Article Figures & Data
Figures
- FIG. 1.
Survival of H. polygyrus-infected mice after T. gondii challenge. (A) Photomicrographs of gut and liver of dually infected mice. Five- to eight-week-old female C57BL/6 mice were infected with H. polygyrus L3, and subsequently 8 days later, the mice were inoculated perorally with T. gondii cysts. At day 10 p.i., the mice were sacrificed and tissues were subjected to histopathological analysis: (i) Ileum from a dually infected mouse showing mild enteritis with slight hypercellularity in the lamina propria. (ii) Ileum from a T. gondii-infected mouse showing subacute enteritis of moderate severities, consisting of increased numbers of mononuclear cells in the lamina propria, blunting of the intestinal villi, and parasites in the intestinal mucosa (arrows). (iii) Liver from a dually infected mouse showing the presence of small numbers of focal inflammatory infiltrates (arrow). (iv) Liver from a T. gondii-infected mouse showed multiple medium foci of inflammatory infiltrates consisting of various numbers of mononuclear cells with occasional neutrophils throughout the parenchymal and portal areas together with the presence of perivasculitis and vasculitis of moderate severities in medium to large blood vessels (arrows) (hematoxylin and eosin; bar = 100 mm). (B) Infection with H. polygyrus results in increased survival rates of mice concomitantly infected with T. gondii (Toxo). Age-matched C57BL/6 mice were coinfected with H. polygyrus and T. gondii as described above, and survival was monitored. There were six animals per group, and data are representative of one experiment. (C) Survivors from the experiment shown in panel B were sacrificed at day 30 post-T. gondii infection, and brains were evaluated for cyst number. Data are presented as means ± standard deviations.
- FIG. 2.
Serum IFN-γ levels of dually infected mice. C57BL/6 mice carrying worm infection were challenged perorally with 10 cysts of T. gondii (Toxo). At day 10 p.i., the serum collected from dually infected and control mice were pooled (three mice per group) and analyzed by ELISA for levels of IFN-γ.
- FIG. 3.
Antigen-specific response (proliferation and IFN-γ production) by T-cell subsets in dually infected mice at day 10 post-T. gondii infection. (A and C) Proliferation. Five- to eight-week-old C57 BL/6 mice were infected with T. gondii (Toxo) and H. polygyrus. At day 10 post-T. gondii infection, mice were sacrificed and spleens and MLNs were collected and pooled (three mice per group). CD4 and CD8+ T cells from the tissues isolated by affinity purification (>95% pure as determined by fluorescence-activated cell sorter analysis; 1 × 105 cells/well) were cultured with 1 × 105 irradiated feeder cells and stimulated with 15 μg/ml of TLA or 20 μg/ml of HpAg. Lymphoproliferation was indicated by [3H]thymidine incorporation after 72 h of incubation. The experiment was performed twice with similar results, and data are representative of one experiment. (B) IFN-γ-positive cells. Splenocytes and MLNs were harvested at day 10 after T. gondii infection, pooled (n = 3), and cultured in vitro with phorbol myristate acetate, ionomycin, and monesin for 4 h. The cultured cells were surface stained for CD4 or CD8 before intracellular staining for IFN-γ. Data are expressed as means ± standard deviations (per lymphoid tissue) and are representative of two independent experiments.
- FIG. 4.
Antigen-specific response in dually infected mice at day 30 post-T. gondii infection. (A) Proliferation. Dually infected mice were sacrificed at day 30 post-T. gondii (Toxo) infection, and antigen-specific proliferation of T-cell subsets was determined. The experiment was performed twice with similar results, and the data are representative of one experiment. (B) IFN-γ-positive cells. The absolute number of IFN-γ-positive CD4 and CD8+ T-cell subsets was determined at day 30 post-T. gondii infection as previously described. Data are expressed as means ± standard deviations (per lymphoid tissue) and are representative of two separate experiments.
- FIG. 5.
Effector response of CD8+ T cells from dually infected mice. (A) Antigen-specific cytotoxicity. MLNs from the mice carrying dual infection or T. gondii (Toxo) or worm infection alone were isolated at day 14 post-T. gondii infection. The tissues (three mice per group) were pooled, and CD8+ T cells were isolated and cultured in presence of 15 μg/ml of TLA and irradiated feeder cells. After 5 days of culture, CD8+ T cells were collected and incubated with 51Cr-labeled T. gondii-infected macrophages at various effector/target ratios. Four hours later, cytotoxic activities were measured by radioactive release. Data are representative of two separate experiments. (B) Adoptive transfer. Purified CD8+ T cells from the MLNs of dually infected mice or mice carrying T. gondii or worm infection were isolated at day 14 p.i. Purified cells (5 × 106) from each group were adoptively transferred to naïve mice (six animals per group). Twenty-four hours later, the mice were challenged perorally with 35 T. gondii cysts. Each experiment was performed twice with similar results, and the data are representative of one experiment.
- FIG. 6.
CD4+ T-cell neutralization of mice carrying chronic T. gondii infection. C57BL/6 mice infected with T. gondii and H. polygyrus or T. gondii alone were administered 0.5 mg of anti-CD4 starting at day 31 after T. gondii infection. The control mice received similar doses of rat IgG. The antibody treatment was continued for 3 successive days and twice a week thereafter. (A) Mortality. The mice were monitored daily for morbidity or mortality until the termination of the experiment. There were six mice per group, and the experiment was performed twice with similar results. (B) Parasite load. Some of the antibody-treated mice were sacrificed at day 17 post-antibody treatment, and brain, liver, spleen, and lung were evaluated for the parasite load by quantitative PCR. (C) Histopathological analysis. Brains of some of the infected mice were analyzed by histopathological studies. Reactivation is shown. (i and ii) Sections of brain from mice infected with T. gondii and treated with control antibody (×20). In panel ii, an arrow points to a tissue cyst. (ii) Cysts were rare and there was minimal inflammation present in these sections. (iii) Sections of H. polygyrus-infected mice treated with antibody to CD4 showed no inflammation or abnormalities in tissue sections (×20). (iv) Tissue sections prepared from T. gondii-infected mice treated with antibody to CD4 were similar to those in mice infected with T. gondii, with rare foci of inflammatory cells seen (×20). (v) Sections from mice infected with H. polygyrus alone showed no inflammation or abnormalities (×20). (vi, vii, and viii) Sections from mice infected with H. polygyrus and T. gondii and treated with control IgG showed a marked increase in the number of cysts in the tissue (vi, ×10; vii and viii, ×40). Arrows in panel vi point to groups of multiple cysts seen at low power (×10). Panel vii demonstrates a cyst in the process of rupturing, releasing T. gondii (arrow) that can invade adjacent cells. Lymphocyte infiltration is present in the vicinity of the rupturing cyst. Panel viii demonstrates focal infiltrates and a developing glial nodule (arrows). Such infiltrates were very common in the brains of dually infected mice. (ix and x) Sections (×20) from T. gondii-infected mice previously infected with H. polygyrus and treated with antibody to CD4 showed a decrease in the number of cysts and areas of inflammation (arrow) compared to dually infected mice treated with control antibody (vi, vii, and viii).
