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Experimental African Trypanosomiasis: A Subset of Pathogenic, IFN--Producing, MHC Class II-Restricted CD4⁺ T Cells Mediates Early Mortality in Highly Susceptible Mice¹

Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada

	Abstract

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Infections of highly susceptible BALB/c mice with virulent strains of Trypanosoma congolense or Trypanosoma brucei result in rapid death (8 days). We have previously shown that this mortality is IFN- dependent. In this study we show that IFN- is produced predominantly by CD3⁺Thy1.2⁺TCR⁺CD4⁺ T cells shortly before the death of infected mice. Mortality may therefore be dependent on IFN--producing CD4⁺ T cells. Surprisingly, infected CD4^+/+ and CD4^–/– BALB/c mice have similar parasitemia and survival time. In infected CD4^–/– mice, the production of both IFN- and IL-10 is very low, suggesting that both cytokines are predominantly produced by CD4⁺ T cells and that the outcome of the disease might depend on the balance of their effects. Infected BALB/c mice partially depleted of CD4⁺ T cells or MHC class II function have lower parasitemia and survive significantly longer than infected normal BALB/c mice or infected BALB/c mice whose CD4⁺ T cells are fully depleted. Partial depletion of CD4⁺ T cells markedly reduces IFN- secretion without a major effect on the production of IL-10 and parasite-specific IgG2a Abs. Based on our previous and current data, we conclude that a subset of a pathogenic, MHC class II-restricted CD4⁺ T cells (Tp cells), activated during the course of T. congolense infection, mediates early mortality in infected BALB/c mice via excessive synthesis of IFN-. IFN-, in turn, exerts its pathological effect by enhancing the cytokine release syndrome of the macrophage system activated by the phagocytosis of parasites. We speculate that IL-10-producing CD4⁺ T cells might counteract this effect.

	Introduction

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African trypanosomes are single-cell parasites that infect both humans and livestock. The parasites survive in the bloodstream of the mammalian host and can cause severe disease and death. BALB/c mice are highly susceptible to Trypanosoma congolense and Trypanosoma brucei infections, whereas C57BL/6 mice are relatively resistant, as measured by levels of parasitemia, immunosuppression, and survival time (1, 2, 3). The mechanisms of susceptibility and relative resistance in these two mouse strains are still poorly understood. There is good evidence that resistance/susceptibility to T. congolense infections is controlled by at least five different quantitative trait loci on chromosomes 17, 5, and 1 (4, 5). The relevant genes and their products are unknown. The early mortality of susceptible BALB/c mice to infection with T. congolense or T. brucei is associated with enhanced synthesis of IFN- and IL-10 (6, 7, 8). The infection of relatively resistant C57BL/6 mice is accompanied by significantly higher plasma levels of parasite-specific IgG, in particular, IgG2a Abs (9). The susceptible mice die of a systemic inflammatory response syndrome (SIRS)⁴ (6). Administration of anti-IFN- Abs to susceptible BALB/c mice infected with T. congolense prevents early mortality (6, 7). Blocking the IL-10R of normally relatively resistant C57BL/6 mice infected with T. congolense leads to early death. These mice have excessively elevated plasma levels of IFN- and monokines (6). The early mortality of C57BL/6 mice infected with T. congolense and treated with anti-IL-10R can be reversed by administration of anti-IFN- Abs (6). Therefore, the early mortality of these infected mice is mediated in part by IFN- produced in excessive amounts (6). The fact that the early death of infected susceptible BALB/c mice was prevented by administration of anti-IFN- led us to suggest a central role for a subset of IFN--producing cells, possibly NK cells, NKT cells, or T cells (6, 10). A novel plastic-adherent suppressor T cell population(s) in the spleen of T. congolense-infected mice has been found to synthesize, in synergy with trypanosome-pulsed Thy1.2^– spleen cells, very large amounts of IFN- (11). We previously speculated that the trypanosomal GPI, the membrane anchor of the variant surface glycoprotein (VSG), might be the major Ag involved (6). We also speculated and started this study with the premise that the IFN--producing cells might be CD1-restricted NKT cells (6). We now have good evidence that the IFN--producing, pathology-inducing T cells are not CD1 restricted (M. Shi, C.-R. Wang, G. Wei, W. Pan, G. Appleyard, and H. Tabel, submitted for publication). The main aim of the present study was to further characterize the subset of IFN--producing cells that mediate a lethal process via IFN- in BALB/c mice infected with T. congolense (6). In the present study, we use immunocytochemistry and FACS analysis to characterize the IFN--producing T cells of BALB/c mice succumbing early to T. congolense infection. We also test the hypothesis that the process leading to early mortality in susceptible mice infected with a virulent strain of T. congolense might be mediated by CD4⁺ T cells that are restricted by the MHC class II (MHC-II).

