Infection with the parasite
Trypanosoma cruzi leads to repeated cycles of obligatory intracellular parasite replication by the amastigote form followed by eventual dissemination throughout the host and infection of various cell types by the morphologically distinct trypomastigote. As this process iterates, the number of parasites circulating in the bloodstream increases. This allows for systemic infection and widespread tissue parasitism. Infection is rarely lethal in immunocompetent hosts and, after a characteristic period of acute parasitemia, is usually controlled by a potent immune response. However, sterile cure of infection seldom occurs. Instead, parasites persist in muscle and nerve tissues, leading to chronic inflammation and the formation of Chagas' disease in humans (
24). Of the estimated 18 to 20 million people who are infected, 30 to 40% will develop severe chronic disease symptoms (
9,
41), and 50,000 will die annually. Currently, chemotherapy is problematic, and no effective vaccine exists (
23,
31).
Despite a strong antiparasite immune response,
T. cruzi is able to persist in the majority of hosts. Various groups have reported potential mechanisms of escape from immunity (
8,
25,
30,
42,
43), although the causes of initial
T. cruzi immune escape and ultimate persistence are unknown and debatable. In several infectious disease models, one demonstrated mechanism of parasite escape from host immune responses involves the induction or exploitation of at least one component of the profound regulatory network known as “peripheral tolerance.” Peripheral tolerance is actively sustained by CD4
+ CD25
+ regulatory T cells (Treg cells), which express the regulatory lineage factor Foxp3, comprise 5 to 10% of peripheral CD4
+ T cells in healthy individuals, and are thus known as “natural” or “endogenous” regulatory T cells (
36). Treg cells modulate immune responses not only to self and tumor antigens (
29,
35,
37) but also to exogenous antigens (
19) and infectious agents (
3,
13,
26,
40). Treg cells have the ability to suppress proliferation and IFN-γ production of both CD4
+ and CD8
+ T-cell subsets (
48), downregulate the activation and cytolytic ability of CD8
+ T cells (
32), and modulate humoral immune responses (
54). In suppressing potentially pathological effector T-cell responses during infection, Treg cells serve to limit collateral tissue damage but may also ultimately prevent the clearance of infection by suppressing crucial immune responses (
5).
MATERIALS AND METHODS
Mice, parasites, and infections.
C57BL/6 (Ly 5.2+), B6.SJL (Ly 5.1+), and C3H/HeSnJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were bred and maintained in the AAALAC-approved University of Georgia animal facility in microisolator cages. At 6 to 12 weeks of age, female mice were infected intraperitoneally with blood-form or tissue culture-derived trypomastigotes of the Brazil strain or with blood-form strain Tulahuen parasites (the parasite strain and quantities used are indicated for each experiment). Blood-form Tulahuen trypomastigotes were maintained via serial passage through C3H/HeSnJ mice, and tissue culture-derived Brazil trypomastigotes were created by passage through Vero cell monolayers cultured in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT). Chronically infected mice were used at 5 to 15 months postinfection.
Hybridomas and in vivo depletion with anti-CD25 antibodies.
Anti-interleukin-2 (IL-2) receptor alpha chain-specific B-cell hybridomas (PC61.5.3 and 7D4) were grown in protein-free hybridoma medium II (Invitrogen, Carlsbad, CA). Supernatants were routinely harvested, spun at 400 × g to remove cells and debris, filtered through a 0.45-μm filter, and stored for less than 2 months at 4°C. To precipitate antibodies, an equal amount of ice-cold saturated ammonium sulfate solution was added to the hybridoma supernatant, mixed at 4°C with a stir bar for 12 h, and then centrifuged at 1,500 × g for 30 min at 4°C. Protein pellets were resuspended and dialyzed until isotonic with phosphate-buffered saline (PBS), filter sterilized with a 0.2-μm filter, and stored at −20°C at a concentration of >1.0 mg/ml until use. Mice were depleted of CD25+ cells by intraperitoneal (i.p.) injections of 250 μg to 1,000 μg anti-CD25 monoclonal antibodies (MAbs) prior to or at different intervals during infection (dose frequency and quantity are indicated for each experiment). Flow cytometric assessment of PC61.5.3 MAb-mediated depletion was conducted using fluorescein isothiocyanate (FITC)-conjugated anti-CD25 MAb (7D4 clone; BD Pharmingen, San Diego, CA), and assessment of 7D4 MAb-mediated depletion was conducted using allophycocyanin (APC)-conjugated anti-CD25 MAb (PC61 clone; Caltag, Burlingame, CA).
