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Infection and Immunity, August 2004, p. 4486-4493, Vol. 72, No. 8
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.8.4486-4493.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of an I-E-Restricted, gp63-Specific, CD4-T-Cell Clone from Leishmania major-Resistant C3H Mice That Secretes Type 2 Cytokines and Exacerbates Infection with L. major

Cynthia M. Theodos,1 Robin V. Morris,2 Jeanette V. Bishop,2 Jeremy D. Jones,2 W. Robert McMaster,3 and Richard G. Titus2*

Biogen, Inc., Cambridge, Massachusetts 02142,1 Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523,2 Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada3

Received 9 February 2004/ Returned for modification 21 March 2004/ Accepted 19 April 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A T-cell clone (designated KLmB-3) was derived from resistant C3H mice 2 weeks after infection with Leishmania major. KLmB-3 was a CD4-T-cell clone that utilized the Vß8.1 T-cell receptor. When adoptively transferred to naive C3H mice, KLmB-3 unexpectedly exacerbated infection with L. major (it increased the cutaneous lesion size and the parasite burden within the lesion). The ability of KLmB-3 to exacerbate disease correlated with its ability to produce the type 2-associated cytokines interleukin-4 (IL-4), IL-5, IL-10, and transforming growth factor beta. Interestingly, KLmB-3 was specific for an epitope in the amino-terminal end of the L. major surface gp63 zinc metalloproteinase (leishmanolysin) that has been shown to be capable of inducing a protective immune response. Moreover, KLmB-3 was activated when this epitope was presented in the context of H-2 I-E rather than H-2 I-A.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leishmania major is a protozoan parasite that causes cutaneous leishmaniasis. Our knowledge of the immune response generated against this parasite has been significantly advanced through the use of suitable murine models of infection. Infection of highly susceptible BALB/c mice, which succumb to L. major, represent one end of the spectrum. In contrast, the other end of the spectrum is represented by the majority of mouse strains, including C57BL/6 and C3H, which are relatively resistant and heal their cutaneous lesions (reviewed in references 8, 19, and 34).

Of the T-cell lineages, CD4 T cells play an important role in determining the course of cutaneous leishmaniasis. Upon adoptive transfer into BALB/c mice, CD4 parasite-specific T cells can have either a beneficial (12, 28, 38, 39) or a detrimental (12, 39, 48, 49) effect on the outcome of infection. The observation that a single subset of parasite-specific T cells can have varied effects on the outcome of infection led to the current concept that L. major-specific CD4 Th1 T cells which secrete gamma interferon (IFN-{gamma}) are protective in cutaneous leishmaniasis, while parasite-specific Th2 T cells which secrete interleukin-4 (IL-4) are detrimental in the disease (8, 19, 26, 34).

In contrast to adoptive transfer studies performed with BALB/c mice, relatively little work has been done with resistant mice, especially with highly resistant mice such as C3H (2). This is surprising since these mice more accurately mimic L. major infections that occur in humans. Thus, the present study was designed to characterize T-cell lines or clones derived from L. major-infected resistant C3H mice. The results presented here describe a CD4-T-cell clone (L. major-specific CD4 T-cell clone B-3 from C3H [k haplotype] mice, designated KLmB-3) that unexpectedly has the Th2 phenotype and thus exacerbates infection with L. major. Interestingly, KLmB-3 is restricted to H-2 I-E, not H-2 I-A. In contrast to Th2-T-cell clones obtained from susceptible BALB/c mice, which are frequently specific for LACK (Leishmania homologue of receptors for activated C kinase [20, 27, 32]), KLmB-3 is specific for an epitope in the N terminus of the gp63 zinc metalloproteinase (leishmanolysin; promastigote surface protease) of L. major. This observation is significant since previous studies have shown that immunization with gp63 can lead to a protective Th1 immune response in resistant CBA mice (5), and thus gp63 is currently regarded as a vaccine candidate for leishmaniasis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. In all cases young (6- to 10-week-old) adult female mice were used. C3H/HeN mice were purchased from the National Cancer Institute (Frederick, Md.). B10.BR and B10.A (4R) mice were purchased from Jackson Laboratories (Bar Harbor, Maine). B10.AQR, B10.RFB3, and B10.E{alpha}+ mice were obtained from the Mayo Clinic (Rochester, Minn.).

Parasites and antigens. Stationary-phase promastigotes of the L. major LV39 isolate (MRHO/Sv/59/P) were used and maintained as previously described (48). The metacyclic form of L. major promastigotes was produced as described elsewhere (36). In some experiments L. major strain A2 and a form of A2 in which all seven copies of the gp63 gene had been knocked out [L. major A2/gp63(1-7)KO (16)] were used. These parasites were cultured like strain LV39.

Recombinant gp63 (rgp63) and deletion polypeptides of the molecule were produced as described previously (41). Ovalbumin (OVA) (grade V; catalog no. A-5503) was purchased from Sigma, St. Louis, Mo.

Infection of mice and derivation and maintenance of T-cell lines and clones. C3H mice were infected subcutaneously with 5 x 106 L. major cells in one hind footpad, and 1 to 2 weeks later (a time when cutaneous lesions were progressing) the draining popliteal and inguinal lymph nodes were removed. A single-cell suspension was prepared, and the cells (5 x 106 cells/ml) were stimulated with L. major promastigotes (106 promastigotes/ml) in vitro to generate T-cell lines and subsequently T-cell clones (48). In certain cases, T-cell lines were derived by stimulating the cells with L. major rgp63 (10 µg/ml) rather than with L. major promastigotes.