	Materials and Methods

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Mice

Female, 8- to 10-wk-old BALB/c AnNCrlBR (BALB/c) and 5- to 6-wk-old female outbred Swiss white mice (CD1) were purchased from the Animal Resource Center of University of Saskatchewan. CD4 (12) knockout mice were generated in T. Mak’s laboratory (Ontario Cancer Institute and Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada). The CD4 deficiency was subsequently bred into mice with the BALB/c background in the same laboratory. The CD4^–/– BALB/c mice used in this study originated from the above colony and were bred in the animal care facilities of the Western College of Veterinary Medicine, University of Saskatchewan. The mice were kept in polycarbonate cages on sawdust and allowed free access to food and water throughout the experiments according to the recommendations of the Canadian Council of Animal Care.

Parasites

T. congolense, Trans Mara strain, variant antigenic type (VAT) TC13 was used in this study. The origin of this parasite strain has been described previously (13). Frozen stabilates of parasites were used for infecting CD1 mice immunosuppressed with cyclophosphamide, and passages were made every third day as described previously (13). The parasites purified from the blood of infected CD1 mice by DEAE-cellulose chromatography (14) were used for infecting BALB/c mice.

Hybridomas and Abs

The rat hybridomas GK1.5 (specific for CD4) and HB3 (specific for I-A^d) were purchased from American Type Culture Collection. The following mAbs were purchased from BD Pharmingen: purified anti-mouse CD16/CD32 (FcIII/IIR, clone 2.4G2), purified rat anti-mouse CD3 (clone 17A2), biotin-conjugated rat anti-mouse CD4 (RM4-5), biotin-conjugated rat anti-mouse CD8 (53-6.7), biotin-conjugated hamster anti-mouse TCR (H57-597), biotin-conjugated anti-mouse Pan-NK cells (DX5), biotin-conjugated rat anti-mouse IFN- (XMG1.2), and FITC-conjugated rat anti-mouse IFN- (XMG1.2). The following Abs were purchased from Cedarlane Laboratories: biotin-conjugated goat anti-rat IgG (H+L) mouse adsorbed, biotin-conjugated anti-mouse Thy1.2 (5a-8), biotin-conjugated rat anti-mouse CD11b (M1/70.15), biotin-conjugated hamster anti-mouse TCR (GL3), purified rat IgG2b, biotin-conjugated rat IgG2a, biotin-conjugated rat IgG2b, biotin-conjugated rat IgG1, biotin-conjugated rat IgM, biotin-conjugated hamster IgG, and FITC-conjugated rat IgG1.

Infections, treatment of mice with mAbs, estimation of parasitemia, and survival time of mice

Mice were infected i.p. with 10³ T. congolense VAT TC13. Some groups of infected BALB/c mice were i.v. injected with a single dose of 4 mg, 500 µg, 100 µg, or 30 µg of anti-CD4 mAb on day 0 after infection. Other groups of infected mice were injected with 400 µg of anti-I-A^d on days 0, 3, 5, and 7. A drop of blood was taken from the tail of each infected mouse. Parasitemia was estimated by counting the number of parasites present in at least 10 fields at x400 magnification by phase contrast microscopy. The survival time was defined as the number of days after infection that the infected mouse remained alive.

Immunocytochemistry

Spleen cells were isolated from T. congolense-infected BALB/c mice on day 7 after infection or from uninfected BALB/c mice (as control). The cells (10⁷/ml) in complete medium were seeded into tissue culture-treated plastic petri dishes (Falcon 3001; VWR International, Edmonton Alberta, Canada) and incubated at 37°C in a 5% CO₂ incubator. After 3 h, the nonadherent cells were removed. The adherent cells were carefully washed and then dislodged with ice-cold versene (0.02% EDTA in PBS (pH 7.2)) and gentle scraping. The cells were diluted at 2.5 x 10⁶ cells/ml. Four hundred and twenty-five microliters of the cell solution was put in each chamber of eight-chamber Lab-Tek slides (Miles Scientific) and cultured for 48 h at 37°C in a 5% CO₂ atmosphere. Then the chamber slides were disassembled. Immunofluorescent double staining for cell surface markers and intracellular IFN- was performed on ice. In brief, the cells were rinsed with PBS, blocked with Fc block (purified anti-mouse CD16/CD32 (FcIII/IIR), clone 2.4G2) and 2% BSA, then incubated with biotin-conjugated anti-mouse Thy1.2, CD3, CD4, CD8, TCR, TCR, CD11b, or pan NK cell mAb or biotin isotype controls for 30 min. After three washes in PBS, the slides were incubated with streptavidin AlexaFluor 488 (Molecular Probes) for 30 min, rinsed with PBS, then fixed with 5% formalin. The cells were incubated with avidin D solution for 15 min, rinsed with PBS, then incubated with biotin solution (avidin/biotin blocking kit; Vector Laboratories) for 15 min. The slides were rinsed with PBS, then incubated with biotinylated rat anti-mouse IFN- (XMG1.2) or rat IgG1 conjugated with biotin (isotype control) in PBS containing 0.1% saponin (cell permeabilization) for 30 min, rinsed with PBS, then stained with streptavidin AlexaFluor 546.