Extra- and intracellular immunophenotyping, tetramer staining, and flow cytometry.
For direct ex vivo phenotyping, 100 μl of whole blood obtained from retro-orbital puncture using heparinized Natelson blood collection tubes (Fisher, Pittsburgh, PA) or 1 × 10
6 red blood cell (RBC)-depleted single-cell suspensions from spleens, lymph nodes (LN), or muscles were first washed using PAB buffer (1× PBS, pH 7.4, 0.05% azide, and 1% bovine serum albumin [wt/vol]) and then centrifuged at 400 ×
g for 10 min (as for all additional washing steps). In the case of MHC tetramer staining, cells were first incubated for 30 min at 37°C with MHC class I (H2K
b) tetramers synthesized at the Tetramer Core Facility (Emory University, Atlanta, GA) loaded with TSKb18 (peptide sequence, ANYDFTLV) or TSKb20 (sequence, ANYKFTLV) peptide (
25) conjugated to APC or phycoerythrin (PE), respectively. Cells were ultimately stained for surface markers in a final volume of 100 μl PAB containing a 1/50 dilution of FcBlock (BD Pharmingen) and appropriate combinations of vendor-purchased fluorescently labeled monoclonal antibodies (diluted 1/100) specific for the murine surface markers CD3, CD4, CD8, CD11b, CD25, CD45, CD45.1, CD45.2, and CD45R (BD Pharmingen, Caltag) for 20 min on ice in the dark, followed by two washes in PAB and fixation in 2% formaldehyde for at least 15 min and up to 20 h at 4°C. Erythrocytic whole blood was lysed in 500 ml lysis buffer (10 mM HEPES, 0.83% ammonium chloride) for 5 min, followed by two washes in PAB prior to fixation. In some cases, following surface staining, intracellular scurfin was detected using PE-conjugated anti-scurfin antibodies (1/50 dilution) and a staining kit according to the manufacturer's instructions (eBioscience, San Diego, CA). A Cyan (Dako Cytomation, Fort Collins, CO) or FACSCalibur (BD Pharmingen) flow cytometer and FlowJo software (Tree Star, Inc., Ashland, OR) were used for cytometric data collection and analysis.
Lymphocyte isolation from secondary and nonlymphoid tissues.
In most experiments, animals were exsanguinated and, in some cases (as noted), perfused with 10 ml sterile Alsever's buffered salt solution (pH 7.2). Spleens and lymph nodes (superficial inguinal, popliteal, and mesenteric LN) were removed, and sterile, single-cell, RBC-depleted suspensions were prepared as described previously (
22). To aid in the isolation of lymphocytes from muscle tissue after mechanical disruption with forceps and mincing with blades, in most cases muscle tissue was then prepared as stated previously (
26), with minor modifications. Briefly, muscle tissue was incubated in serum-free RPMI medium containing Liberase Blendzyme II (70 μg/ml; Roche, Indianapolis, IN) for 1 h at 37°C. Muscle tissue was dissociated in RPMI medium containing 10% FBS and 80 U/ml DNase I (Roche), and supernatants were washed through 40-μm nylon cell strainers (BD Biosciences, Bedford, MA) to filter debris, followed by staining with PE-conjugated anti-CD45 antibodies (BD Pharmingen) (15 min on ice, followed by two washes with RPMI medium). CD45
+ cells (white blood cells) were enriched to as least 95% purity by fluorescence-activated cell sorting (FACS) using a MoFlo FACS flow cytometer (Dako Cytomation, Fort Collins, CO).
Cell stimulation, intracellular cytokine staining, and analysis.
