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The Journal of Immunology, 2001, 166: 4596-4603.
Copyright © 2001 by The American Association of Immunologists

Antigen-Specific Th1 But Not Th2 Cells Provide Protection from Lethal Trypanosoma cruzi Infection in Mice1

Sudesh Kumar and Rick L. Tarleton2

Department of Cellular Biology, University of Georgia, Athens, GA 30602


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4+ T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 107 OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The protozoan parasite Trypanosoma cruzi causes Chagas’ disease in humans and is a major public health problem in Latin America. It is estimated that 18 million people are infected with the parasite and 90 million people are at risk of infection (1). Although vector control programs and the application of chemotherapeutics are partial solutions to control of the infection and disease, these approaches alone are unlikely to be long-term solutions to combating Chagas’ disease. Vaccination also has potential for reducing the severity of T. cruzi infection Chagas’ disease (2, 3, 4). The design of appropriate vaccination strategies against T. cruzi requires the elucidation of the mechanisms for immune protection. Experimental T. cruzi infection in murine models has provided the means for the identification of these protective immune mechanisms operating against the parasite.

In infected hosts, T. cruzi circulates in the blood as nonreplicating trypomastigote forms that invade a wide variety of cells and subsequently multiply intracellularly as amastigotes. Both amastigotes and trypomastigotes elicit a complex pattern of immune responses including substantial Ab production and cellular responses mediated by CD4+ and CD8+ T cells (5). In the absence of B cells, CD4+ T cells, or CD8+ T cells, mice infected with T. cruzi develop high tissue parasite burden and die early in infection (6, 7, 8, 9, 10, 11, 12). Similar to infections with other intracellular pathogens (Leishmania (13), Mycobacterium (14), and Listeria (15)), where a strong Th1 response protects whereas a Th2 response increases susceptibility to infection (16, 17), there is some evidence for a protective role of Th1 cells (18) and an exacerbative role for Th2 cells (19) in T. cruzi infection. Production of the type 1 cytokine, IFN-{gamma}, in the acute phase of T. cruzi infection is associated with resistance (20, 21, 22) and depletion of IFN-{gamma} exacerbates parasitemia and results in increased mortality in T. cruzi-infected mice (23, 24). Similarly, IL-12, an inducer of the type 1 cytokine response, promotes resistance to T. cruzi in murine models (25). In contrast, IL-10, a cytokine suggested to induce type 2 response, has been linked to susceptibility to T. cruzi in several murine models (26) with an elevation of IL-10 production in susceptible mice strains compared with resistant strains (27).

To investigate further the role of Ag-specific Th1/Th2 cells in T. cruzi infection, we have developed a system by which we can generate and transfer parasite-specific Th1 and Th2 cell populations and determine their ability to protect naive mice from lethal T. cruzi infection. This system uses T. cruzi lines expressing chicken OVA as a source of infective parasites and OVA-specific Th1 and Th2 cells from DO11.10 TCR transgenic mice as the source of parasite-specific T cells (28). The adoptive transfer of OVA-specific Th1 cells protected mice, whereas transfer of Th2 cells reversed the protective effect of Th1 cells transfer in mice infected with a lethal dose of OVA-expressing T. cruzi. Immunohistochemical analysis of spleens, lymph nodes, and skeletal muscle of recipient mice showed that OVA-specific Th1 and Th2 cells persisted and expanded in vivo in response to OVA-expressing T. cruzi and not in response to wild-type parasites. These results suggest that a primed Th1 response and the absence of a Th2 response provide optimal control of T. cruzi infection.


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

Mice transgenic for the DO11.10 TCR (I-Ad restricted and OVA specific) were obtained from Dr. D. Loh (Washington University School of Medicine, St. Louis, MO) and wild-type BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Epimastigotes of T. cruzi (Brazil strain) were cultured at 28°C in liver infusion tryptose (LIT)3 broth supplemented with 5% heat-inactivated FBS (HyClone, Logan, UT). Infective metacyclic trypomastigotes were obtained from 4- to 5-wk-old stationary-phase cultures of epimastigotes. Vero cells (African green monkey kidney fibroblasts; American Type Culture Collection, Manassas, VA) were infected with the infective metacyclic trypomastigotes to obtain fibroblast-derived trypomastigotes. Blood-form trypomastigotes (BFTs) were maintained by biweekly passages in C3HHe/SnJ mice and were used for infection of mice. Trypomastigotes were converted to amastigotes extracellularly in LIT broth for flow cytometric analysis (29).