Flow cytometry

Spleen cells were collected from T. congolense-infected BALB/c mice on day 7 after infection or from uninfected BALB/c mice (as a negative control). The cells were diluted to 5 x 10⁶ cells/ml and cultured (200 µl/well) in a 96-well plate for 48 h. Spleen cells from uninfected mice were stimulated with 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma-Aldrich) as a positive control. After 44 h of incubation, 2 µM monensin (GolgiStop; BD Pharmingen) was added to the cultures. Four hours later, cells were harvested and washed twice in staining buffer (BD Pharmingen). The cells were incubated (15 min, 4°C) with purified mAb 2.4G2 to block nonspecific binding of Abs to FcRs, washed with staining buffer, resuspended in staining buffer, and surface stained with the relevant biotinylated Abs. Cells were washed with staining buffer, labeled with PE-conjugated strepavidin (BD Pharmingen), and washed twice. Intracellular staining was performed using the Cytofix/CytoPerm kit (BD Pharmingen) in accordance with the manufacturer’s recommendations. Briefly, cells were treated with formaldehyde and saponin to fix and permeabilize the cells. Intracellular staining was then performed using FITC-conjugated anti-IFN- mAb (XMG1.2) and FITC-conjugated rat IgG1 (isotype-matched control Ab). Samples were resuspended in PBS containing 1% formaldehyde, tested by FACS, and analyzed using CellQuest software (BD Biosciences).

Splenocyte cultures for measurement of cytokine synthesis

Splenocytes were collected from T. congolense-infected CD4^–/– or wild-type of BALB/c on day 7 after infection as well as from relevant uninfected mice. Cells were cultured at a concentration of 5 x 10⁶ cells/ml (200 µl/well) in 96-well tissue culture plates in a humidified incubator containing 5% CO₂ in the air. The culture supernatant fluids were collected after 48 h and centrifuged at 1500 x g for 10 min, and the supernatant fluids were stored for cytokine assays at –35°C until used.

Cytokine assays

Recombinant murine cytokines (IFN-, IL-10, and IL-12p40) and Abs to these cytokines for use in ELISA were purchased from BD Pharmingen or R&D Systems. The levels of cytokines in culture supernatant fluids or plasma were determined by routine sandwich ELISA using Immuno-4 plates (Dynax Technologies) according to the manufacturer’s protocols. Each sample was tested for each cytokine in triplicate.

ELISA for trypanosome-specific Abs

Whole trypanosome lysate was prepared by three cycles of freezing and thawing of freshly isolated T. congolense VAT TC14 in the presence of 5 mM N-tosyl-L-lysine chloromethylketone (Sigma-Aldrich), and the total protein content was determined using a protein assay kit (Bio-Rad). ELISA plates were coated overnight at 4°C with 50 µl of the lysate containing 25 µg/ml total protein. The plates were washed twice with PBS/Tween 20, and nonspecific binding sites were blocked for 2 h at room temperature with 200 µl of PBS containing 10% heat-inactivated FBS (PBS-FBS). Serum samples (100 µl), diluted 1/50 in PBS-FBS were added to each well and incubated for 2 h at 37°C. After washing (four times), 100 µl of previously determined dilutions of peroxidase-conjugated goat anti-mouse isotype-specific Abs (Southern Biotechnology Associates) in PBS-FBS were added to each well and incubated for 2 h at room temperature. The plates were washed eight times, and color development was achieved by adding 100 µl of ABTS (Kirkegaard & Perry) and incubating for 15–30 min at room temperature. ODs were read in a microtiter plate reader at a wavelength of 405 mm.

Statistical analysis

Data are represented as the mean ± SE. Significance of differences was determined by ANOVA using StatView SE 1988 software (Abacus Concepts) or a log-rank test for curve comparison using a PRISM computer program (GraphPad).

	Results

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IFN- is produced predominantly by CD3⁺Thy1.2⁺TCR⁺CD4⁺ T cells in BALB/c mice infected with T. congolense

To characterize the IFN--producing cells, immunocytochemical double staining for cell surface markers and intracellular IFN- was performed. All cell cultures stained with control Abs of the same isotype were negative. We found IFN--producing cells in spleen cultures of mice infected with T. congolense. In contrast, no IFN--positive cells were detected in spleen cultures of uninfected mice. The IFN--producing cells were glass adherent and frequently located close to large cells that had the morphology of macrophages or dendritic cells (see insets of Fig. 1A). The IFN--producing cells were DX5^– (Fig. 1B) and CD11b^– (Fig. 1D). This indicated that they were neither NK cells nor macrophages. All IFN--positive cells expressed CD3 (Fig. 1A) and Thy1.2 (Fig. 1C) marker. Thus, the IFN--secreting cells were T cells. We also found that IFN--producing cells were TCR⁺ (Fig. 1E) and ^– (Fig. 1F). The vast majority (90%) of the IFN--producing cells were CD4⁺ (Fig. 1G). However, some IFN--producing cells were CD4^– (Fig. 1G). Four percent of IFN--producing cells were CD8⁺ (Fig. 1H). Thus, the immunocytochemical analysis showed that IFN- was predominantly produced by CD3⁺Thy1.2⁺TCR⁺CD4⁺ cells. When in contact with a CD11b⁺ cell, IFN--producing T cells were polarized, i.e., the intracellular IFN- was concentrated at the area of cell-cell contact (Fig. 1D).