At 2 days of culture, supernatants were removed from cells stimulated with rgp63 and from some of the cells stimulated with L. major promastigotes to determine their cytokine contents (for measurement methods see below). At 5 days of culture, blast cells in the remaining wells stimulated with L. major promastigotes were recovered by using Percoll gradients (48). These blast cells were rested for 7 to 10 days with irradiated syngeneic spleen cells as previously described (4). After resting, viable blast cells were recovered on Ficoll gradients (40). Clones were obtained from the rested T-cell blasts by the limiting dilution approach (28) in 96-well round-bottom microtiter plates (catalog no. 3799; Costar, Corning, N.Y.). The culture conditions for cloning were as follows: 0.3 rested T-cell blast, 106 irradiated syngeneic spleen cells, and 105 L. major promastigotes per well in complete Dulbecco's modified Eagle's medium (48) containing 10% fetal bovine serum (HyClone, Logan, Utah) and 5% P388D1 supernatant (30).

Wells that contained replicating T-cell blasts were identified by light microscopy and were expanded for further analysis. Complete characterization of one clone (KLmB-3) is described here. KLmB-3 was maintained through alternating cycles of stimulation in the presence of 10% (final concentration) concanavalin A supernatant (9) and resting. For all experiments, cells were collected from rested cultures and used as described below.

Fluorescence-activated cell sorter analyses. The surface phenotype (CD4 versus CD8, Vß usage) of T cells was determined by using previously described techniques (40).

Adoptive transfer of KLmB-3 and its effect on infection with L. major. To determine the effect that KLmB-3 had on infection with L. major in mice, 2.5 x 105 KLmB-3 cells were coinjected with 2.5 x 105 L. major metacyclic promastigotes subcutaneously into one hind footpad of syngeneic C3H mice. Lesion development was monitored as previously described (46). Control mice were injected with L. major metacyclics alone.

Parasite burdens in footpad lesions of L. major were determined by using a limiting dilution assay as described previously (22).

KLmB-3 proliferation and cytokine production and measurement. As a source of antigen-presenting cells (APCs), 106 irradiated (1,500 rads) syngeneic C3H spleen cells (or congenic cells [see Fig. 5]) were seeded into the wells of 96-well flat-bottom microtiter plates (Costar 3997), and antigens (as described below) were added. The plates were incubated overnight at 37°C, and then KLmB-3 cells (40,000 cells/well; final volume, 100 µl) were added to the wells. Twenty-four, 48, or 72 h later, the wells were pulsed for 18 h with 1 µCi of [3H]thymidine (Amersham, Arlington Heights, Ill.) to determine the degree of cell proliferation (48).



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  		FIG. 5. Genetic restriction of clone KLmB-3. Cultures were constructed a described in the legend to Fig. 3. Cultures were stimulated for 48 h and then pulsed with tritiated thymidine for 18 h.

 
To determine the cytokines elicited by KLmB-3, 1 x 106 KLmB-3 cells, 7 x 106 irradiated syngeneic C3H spleen cells, and 106 L. major promastigotes were plated into the wells (1 ml/well) of 24-well plates (Costar 3524). Supernatants were collected 12, 24, 48, and 72 h later, and their cytokine contents were assessed by using previously described techniques and commercial enzyme-linked immunosorbent assays (ELISAs) for cytokines (43).

Determining the isotypes of anti-L. major antibodies induced by KLmB-3. On the days of infection indicated below, sera were collected from mice infected with L. major alone or with L. major plus KLmB-3. The isotypes of the anti-L. major antibodies present in the sera were determined by an ELISA. Briefly, ELISA plates were coated with a frozen and thawed lysate of L. major promastigotes (10 µg of L. major protein/ml [48]) by using standard techniques (40). The test sera were added to the plates, and the plates were then developed by using a monoclonal antibody isotyping kit (to quantify immunoglobulin G1 [IgG1], IgG2b, and IgG2a; catalog no. 37501; Pierce, Rockford, Ill.) and the manufacturer's directions. To detect IgE, the plates were incubated with biotin-labeled anti-IgE (catalog no. RMGE15; Caltag, Burlingame, Calif.), followed by avidin peroxidase (catalog no. A-3151; Sigma) and a TMB peroxidase development system (catalog no. 50-76-00; Kirkegaard & Perry, Gaithersburg, Md.).

Treatment of mice with anti-IFN-{gamma}. Anti-IFN-{gamma} antibodies were purified from ascites fluid as described elsewhere (7). Normal rat Ig was prepared from normal rat serum (Accurate, Westbury, N.Y.) by using the techniques employed to purify anti-IFN-{gamma} antibodies. Mice were injected intraperitoneally with 2 mg of either normal rat Ig or anti-IFN-{gamma} 3 h before infection with L. major.

Statistical analyses. Data for lesion progression were analyzed by using analysis of variance for repeated measure and for parasite burdens by using unpaired t tests. All experiments were repeated at least twice; most of the results presented below are representative of the results of three or four independent experiments.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
L. major-infected resistant C3H mice produce less type 1 cytokines and more type 2 cytokines in response to gp63 in the first 2 weeks of infection. Initially, we infected resistant C3H mice with L. major promastigotes in one hind footpad and removed the draining popliteal and inguinal lymph node cells to restimulate the cells in vitro. As reported by others (8, 19, 34), the cells produced little IL-4 or IL-5 when they were restimulated with L. major promastigotes in vitro (Table 1). However, curiously, when the cells were restimulated with L. major gp63, a quite different response was observed (Table 1). gp63, or leishmanolysin (25), is the major glycoprotein expressed on the surface of L. major promastigotes. Lymph node cells stimulated with gp63 produced 15- to 24-fold more IL-4 or IL-5 than cells stimulated with L. major promastigotes produced. The production of IL-4 by the cells was not as consistent as the production of IL-5, possibly due to consumption of the IL-4. gp63 also induced approximately 10-fold less IFN-{gamma} (Table 1).