Plasmid

Plasmid pHD421{beta} containing the Trypanosoma brucei {beta}-tubulin gene (a gift from Dr. Elizabeth Wirtz, Rockefeller University, NY) was modified by replacing the T. brucei {beta}-tubulin gene with ~0.7 kb of the {beta}-tubulin gene of T. cruzi (Brazil strain). A forward primer (5'-GGTACCTGTATTGAAATGAAGCCCTGT-3') was designed at position 601 of the T. cruzi {beta}-tubulin gene to add a KpnI site and a reverse primer (5'-CTCGACCTTCCTCCTCAATGGTGGCGGTC-3') was designed at position 1300 to add a XhoI site. These primers were used to amplify the {beta}-tubulin gene from T. cruzi (Brazil strain) genomic DNA and the amplified product was cloned in pHD421{beta} at KpnI and XhoI sites. The luciferase gene from the resulting plasmid was then removed by restriction digestion with HindIII and BamHI enzymes and replaced by a G-OVA.GPI construct (30), encoding the N-terminal signal sequence of T. cruzi glycoprotein gp-72 (aa 1–47); aa 139–357 of chicken OVA followed by 45 amino acids of amastigote surface protein 1 providing a C-terminal GPI cleavage/attachment site to yield pHD421{beta} G-OVA.GPI.

Transfection of T. cruzi and generation of T. cruzi G-OVA.GPI

Mid-log phase T. cruzi epimastigotes were transfected with 25 µg of the pHD421{beta} G-OVA.GPI plasmid linearized at a unique NotI site in the T. cruzi {beta}-tubulin gene to allow for homologous recombination in one of the T. cruzi {beta}-tubulin genes loci. Hygromycin was added to a final concentration of 1.0 mg/ml after 48 h of incubation at 28°C for selection of transfectants. Clones of T. cruzi G-OVA.GPI were selected that continued to grow in the presence of hygromycin (1.0 mg/ml) at rates similar to those of wild-type T. cruzi growing in drug-free medium. After 4 wk of drug selection, the parasites were moved to drug-free medium.

Flow cytometric analysis of T. cruzi G-OVA.GPI

Amastigotes of T. cruzi were washed in PBS containing 0.1% sodium azide and 0.1% casein (PAC). In some experiments, parasites were also treated with 1 x 10-2 U of Bacillus cereus phosphatidylinositol-specific phospholipase C (PIPLC; Boehringer Mannheim, Indianapolis, IN) in 100 µl of PIPLC buffer (30). For flow cytometric analysis, 1 x 106 parasites were suspended in 50 µl of PAC containing rabbit anti-OVA Ab (1/200) (Sigma, St. Louis, MO) for 30 min at 4°C. After washing with 1 ml of PAC containing 0.01% Tween 20, the parasites were incubated with FITC-labeled goat F(ab')2 anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL) (1/50 dilution in PAC) at 4°C for 30 min in the dark. Cells were then washed once with PAC containing Tween 20, resuspended in 250 µl of PAC, and analyzed by flow cytometry on an EPICS Elite Analyzer (Coulter Pharmaceutical, Hialeah, FL).

Generation of OVA-specific Th1 and Th2 cells

Splenocytes from DO11.10 transgenic mice were depleted of RBCs by hypotonic lysis and were cultured at 5 x 106 cells/well in 2 ml complete RPMI 1640 medium (Mediatech, Herndon, VA) containing 10% FBS (HyClone) in 24-well plates. IL-2 (20 U/ml; Cetus Corporation, Emeryville, CA), IL-12 (10 µg/ml; Genetics Institute, Cambridge, MA), and anti-IL-4 mAb 11B11 (10 µg/ml) were added to cultures to generate Th1 cells, and IL-4 (100 U/ml; DNAX, Palo Alto, CA) and anti-IFN-{gamma} mAb R4-6AB (75 µg/ml) were added to generate Th2 cells. All wells also received OVA peptide (0.3 µM) containing aa 323–339 (SQAVHAAHAEINEAGRE) of chicken OVA protein (31). After 4 days of stimulation, the frequency of cells expressing OVA TCR was determined by flow cytometric analysis using OVA TCR-specific mAb KJ1-26 (32) (obtained from Dr. John Kappler, University of Colorado Health Science Center, Denver, CO). Th1 and Th2 cells (2.5 x 105) were restimulated in 2-ml cultures with 4.5 x 106 of RBC-depleted irradiated (2600 rad) H-2d BALB/c splenocytes and OVA peptide. Supernatants from these cultures were collected after 48 h and cytokine levels assayed by ELISA for IL-4 and IFN-{gamma}. IFN-{gamma} in the supernatant fluids was measured by ELISA as previously described (21), and IL-4 levels were determined using a commercial kit (BD PharMingen, San Diego, CA) following the manufacturers instructions.