View larger version (80K): [in this window] [in a new window]   FIGURE 1. Characterization of the IFN--producing cells in spleen cultures of mice infected with T. congolense by immunofluorescent double staining. BALB/c mice were infected with 103 T. congolense. Plastic-adherent spleen cells were collected, as described in Materials and Methods, from infected mice on day 7 after infection and cultured at concentration of 2.5 x 106 cells/ml in chamber slides. Immunofluorescent double staining for cell surface phenotype and intracellular IFN- was performed as described in Materials and Methods. Original magnification, x400; for insets, x1000. IFN--producing cells (red, arrows) were CD3+ (green; A) and DX5– (green; B): Note that the intracellular IFN- is polarized toward the neighboring cell. C, Thy1.2+ (green); D, CD11b– (green). Note that the IFN- within the T cell is polarized toward the area of contact with the CD11b+ cell. E, TCR+ (green); F, TCR– (green). G, The vast majority of IFN--producing cells (90%) (red, arrows) had the CD4 marker (green), although some IFN-+ CD4– cells were detected. H, A small portion of IFN--producing cells was CD8+ (4%; green).

View larger version (24K): [in this window] [in a new window]   FIGURE 2. IFN--producing cells in cell cultures of whole spleen from mice infected with T. congolense: characterization by flow cytometry. Groups of six BALB/c mice were infected with 103 T. congolense. Spleen cells collected on day 7 after infection from infected mice were cultured at concentration of 5 x 106 cells/ml in 96-well plates (200 µl/well) for 48 h, and 2 µM monensin (GolgiStop; BD Pharmingen) was added to the culture at 44 h. Double staining for cell surface phenotype and intracellular IFN- was performed as described in Materials and Methods. IFN--producing cells are Thy1.2+ (D; isotype control, A), TCR+ (E; isotype control, B), and CD4+ (F; isotype control, C). The results presented are representative of two separate experiments.

We formulated the hypothesis that CD4^–/– BALB/c mice infected with T. congolense would have lower parasitemia and survive longer than infected wild-type BALB/c mice, because IFN- was predominantly produced by CD4⁺ T cells, and we had demonstrated that IFN- mediated early mortality (7, 11). Examination of CD4^–/– and wild-type BALB/c mice after infection with T. congolense showed that there were no significant differences in either parasitemia (Fig. 3A) or survival time (Fig. 3B). Thus, our observations did not support our hypothesis. We concluded that infections of CD4 knockout mice are an experimental design of limited usefulness and might yield misleading results. One has to keep in mind that CD4 molecules are expressed on a number of subsets of T cells with different, if not opposing, functions. These CD4⁺ T cells would include IFN--producing Th1 cells (15), IL-10-producing regulatory T cells (Tr1) (16), and, as postulated in this study, IFN--producing pathogenic T cells. This led us to explore the possibility that the IFN-, shown to be critical for mediating death, was produced by a special subset of CD4⁺ T cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. In CD4–/– BALB/c mice infected with T. congolense, parasitemia and survival were not significantly different, but secretion of IFN- and IL-10 was decreased by >97%. Groups of 10 CD4–/– () or wild-type BALB/c (•) mice were infected with 103 TC13. Mice were monitored for parasitemia (A) and survival (B). Groups of four CD4–/– () or wild-type () BALB/c mice were infected with 103 TC13. Spleen cells collected on day 7 after infection from infected mice were cultured at concentration of 5 x 106 cells/ml in 96-well plates (200 µl/well) for 48 h. The culture supernatant fluids were assayed for IFN- and IL-10, as described in Materials and Methods. Data are presented as the mean ± SE. The results presented are representative of two separate experiments.

Synthesis of IFN- and IL-10 is markedly lower in spleen cultures from CD4^–/– BALB/c mice infected with T. congolense than in spleen cultures from infected wild-type BALB/c mice

We assessed whether spleen cells from infected CD4^–/– BALB/c mice secreted IFN- and IL-10. We measured the levels of IFN- as well as IL-10 in spleen cell cultures from CD4^–/– and CD4^+/+ BALB/c mice infected with T. congolense. Besides IFN-, IL-10 is also significantly enhanced in susceptible BALB/c mice infected with T. congolense (8) and is crucial for controlling the cytokine release syndrome and the development of SIRS (6). Spleen cultures from infected mice always contain a certain number of parasites and, hence, parasite Ag. Supernatant fluids of spleen cell cultures derived from infected CD4^+/+ and CD4^–/– BALB/c mice had 583 pg of IFN-/ml and 17 pg of IFN-/ml, respectively (Fig. 3C). Thus, the spleen cell cultures of infected CD4^–/– mice only yielded 3% the IFN- produced by the cultures of infected wild-type mice (p < 0.01). This small amount of IFN- was possibly produced by CD8⁺ T cells (Fig. 1H). A major involvement of IFN--producing CD8⁺ T cells in the IFN--mediated death appeared unlikely, because CD8-deficient BALB/c mice do not survive longer after infection than wild-type BALB/c mice (data not shown). Again, these data support the already drawn conclusion that IFN- is predominantly produced by a small subset of CD4⁺ T cells in infected wild-type BALB/c mice.