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  		TABLE 1. gp63 induces less type 1 cytokine and more type 2 cytokine

 
To corroborate and extend these results, we restimulated cells from L. major-infected C3H mice with either of a pair of L. major parasites, one that expressed gp63 and one that was genetically engineered so that all seven copies of the gp63 gene were deleted and thus did not produce gp63. As Table 1 shows, wild-type (gp63-expressing) L. major (L. major A2) induced more type 2 and less type 1 cytokines than the mutant parasite lacking gp63 [L. major gp63(1-7)KO] induced. Thus, the two experimental approaches (stimulating with rgp63 and stimulating with parasites genetically engineered to either express or not express gp63) yielded the same results: gp63 favored the development of a type 2 immune response.

Derivation of KLmB-3 and analysis of its surface phenotype. To characterize this phenomenon further, we infected C3H mice with 5 x 106 L. major promastigotes and 2 weeks later derived long-term T-cell lines from which we subsequently derived T-cell clones (for details of the techniques used see Materials and Methods). Clone KLmB-3 was selected for further analysis. Using flow cytometry, we determined that KLmB-3 was a CD4-T-cell clone that utilized the Vß8.1 T-cell receptor (data not shown).

Effect that adoptively transferring KLmB-3 has on a subsequent infection with L. major. To determine how KLmB-3 influenced an infection with L. major metacyclic promastigotes, the parasite (2.5 x 105 cells) and KLmB-3 (2.5 x 105 cells) were coinjected into the hind footpads of syngeneic C3H mice. Controls were injected with the parasite alone. Lesion development (including the increase in footpad thickness and the parasite burden in the developing lesions) was then monitored over time. As Fig. 1 shows, lesions induced with promastigotes plus KLmB-3 were 2 to 10 times the size of lesions on control mice (P < 0.001). It is also important to mention that no delayed-type hypersensitivity response (i.e., footpad swelling) was observed at the site of injection of KLmB-3 and L. major promastigotes at either 24, 48, or 72 h postinjection (Fig. 1).



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  		FIG. 1. Clone KLmB-3 exacerbates infection with L. major in C3H mice. Groups (n = 5) of C3H mice received a subcutaneous injection of either 2.5 x 105 L. major metacyclic promastigotes (control) or a mixture of parasites and 2.5 x 105 KLmB-3 cells in one hind footpad. The data show the development of lesions (increase in footpad thickness) over time. There was a significant (P < 0.001) difference in lesion size between groups. This effect of KLmB-3 on infection with L. major was not due to the nonspecific effect of simply adoptively transferring cells to recipient mice since adoptive transfer of a similarly restricted T-cell clone with a different specificity for molecules associated with the surface lipophosphoglycan of L. major had the opposite effect on infection (namely, it protected against challenge with the parasite) (47).

 
To verify that the increased lesion size observed in mice that received both KLmB-3 and parasites was due to an increase in the level of infection, parasite burdens were measured by using a limiting dilution assay (22). Our results indicated that coinjection of KLmB-3 and L. major resulted in a statistically significant increase in the number of parasites (2.6- to 940-fold more parasites) in the lesions of these mice compared to the number in control mice injected with the parasite alone (Table 2) (the P values ranged from <0.05 to < 0.001). Thus, KLmB-3 significantly exacerbated L. major infection both with respect to lesion size and with respect to parasite burden.


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  		TABLE 2. Parasite burden in lesions of C3H mice injected with KLmB-3

 
Functional phenotype of KLmB-3. The fact that KLmB-3 exacerbated infection with L. major in C3H mice (Fig. 1 and Table 2) suggested that the cells might be Th2 cells. To test this hypothesis, we analyzed the cytokines that KLmB-3 induced after stimulation with L. major. As Table 3 shows, activation of KLmB-3 in vitro resulted in the secretion of substantial amounts of the type 2 cytokines IL-4, IL-5, IL-10, and transforming growth factor beta (TGF-ß) into the culture supernatants. In contrast, we could not detect secretion of the type 1-associated cytokines IFN-{gamma} and IL-12.


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  		TABLE 3. Cytokines induced in vitro after activation of KLmB-3

 
To confirm these cytokine results and to obtain evidence that KLmB-3 was behaving like a Th2 cell in vivo, we next determined the isotypes of L. major-specific antibodies produced in mice coinjected with the parasite and KLmB-3. This information was useful since certain cytokines are known to be switch factors for particular antibody isotypes. For example, IL-4 is known to be a cytokine that preferentially induces an isotype switch to IgG1 and IgE, while TGF-ß can induce a switch to IgG2b (14). As indicated above, both of these cytokines were secreted by KLmB-3 following in vitro restimulation with L. major. Therefore, we determined whether the levels of parasite-specific antibodies of these three isotypes were elevated in the sera of mice coinjected with L. major plus KLmB-3. Higher levels of parasite-specific IgG1 and IgE were seen in the sera of mice coinjected with KLmB-3 and the parasite (Fig. 2). Moreover, IgG2b was detected only in the sera of mice coinjected with KLmB-3 and L. major (Fig. 2). Finally, although parasite-specific IgG2a (an isotype stimulated by Th1-associated cytokines [14]) could be detected at days 13, 18, and 28 of infection in mice injected with L. major, this isotype could not be detected in the sera of mice coinjected with L. major and KLmB-3 until day 28 of infection (Fig. 2).



