular killing of Leishmania (34, 35).

Results from experiments using mice transgenic for the {beta}-chain of the LACK-specific TCR have shown that, following infection with L. major, T cells from susceptible and resistant mice, although they are activated and expand with similar kinetics, express low- and high-affinity TCR, respectively (28). Thus it has been suggested that, compared with resistant mice, BALB/c mice have an impaired capacity of selecting high-affinity LACK-reactive cells following infection. Such findings could be related to differences between susceptible and resistant mice either T cell intrinsic (36) or not related to the T cell compartment. In this context, it has been clearly shown that after infection with identical numbers of L. major the early dissemination of parasite Ags from the site of inoculation to the draining lymph nodes is better restrained in resistant than in susceptible mice (37). Low Ag concentrations generally tend preferentially to induce Th1 responses, whereas high concentrations induce Th2 development (38). Thus, the results above could suggest that high antigenic doses favor activation and expansion of low-affinity T cells, resulting in IL-4 production, whereas low antigenic concentrations activate high-affinity T cells, producing preferentially IFN-{gamma}. Other results support the notion that the pathway of Th cell maturation is influenced by the affinity of the specific TCR (39, 40). Although the affinity of the V{beta}4-V{alpha}8 TCR of the CD4+ T cells exhibiting increased IL-4 mRNA expression 16 h after injection of LACK has not been compared in BALB/c and anti-IFN-{gamma}- or -IL-12-treated C57BL/6 mice, our results show that the same dose of Ag induced IL-4 responses of similar magnitude in the two strains. It is rather unlikely that the I-Ab-restricted LACK epitope, recently mapped 120 aa apart from the I-Ad-restricted LACK epitope (aa 156–173) (Ref. 8 and P. Launois, S. Pingel, R. M. Locksley, and J. A. Louis, manuscript in preparation), preferentially selects V{beta}4-V{alpha}8 CD4+ T cells with high-affinity TCR. Immunological interventions that redirect protective Th1 cell development in BALB/c mice do not result in the selection of LACK-specific T cells expressing high-affinity receptor (N. Glaichenhaus, unpublished observations). Thus, the possibility that neutralizing IFN-{gamma} or IL-12 in C57BL/6 mice could rescue LACK-specific T cells with TCR of low affinity is also rather unlikely.

It is noteworthy that the kinetics of IL-4 mRNA expression following infection with L. major in BALB/c and anti-IL-12 or -IFN-{gamma}-treated C57BL/6 mice are different. In mice from both strains a burst of IL-4 transcripts is seen in draining lymph nodes 16 h after infection. After this initial burst, IL-4 mRNA expression returned to baseline values within 2 days of infection in BALB/c mice before the occurrence of a second and permanent wave of IL-4 transcripts. In contrast, after the initial burst, IL-4 transcripts remained elevated in both anti-IL-12 and -IFN-{gamma}-treated C57BL/6 and BALB/c mice. Strikingly, the kinetics of IL-4 mRNA expression after injection of the LACK protein were similar in normal BALB/c and anti-IL-12 or -IFN-{gamma}-treated C57BL/6 mice. These findings could indicate that constituent of L. major, other than LACK, are able to set in motion in BALB/c mice mechanisms capable of down-regulating, 24 h after infection, the early increase in IL-4 transcripts. The possibility that treatment with anti-IL-12 or -IFN-{gamma} interferes with the induction or manifestation of this regulatory mechanism is currently being investigated.