Adoptive transfer of OVA-specific Th1 and Th2 cells

OVA-specific Th1 and Th2 cells were purified over lymphocyte separation medium (ICN Biochemicals, Aurora, OH), and 107 cells resuspended in 0.5 ml of DMEM (Life Technologies, Grand Island, NY) were transferred in naive BALB/c mice by injection into the tail vein. Control animals received DMEM alone. Mice (eight in each group) were infected with 5 x 104 or 105 BFT of T. cruzi by i.p. injection 12 h after the injection of T cells. Parasitemias were monitored at weekly intervals by hemacytometer counts of parasites in tail blood and mortality was recorded daily.

Flow cytometric analysis of cells for intracellular cytokines

Flow cytometric analysis of splenocytes from recipients of Th1 or Th2 cells was accomplished using biotinylated KJ1-26 mAb plus streptavidin-cy7 (Molecular Probes, Eugene, OR) to identify OVA-specific CD4+ T cells. Detection of intracellular IFN-{gamma} and IL-4 was done using PE-labeled anti-IFN-{gamma} mAb R46A-2 and anti-IL-4 mAb 11B11, respectively (BD PharMingen), in single cell suspension of splenocytes in PAC buffer using Cytoperm/Cytofix (with GolgiPlug) kit (BD PharMingen) per the manufacturer’s instructions.

Histology and immunohistochemistry

Cardiac and skeletal muscle tissues were collected at 15 and 30 days postinfection in PBS and fixed in 10% buffered formalin. Sections (5 µm) from paraffin-embedded tissues were stained with hematoxylin and eosin for histopathological analysis. To detect OVA-specific Th1 and Th2 cells posttransfer, lymph nodes, spleens, and skeletal muscle tissues from recipient mice were frozen in liquid nitrogen and 5- to 10-µm-thick sections were analyzed as previously described with some modifications (33). Briefly, acetone-fixed tissue sections were quenched with PBS containing 0.3% of H2O2 and 0.1% of sodium azide and incubated with biotinylated KJ1-26 Ab in PBS at 4°C overnight. Enhanced color development was achieved using HRP and biotinyl tyramide (TSA-Indirect; NEN, Boston MA) following the manufacturer’s instructions. Color was developed using diaminobenzidine (Sigma).

Quantitation of T. cruzi DNA in the tissues

DNA was isolated from proteinase K-treated (0.3 mg/ml in proteinase K buffer) skeletal muscle tissue by phenol-chloroform-isoamyl alcohol (Sigma) extraction and ethanol precipitation as described previously (34). DNA in the tissues was quantitated by a real-time PCR protocol (K. Cummings and R. L. Tarleton, manuscript in preparation) using a LightCycler (Roche Diagnostic Systems, Indianapolis, IN). Data acquisition and analysis was performed using LightCycler version 3.0 software. Standards of serially diluted T. cruzi DNA mixed with skeletal muscle DNA were used for quantification of samples. Standard curves generated were then used to determine parasite equivalents per 50 µg of tissue DNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the absence of a clonal population of CD4+ T cells that can recognize specific Ags expressed by T. cruzi, it is very difficult to directly analyze the role of Ag-specific Th1 and Th2 cells in susceptibility or exacerbation of T. cruzi infection and Chagas’ disease. Therefore, we developed a system that made use of DO11.10 OVA TCR transgenic mice as donors of Th cells and T. cruzi expressing the OVA protein as targets of OVA-specific Th1 and Th2 cells.