Our observations had led us to conclude that the T cells centrally involved in causing early death in infected BALB/c mice are most probably CD4⁺ T cells as described above (Fig. 1). Our observations also show that the parasitemia and survival time are similar in CD4^–/– and CD4^+/+ mice after infection (Fig. 3). To try to resolve this paradox, we measured the production of IL-10, because IL-10 is crucial in preventing SIRS in T. congolense-infected mice (6). The spleen cell cultures of infected CD4^–/– BALB/c mice produced 4.5-fold less IL-10 (p < 0.01) than those from infected wild-type BALB/c mice, amounts similar to those produced by uninfected spleen cell cultures (Fig. 3D). Thus, our data demonstrate that the synthesis of IL-10 in T. congolense-infected BALB/c mice is CD4⁺ T cell dependent.

Production of parasite-specific IgG2a, but not IgM, Abs is lower in infected CD4^–/– BALB/c than in infected wild-type mice

We measured the plasma levels of Abs specific for T. congolense, because anti-VSG Abs play a significant role in the clearance of the parasitemia (17, 18, 19), and Abs to common Ags also mediate a protective role in African trypanosomiasis (20, 21). We did not find any differences in plasma levels of IgM Abs specific for T. congolense (Fig. 4A) in CD4^–/– and wild-type BALB/c mice infected with T. congolense. However, IgG2a Abs (Fig. 4C) to T. congolense were abrogated, and total parasite-specific IgG Abs (Fig. 4B) were significantly reduced in CD4^–/– mice compared with infected wild-type mice (p < 0.01). Thus, infected CD4^–/– mice are expected not only to lack the subset of IFN--producing CD4⁺ T cells mediating early death, but also a subset of parasite-specific CD4⁺ Th cells that produce IFN- and support the switch from production of IgM Abs to parasite-specific IgG2a Abs.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Synthesis of IgG2a (but not IgM) parasite-specific Abs was abrogated in CD4–/– BALB/c mice infected with T. congolense. Groups of four CD4–/– () or wild-type (•) BALB/c mice were infected with 103 TC13. Plasma samples were collected from the infected mice on days 0, 6, and 7 after infection. The Ab levels in plasma were measured by ELISA as described in Materials and Methods. Data are presented as the mean ± SE. The results presented are representative of two separate experiments.

Both CD4^–/– and CD4^+/+ BALB/c mice infected with T. congolense succumbed to the infection without control of the first parasitemia (Fig. 3, A and B). Infected CD4^–/– BALB/c mice produced neither IFN- nor significant amounts of IL-10 (Fig. 3, C and D). We asked ourselves: why did the CD4^–/– mice die in the absence of IFN-? One can make the following arguments: 1) The primary excessive activation of the macrophage system happens by phagocytosis of trypanosomes mediated by anti-VSG Abs of IgM class. 2) Whether the development of SIRS and death occurs does not only depend on the superimposed effect of IFN-, but also the counterbalancing effect of IL-10 that down-regulates macrophage activity (22). In fact, we and other investigators (6, 23) have shown the absolute requirement for IL-10 activity to prevent trypanosome-induced death. If the above reasoning is correct, plasma levels of monokines should be high in infected CD4^–/– BALB/c mice that lack significant amounts of IFN- and IL-10. Thus, we measured the plasma levels of the monokine IL-12p40 in infected CD4^–/– BALB/c mice. The result showed that plasma levels of IL-12p40 are, indeed, very high (Fig. 5).

View larger version (13K): [in this window] [in a new window]   FIGURE 5. Plasma levels of IL-12p40 in CD4–/– BALB/c mice infected with T. congolense are as high as those in infected CD4+/+ BALB/c mice. Groups of five mice were infected with 103 TC13, and blood samples were collected 7 days after infection. Concentrations of IL-12p40 in the plasma were measured by ELISA as described in Materials and Methods. Data are presented as the mean ± SE. The results presented are representative of two separate experiments.

