Role of CD4+ Foxp3+ Regulatory T Cells in Protection Induced by a Live Attenuated, Replicating Type I Vaccine Strain of Toxoplasma gondii

Abstract

Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii type I and type II strains. The involvement of regulatory T cells (Tregs) in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4(+) Foxp3(+) T regulatory cells followed by recruitment/expansion of CD4(+) Foxp3(-) CD25(+) effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Tregs were of the TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4(+) Foxp3(+) cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of the TH1/TH17-like type. In addition, in vivo Treg induction in RH-infected mice with interleukin-2 (IL-2)/anti-IL-2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Tregs may contribute to the protective response after vaccination with Mic1.3KO.

FIG 1

A lack of parasite control in the RH group is not correlated with low levels of IFN-γ secretion. (A) Parasites were counted directly after peritoneal lavage at the indicated times postinfection with 100 RH or Mic1.3KO tachyzoites. Results are expressed as the median plus range. Cumulative data from four different experiments are shown (4 to 6 mice per group per time point per experiment). (B) Sera were recovered at the indicated times postinfection, and the IFN-γ levels were quantified. Results are representative of those from three independent experiments with 5 or 6 mice per group per time point in which mice were injected with 100 tachyzoites of RH or Mic1.3KO. **, P < 0.01 using the Kruskal-Wallis test followed by Dunn's posttest; ***, P < 0.001 using the Kruskal-Wallis test followed by Dunn's posttest.

FIG 2

CD4⁺ Foxp3⁺ Tregs were recruited/expanded before CD4⁺ Foxp3⁻ CD25⁺ T effector cells at the local site after infection with Mic1.3KO but not at the systemic level. PECs recovered at the indicated times postinfection and splenocytes recovered at 7 dpi were stained with anti-CD4 anti-CD25 and anti-Foxp3 MAbs and analyzed by flow cytometry. (A) Percentage of the total Foxp3⁺ population among CD4⁺ cells recovered at 4 dpi. *, P < 0.05 using the Kruskal-Wallis test. (B and C) For calculation of the count, the absolute number of lymphocytes was evaluated by multiplying the total PEC count by the percentage of the lymphocyte gate compared to the whole population. Tregs were defined as Foxp3⁺ CD25⁺ and Fopx3⁺ CD25⁻ cells within the CD4⁺ population (B), and T effector cells (T eff) were defined as Foxp3⁻ CD25⁺ cells within the CD4⁺ population (C). The data presented are representative of those from two independent experiments with 5 mice per group per injection time point. *, P < 0.05 using the Kruskal-Wallis test followed by Dunn's posttest; **, P < 0.01 using the Kruskal-Wallis test followed by Dunn's posttest. (D) Percentage of the total Foxp3⁺ and CD25⁺ Foxp3⁺population within CD4⁺ splenocytes at 7 dpi with RH or Mic1.3KO tachyzoites. Results are representative of those from four independent experiments with 4 to 6 mice per group. *, P < 0.05 using the Kruskall-Wallis test followed by Dunn's posttest. (E) Ratio of the percentage of Foxp3⁺ cells expressing CD25 compared to the total percentage of Foxp3⁺ cells. Results are cumulative data from 4 different experiments. *, P < 0.05 using the Kruskall-Wallis test followed by Dunn's posttest.

FIG 3

Higher chemokine levels in the RH group. Chemokines (CCL2, CCL3, CCL20) in sera (A) and peritoneal washes (B to D) were quantified at 7 dpi with RH or Mic1.3KO tachyzoites. The levels of all chemokines were below the detection threshold in control naive mice. The data presented are representative of those from five independent experiments with 5 to 6 mice per group. **, P < 0.01 using the Wilcoxon-Mann-Whitney exact test with strata; ***, P < 0.001 using the Wilcoxon-Mann-Whitney exact test with strata.

FIG 4

Higher proinflammatory cytokine levels in the RH group. (A to D) IL12p40, IL12p70, and IL-23 were quantified by ELISA in peritoneal washes (IL12p40 only) (A) and in sera (B to D) at 7 dpi. (E to H) IFN-γ and IL-6 levels in peritoneal washes (E, G) and sera (F, H) at 7 dpi were quantified by ELISA. The levels of all cytokines were below the detection threshold in control naive mice. The data presented are representative of those from four independent experiments with 4 to 6 mice per group. **, P < 0.01 using the Wilcoxon-Mann-Whitney exact test with strata; ***, P < 0.001 using the Wilcoxon-Mann-Whitney exact test with strata.

FIG 5

Strong systemic, nonspecific IFN-γ production and low levels of IL-10 production induced by infection with RH. Splenocytes were recovered at 7 dpi with RH or Mic1.3KO tachyzoites and stimulated with medium or with parasite extract (TE). Culture supernatants were collected after a 48-h stimulation period. IFN-γ (A) and IL-10 (B) levels were determined by ELISA. The data presented are representative of those from four independent experiments with 4 to 6 mice per group.*, P < 0.05 using the Kruskall-Wallis test followed by Dunn's posttest; **, P < 0.01 using the Kruskall-Wallis test followed by Dunn's posttest.

FIG 6

Higher chemokine and proinflammatory cytokine levels in the RH-infected control group than the RH-infected and IL-2-treated group. (A and B) The chemokine CCL2 (A) and CCL3 (B) levels in peritoneal washes were quantified at 7 dpi. (C to F) IFN-γ (C, D) and IL-6 (E, F) levels in peritoneal washes (C, E) and sera (D, F) were quantified at 7 dpi by ELISA. The control and IL-2 groups were injected by the i.p. route with PBS and IL-2/anti-IL-2 antibody, respectively. Results are expressed as the median plus range. The data presented are representative of those from two independent experiments with 5 to 6 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using Wilcoxon-Mann Whitney exact test with strata on the cumulative data of the two experiments.

FIG 7

Systemic, antigen-specific IFN-γ and IL-10 production after treatment with IL-2/anti-IL-2 in RH-infected mice. Splenocytes were recovered at 7 dpi with RH tachyzoites and stimulated for 72 h with TE. Culture supernatants were collected after a 48-h stimulation period. The data presented are representative of those from two independent experiments with 5 to 6 mice per group. The control and IL-2 groups were injected by the i.p. route with PBS and IL-2/anti-IL-2 antibody, respectively. Values were corrected by subtraction of the level in control wells without antigen. IFN-γ (A) and IL-10 (B) levels were measured by ELISA.*, P < 0.05 using the Wilcoxon-Mann-Whitney exact test with strata on the cumulative data from the two experiments.