Generation of OVA-expressing T. cruzi G-OVA.GPI

We have previously reported the generation of T. cruzi expressing both GPI-anchored (surface expressed) and secreted forms of OVA (30). In addition, OVA secreted by intracellular parasites was processed and presented in association with class I MHC on the surface of infected host cells (30). However, these OVA-expressing T. cruzi were not deemed useful for in vivo infection because continuous OVA expression depended upon continuous drug pressure on the parasites. To generate stable OVA-expressing T. cruzi, a plasmid pHD421{beta}G-OVA.GPI containing the T. cruzi (Brazil strain) {beta}-tubulin gene and G-OVA.GPI was constructed and electroporated into epimastigotes of T. cruzi for stable integration into one of the {beta}-tubulin gene loci (Fig. 1GoA). Trypomastigotes of T. cruzi G-OVA.GPI were generated by infection of VERO cells by metacyclic phase parasites and were converted into amastigotes by overnight incubation in LIT medium. Surface expression of OVA by the amastigotes of T. cruzi G-OVA.GPI was confirmed by FACS analysis using a polyclonal rabbit anti-chicken OVA Ab (Fig. 1GoB). The attachment of OVA via a GPI anchor was indicated by the fact that treatment of T. cruzi G-OVA.GPI with PIPLC resulted in loss of surface expression of OVA (Fig. 1GoB). Stable expression of OVA was also documented by FACS analysis of parasites after passage of T. cruzi G-OVA.GPI through mice, after 5 mo of culture in drug-free medium, and by the presence of anti-OVA Abs in mice infected with T. cruzi G-OVA.GPI (data not shown). These results established that OVA was being expressed by T. cruzi G-OVA.GPI and, thus, could potentially be presented to OVA-specific Th1 and Th2 cells in vivo during infection.



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  		FIGURE 1. Generation of OVA-expressing T. cruzi. A, The plasmid pHD421{beta} G-OVA.GPI containing G-OVA.GPI insert (flanked by 5'- and 3'-untranslated region of T. brucei aldolase) and the hygromycin resistance gene (flanked by 5'- and 3'-untranslated region of T. brucei actin) was linearized at a unique NotI site in {beta}-tubulin sequence and electroporated into epimastigotes of T. cruzi (Brazil strain) to allow for stable integration. B, Amastigotes of wild-type T. cruzi or OVA-expressing (G-OVA. GPI) T. cruzi were stained with a rabbit anti-OVA Ab followed by FITC-labeled goat anti-rabbit IgG and analyzed by FACS. Staining of OVA-expressing T. cruzi with FITC-labeled goat anti-rabbit IgG is shown for reference. Some parasites were treated with PIPLC to cleave the GPI anchor, thus releasing OVA from the parasite surface.

 
Generation of OVA-specific Th1 and Th2 cells

CD4+ T cells in DO11.10 TCR transgenic mice express a clonotypic TCR that recognizes peptide fragment 323–339 from chicken OVA protein in association with class II MHC molecules (H-2d). Th1 and Th2 subpopulations of OVA-specific CD4+ T cells were generated by in vitro stimulation of DO11.10 splenocytes with OVA peptide plus IL-2, IL-12, and anti-IL-4 Ab (for Th1) or IL-4 and anti-IFN-{gamma} Ab (for Th2). FACS analysis of cells after 4 days of culture using the TCR-specific KJ1-26 mAb showed that >90% of the cells expressed the clonotypic TCR (Fig. 2GoA). Phenotypic analysis of Th1 and Th2 cells was accomplished by measuring production of IFN-{gamma} and IL-4 cytokines, after incubation with irradiated APCs. OVA peptide-stimulated Th1 cells produced predominantly IFN-{gamma} but no IL-4, whereas Th2 cells produced IL-4 but not IFN-{gamma} (Fig. 2Go, B and C). No IFN-{gamma} and IL-4 was produced by Th1 and Th2 cells in the absence of OVA peptide stimulation (Fig. 2Go, B and C).



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  		FIGURE 2. Generation and phenotypic analysis of OVA-specific Th1 and Th2 cells. DO11.10 splenocytes were stimulated with IL-2 plus IL-12 and anti-IL-4 Ab 11B11 to generate Th1 cells or IL-4 and anti-IFN-{gamma} Ab R4-6A2 to generate Th2 cells. OVA peptide was added to all cultures. A, After 4 days of culture, >90% of the cells expressed the OVA-specific clonotypic TCR. Control Ab is FITC-labeled goat anti-rabbit IgG. B and C, Th1 and Th2 cells incubated with irradiated splenocytes plus OVA peptide secreted IFN-{gamma} or IL-4, respectively (mean ± SE).