It seemed to us that more complex mechanisms than we initially had anticipated must determine the outcome of T. congolense infections of CD4^–/– BALB/c mice. We thought that administration of a certain optimal amount of anti-CD4 mAb might remove sufficient numbers of the IFN--producing, death-inducing T cells and hence might reduce the production of IFN- below a pathology-inducing level. Such treatment, of course, could only work if it did not detrimentally affect IL-10-producing T cells and did not affect the protective T cells that may act by helping the generation of protective antiparasite-specific IgG2a Abs. We therefore treated groups of T. congolense-infected CD4^+/+ BALB/c mice with 4 mg, 500 µg, 100 µg, or 30 µg of anti-CD4 mAb (GK1.5) on day 0. The injection of 30 µg of anti-CD4 mAb had no effect (not shown). The infected mice treated with the high dose (4 mg) of anti-CD4 showed a pattern of parasitemia (Fig. 6A) and survival (Fig. 6B) similar to that of infected CD4^–/– BALB/c mice (Fig. 3, A and B). FACS analysis demonstrated that >99% of CD4⁺ T cells in these mice were eliminated (not shown). The groups of infected mice treated with 500 or 100 µg of anti-CD4 mAb, however, had lower parasitemia (Fig. 6A) and significantly longer survival time (Fig. 6B) than the untreated infected BALB/c mice. As determined by FACS, infected BALB/c mice injected with 100 µg of anti-CD4 mAb had 60% less CD4⁺ T cells than untreated infected mice on day 6. In addition, these T cells expressed, on the average, 10-fold fewer CD4 molecules per cell than T cells from untreated mice (Fig. 6C).

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Low-dose, but not high-dose, anti-CD4 treatment enhances the survival of highly susceptible BALB/c mice infected with T. congolense. Groups of five BALB/c mice were infected with 103 TC13 and treated with 4 mg (), 0.5 mg (), or 0.1 mg () of anti-CD4 or were left untreated (•) as a control on day 0 after infection, respectively. Mice were monitored for parasitemia (A) and survival (B). FACS analysis was performed for CD4+ spleen cells of infected mice untreated or treated with 0.1 mg/ml anti-CD4 Abs (C). The results presented are representative of two separate experiments.

Partial depletion of CD4⁺ T cells in vivo significantly reduces IFN- secretion in spleen cell cultures without strongly affecting secretion of IL-10 and plasma levels of IgG2a Abs specific for T. congolense

Next, we assessed the IFN- and IL-10 secretion in spleen cultures and plasma levels of IgG2a Abs specific for T. congolense on day 7 after infection after treatment with anti-CD4 mAb. As shown in Fig. 7A, IFN- secretion was significantly reduced by treatment with a low dose (0.1 mg) of mAb and was almost abrogated by treatment with a high dose (4 mg) of mAb. In contrast, there was no significant difference between untreated mice and mice treated with an optimal dose (0.1 mg) of mAb regarding the plasma levels of IgG2a Abs specific for T. congolense. The plasma levels of IgG2a Abs specific for T. congolense, however, were significantly reduced by treatment with a high dose (4 mg) of anti-CD4 mAb (Fig. 7B). After treatment with the low dose of anti-CD4 (0.1 mg), secretion of IL-10 by spleen cell cultures was not strongly reduced (Fig. 7C). Taken together, these results suggest that removal of a certain number of pathogenic CD4⁺ T cells by treatment with an optimal amount of anti-CD4 enhances the survival of the susceptible mice infected with T. congolense. Such treatment prevents the excessive synthesis of IFN-. At the same time, this treatment allows the production of sufficient down-regulatory IL-10 as well as synthesis of sufficient parasite-specific IgG2a Abs.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Partial depletion of CD4+ T cells in T. congolense-infected BALB/c mice markedly reduces IFN- secretion in spleen cell cultures without a strong effect on IL-10 secretion and plasma levels of parasite-specific IgG2a Abs. Groups of five BALB/c mice were infected with 103 TC13 and treated with 4 or 0.1 mg of anti-CD4 or were left untreated on day 0. On day 7 after infection, spleen cells were collected from infected mice and cultured (5 x 106 cells/ml) in 96-well plates (200 µl/well) for 48 h. The culture supernatant fluids were assayed for IFN- and IL-10 as described in Materials and Methods. Plasma levels of IgG2a Abs specific for T. congolense were also assessed as described in Materials and Methods. Data are presented as the mean ± SE. The results presented are representative of two separate experiments.

Partial blocking of MHC-II leads to control of the first parasitemic wave, increases the survival, and markedly reduces the production of IFN-, but not IL-10, of highly susceptible BALB/c mice infected with T. congolense

To test whether the pathogenic, IFN--producing T cells might be dependent on MHC-II, we treated infected BALB/c mice with anti-I-A^d mAbs (24). Whereas the infected control mice died with fulminating parasitemia (Fig. 8A), the majority of infected mice treated with anti-I-A^d mAbs controlled the first wave of parasitemia (Fig. 8A). The survival time of the infected mice treated with anti-I-A^d mAbs was significantly enhanced (Fig. 8B). In the spleen cultures of infected mice treated with anti-I-A^d mAbs, when tested on day 7, the production of IFN- was reduced by >10-fold (Fig. 8C), and the synthesis of IL-12p40 was reduced by 50% (not shown), compared with infected control mice. The production of IL-10 by the spleen cell cultures did not seem to be altered very much by anti-I-A^d mAb treatment of the infected mice (Fig. 8D).