A previously described method (
22) was used for IFN-γ detection. Briefly, 1 × 10
6 RBC-depleted splenocytes were cultured overnight in 0.2 ml RPMI 1640 supplemented with 10% FBS in 96-well flat-bottomed plates that had been coated previously with 30 μg/ml anti-CD3 (145-2C11). In the case of peptide stimulations, splenocytes were incubated for 5 h in 96-well round-bottomed plates in a total volume of 0.2 ml RPMI medium plus 10% FBS containing a 1:1,000 dilution of Golgi Plug (BD Pharmingen) and a 2.5 μM concentration of the indicated peptide, phorbol myristate acetate (PMA)-ionophore (50 ng/ml and 500 ng/ml, respectively), or medium only. In vitro-cultured splenocytes were assayed for intracellular IFN-γ, using a Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer's instructions. Briefly, FcRγIII and -II were blocked using FcBlock (BD Pharmingen), and cells were stained for surface expression of CD8, using PE-conjugated anti-CD8α diluted 1/100 (BD Pharmingen) for 30 min on ice. Cells were fixed using Cytofix/Cytoperm (BD Pharmingen) on ice for 15 min, washed twice with PermWash (BD Pharmingen), and stained with anti-IFN-γ-APC for 20 min on ice in the dark. In some cases, surface and intracellular staining was performed simultaneously. Cells were fixed and analyzed as described above.
In vivo cytotoxicity assay.
Red blood cell-depleted spleen cells from naïve mice were pulsed separately with 10 μM cruzipain-9 (CZKb9; amino acid sequence, VPLNKCNRL) or TSKb20 peptide or with no peptide for 1 h at 37°C. Cells were washed twice in PBS and labeled (1 × 107 cells/ml) with 5 μM, 1 μM, or 0.2 μM (CZKb9-treated, TSKb20-treated, and unpulsed cells, respectively) carboxyfluorescein diacetate succinimidyl ester (CFSE) for 3 min at room temperature. CFSE labeling was quenched with an equal volume of cold FBS, and cells were washed thrice with RPMI medium containing 10% FBS. The 5 μM (CZKb9), 1 μM (TSKb20), and 0.2 μM (unpulsed) CFSE-labeled cells were combined in equal quantities and transferred intravenously to naïve and acutely infected CD25-depleted or control mice. After 12 h, recipient mice were sacrificed, and single-cell suspensions were prepared from spleens and fixed. CFSE-labeled cells were detected using a Cyan flow cytometer and analyzed using FlowJo software. The percentage of specific killing for each peptide-condition combination was calculated using the following formula: {1 − [(% CFSENO PEPTIDE labeling for naïve mice/% CFSEPEPTIDE labeling for naïve mice)/(% CFSENO PEPTIDE labeling for acutely infected mice/% CFSEPEPTIDE labeling for acutely infected mice)]} × 100.
RNA isolation, reverse transcriptase PCR, and qualitative PCR with cDNA.
Total RNAs were isolated from 1 × 105 to 1 × 106 FACS-enriched CD45+ splenocytes and muscle-derived lymphocytes and also from directly ex vivo-isolated lymph node-derived lymphocytes, using a HighPure RNA isolation kit, and were treated with DNase I according to the manufacturer's protocol (Roche). First-round cDNA products were obtained from 500 ng purified total RNA primed with a mixture of oligo(dT)VN (5′-T24VN-3′) and a cDNA primer specific to murine 18S rRNA (5′-TAATGATCCTTCCGCAGGTTC-3′). Briefly, 0.5 μg oligo(dT)VN and 5 pmol of 18S rRNA primer were added to RNA samples, and the primer-template mixes were heated to 70°C for 10 min to denature. The samples were then cooled to 42°C, and 20-μl reaction mixes were created which contained 1× first-strand buffer, 10 mM dithiothreitol, a 0.5 mM concentration of each deoxynucleoside triphosphate, 40 U RNasin, and 200 U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). The first-round cDNA reaction mixes were incubated for 1.5 h at 42°C. For each RNA sample, parallel control cDNA reaction mixes were prepared in which the reverse transcriptase was omitted. After first-strand synthesis, the reactions were inactivated by incubation for 10 min at 70°C and then treated with 2 U RNase H (Invitrogen) for 45 min at 37°C to degrade RNA.
