Lack of CD4(+) T cells does not affect induction of CD8(+) T-cell immunity against Encephalitozoon cuniculi infection

Abstract

Cell-mediated immunity has been reported to play an important role in defense against Encephalitozoon cuniculi infection. Previous studies from our laboratory have underlined the importance of cytotoxic CD8(+) T lymphocytes (CTL) in survival of mice infected with E. cuniculi. In the present study, immune response against E. cuniculi infection in CD4(+) T-cell-deficient mice was evaluated. Similar to resistant wild-type animals, CD4(-/-) mice were able to resolve E. cuniculi infection even at a very high challenge dose (5 x 10(7) spores/mouse). Tissues from infected CD4(-/-) mice did not exhibit higher parasite loads in comparison to the parental wild-type mice. Conversely, at day 21 postinfection, susceptible CD8(-/-) mice had 10(14) times more parasites in the liver compared to control wild-type mice. Induction of the CD8(+) T-cell response in CD4(-/-) mice against E. cuniculi infection was studied. Interestingly, a normal antigen-specific CD8(+) T-cell response to E. cuniculi infection was observed in CD4(-/-) mice (precursor proliferation frequency, 1/2.5 x 10(4) versus 1/10(4) in wild-type controls). Lack of CD4(+) T cells did not alter the magnitude of the antigen-specific CTL response (precursor CTL frequency; 1/1.4 x 10(4) in CD4(-/-) mice versus 1/3 x 10(4) in control mice). Adoptive transfer of immune CD8(+) T cells from both CD4(-/-) and wild-type animals prevented the mortality in CD8(-/-) mice. E. cuniculi infection thus offers an example of an intracellular parasitic infection where CD8(+) T-cell immunity can be induced in the absence of CD4(+) T cells.

FIG. 1

Survival of gene knockout mice challenged with a high dose of E. cuniculi spores. Five- to six-week-old female CD4^−/−, CD8^−/−, and wild-type (WT) C57BL/6 mice were infected i.p. with 5 × 10⁷ spores of E. cuniculi. Animals were monitored on a daily basis. The study was performed twice with similar results.

FIG. 2

Standardization of quantitative PCR for detection of E. cuniculi parasites. (A) Correlation of number of parasites with amount of internal standard. A competitive PCR was performed as described in Materials and Methods with a known amount of parasites and different dilutions of the internal standard. The 50 ng of DNA used in the quantitative PCR was obtained by adding a known number of parasites to 75 mg of liver from a naive mouse before DNA extraction. The range of the internal standard indicates the highest and lowest possible values for each number of parasites tested. (B) Detection of E. cuniculi DNA by competitive PCR. A competitive PCR was done using various dilutions of internal standard added to samples with a constant amount of DNA. The result shows a decrease of the signal of the genomic DNA as the concentration of the internal standard increases. The point at which the intensities of the two bands are equal is considered the concentration of the internal standard used to determine the number of parasites in each sample.

FIG. 3

Levels of parasite DNA in organs of E. cuniculi-infected knockout (CD4^−/− and CD8^−/−) and parental C57BL/6 mice. CD4^−/−, CD8^−/−, and wild-type (WT) C57BL/6 mice were infected i.p. with 10⁷ spores of E. cuniculi. At days 0, 7, 14, and 21 p.i., the mice were sacrificed and the livers were isolated. The liver DNA was extracted as described in Materials and Methods; 3 μg of purified DNA was amplified by PCR using E. cuniculi-specific primers. Each time point comprised three mice, and data are expressed as mean ± SD. The experiment was performed twice with similar results.

FIG. 4

Antigen-induced proliferation of T cells from E. cuniculi-infected mice in an LDA. Five- to eight-week-old female CD4^−/−, CD8^−/−, and parental wild-type (WT) C57BL/6 mice were infected i.p. with 10⁷ spores of E. cuniculi. At day 15 p.i., total T cells (>95% pure) from the pooled splenocytes (n = 3 mice/group) were isolated and cultured in the presence of E. cuniculi spores and irradiated feeder cells. After 1 week in culture, PPF of T cells was determined. Data are representative of one of two separate experiments.

FIG. 5

In an LDA, E. cuniculi-infected mice generate pCTLs when stimulated in vitro with E. cuniculi spores. Five- to eight-week-old CD4^−/−, CD8^−/−, and wild-type (WT) C57BL/6 mice were infected with E. cuniculi as described in Materials and Methods. At day 17 p.i., splenocytes from each group of mice were isolated, pooled (three mice/group), and cultured by LDA in the presence of spores and irradiated feeder cells. After 1 week in culture, pCTL frequency of spleen cells was determined. Data shown are representative of one of the two separate experiments performed.

FIG. 6

Detection of cytokine production by intracellular staining. Five- to six-week-old wild-type C57BL/6 and age-matched knockout mice were infected with 10⁷ spores of E. cuniculi as described in Materials and Methods. At day 15 p.i., total splenocytes from CD4^−/− (D), CD8^−/− (B), and wild-type (A and C) infected mice were cultured in vitro with PMA, ionomycin, and monensin for 4 h. Cultured cells were then labeled for CD4⁺ (A and B) or CD8⁺ (C and D) T cells before intracellular staining for IFN-γ, IL-4, and IL-10. Values are the mean percentage of cells positive for IFN-γ, IL-4, or IL-10. Error bars represent the SD for four mice per group. Statistical significance was determined using the Student t test (∗, P < 0.05).

FIG. 7

Adoptive transfer of immune CD8⁺ T cells from wild-type (WT) E. cuniculi-infected mice protects naive CD8^−/− mice against a lethal E. cuniculi challenge. CD8⁺ T cells from pooled splenocytes (n = 3/group) from E. cuniculi-infected parental C57BL/6 mice were isolated by magnetic separation at day 17 after challenge. A total of 10⁷ CD8⁺ T cells (>95% pure) were injected intravenously into CD8^−/− mice (n = 6 mice/group). Control animals received an equal amount of cells from uninfected mice. After 24 h, mice were challenged i.p. with 10⁷ spores of E. cuniculi, and survival was monitored until the end of the experiment. The experiment was performed twice with similar results.

FIG. 8

Adoptive transfer of immune CD8⁺ T cells from CD4^−/− E. cuniculi-infected mice protect naive CD8^−/− mice against a lethal E. cuniculi challenge. CD8⁺ T cells from pooled splenocytes (n = 3 mice/group) from E. cuniculi-infected CD4^−/− and parental wild-type (WT) C57BL/6 mice were isolated by magnetic separation at day 17 after challenge. A total of 10⁷ CD8⁺ T cells (>95% pure) were injected intravenously to CD8^−/− mice (n = 6 mice/group). Control animals received an equal amount of cells from uninfected mice. After 24 h, mice were challenged i.p. with 10⁷ spores of E. cuniculi, and survival was monitored until the end of the experiment. The experiment was performed twice with similar results.