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Partial blocking of MHC-II leads to increased survival of highly susceptible BALB/c mice infected with T. congolense, associated with control of the first parasitemic wave and reduced production of IFN- without significantly affecting IL-10 production. Groups of five BALB/c mice were infected with 103 TC13 and treated with 400 µg of Ab specific for I-Ad or rat IgG on days 0, 3, 5, and 7 after infection and compared with infected mice that did not receive Ab treatment. Mice were monitored for parasitemia (A) and survival (B). Spleen cell cultures were performed as described in Materials and Methods. The concentrations of IFN- (C) and IL-10 (D) in the supernatant fluids were determined by ELISA. Data are presented as the mean ± SE. The results presented are representative of two separate experiments.

	Discussion

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Because we found that CD4⁺ T cells produced most of the IFN- that mediated early mortality during the course of infection with T. congolense, we anticipated that CD4^–/– BALB/c mice infected with T. congolense would survive longer than the infected wild-type BALB/c mice. To our surprise, the total deficiency of CD4⁺ T cells did not alter the parasitemia and survival time after infection (Fig. 3, A and B). Why would infected CD4^–/– BALB/c mice still die when IFN--producing CD4⁺ T cells were absent? It appeared to us that the operating mechanisms were more complex than we had initially anticipated. We concluded that infections of CD4 knockout mice are an experimental design of limited usefulness for this study. Our reasoning was based on the observation that CD4 molecules are expressed on a number of subsets of T cells with different, if not opposing, functions. The population of CD4⁺ T cells include subsets of IFN--producing Th1 cells (15), IL-10-producing Tr1 (16), and, as postulated in this study, IFN--producing pathogenic T cells. It is plausible that the excessive amount of IFN- is only one of many pathogenic factors. The pathogenic effect of IFN- can be viewed as an amplifier of the cytokine release syndrome of trypanosome-pulsed macrophages that we reported and discussed previously (6). We found that CD4^–/– mice infected with T. congolense produced significantly less total parasite-specific IgG Abs and produced almost no parasite-specific IgG2a Abs compared with infected wild-type mice (Fig. 4). The levels of parasite-specific IgM Abs were not affected. These results support the idea that IgM production is predominantly CD4⁺ T cell independent (26, 27, 28). Presumably, the switch from IgM to IgG (in particular, IgG2a) requires help from CD4⁺ Th1 cells. We suggest that in T. congolense infections of CD4^–/– BALB/c mice, the beneficial effect of a substantial decrease in IFN- is partially abrogated by 1) lack of the protective parasite-specific IgG Abs, and 2) lack of down-regulatory IL-10.

The development of IgG Abs to trypanosomal cysteine proteinase is associated with resistance of cattle to the disease caused by T. congolense infections (20, 21). For the time being, the above explanation is incomplete, because presently we do not know the nature of the murine IgG anti-parasite Abs measured by ELISA. We have evidence that antiparasite Abs of IgG3 and IgG2a isotype are correlated with resistance to T. congolense infections of mice (9). In a study of IgG2a vs IgM Abs to VSG of T. congolense, we found that both types of Abs were equally effective in mediating phagocytosis of T. congolense by macrophages. IgG2a anti-VSG-mediated phagocytosis, however, induces the production of more NO by trypanosome-pulsed macrophages than IgM anti-VSG-mediated phagocytosis (29). NO produced by macrophages is known to be cytotoxic to T. brucei (30) and T. congolense (29).

T. congolense-infected CD4^–/– BALB/c mice lacked IL-10 production (Fig. 3), indicating that most of the elevated IL-10 production in infected mice is dependent on CD4⁺ T cells. We suggest that T. congolense-infected CD4^–/– BALB/c mice might lack Tr1 producing IL-10. IL-10-producing Tr1 have been shown to play a role in infections of Bordetella pertussis (16, 31) and Leishmania major (32). We and others (6, 23) have shown that IL-10 function is absolutely required to prevent early death in trypanosome-infected, relatively resistant mice. IL-10 predominantly exerts its down-regulatory activity on T cells by down-regulating APCs (22), such as macrophages. The lack of high levels of IL-10 (Fig. 3) might explain the presence of highly activated macrophages, as shown by high plasma levels of monokines (Fig. 5) and the development of SIRS in infected CD4^–/– BALB/c mice (Fig. 3) despite the lack of activity of Tp cells (Fig. 3).