The causal relationship between a rapid accumulation of IL-4 transcripts and the susceptible phenotype has been questioned, particularly in one study showing that L. major also induced a rapid production of IL-4 in resistant B10.D2 mice (41). However, in this study the fold increases in IL-4 mRNA expression in response to L. major were singularly modest in mice from both strains (<10). It is also noteworthy that the control values of IL-4 mRNA expression in noninfected mice, used to calculate the increase in IL-4 transcripts following infection with L. major in both strains, were arbitrarily fixed as 1. Thus it is likely that, compared with B10.D2 mice, BALB/c mice overproduced IL-4 in response to L. major and in amounts sufficient to exceed the threshold required for Th2 cell maturation. Nonetheless, these data contrast with our unpublished results showing that, regardless of their MHC haplotype, genetically susceptible mice mount an early IL-4 response to L. major and genetically resistant mice fail to express this response unless IL-12 or IFN-{gamma} are neutralized at the onset of infection (P. Launois, S. Pingel, R. M. Locksley, and J. A. Louis, manuscript in preparation). Although the reason for this discrepancy is not known, it is noteworthy that different strains and numbers of L. major were used. Remarkably, deletion of V{beta}4 CD4+ T cells in I-E transgenic C57BL/6 mice by prior infection with MMTV(SIM) abrogated the capacity of treatment with anti-IFN-{gamma} to allow the expression of an early IL-4 response to L. major, to redirect Th2 cell maturation, and to modify their resistant phenotype. The data indicating a requirement for V{beta}4-V{alpha}8 CD4+ T cells for the manifestations of the effects of treatment with anti-IFN-{gamma} on Th2 cell maturation and disease progression in resistant mice provide further support for the essential role of these cells and the IL-4 they produce for effector Th2 lineage commitment in response to L. major. Admittedly, however, the lesions developing in resistant mice treated with anti-IL-12 or -IFN-{gamma} Abs during the early stage of infection never reached the magnitude of lesions seen in genetically susceptible BALB mice. Thus, although the early production of IL-4 by V{beta}4-V{alpha}8 CD4+ T cells in anti-IL-12- or -IFN-{gamma}-treated resistant mice appears to be required for alteration of the resistant phenotype, it is not in itself sufficient to generate a fully susceptible phenotype. The demonstration that the genetically determined resistance to infection with L. major is under the control of several genes (42) provides a basis for these findings.

Finally, it is puzzling that the early IL-4 response to L. major seen in C57BL/6 mice as a result of treatment with anti-IL-12 or -IFN-{gamma} at the initiation of infection also occurs in CD4+ T cells that express V{beta}-V{alpha} TCR chains identical to the CD4+ T cells where the early IL-4 transcripts initiated by infection in BALB/c mice were localized (8). Extending these studies to other H-2b or H-2d mice, we have recently observed that the CD4+ T cells responsible for the early IL-4 response to L. major, occurring either spontaneously in susceptible mice or as a result of treatment with anti-IL-12 or -IFN-{gamma} in resistant mice, reacted to either an I-Ab (C57BL/6 or BALB.B)- or an I-Ad (BALB/c or B10.D2)-restricted epitope of LACK and always expressed the V{beta}4-V{alpha}8 TCR chains (P. Launois, S. Pingel, R. M. Locksley, and J. A. Louis, manuscript in preparation). Together these baffling results could suggest that the CD4+ T cells reacting either to the I-Ab- or I-Ad-restricted LACK epitope, although clearly not belonging to the NK1.1+ minor subset (7, 43, 44), represent a peculiar lineage with restricted TCR usage and special functional characteristics.


   Footnotes
 
1 This work was supported by grants from the Swiss National Science Foundation, the Sandoz Research Foundation, the Deutsche Forschungsgemeinschaft, the National Institutes of Health, and the Howard Hughes Medical Institute. Back

2 Current address: Pasteur Institute, Cayenne, French Guyana. Back

3 Address correspondence and reprint requests to Dr. Jacques A. Louis, World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, 150 Chemin des Boveresses, CH-1066 Epalinges, Switzerland. E-mail address: jacques.louis{at}ib.unil.ch Back

4 Abbreviations used in this paper: LACK, Leishmania homolog of mammalian RACK1; MMTV, mouse mammary tumor virus. Back

Received for publication November 1, 2001. Accepted for publication February 19, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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