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  		FIG. 2. Clone KLmB-3 modulates production of L. major-specific IgG1, IgG2b, IgE, and IgG2a in vivo. At 13, 18, and 28 days of infection, sera were collected from mice inoculated with L. major alone or L. major plus KLmB-3. The levels of parasite-specific IgG1, IgG2b, IgE, and IgG2a were then determined as described in Materials and Methods. IgG1, IgG2b, and IgG2a results were obtained with sera diluted 1:100; IgE results were obtained with undiluted sera. IgG2b was detected only in day 28 sera from mice with adoptively transferred KLmB-3; IgG2a was not detected until day 28 of infection in sera from mice with adoptively transferred KLmB-3. ND, not detected; OD, optical density.

 
Antigenic specificity of KLmB-3. To determine the antigenic specificity of KLmB-3, we stimulated the cells in vitro with the unrelated and non-cross-reactive antigen OVA (49), with L. major promastigotes, or with a panel of gp63 recombinant proteins. The cultures were subsequently pulsed at different times with tritiated thymidine to determine the degree of proliferation in response to the stimuli. As expected, KLmB-3 did not respond to the non-cross-reactive antigen OVA (Fig. 3). In contrast, KLmB-3 proliferated vigorously in response to stimulation with L. major promastigotes. Importantly, KLmB-3 cells also responded vigorously to gp63 (Fig. 3), which is the same result which we found when we used gp63 to stimulate lymph node cells obtained from C3H mice infected with L. major for 1 to 2 weeks (Table 1).



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  		FIG. 3. Specificity of clone KLmB-3. KLmB-3 was cocultured with irradiated syngeneic spleen cells as a source of APCs for different times. The cultures were stimulated with nothing (control, medium alone), OVA (200 µg/ml), L. major promastigotes (2 x 105 promastigotes/ml) (Lm), or rgp63 (10 µg/ml) (gp63). At the end of the times indicated the cultures were pulsed with tritiated thymidine to determine the degree of proliferation of KLmB-3.

 
To define the exact specificity of KLmB-3, we used a series of recombinant deletion peptides of gp63 (Fig. 4) (41). TEX51, TEX82, and TEX84 are peptides of gp63 in which various amounts of the carboxy-terminal end have been deleted. In contrast, TEX50, TEX79, and TEX80 are peptides of gp63 in which various amounts of the amino-terminal end of the protein have been deleted. As a positive control we included TEX48, which is the full-length gp63 molecule. Similar to the data shown in Fig. 3, KLmB-3 did not proliferate in response to stimulation with OVA, but it proliferated vigorously to response to stimulation with L. major promastigotes. Deleting various amounts of the C terminus of gp63 had no discernible effect on the proliferation of KLmB-3 (Fig. 4A). In contrast, deleting the N terminus of gp63 profoundly inhibited the proliferation of KLmB-3 (Fig. 4B). Based on the data shown in Fig. 4 as a whole, it appeared that the epitope that KLmB-3 recognizes lies somewhere between amino acids 101 and 206 in the N-terminal region of the gp63 molecule.



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  		FIG. 4. Fine specificity of clone KLmB-3. Cultures were constructed as described in the legend to Fig. 3. All TEX polypeptides were added to the cultures at a final concentration of 10 µg/ml. Lm, L. major promastigotes.

 
MHC restriction of KLmB-3. Finally, we analyzed the major histocompatibility complex (MHC) restriction of KLmB-3. For these experiments we utilized a series of B10 congenic mice or F1 mice derived from crosses between various B10 congenic mice. The resultant mice varied in the ability to express functional H-2 MHC class I or MHC class II (I-Ak or I-Ek) molecules of the k haplotype. KLmB-3 was derived from a C3H mouse and thus was H-2k restricted. We first determined the proliferative response of KLmB-3 using a k haplotype B10 series mouse (B10.BR) as a source of APCs. We found that KLmB-3 proliferated vigorously when it was stimulated with L. major promastigotes and B10.BR spleen cells as a source of APCs (Fig. 5). KLmB-3 also proliferated vigorously in response to L. major when spleen cells from B10.AQR mice were used as the APC source. These mice are of the k haplotype at the MHC class II loci but not at the MHC class I loci. Therefore, these results demonstrate that KLmB-3 is not restricted to MHC class I and suggest that KLmB-3 cells, as expected, are restricted by MHC class II (Fig. 5). To investigate this further, we used spleen cells from B10.A (4R) mice as a source of APCs to restimulate KLmB-3. These mice are of the k haplotype in the I-A region of MHC class II but not in the I-E region. Our results indicated that spleen cells from B10.A (4R) mice did not stimulate KLmB-3 proliferation, suggesting that KLmB-3 was restricted to I-Ek (Fig. 5). To test this hypothesis, we used APCs from two F1 mice (Fig. 5) that expressed a functional I-Ek (but not I-Ak) molecule through transcomplementation. To generate these mice, crosses were made between B10.A (4R) mice and either a B10.RFB3 mouse (1) or a B10 mouse transgenic for the k haplotype I-E{alpha} molecule (B10.E{alpha}+ mouse) (21). Both of these types of APCs supported vigorous proliferation by KLmB-3 which was equivalent to that seen with APCs from the B10.BR H-2k mouse. Together, these data demonstrate that KLmB-3 cells are CD4 T cells that are surprisingly restricted to I-Ek rather than I-Ak.

