Murine dendritic cells pulsed in vitro with Toxoplasma gondii antigens induce protective immunity in vivo

Abstract

The activation of a predominant T-helper-cell subset plays a critical role in disease resolution. In the case of Toxoplasma gondii, the available evidence indicates that CD4(+) protective cells belong to the Th1 subset. The aim of this study was to determine whether T. gondii antigens (in T. gondii sonicate [TSo]) presented by splenic dendritic cells (DC) were able to induce a specific immune response in vivo and to protect CBA/J mice orally challenged with T. gondii cysts. CBA/J mice immunized with TSo-pulsed DC exhibited significantly fewer cysts in their brains after oral infection with T. gondii 76K than control mice did. Protected mice developed a strong humoral response in vivo, with especially high levels of anti-TSo immunoglobulin G2a antibodies in serum. T. gondii antigens such as SAG1 (surface protein), SAG2 (surface protein), MIC1 (microneme protein), ROP2 through ROP4 (rhoptry proteins), and MIC2 (microneme protein) were recognized predominantly. Furthermore, DC loaded with TSo, which synthesized high levels of interleukin-12 (IL-12), triggered a strong cellular response in vivo, as assessed by the proliferation of lymph node cells in response to TSo restimulation in vitro. Cellular proliferation was associated with gamma interferon and IL-2 production. Taken together, these results indicate that immunization of CBA/J mice with TSo-pulsed DC can induce both humoral and Th1-like cellular immune responses and affords partial resistance against the establishment of chronic toxoplasmosis.

FIG. 1

Phenotypic profile of DC. Splenic DC were purified and stained with MAbs as described in Materials and Methods.

FIG. 2

Assay for protection against oral challenge. CBA/J mice injected with 3 × 10⁵ unpulsed DC (DC) or TSo-pulsed DC (DC∼TSo) or with TSo alone (TSo) and untreated mice were orally infected with 100 cysts of T. gondii 76K 28 days later. The brain cyst load was evaluated 1 month after challenge. The results are the means of the numbers of brain cysts in each mouse ± standard deviations (SD). Results from one of three similar experiments are shown.

FIG. 3

Isotype analysis of TSo-specific responses. Mice were injected with 3 × 10⁵ TSo-pulsed DC (DC∼TSo) or with unpulsed DC (DC). Control mice were injected with TSo alone (TSo) or untreated. The total specific response (all isotypes) and specific antibodies of IgG1 and IgG2a isotypes were tested on day 24 after immunization by ELISA for each mouse serum (dilution of 1:150). O.D., optical density. The results are representative of three separate experiments.

FIG. 4

Western blot analysis of T. gondii antigens recognized by serum IgG antibodies and MAbs. Mice were injected with TSo-pulsed DC (lane 1), TSo alone (lane 2), or unpulsed DC (lane 3) or were untreated (lane 4). The following MAbs were used: 3G11 (anti-p22 [SAG2]) (lane a), 1E5 (anti-p30 [SAG1]) (lane b), 1F7 (anti-gp60 [MIC1]) (lane c), 4A7 (anti-55- and 60-kDa [ROP2 to ROP4]) (lane d), and 4A11 (anti-p100 [MIC2]) (lane e). The molecular masses (in kilodaltons) of protein standards are given on the left.

FIG. 5

In vitro proliferation of lymph node cells from mice injected in the fore and hind footpads with 3 × 10⁵ TSo-pulsed DC (DC∼TSo) or unpulsed DC (DC) per footpad. Control mice were injected with TSo alone (TSo) or untreated. Results are expressed as Δcpm. The results are representative of at least three experiments.