 
Adoptive transfer of OVA-specific Th1 cells but not Th2 cells protects naive mice from infection with T. cruzi G-OVA.GPI

The ability of Ag-specific Th1 and Th2 cells to modulate the outcome of a lethal T. cruzi infection was investigated by adoptive transfer of Th1 and Th2 cells to naive BALB/c mice and then challenge of these mice with a lethal dose of T. cruzi G-OVA.GPI. Mice infected with 5 x 104 BFT of either T. cruzi G-OVA.GPI or wild-type T. cruzi developed similar parasitemia levels and died by day 42 postinfection suggesting that the OVA-transgenic parasites were equally virulent as wild-type parasites (Fig. 3Go, A and B). Transfer of OVA-specific Th1 cells protected mice from lethal infection with T. cruzi G-OVA.GPI but not from infection with wild-type T. cruzi. Parasitemias in these protected animals were below the level of detection by 49 days after infection and 80% of the mice survived the infection until 200 days postinfection when the experiment was terminated. In contrast, mice receiving Th2 cells developed high parasitemias and died between days 23 and 43 after infection with either T. cruzi G-OVA.GPI or wild-type T. cruzi (Fig. 3Go, A and B). Thus, transfer of OVA-specific Th1 cells conferred resistance in susceptible BALB/c mice to lethal T. cruzi infection. This protection occurred in an Ag-specific manner because mice receiving Th1 cells and infected with T. cruzi G-OVA.GPI were protected, whereas Th1 cells recipients infected with wild-type T. cruzi were not.



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  		FIGURE 3. Protection conferred by adoptive transfer of OVA-specific Th1 or Th2 cells in mice followed 12 h later by infection with 5 x 104 BFT of T. cruzi G-OVA.GPI or wild-type (WT) T. cruzi. Five mice per group receiving 107 Th1 or Th2 cells each were challenged with T. cruzi. Parasites in the blood were counted weekly (A) and mortality recorded daily (B).

 
Histology

Histopathological analysis of tissues at 30 days postinfection revealed higher tissue parasitism and more severe pathology in skeletal muscles than in the hearts of mice in all groups (data not shown). Skeletal muscle of Th1 cell-recipient mice showed mild to moderate inflammation with a predominant lymphocytic infiltration and relatively lower tissue parasitism (Fig. 4GoA). In contrast, the skeletal muscles of Th2 cell-recipient mice were heavily parasitized with severe inflammation consisting predominantly of polymorphonuclear cells and significant tissue destruction and necrosis (Fig. 4GoB). Mice that received Th1 or Th2 cells and were then infected with wild-type T. cruzi showed high tissue parasitism and moderate to severe inflammation in the skeletal muscles (Fig. 4Go, C and D). The differential control of tissue parasite load in Th1- and Th2-recipient mice infected with T. cruzi GOVA.GPI was confirmed by quantitation of parasite DNA by real-time PCR. Recipients of Th2 cells had significantly higher amounts of T. cruzi DNA in the skeletal muscle tissue compared with Th1 cell recipients after infection with T. cruzi GOVA.GPI (Fig. 5Go). Thus, although the Th2 cell-recipient mice mounted an intense inflammatory response, this response was clearly less effective in controlling parasites in the tissues of these mice. We conclude from these results that Ag-specific Th2 cells were incapable of mediating clearance of infection with T. cruzi from the tissues despite eliciting a strong inflammatory response.



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  		FIGURE 4. Histological analysis of skeletal muscle tissue 30 days postinfection in mice receiving Th1 or Th2 cells. Hematoxylin and eosin stained tissues from Th1 (A) or Th2 (B) cell recipient mice infected with T. cruzi G-OVA.GPI. Tissues from Th1 (C) or Th2 (D) cell recipient mice infected with wild-type T. cruzi. Arrows in B–D indicate pseudocysts of intracellular amastigotes.

 


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  		FIGURE 5. Parasite load in skeletal muscle tissue. At 30 days postinfection, T. cruzi kDaNA was quantitated (mean ± SE) by real-time PCR from skeletal muscle tissue of mice receiving Th1 cells, Th2 cells, or no cells and infected with T. cruzi G-OVA.GPI or wild-type (WT) T. cruzi.

 
OVA-specific Th1 and Th2 cells persist and expand in response to T. cruzi G-OVA.GPI infection in vivo

The OVA system provided a tool to monitor the distribution and expansion of parasite-specific T cells in infected mice. For this purpose, the OVA-TCR-specific mAb KJ1-26 was used to detect OVA-specific Th1 and Th2 cells in the spleen and lymph nodes of mice infected either with T. cruzi G-OVA.GPI or wild-type T. cruzi. KJ1-26-positive cells were present in small clusters around the arterioles at day 4 (Fig. 6Go, C and E) and day 15 postinfection (data not shown) in spleens of mice receiving Th1 cells and infected with either OVA-expressing or wild-type T. cruzi. At 30 days postinfection, there was an increase in the frequency of KJ1-26-positive cells in spleens of mice receiving Th1 cells and infected with T. cruzi G-OVA.GPI and these cells were localized in T cell-rich areas around the lymphoid follicles (Fig. 6GoD). However, KJ1-26-positive cells could not be detected in the spleens of Th1 cell recipients infected with wild-type T. cruzi at 30 days postinfection (Fig. 6GoF). Similar persistence and expansion of OVA-specific cells was observed in the spleens of recipients of Th2 cells challenged with T. cruzi G-OVA.GPI (Fig. 7Go, A and B) but not in mice receiving Th2 cells and challenged with wild-type T. cruzi (Fig. 7Go, C and D).