Does gp63 contribute to the progression of disease in C3H mice treated with anti-IFN-{gamma}? Treating mice that resist infection with anti-IFN-{gamma} reverses the phenotype of the mice, causing the mice to develop a progressive disease similar to that seen in susceptible BALB/c mice (3, 37). To determine whether gp63 drives a Th2 response in C3H mice treated with anti-IFN-{gamma}, we challenged treated mice with L. major and 1 week later harvested the draining lymph nodes. The lymph node cells were stimulated in the fashion shown in Table 1, and the supernatants of the cultures were tested to detemine their IL-4, IL-5 and IFN-{gamma} contents. The results of these experiments (data not shown) were very similar to the results shown in Table 1. Next, we infected anti-IFN-{gamma}-treated C3H mice with L. major and removed the draining lymph nodes 6 weeks later, a time when the infection was completely uncontrolled in the mice. The lymph node cells were again stimulated with either L. major or gp63, and the levels of IL-4, IL-5, and IFN-{gamma} present in the culture supernatants were determined 48 h later. The results are presented in Table 4. At this point in the infection control mice (normal rat Ig-treated mice) produced considerably more IFN-{gamma} (~1,000-fold more) than they produced after 1 week of infection (compare Tables 1 and 4). Anti-IFN-{gamma} treatment led to a greatly decreased ability to produce IFN-{gamma} and to a modest increase in production of the Th2 cytokines IL-4 and IL-5 when cultures were stimulated with L. major (Table 4). Similar observations have been reported by other workers (37). Approximately the same amounts of IL-4 and IL-5 were produced when the cultures were stimulated with gp63. Thus, the capacities of L. major and gp63 to induce Th2 cytokines in anti-IFN-{gamma}-treated C3H mice were similar. Perhaps the most striking aspect of the data shown in Table 4 is that gp63 is a very poor inducer of the Th1 cytokine IFN-{gamma} (gp63 induced 431-fold less IFN-{gamma} than L. major induced in anti-IFN-{gamma}-treated mice and 4,128-fold less IFN-{gamma} than L. major-stimulated cells from normal rat Ig-treated mice induced [Table 4]). Finally, it should be mentioned that since the strain of L. major used in these studies, LV39, can induce IL-13 (29), we also measured IL-13 levels in culture supernatants but found no consistent differences between groups (data not shown).


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  		TABLE 4. Response to gp63 in mice treated with anti-IFN-{gamma}

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe here the derivation and characterization of a L. major-specific Th2 CD4-T-cell clone (KLmB-3) from resistant C3H mice. C3H mice were selected for this study since they are highly resistant (2) to infection with L. major and therefore behave as most humans do when they are infected with the parasite. Thus, C3H mice normally develop a protective Th1 response when they are infected with L. major. The fact that KLmB-3 was an exacerbative Th2-T-cell clone was unexpected. Although this phenotype of KLmB-3 was not expected, this observation is at least consistent with previously described results. Susceptible BALB/c mice and resistant C57BL/6 mice do not become polarized to a Th2 or Th1 phenotype until after 1 and 2 weeks of infection with L. major (31). Since KLmB-3 was isolated from infected resistant C3H mice after 2 weeks of infection, this isolation may correlate with a time when L. major-specific Th2 cells were still active in these mice. The functional capacity of KLmB-3 is clearly that of a classical Th2-T-cell clone; that is, this cell clone does not mediate a delayed-type hypersensitivity reaction (6, 13), secretes exacerbative type 2 cytokines (IL-4, IL-5, IL-10, TGF-ß) but not protective type 1 cytokines (IFN-{gamma}, IL-12), and stimulates the production of type 2-associated Ig isotypes.

Despite the fact KLmB-3 is a classical Th2-T-cell clone, the cells were still only capable of exacerbating infection for the first 4 weeks following adoptive transfer; beyond this time lesions began to heal. It has been shown in L. major-infected mice that adoptively transferred parasite-specific T cells migrate to developing cutaneous lesions of L. major, where they are functionally active for the first few weeks of infection. After this the cells can no longer be detected (49). It appears that the same phenomenon occurred in the experiments described here. In mice that received KLmB-3, lesions began to heal and the Th1-associated antibody isotype IgG2a was produced by 4 weeks of infection. This suggests that KLmB-3 was not active beyond 4 weeks of infection.

Considerable work has been done to characterize the specificity of Th2 T cells that respond to infection to L. major in susceptible BALB/c mice. These cells utilize a Vß4 T-cell receptor and are specific for the leishmanial LACK antigen (19). In contrast, KLmB-3 utilizes a Vß8.1 T-cell receptor and is specific for an epitope in the amino terminus of the surface gp63 molecule of L. major promastigotes. Thus, the parasite epitopes that can trigger Th2 cells and the T-cell receptor utilized by the T cells can differ in BALB/c and C3H mice.

