Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection

Abstract

It was previously demonstrated that immunizing mice with spleen dendritic cells (DCs) that had been pulsed ex vivo with Toxoplasma gondii antigens triggers a systemic Th1-biased specific immune response and induces protection against infection. T. gondii can cause severe sequelae in the fetuses of mothers who acquire the infection during pregnancy, as well as life-threatening neuropathy in immunocompromised patients, in particular those with AIDS. Here, we investigate the efficacy of a novel cell-free vaccine composed of DC exosomes, which are secreted antigen-presenting vesicles that express functional major histocompatibility complex class I and II and T-cell-costimulatory molecules. They have already been shown to induce potent antitumor immune responses. We investigated the potential of DC2.4 cell line-derived exosomes to induce protective immunity against toxoplasmosis. Our data show that most adoptively transferred T. gondii-pulsed DC-derived exosomes were transferred to the spleen, elicited a strong systemic Th1-modulated Toxoplasma-specific immune response in vivo, and conferred good protection against infection. These findings support the possibility that DC-derived exosomes can be used for T. gondii immunoprophylaxis and for immunoprophylaxis against many other pathogens.

FIG. 1.

Immunofluorescence analysis of DC2.4 cell markers. TAg-pulsed DCs express more MHC class II molecules and CD80 than unpulsed DCs. Both express the same level of CD86, and neither expresses CD8α, CD40, MAC-1, or CD90.2. Shaded peaks represent the results of cell staining with the isotype standard only. These data are representative of three experiments with similar results. FSC, forward scatter; SSC, side scatter; FL1 and -2, fluorescence experiments 1 and 2, respectively.

FIG. 2.

Negative staining of DC-derived exosomes obtained by differential ultracentrifugation of a 24-h culture of DC2.4 cell supernatant; 60- to 150-nm-diameter vesicles were observed in an isolated (A) or congregated (B) form.

FIG. 2.

Negative staining of DC-derived exosomes obtained by differential ultracentrifugation of a 24-h culture of DC2.4 cell supernatant; 60- to 150-nm-diameter vesicles were observed in an isolated (A) or congregated (B) form.

FIG. 3.

Fifty micrograms of TAg-pulsed-DC2.4 cell-derived exosomes (A) or 5 × 10⁶ TAg-pulsed-DC2.4 cells (B) were incubated with ⁵¹Cr before injection into C57BL/6 mice. At 2 h (first white bar for each sample), 6 h (black bar), or 24 h (second white bar for each sample) after the transfer, radioactivity was measured in each organ. Levels of homing to different organs were compared. Results are expressed as the percentages of radioactivity recovered from a specific organ out of the total radioactivity in the mouse. Results from one of three similar experiments are shown and are expressed as means ± standard errors of the means (P < 0.05). CLN, cervical lymph node; ILN, inguinal lymph node; PLN; popliteal lymph node; BLN, brachial lymph node; MLN, mesenteric lymph node.

FIG. 4.

Western blot analysis of T. gondii antigens recognized by serum IgM (A) and serum IgG (B) antibodies. Sera were collected from untreated mice (lane 1); from mice treated with TAg antigen (lane 2), unpulsed DC2.4 cells (lane 3), TAg-pulsed DC2.4 cells (lane 4), unpulsed-DC2.4 cell-derived exosomes (lane 5), and TAg-pulsed-DC2.4 cell-derived exosomes (lane 6); and from mice infected 21 days earlier with T. gondii cysts (strain 76K) (lane 7). The MAb 1E5 (anti-P30 [SAG1]) was used as a control (lane 8). The molecular masses (in kilodaltons) of protein standards are given on the left.

FIG. 5.

Cellular proliferative response and cytokine production following intravenous immunization with 10 μg of TAg-pulsed- or unpulsed-DC2.4 cell-derived exosomes or with 2.5 × 10⁵ TAg-pulsed or unpulsed DC2.4 cells, before challenge with 30 cysts of T. gondii 76K. At 10 days after immunization, the spleen cells were isolated and stimulated in vitro with TAg (10 μg/ml). Proliferation was assessed after 4 days. Values were measured at 24 h of culture for IL-2, at 48 h for IL-5, at 72 h for IFN-γ, and at 96 h for IL-10. Results are the mean cytokine concentrations or counts per minute for proliferation assays with spleen and mesenteric lymph node cells from three mice per experimental group. Results from one of three similar experiments are shown and are expressed as means ± standard errors of the means (P < 0.001).

FIG. 6.

Survival of C57BL/6 mice following immunization with TAg, unpulsed-DC2.4 cell-derived exosomes, and TAg-pulsed-DC2.4 cell-derived exosomes (A), and with TAg, unpulsed DC2.4 cells, and TAg-pulsed DC2.4 cells (B). Mice immunized twice with 10 μg of TAg, with 2.5 × 10⁵ TAg-pulsed or unpulsed DC2.4 cells, or with 10 μg of TAg-pulsed- or unpulsed-DC2.4 cell-derived exosomes were orally infected with 30 cysts of T. gondii 10 days later. Mice were observed daily for mortality. These data are representative of three experiments with similar results.

FIG. 7.

Assay for protection against chronic toxoplasmosis (oral challenge). C57BL/6 mice were immunized twice with 2.5 × 10⁵ Tag-pulsed or unpulsed DC2.4 cells, with 10 μg of TAg-pulsed- or unpulsed-DC-derived exosomes, or with 10 μg of TAg only. C57BL/6 mice were orally infected with 10 cysts of T. gondii 10 days later. The cyst burden was determined by counting brain cysts at 30 days postchallenge; standard deviations are noted. These data are representative of three experiments with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001.