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  		FIGURE 6. OVA-specific Th1 cells expand in vivo in response to infection with T. cruzi G-OVA.GPI. Spleens from Th1 cell-recipient mice at 4 and 30 days posttransfer and infection were stained for OVA-specific Th1 cells using anti-OVA TCR Ab KJ1-26. Spleens from OVA TCR transgenic DO11.10 mice stained with KJ1-26 MAb (A) or with mouse anti-rat IgG Ab (B) were used as positive and negative staining controls, respectively. KJ1-26-positive cells were apparent in the spleens of Th1-recipient mice infected with either T. cruzi G-OVA.GPI (C) or wild-type T. cruzi (E) at 4 days postinfection. Further expansion of the KJ1-26-positive cells are obvious 30 days postinfection in the spleens of Th1 cell recipients infected with T. cruzi G-OVA (D) but no KJ1-26-positive cells were detectable in spleens of Th1-recipient infected with wild-type T. cruzi (F).

 


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  		FIGURE 7. Th2 cells persist and expand in the spleen selectively in response to OVA-expressing parasites. Similar to Fig. 5Go, at 4 days postinfection, Th2 cells are detected in the spleens of mice infected with T. cruzi G-OVA.GPI (A) and wild-type T. cruzi (C). But at 30 days postinfection, expansion of these cells could only be detected in the spleens of T. cruzi G-OVA.GPI-infected mice (B) and not in the spleens of mice infected with wild-type T. cruzi (D).

 
To assess whether the transferred Th1 or Th2 cells continued to maintain their respective cytokine production phenotype after transfer, flow cytometric analysis was used to monitor intracellular IFN-{gamma} and IL-4 production in KJ1-26+ cells from spleens (Fig. 8Go). At 4 days postinfection, KJ1-26+ Th1 or Th2 cells largely retained their respective restriction in production IFN-{gamma} or IL-4, respectively. This was also the case at day 15 postinfection; however, at this time point, an increasing proportion of KJ1-26+ cells from Th1 or Th2 recipients produced IL-4 or IFN-{gamma}, respectively. It is also noteworthy that in the Th2 recipients, a lower percentage of KJ1-26-negative cells produced IFN-{gamma} compared with Th1 recipients. One interpretation of these data is that the transferred Th1 and Th2 cells influenced the cytokine production pattern of cells responding to parasite Ags other than OVA.



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  		FIGURE 8. Cytokine production by OVA-specific Th1 and Th2 cells in vivo. Splenocytes collected from Th1 and Th2 cell-recipient mice infected with T. cruzi GOVA.GPI at 4 and 15 days postinfection were stimulated with OVA peptide in the presence of GolgiPlug and then stained for the expression of the OVA TCR (KJ1-26) and IFN-{gamma} or IL-4 expression. Data shown are representative of two mice in each group.

 
To determine whether OVA-specific Th1 and Th2 cells migrated to the sites of parasite multiplication in muscle tissue, skeletal muscle from mice at 30 days postinfection were stained for the presence of OVA-specific T cells (Fig. 9Go). Tissues from mice infected with T. cruzi G-OVA.GPI (Fig. 9Go, A and B) but not those from mice infected with wild-type T. cruzi (Fig. 9Go, C and D) revealed the presence of OVA-specific Th1 or Th2 cells. These results demonstrate the Ag-driven proliferation of OVA-specific Th1 and Th2 cells in the lymphoid organs, the maintenance of type1 and type 2 phenotypes, and the homing of these cells to sites of active infection.



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  		FIGURE 9. OVA-specific Th1 and Th2 cells are present in the skeletal muscle lesions in T. cruzi G-OVA.GPI-infected mice. At 30 days after infection, KJ1-26-positive cells are detected in the skeletal muscles of Th1 (A) and Th2 (B) cell-recipient mice infected with T. cruzi G-OVA.GPI but not in mice receiving Th1 (C) or Th2 (D) cells and infected with wild-type T. cruzi.