Although it is clear that gp63 can elicit a type 2 response in C3H mice infected with L. major and that gp63-specific Th2 T cells can be isolated from infected C3H mice, our results suggest that these cells are not able to completely skew the immune response in these mice to a Th2 phenotype and thus cause the ultimate death of the animals. The reason(s) for this is not known, but it may be due to the inability of gp63-specific cells to elicit an IL-4 response that has the same kinetics and is of the same magnitude as the response seen with LACK-specific cells in susceptible BALB/c mice infected with L. major. In response to LACK, BALB/c mice produce IL-4 within hours of infection with L. major, but this does not occur in resistant mice infected with the parasite. Thus, BALB/c mice ultimately die of infection, whereas resistant mice (e.g., C3H mice) cure their infection (2, 19). Since resistant mice can be made susceptible to L. major by treatment with anti-IL-12 (11, 45) or anti-IFN-{gamma} (3, 37), we treated C3H mice with anti-IFN-{gamma} to determine whether gp63 could become a strong inducer of a Th2 response in this Th2 environment. We found that gp63 induced Th2 cytokines with the same potency as L. major. Since we and other workers (37) have found that the principal effect of anti-IFN-{gamma} treatment is to reduce IFN-{gamma} production rather than increase Th2 cytokine production, there may be no epitope of L. major that induces a strong Th2 response in C3H mice. In this vein, however, Launois et al. (18) recently demonstrated that the LACK antigen becomes a strong inducer of IL-4 in C57BL/6 mice treated with anti-IFN-{gamma} antibody.

KLmB-3 is activated when the amino-terminal epitope of gp63 is presented in the context of the MHC class II protein I-E and not in the context of I-A. Louis et al. (23, 24) were the first workers to report the derivation of L. major-specific T-cell lines and clones. An analysis of the MHC restriction of these cells revealed that they were restricted to I-A and not I-E. Subsequently, T-cell clones were derived from susceptible BALB/c mice and semiresistant C57BL/6 mice by a number of laboratories (28, 32, 38, 39, 49). The MHC restriction of these cells was not examined, but many of the clones were found to be LACK specific and to utilize the Vß4 T-cell receptor (32). Thus, to the best of our knowledge, the MHC restriction (I-E), antigenic specificity (gp63), and T-cell receptor usage of KLmB-3 (Vß8.1) of KLmB-3 are unique.

Previous immunization studies with gp63 have had diverse outcomes. gp63 has been shown either to induce protection (33, 44) or to not induce protection (10) in mice, and adjuvant has been shown to augment protection (15, 44) or to reverse protection (33, 35). In addition, when gp63 is transfected into bacterial delivery systems, this can lead to protection (5, 51). Thus, as pointed out by Soares et al. (42), it is crucial to carefully select a proven protective epitope of gp63 or any potential immunogen. The present studies show that following infection with L. major promastigotes, gp63 can induce an exacerbating Th2 response. Taken together, the results imply that the context of the initial exposure to gp63, the epitope of gp63 presented, and the manner in which it is presented may determine whether a (protective) Th1 or a (exacerbative) Th2 response evolves following activation by this molecule.

Finally, the fact that C3H mice (the mouse model used in this study) are resistant to infection with L. major (2) and thus accurately mimic L. major infection in most humans adds particular significance to the results presented in this paper. gp63 is currently considered a vaccine candidate for L. major infection of humans. The results presented in this report indicate that it is possible that gp63 may not induce the desired protective type 1 response in some humans. Indeed, peripheral blood mononuclear cells from patients with visceral leishmaniasis produce IL-4 when they are restimulated with gp63 in vitro (17, 50). Therefore, gp63 may be an inappropriate choice for a globally effective vaccine formulation for humans.


   ACKNOWLEDGMENTS
 
We thank C. David (Mayo Clinic, Rochester, Minn.) for providing the B10.AQR, B10.RFB3, and B10.E{alpha}+ mice. We thank Joseph Sypek (Genetics Institute) for helpful suggestions and for critically reading the manuscript.

This work was supported by NIH grants AI 27511 and AI29955 (to R.G.T.) and in part by grant MT-7399 from CIHR (to W.R.M.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Immunology and Pathology, CVMBS, Colorado State University, 1619 Campus Delivery, Fort Collins, CO 80523-1619. Phone: (970) 491-1607. Fax: (970) 491-0603. E-mail: richard.titus{at}colostate.edu. Back