The following specific primers and conditions were then used to perform separate qualitative PCRs, using equal amounts of first-round cDNA as a template: 18S forward (5′-GATGGTTTAGTGAGGCCCTCGG-3′) and reverse (5′-ACCTACGGAAACCTTGTTACGACTTTTA-3′) primers, with a 60°C annealing temperature; Foxp3 primers and conditions, as previously described (
14); and CD8α and CD4 primers and conditions, as previously described (
1). PCR amplifications were carried out separately in 50-μl reaction mixtures containing 2 μl cDNA and final concentrations of 2.5 U Jumpstart
Taq (Sigma), 200 μM deoxynucleoside triphosphates, and 0.5 μM specific primer mixes, utilizing the following program: 2.5 min of denaturation at 94°C, followed by 34 cycles of 30 s at 94°C, 30 s at the indicated annealing temperature, and 30 s at 72°C. Ten microliters of each reaction mix and a 1-kb DNA ladder were loaded in a 1.4% agarose-Tris-borate-EDTA gel, electrophoresed at 100 V for 30 min, and stained using ethidium bromide.
Statistical analysis.
Student's t test was used to determine statistical significance between appropriate groups.
DISCUSSION
While much literature regarding Treg cells and infectious disease has focused on their suppressive effects against effector CD4
+ T cells (
4,
26,
35), accumulating evidence demonstrates that Treg cells also have the ability to suppress CD8
+ T-cell cytokine production and proliferation (
7,
32) and that depletion of Treg cells before or during infection can lead to increased CD8
+ T-cell cytotoxicity, IFN-γ secretion, and clonal expansion and to enhanced formation of immunological memory (
12,
40,
49). Since induction of Treg-cell function can sometimes favor the microbe rather than the host, this may be a mechanism of immune evasion by some parasites, or in some cases, when bystander tissue damage is prevented, both the host and the parasite may experience a mutual benefit (
5,
34).
In vivo injection of anti-IL-2 receptor alpha chain-specific monoclonal antibodies depletes Treg cells and thus abrogates their suppressive effects. Apparent roles for Treg cells in allowing parasite escape from host immunity have been demonstrated in various acute and chronic infection models by depleting CD4
+ CD25
+ Treg cells with anti-CD25 MAbs (for a review, see reference
27). This approach has been used to demonstrate a role for Treg cells in allowing
Leishmania (
4) to escape from host immunity, as well as uncovering Treg-cell roles in shaping the immunodominance of CD8
+ T-cell responses (
12). Other studies have demonstrated that CD8
+ T-cell-mediated primary immune responses are enhanced when CD4
+ CD25
+ regulatory T cells are depleted prior to infection with
Plasmodium (
13) or prior to vaccination and then infection with hepatitis B virus or herpes simplex virus (
11,
40).
In this study, similar experiments were performed to determine the role of Treg cells in the acute immune response to T. cruzi infection. While depletion of Treg cells prior to a lethal parasite challenge did not enhance the survival of depleted versus nondepleted control animals, a modest difference in the magnitude of antigen-specific CD8+ T-cell clonal expansion was observed during a period of high parasitemia. While Treg cells may play a role in limiting the magnitude of CD8+ T-cell immune responses in some situations, as suggested by the increased magnitude of TSKb20-specific CD8+ T cells in the Treg-cell-depleted animals, this was not sufficient to improve the outcome of the infection. This indicates that while Treg cells may play a part in modulating immune responses during infection with T. cruzi, Treg cells do not play a pivotal role in suppressing either beneficial or detrimental immune responses, which could benefit the parasite or host, respectively.
To further investigate the role of Treg cells in modulating the acute immune response to
T. cruzi, Treg cells were depleted prior to and during acute infection with a nonlethal dose/strain of parasite. In our hands, depletion of CD4
+ CD25
+ T cells had neither beneficial nor detrimental effects on the outcome of the acute immune response. Interestingly, while anti-CD25 treatments delivered throughout the course of infection did deplete CD4
+ CD25
+ T cells, this did not appear to reduce the overall magnitudes of antigen-specific CD8
+ effector T cells compared to those in nondepleted infected animals, suggesting that anti-CD25 MAb treatments preferentially target CD4
+ CD25
+ T cells. Had Treg cells been suppressing the priming, activation, or effector function of CD8
+ T cells during acute infection, it was hypothesized that their removal would lead to enhanced antigen-specific immune responses, perhaps followed by decreased parasitemia and increased longevity. These outcomes were not observed. Conversely, if Treg cells were playing an overall beneficial role during the immune response by dampening immune hyperactivity, it was hypothesized that depletion of Treg cells would lead to earlier mortality or cachexia, such as occurs during acute
T. cruzi infection in the absence of the immunomodulatory cytokine IL-10 (
17). This was not the case, as differential mortality and morbidity were not observed for depleted versus nondepleted animals. Therefore,