 
Adoptive transfer of OVA-specific Th2 cells abrogates the protective effect of Th1 cells against T. cruzi G-OVA.GPI infection

The data above demonstrated that Th1 but not Th2 cells help mediate protection in T. cruzi-infected mice. The Th cell response to T. cruzi in both susceptible and resistant strains of mice normally exhibits a mixed Th1/Th2 cytokine production profile (33). Thus, it was of interest to determine whether the adoptive transfer of a combination of Th1 and Th2 cells or of naive DO.11.10 T cells (that could differentiate into Th1 and Th2 cells in vivo) could confer protection to T. cruzi infection equivalent to that of Th1 cells alone. Adoptive transfer of OVA-specific Th1 plus Th2 cells (5 x 106 each) or 107 naive DO.11.10 cells failed to provide protection in mice challenged with 105 BFT of T. cruzi G-OVA.GPI (Fig. 10GoA). One possible explanation for these results is that 5 x 106 Th1 cells may not be sufficient to provide the level of protection that 107 Th1 cells do. To address this possibility, we compared the effect of transfer of 5 x 106 Th1 or Th2 cells with that of Th1 plus Th2 cells (5 x 106 each). Mice receiving 5 x 106 Th1 or Th2 cells developed similar levels of parasitemia and mortality as mice receiving 107 Th1 or Th2 cells, whereas mice receiving Th1 plus Th2 cells again succumbed to the acute infection (Fig. 10GoB). These results indicate that adoptive transfer of parasite-specific Th1 cells provides significant protection from lethal T. cruzi infection in naive mice but cotransfer of Th2 cells abrogated this protective ability of Th1 cells. The protection provided by Th1 cells was again Ag specific and Ag dependent as demonstrated by the fact that mice receiving OVA-specific Th1 cells and challenged with wild-type T. cruzi were not protected.



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  		FIGURE 10. Adoptive transfer of Th2 cells abrogates the protective effect of Th1 cells in mice from T. cruzi G-OVA.GPI infection. Groups of five mice were challenged with 105 BFT of T. cruzi G-OVA.GPI or T. cruzi wild type (WT). A, Mortality in mice receiving no cells, 5 x 106 Th1 cells, 5 x 106 Th2 cells, or 5 x 106 Th1 plus 5 x 106 Th2 cells. B, Mortality in mice receiving no cells, 107 Th1 cells, 107 Th2 cells, 107 naive DO.11 splenocytes, or 5 x 106 Th1 plus 5 x 106 Th2 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The requirement for CD4+ T cells as critical components of a protective immune response against T. cruzi has been established by studies in mice deficient in genes encoding class II MHC or CD4 molecules and thereby lacking CD4+ T cells (7, 8, 11, 35). Mice deficient in CD4+ T cells are highly susceptible to infection with T. cruzi, develop high tissue parasite burden, and die early in the acute phase of the infection with negligible inflammation (11, 36). Resistance to infection with other intracellular pathogens, including Leishmania major (37, 38, 39, 40, 41, 42, 43), Mycobacterium tuberculosis (44, 45, 46), and Listeria monocytogenes (15), has been associated with a strong and polarized Th1 response, whereas a Th2 response has been shown to associate with susceptibility (16, 17). In contrast, a linkage between Th1 responses and resistance has not been so firmly established with respect to T. cruzi infection. The results of some studies suggest a requirement for a balanced type 1 and type 2 response to control infection with T. cruzi (23, 26, 47), while other studies attribute protection to the production of type 1 but not type 2 cytokines in vivo in different mouse and parasite strain combinations (18, 19, 20, 24, 48, 49).

To investigate directly the role of primed Ag-specific Th1 and Th2 cells in modulating the host immune response to T. cruzi, we developed a system by which Th1 or Th2 cells specific for a parasite-expressed Ag could be generated and tested for their ability to protect naive mice from a lethal infection with T. cruzi. We were constrained by the lack of a source for a clonal population of Th1 and Th2 cells specific for a bona fide T. cruzi-derived class II MHC epitope; therefore, we generated T. cruzi that expressed the model Ag chicken OVA and used DO.11 TCR transgenic mice (28) as a source of OVA-specific CD4+ T cells. CD4+ T cells in these mice express a clonotypic TCR that recognizes OVA peptide aa 323–339 in the context of the class II molecules (H-2d). Using the DO.11.10 TCR-specific monoclonal Ab KJ1-26 (32), we were also able to follow the transferred cells in vivo and monitored persistence and expansion of these cells in response to infection with T. cruzi G-OVA.GPI.