Editor: W. A. Petri, Jr.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1. Anderson, G. D., and C. David. 1989. In vivo expression and function of hybrid Ia dimers (E{alpha}Aß) in recombinant and transgenic mice. J. Exp. Med. 170:1003-1008.[Abstract/Free Full Text]
2. Behin, R., J. Mauel, and B. Sordat. 1979. Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice. Exp. Parasitol. 48:81-91.[CrossRef][Medline]
3. Belosevic, M., D. S. Finbloom, P. H. Van Der Meide, M. V. Slayter, and C. A. Nacy. 1989. Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. J. Immunol. 143:266-274.[Abstract]
4. Boom, W. H., L. Liebster, A. K. Abbas, and R. G. Titus. 1990. Patterns of lymphokine secretion in murine leishmaniasis: correlation with disease progression or resolution. Infect. Immun. 58:3863-3870.[Abstract/Free Full Text]
5. Connell, N. D., H. Medina-Acosta, W. R. McMaster, B. R. Bloom, and D. G. Russell. 1993. Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guerin expressing the Leishmania surface proteinase gp63. Proc. Natl. Acad. Sci. USA 90:11473-11477.[Abstract/Free Full Text]
6. Convit, J., M. E. Pinardi, and A. J. Rondon. 1972. Diffuse cutaneous leishmaniasis: a disease due to an immunological defect of the host. Trans. R. Soc. Trop. Med. Hyg. 66:603-610.[CrossRef][Medline]
7. DeKrey, G. K., H. C. Lima, and R. G. Titus. 1998. Analysis of the immune responses of mice to infection with Leishmania braziliensis. Infect. Immun. 66:827-829.[Abstract/Free Full Text]
8. Etges, R., and I. Muller. 1998. Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions. J. Mol. Med. 76:372-390.[CrossRef][Medline]
9. Fitch, F. W., and T. F. Gajewski. 1994. Production of T cell clones, p. 3.13.6-3.13.8. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology. John Wiley and Sons, Inc., New York, N.Y.
10. Handman, E., L. L. Button, and R. W. McMaster. 1990. Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice. Exp. Parasitol. 70:427-435.[CrossRef][Medline]
11. Heinzel, F. P., R. M. Rerko, F. Ahmed, and E. Pearlman. 1995. Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice. J. Immunol. 155:730-739.[Abstract]
12. Holaday, B. J., M. D. Sadick, Z.-E. Wang, S. I. Reiner, F. P. Heinzel, T. G. Parslow, and R. M. Locksley. 1991. Reconstitution of Leishmania immunity in severe combined immunodeficiency mice using Th1- and Th2-like cell lines. J. Immunol. 147:1653-1658.[Abstract]
13. Howard, J. G., C. Hale, and F. Y. Liew. 1980. Immunological regulation of experimental cutaneous leishmaniasis. III. Nature and significance of specific suppression of cell-mediated immunity in mice highly susceptible to Leishmania tropica. J. Exp. Med. 152:594-607.[Abstract/Free Full Text]
14. Janeway, C. A., and P. Travers. 1997. The humoral immune response, p. 8.8-8.9. In Immunobiology, the immune system in health and disease. Current Biology Ltd./Garland Publishing, New York, N.Y.
15. Jardim, A., J. Alexander, H. S. The, D. Ou, and R. W. Olafson. 1990. Immunoprotective Leishmania major synthetic T cell epitopes. J. Exp. Med. 172:645-648.[Abstract/Free Full Text]
16. Joshi, P. B., B. L. Kelly, S. Kamhawi, D. L. Sacks, and W. R. McMaster. 2002. Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor. Mol. Biochem. Parasitol. 120:33-40.[CrossRef][Medline]
17. Kurtzhals, J. A., A. S. Hey, A. Jardim, M. Kemp, K. U. Schaefer, E. O. Odera, C. B. Christensen, J. I. Githure, R. W. Olafson, T. G. Theander, and A. Kharazmi. 1994. Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2-like response to gp63 and Th1-like response to lipophosphoglycan-associated protein in cells from cured visceral leishmaniasis patients. Clin. Exp. Immunol. 96:416-421.[Medline]
18. Launois, P., A. Gumy, H. Himmelrich, R. M. Locksley, M. Rocken, and J. A. Louis. 2002. 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 lesions. J. Immunol. 168:4628-4635.[Abstract/Free Full Text]
19. Launois, P., F. Tacchini-Cottier, C. Parra-Lopez, and J. A. Louis. 1998. Cytokines in parasitic diseases: the example of cutaneous leishmaniasis. Int. Rev. Immunol. 17:157-180.[Medline]
20. Launois, P., I. Maillard, S. Pingel, K. G. Swihart, I. Xenarios, H. Acha-Orbea, H. Diggelmann, R. M. Locksley, H. R. MacDonald, and J. A. Louis. 1997. IL-4 rapidly produced by Vß4 V{alpha}8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541-549.[CrossRef][Medline]
21. LeMeur, M., P. Gerlinger, C. Benoit, and D. Mathis. 1985. Correcting an immune-response deficiency by creating E{alpha} gene transgenic mice. Nature 316:38-42.[CrossRef][Medline]
22. Lima, H. C., J. Bleyenberg, and R. G. Titus. 1997. A simple method for quantifying Leishmania in tissues of infected animals. Parasitol. Today 13:80-82.[CrossRef][Medline]
23. Louis, J. A., E. Moedder, H. R. MacDonald, and H. D. Engers. 1981. Recognition of protozoan parasites by murine T lymphocytes. II. Role of the H-2 gene complex in interactions between antigen-presenting macrophages and Leishmania-immune T lymphocytes. J. Immunol. 126:1661-1666.[Abstract]
24. Louis, J. A., R. H. Zubler, S. G. Coutinho, G. Lima, R. Behin, J. Mauel, and H. D. Engers. 1982. The in vitro generation and functional analysis of murine T cell populations and clones specific for a protozoan parasite, Leishmania tropica. Immunol. Rev. 61:215-243.[CrossRef][Medline]
25. McMaster, W. R., C. J. Morrison, M. H. MacDonald, and P. B. Joshi. 1994. Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases. Parasitology 108(Suppl.):S29-S36.
26. Mosmann, T. R., and R. L. Coffman. 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145-173.[CrossRef][Medline]
