Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4-deficient mice

Abstract

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.

FIG. 1.

(A) Survival of CD4-deficient mice and B6 mice infected p.o. with 10 ME49 cysts. Groups of six B6 and CD4-deficient mice were given 10 ME49 cysts p.o. The survival time of CD4-deficient mice was significantly shorter (P < 0.05). (B) Brain cyst burdens in infected CD4-deficient B6 and B10.BR mice. Groups of four (B6 background) or five (B10.BR background) mice were infected with 10 ME49 cysts p.o. Cyst burdens in brains were determined 5 weeks later. Significantly more cysts were found in brains of CD4-deficient mice of each strain than in brains of corresponding controls (P < 0.05). WT, wild type.

FIG. 2.

Survival of ts-4-vaccinated (Vacc.) B6 and CD4-deficient mice after a challenge with RH tachyzoites. Groups of five B6 or CD4-deficient female mice were vaccinated i.p. with 2 × 10⁴ ts-4 tachyzoites and challenged, along with groups of five unvaccinated (Unvacc.) controls, by i.p. injection of 2 × 10³ RH tachyzoites 56 days later. Vaccinated CD4-deficient mice had significantly shorter survival times than did vaccinated B6 controls (P < 0.05). Vaccinated CD4-deficient mice survived significantly longer than did unvaccinated CD4-deficient mice (P < 0.05).

FIG. 3.

(A) Percentage of CD45RB^lo CD8⁺ peripheral blood lymphocytes in vaccinated and unvaccinated CD4-deficient and control B6 mice. Groups of five B6 or CD4-deficient male mice were vaccinated i.p. with 2 × 10⁴ ts-4 tachyzoites and bled 8 weeks later. Peripheral blood leukocytes were isolated and analyzed by flow cytometry. Bars show the percentages of CD8⁺ lymphocytes that exhibited the CD45RB^lo phenotype. These percentages were not significantly different between vaccinated CD4-deficient mice and B6 controls but were significantly greater in vaccinated mice of each strain than in corresponding unvaccinated controls (P < 0.05). (B) Numbers of CD45RB^lo CD8⁺ splenic lymphocytes in ts-4-vaccinated and unvaccinated CD4-deficient and control B6 mice. Groups of four B6 or CD4-deficient mice were vaccinated with 2 × 10⁴ ts-4 tachyzoites i.p., and their spleens were harvested 8 weeks later and analyzed by flow cytometry. Numbers of activated CD8⁺ splenic lymphocytes were not significantly different between vaccinated B6 and CD4-deficient mice, but numbers of those cells were significantly greater in spleens of vaccinated mice than in those of corresponding unvaccinated controls (P < 0.05).

FIG. 4.

IL-12p40 levels in cultures of spleen cells from vaccinated CD4-deficient mice and B6 controls. Groups of four B6 or CD4-deficient mice were immunized by i.p. injection of 2 × 10⁴ ts-4 tachyzoites. Spleen cells were harvested 8 weeks later, plated, and stimulated in vitro with ts-4 tachyzoites as described in Materials and Methods. Culture media were harvested after 72 h and analyzed for content of IL-12p40 by ELISA. IL-12 levels were not significantly different between vaccinated B6 and CD4-deficient spleen cell cultures, but levels of each were significantly higher than levels in corresponding cultures from control unvaccinated mice (P < 0.05).

FIG. 5.

IFN-γ levels in cultures of spleen cells from vaccinated CD4-deficient mice and B6 controls. Groups of four B6 or CD4-deficient mice were immunized by i.p. injection of 2 × 10⁴ ts-4 tachyzoites. Spleen cells were harvested 8 weeks later and stimulated in vitro with ts-4 tachyzoites as described in Materials and Methods. Culture media were harvested after 72 h and analyzed for IFN-γ content by ELISA. IFN-γ levels were not significantly different between vaccinated B6 and CD4-deficient spleen cell cultures, but levels of each were significantly higher than levels in corresponding cultures from control unvaccinated mice (P < 0.05).

FIG. 6.

T. gondii-specific serum IgG2a levels in ts-4-vaccinated CD4-deficient and B6 mice. B6 and CD4-deficient mice were immunized by i.p. injection of 2 × 10⁴ ts-4 tachyzoites and bled 8 weeks later. T. gondii-specific serum IgG2a titers were determined by ELISA. Antibody titers in immunized B6 mice were significantly (P < 0.05) higher than those in immunized CD4-deficient mice. Titers in unimmunized mice of both strains were beneath the limit of detection (log₁₀ titer of 1.70).

FIG. 7.

Changes in body weight and survival of ts-4-immune CD4-deficient mice given immune or nonimmune serum and challenged with RH tachyzoites. Two groups of five CD4-deficient mice and one group of five B6 mice were immunized by i.p. injection of 2 × 10⁴ ts-4 tachyzoites. Six weeks later, CD4-deficient mice were given injections of 0.5 ml of immune (log₁₀ T. gondii-specific IgG2a titer, 4.20) or nonimmune serum on days −1, 0, 2, 4, 7, and 10 relative to an i.p. challenge with 2 × 10³ RH tachyzoites. CD4-deficient mice given nonimmune serum survived for a significantly shorter time than did CD4-deficient mice given immune serum (P < 0.05).

FIG. 8.

Survival of chronically infected CD4-deficient mice given serotherapy. Groups of CD4-deficient mice were infected with 10 ME49 cysts p.o. Mice were given i.p. injections of 0.5 ml immune or nonimmune serum on days 32, 36, 40, 42, 44, 46, 50, and 54. Mice given immune serum survived significantly longer than did mice given nonimmune serum (P < 0.05).