T. cruzi has adopted a mechanism of immune evasion that appears to be independent of Treg-cell-mediated suppression of
T. cruzi-specific immune responses.
The impetus for the second part of this investigation was our recent report that effector/memory CD8
+ T cells isolated from the muscles of mice with chronic
T. cruzi infection have little cytolytic ability and are poor producers of IFN-γ compared to effector/memory CD8
+ T cells from the spleens of the same chronically infected animals (
22). Since dysfunctional CD8
+ T cells reside in muscle, a site of parasite persistence in chronically infected mice, investigating the cause or maintenance of the observed CD8
+ T-cell dysfunction was of great interest. Thus, we asked if Treg-cell-mediated suppression was an explanation for the poor effector function of CD8
+ T cells in muscle tissue. Since the infiltration and retention of Treg cells in chronically inflamed tissues are a requirement for Treg-cell-mediated suppression of tissue-resident effector cells (
16,
38,
39), it was hypothesized that if Treg cells were suppressing CD8
+ T-cell responses in chronically infected muscle tissue, then Treg cells should be detectable in this tissue. While CD4
+ CD25
+ T cells were detected in chronically infected muscle tissue, as well as other tissues, one caveat of this phenotypical approach is that CD25 is not expressed exclusively by natural Treg cells but is also transiently expressed by other lymphoid cells upon activation during autocrine IL-2 signaling. Thus, we measured the expression of the Foxp3 gene (and its protein product, scurfin), which has been identified as the best-defined marker of natural Treg cells (
36). It was hypothesized that if Treg cells were playing a dominant suppressive role in chronically infected muscle, we would expect to see a relative increase in the frequency of CD4
+ Foxp3
+ T cells in the muscle, in contrast to the case for the spleen, where CD8
+ effector T-cell responses appear to be robust. However, the CD4
+ T-cell expression of scurfin and total lymphocyte Foxp3 mRNA expression were relatively lower in the muscle tissues than in the spleens of chronically infected mice.
The observed expression of CD25 by CD4
+ T cells derived from chronically infected muscle tissue may indicate that these CD4
+ T cells are activated effector cells. Class II MHC molecules are expressed in inflamed muscle (
51), in chronically
T. cruzi-infected muscle (
53), and on professional antigen-presenting cells that are found in the inflamed muscles of
T. cruzi-infected mammals (
2). Therefore, MHC class II-positive antigen-presenting cells capable of activating CD4
+ T cells are found in chronically infected muscle tissue.
In at least three separate experiments administering depleting anti-CD25 monoclonal antibodies to chronically infected mice for continuous periods ranging from 6 to 24 days, we observed relative reductions of CD4+ CD25+ T cells of approximately 70 to 90%, comparing depleted versus PBS-treated mice. Ex vivo polyclonal stimulation of lymphocytes isolated from the muscles and spleens of chronically infected-depleted and control treated mice revealed that the frequency of IFN-γ-secreting CD8+ T cells was not increased as a result of depletion, further suggesting that natural Treg cells do not play a role in the suppression of CD8+ T-cell function in muscle tissue. However, the relative reduction of CD4+ CD25+ T cells varied according to the target tissue analyzed. In our hands, the greatest reduction of CD4+ CD25+ T cells achieved in muscle tissue was 76%. While the inability to completely deplete CD4+ CD25+ T cells from the muscle tissue of chronically infected mice might confound interpretation of the resultant functional effects of depletion, the finding that this degree of depletion did not increase the frequency of IFN-γ-producing CD8+ T cells, taken together with the finding that CD4+ Foxp3+ Treg cells are not enriched in muscle tissue, supports our conclusion that in the Brazil strain/B6 mouse model, Treg cells do not play a major role in the regulation of muscle-resident CD8+ T cells of chronically infected mice.
These results rule out one possibility but offer little insight into other possibilities in determining the mechanism of T. cruzi evasion of the immune response during the acute phase of infection or the cause of CD8+ T-cell dysfunction in the muscle tissue of mice with chronic T. cruzi infection.