27. Mougneau, E., F. Altare, A. E. Wakil, S. Zheng, T. Coppola, Z. Wang, R. Waldmann, R. M. Locksley, and N. Glaichenhaus. 1995. Expression cloning of a protective Leishmania antigen. Science 268:563-566.[Abstract/Free Full Text]
28. Muller, I., and J. A. Louis. 1989. Immunity to experimental infection with Leishmania major: generation of protective L3T4+ T cell clones recognizing antigen(s) associated with live parasites. Eur. J. Immunol. 19:865-871.[Medline]
29. Noben-Trauth, N., W. E. Paul, and D. L. Sacks. 1999. IL-4- and IL-4 receptor-deficient BALB/c mice reveal differences in susceptibility to Leishmania major parasite substrains. J. Immunol. 162:6132-6140.[Abstract/Free Full Text]
30. Nordan, R. P., C. D. Richards, and J. Gauldie. 1994. Measurement of interleukin 6, p. 6.6.1-6.6.5. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology. John Wiley & Sons, Inc., New York, N.Y.
31. Reiner, S. L., S. Zheng, Z. Wang, L. Stowring, and R. M. Locksley. 1994. Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection. J. Exp. Med. 179:447-456.[Abstract/Free Full Text]
32. Reiner, S. L., Z.-E. Wang, F. Hatam, P. Scott, and R. M. Locksley. 1993. TH1 and TH2 cell antigen receptors in experimental leishmaniasis. Science 259:1457-1460.[Abstract/Free Full Text]
33. Rivier, D., P. Bovay, R. Shah, S. Didisheim, and J. Mauel. 1999. Vaccination against Leishmania major in a CBA mouse model of infection: role of adjuvants and mechanism of protection. Parasite Immunol. 21:461-473.[CrossRef][Medline]
34. Rogers, K. A., G. K. DeKrey, M. L. Mbow, R. D. Gillespie, C. I. Brodskyn, and R. G. Titus. 2002. Type 1 and type 2 responses to Leishmania major. FEMS Microbiol. Lett. 209:1-7.[CrossRef][Medline]
35. Russell, D. G., and J. Alexander. 1988. Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes. J. Immunol. 140:1274-1279.[Abstract]
36. Sacks, D. L., S. Hieny, and A. Sher. 1985. Identification of cell surface carbohydrate and antigenic changes between noninfective and infective developmental stages of Leishmania major promastigotes. J. Immunol. 135:564-569.[Abstract]
37. Scott, P. 1991. IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis. J. Immunol. 147:3149-3155.[Abstract]
38. Scott, P., P. Caspar, and A. Sher. 1990. Protection against Leishmania major in BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular weight antigen released by promastigotes. J. Immunol. 144:1075-1079.[Abstract]
39. Scott, P., P. Natovitz, R. L. Coffman, E. Pearce, and A. Sher. 1988. Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper cell subsets and respond to distinct parasite antigens. J. Exp. Med. 168:1675-1684.[Abstract/Free Full Text]
40. Shankar, A. H., and R. G. Titus. 1995. T cell and non-T cell compartments can independently determine resistance to Leishmania major. J. Exp. Med. 181:845-855.[Abstract/Free Full Text]
41. Shreffler, W. G., J. M. Burns, R. Badaro, H. W. Ghalib, L. L. Button, W. R. McMaster, and S. G. Reed. 1993. Antibody responses of visceral leishmaniasis patients to gp63, a major surface glycoprotein of Leishmania. J. Infect. Dis. 167:426-430.[Medline]
42. Soares, L. R., E. E. Sercarz, and A. Miller. 1994. Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression. Int. Immunol. 6:785-794.[Abstract/Free Full Text]
43. Soares, M. B. P., J. R. David, and R. G. Titus. 1997. An in vitro model for infection with Leishmania major that mimics the immune response in mice. Infect. Immun. 65:2837-2845.[Abstract]
44. Spitzer, N., A. Jardim, D. Lippert, and R. W. Olafson. 1999. Long-term protection of mice against Leishmania major with a synthetic peptide vaccine. Vaccine 17:1298-1300.[CrossRef][Medline]
45. Sypek, J. P., C. L. Chung, S. E. Mayor, J. M. Subramanyam, S. J. Goldman, D. S. Sieburth, S. F. Wolf, and R. G. Schaub. 1993. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J. Exp. Med. 177:1797-1802.[Abstract/Free Full Text]
46. Theodos, C. M., J. M. C. Ribeiro, and R. G. Titus. 1991. Analysis of the enhancing effect of sand fly saliva on Leishmania infection in mice. Infect. Immun. 59:1592-1598.[Abstract/Free Full Text]
47. Theodos, C. M., D. L. Sacks, W. R. McMaster, and R. G. Titus. 1992. Analysis of the antigenic specificity of T cell clones derived from L. major-infected, genetically-resistant C3H mice. FASEB J. 6:A1797.
48. Titus, R. G., G. Lima, H. D. Engers, and J. A. Louis. 1984. Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity. J. Immunol. 133:1594-1600.[Abstract]
49. Titus, R. G., I. Muller, P. Kimsey, A. Cerny, R. Behin, R. M. Zinkernagel, and J. A. Louis. 1991. Exacerbation of experimental murine cutaneous leishmaniasis with CD4+ Leishmania major-specific T cell lines or clones which secrete interferon-{gamma} and mediate parasite-specific delayed-type hypersensitivity. Eur. J. Immunol. 21:559-567.[Medline]
50. Vinhas, V., M. Freire, O. Bacellar, S. Cunha, H. Rocha, and E. M. Carvalho. 1994. Characterization of T cell responses to purified leishmania antigens in subjects infected with Leishmania chagasi. Braz. J. Med. Biol. Res. 27:1199-1205.[Medline]
51. Yang, D. M., N. Fairweather, L. L. Button, W. R. McMaster, L. P. Kahl, and F. Y. Liew. 1990. Oral Salmonella typhimurium (AroA–) vaccine expressing a major leishmanial surface protein (gp63) preferentially induces T helper 1 cells and protective immunity against leishmaniasis. J. Immunol. 145:2281-2285.[Abstract]

Infection and Immunity, August 2004, p. 4486-4493, Vol. 72, No. 8
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.8.4486-4493.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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