The unexpected results of infection of CD4^–/– BALB/c mice prompted us to investigate whether partial depletion of CD4⁺ T cells might decrease the secretion of IFN- without significantly reducing the production of IgG Abs and IL-10. We reasoned that an optimal dose of anti-CD4 might sufficiently affect the small, but powerful, population of Tp cells without strongly affecting the other subsets of CD4⁺ T cells. To this end, we treated infected wild-type BALB/c mice with varying doses of anti-CD4 Abs. The administration of 4 mg of anti-CD4 Abs to infected BALB/c mice eliminated >99% of the CD4⁺ T cells and thus was similar to the situation in CD4^–/– mice. It did not significantly alter parasitemia or survival time, compared with untreated infected controls (Fig. 6). However, the administration of 0.5 mg, and, even more so, 0.1 mg of anti-CD4 mAb resulted in decreased parasitemia and enhanced survival time (Fig. 6). These results suggest that removal of a certain number of CD4⁺ Tp cells from T. congolense-infected BALB/c mice by treatment with an optimal amount of anti-CD4 enhances the survival of the infected mice. Such treatment prevents the excessive synthesis of IFN- (Fig. 7A). At the same time, this treatment allows the production of sufficient down-regulatory IL-10 (Fig. 7B) as well as the synthesis of sufficient parasite-specific IgG2a Abs (Fig. 7C). The administration of an optimal amount of anti-I-A^d mAbs to T. congolense-infected BALB/c mice had a similar effect as the anti-CD4 treatment. The treated mice controlled the first parasitemic wave (Fig. 8A), had longer survival time (Fig. 8B), and produced significantly less IFN- (Fig. 8C) without affecting the synthesis of IL-10 (Fig. 8D). These results strongly suggest that the IFN--producing Tp cells are MHC class II restricted. This conclusion is supported by the observation that early IFN--mediated death occurs in infected relatively resistant C57BL/6 mice after treatment with anti-IL10R Abs (6), but not in infected MHC-II-deficient C57BL/6 mice treated equally (M. Shi, G. Wei, and H. Tabel, unpublished observations).

Why did the administration of anti-CD4 or anti-MHC-II preferentially abolish the function of CD4⁺ Tp cells? We presently do not know; we can only speculate. We do know from our FACS analysis that the administration of 0.1 mg of anti-CD4 reduced the total CD4⁺ T cell population of the spleen by 60% and the average expression of CD4 molecules per cell by 90% (Fig. 6C). We speculate that the TCRs of CD4⁺ Tp cells might have a low affinity for the parasite Ag(s). Thus, Tp cells might require the binding of more clusters of TCR/CD4/MHC complexes to be activated than the other subsets of CD4⁺ T cells require.

We discuss our data with the use of a hypothetical simplistic diagram. We are aware that the presented synopsis is incomplete, but we suggest that it is coherent and compatible with presently known facts (Fig. 9). 1) IgM anti-VSG, initially produced independently of T cells (26, 27, 28), binds to the circulating trypanosomes, and 2) mediate phagocytosis of the opsonized parasites (19, 33). 3) The trypanosome-pulsed macrophages produce monokines (19, 33, 34). 4) The pulsed macrophages process the parasites and present parasite Ag via surface MHC class II to CD4⁺ T cells. 5) A subset of activated pathogenic CD4⁺ T cells (Tp cells) produce IFN-, which, in turn, further activates the macrophages and induces the macrophages to produce enhanced amounts of monokines, such as TNF-, IL-1, IL-6, IL-12, and NO, etc. (19, 29, 34). 6) The macrophages also produce IL-10 (34), which, via an autocrine pathway, down-regulates macrophages in a negative feedback loop. 7) Presently, we have no direct evidence for the involvement of regulatory T cells. However, because we and others have provided evidence that IL-10 is absolutely necessary for preventing early death in trypanosome-infected, relatively resistant mice (6, 23), and the production of the high amounts of IL-10 in T. congolense infections is dependent on CD4⁺ T cells (Fig. 3D), we speculate that some of the IL-10 might be produced by Tr1. Ag-specific CD4⁺ Tr1 might produce IL-10 down-regulating CD4⁺ Tp cells as well as macrophages (6). When activation of macrophages, predominantly Kupffer cells of the liver (6, 19), is exceeding a certain threshold, excessive SIRS will lead to death. Thus, the state of macrophage activation (25) is determined by at least three major factors: phagocytosis of trypanosomes, enhancing effect of IFN-, and down-regulatory effect of IL-10 (6).

View larger version (26K): [in this window] [in a new window]   FIGURE 9. T. congolense infections: a hypothetical simplistic view of the cascade of events. Hyperactivation of the macrophage system leads to the development of a fatal SIRS. Detailed explanations are provided in Discussion.

In summary, we conclude that MHC-II-restricted CD4⁺ Tp cells exert mortality via secretion of IFN- in susceptible mice infected with a virulent strain of T. congolense.

	Acknowledgments

 
We thank Dr. Tak Mak for providing two breeding pairs of CD8- and CD4-deficient BALB/c mice. We are grateful to Brian Chelack (Prairie Diagnostic Services, Saskatoon, Canada) for his assistance with the FACS analyses. We also thank Juliane Deubner (Western College of Veterinary Medicine) for drawing the diagram of the synopsis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a research grant from the Canadian Institutes of Health Research (to H.T.) and a postdoctoral fellowship from the Health Services Utilization and Research Commission of Saskatchewan (to M.S.).

² M.S. and G.W. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Henry Tabel, Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B4. E-mail address: tabel{at}sask.usask.ca

⁴ Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome; MHC-II, MHC class II; Tp, pathogenic, MHC class II-restricted CD4⁺ T cell; Tr1, regulatory T cell; VAT, variant antigenic type; VSG, variant surface glycoprotein.

Received for publication August 16, 2005. Accepted for publication November 10, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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