Mice receiving OVA-specific Th1 cells controlled the infection with T. cruzi G-OVA.GPI and showed reduced pathology and parasite burden in the skeletal muscles. In contrast, recipients of Th2 cells remained highly susceptible to infection with T. cruzi G-OVA.GPI and developed much higher blood and tissue parasitism than did Th1 recipients. The susceptibility of the Th2 cell-recipient mice was not due to the absence of a potent response to the parasite, either systemically or in infected tissues. Th2-recipient mice exhibited expansion of OVA-specific cells in the lymphoid tissues, the homing of these cells to sites of infection in peripheral tissues, and vigorous inflammatory responses with predominantly polymorphonuclear cells at sites of active infection. However, this response appeared to be relatively unproductive and not sufficient to control parasite replication in the tissues. Consequently, all Th2 cell recipients died in the acute phase of infection, whereas the majority of Th1 cell recipients controlled the infection and became aparasitemic. We demonstrate that this protection occurred in an Ag-specific manner because the mice that received OVA-specific Th1 cells and challenged with OVA-expressing T. cruzi were protected and the mice challenged with wild-type T. cruzi were not.

In naive animals, antigenic stimulation causes an increase in the frequency of clonal populations of T cells specific for their cognate Ags. However, due to the broad repertoire of Ag-specific T cells that are stimulated in a protozoal infection, it is difficult to monitor the change in frequency of individual clones of T cells (50). Hence, the system of adoptive transfer of a clonal population of Ag-specific cells obtained from TCR-transgenic mice and Abs specific for the clonotypic TCR are valuable tools to analyze persistence, activation, and expansion of clonotypic T cells in vivo (51, 52, 53, 54, 55, 56). Using KJ1-26 mAb, we showed persistence and expansion of OVA-specific Th1 and Th2 cells in the spleens, lymph nodes, and skeletal muscle of recipient mice.

A number of studies have suggested that a polyclonal, Ag-nonspecific expansion of the T and B cell compartments occurs in T. cruzi infection in mice (57, 58, 59, 60). These conclusions are based on the normal representation of V{beta} TCR in proliferating CD4+ T cells (60) and on the dramatic expansion of B cells and T cells that does not appear to be specific for T. cruzi Ags (57, 58, 59). The results of the present study suggest that OVA-specific Th1 and Th2 cells did not undergo polyclonal activation but expanded in vivo only in the presence of OVA in an Ag-specific manner. Thus, with respect to primed OVA-specific CD4+ T cells, T. cruzi does not appear to induce a true polyclonal immune response.

The adoptive transfer system described herein provides the means to also follow the fate of naive OVA-specific T cells after infection with wild-type or OVA-expressing T. cruzi to 1) further explore the phenomenon of polyclonal activation and 2) determine whether these cells differentiate into Th1 or Th2 cells during infection. Understanding the development of Th1 and Th2 cells and their relatively ability to control T. cruzi infection and the development of Chagas’ disease is crucial for designing appropriate immune intervention strategies for this infection and disease. Using the Th1/Th2 cell transfer system, we demonstrate that initial priming of a Th1 response is required for control of T. cruzi infection. This conclusion is supported by the recent results of Hoft et al. (48) demonstrating that immunization of mice under conditions that promote a Th1 response results in protection from challenge infection with T. cruzi. In addition, these results confirm and extend the findings from our laboratory that mice lacking the ability to produce Th2 responses (as a result of targeted knock-of the stat6 gene) develop highly efficient immune responses and less sever disease than wild-type mice (61). Thus, Th2 responses are not necessary and in fact are deleterious in the response to T. cruzi. In the future, the Th1/Th2 cell transfer system will allow us to identify ways in which to modulate the course of the immune response to T. cruzi to achieve a strong type-1 biased cytokine response.


   Acknowledgments
 
We thank Nisha Garg for assistance in generation of OVA-expressing T. cruzi, Kara Cummings for help with the real time PCR analysis, and Miriam Postan for histopathological analysis.


   Footnotes
 
1 Supported by National Institutes of Health Grants AI 22070 and AI 33106 (to R.L.T). R.L.T. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology. Back

2 Address correspondence and reprint requests to Dr. Rick L. Tarleton, Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602. Back

3 Abbreviations used in this paper: LIT, liver infusion tryptose; BFT, blood-form trypomastigotes; PAC, PBS containing 0.1% sodium azide and 0.1% casein; PIPLC, phosphatidylinositol-specific phospholipase C. Back

Received for publication February 14, 2000. Accepted for publication January 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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