Figure 1.
Groups of three mice were infected with nonlethal P. yoelii 17XNL or lethal P yoelii YM. Blood smears were made every 2–3 d and stained with Giemsa. The numbers of infected red cells were counted in at least 20 fields of approximately 150 red cells to establish the absence of patent parasitemia. Parasitemias were monitored in every experiment, and this figure is an example of the typical course of infection. Error bars represent mean parasitemia ± standard error of the mean. The parasitemia curves represent one of multiple experiments.
Figure 2.
Maturation Profiles and Phenotype of DCs following Infection with Lethal and Nonlethal P. yoelii
Groups of three mice were infected with either nonlethal P. yoelii 17XNL or lethal P. yoelii YM. DCs were isolated from individual spleens and labelled to detect MHC class II, CD80, and CD86 by flow cytometry 7 d after infection.
(A) The flow cytometry profiles are examples of DCs from naïve mice or infected mice expressing the relevant cell surface molecules. The profiles were similar between three experiments.
(B) The bar charts represent the average mean fluorescence intensity (MFI) of DCs expressing the relevant cell surface molecules from at least three mice per group. The error bars represent the MFI ± standard error of the mean. The bar charts represent one of three experiments that gave similar profiles.
(C) Bar chart shows the absolute numbers of CD11c+CD8+ DCs, CD11c+CD4+ DCs, CD11c+CD4−CD8− DCs, CD11c+CD45R(B220)+ DCs, or F480hi macrophages per spleen in infected and naïve mice for groups of mice ± standard error of the mean. The absolute number of cells for each subpopulation was calculated by multiplying the percentage of cells by the total number of cells isolated from the spleen. Labelling by the control antibody was used to set the gates. The p-values in the bars for a nonlethal infection represent the statistical difference compared to a lethal infection.
Figure 3.
Comparison of Cytokine Profiles from Groups of Mice following Nonlethal and Lethal P. yoelii Infections and Cytokine Secretion by DCs
(A) Groups of three mice were infected with nonlethal P. yoelii 17XNL or lethal P. yoelii YM. After 4 and 7 d, the levels of IL-12 and TNF-α were assessed in the serum using the Becton Dickinson bead array assay. Error bars represent the pg of cytokine per ml ± standard error of the mean. The data represent one of duplicate experiments.
(B) Groups of three or five mice were infected with nonlethal or lethal P. yoelii. After 7 d, total CD11c+ DCs were isolated from individual, naïve, or infected mice, cultured with or without CpG oligonucleotide plus LPS overnight, and then tested for IL-12 secretion by a p70 IL-12–specific ELISPOT assay. Error bars represent the mean number of DCs secreting IL-12 per spleen ± standard error of the mean. The data represent one of multiple experiments.
(C) Total CD11c+ DCs were isolated from groups of three individual, naïve, or infected mice and cultured with CpG oligonucleotide and poly-I:C for 36 h. The supernatants were tested for secretion of α-interferon by ELISA. Error bars represent the mean pg secretion of α-interferon per spleen ± standard error of the mean. The data represent one of triplicate experiments for the nonlethal parasite and duplicate for the lethal parasite.
Figure 4.
Comparison of the Antigen Presentation Function of CD11c+ DCs Isolated from Groups of Mice following Nonlethal and Lethal P. yoelii Infections
Groups of three mice were infected with nonlethal or lethal P. yoelii. After 7 d, CD11c+ DCs were isolated from individual mice, serially diluted, and incubated with a fixed number of allogeneic T cells (BALB/c) or syngeneic T cells from OTII mice with ovalbumin. After 3 d, the cultures were pulsed with 3H-thymidine for 18 h and the uptake of radiolabel measured. The error bars represent mean count ± standard error of the mean. These assays were undertaken three times.
Figure 5.
Protection Is Mediated by DCs and by IL-12
(A) To measure the protection offered by CD11c+ DCs, large groups of mice were infected with nonlethal P. yoelii 17XNL or lethal P. yoelii YM. DCs were isolated from naïve or infected (drug-cured) mice, and 1.5 × 107 DCs were transferred to each naïve mouse in groups of four or five. After 24 h, each mouse was infected with a lethal dose of P. yoelii YM, and survival, parasitemia, and hemoglobin levels monitored every 1–3 d. This experiment was repeated twice with mice given 1.5 × 107 DCs.
(B) To determine the role of IL-12 in DC-mediated protection, groups of mice were infected with nonlethal P. yoelii 17XNL, total CD11c+ DCs were isolated from these infected mice after drug curing, and 1.5 × 107 DCs were transferred to naïve mice in groups of four or five. The cohorts of mice were given control Ig or anti-IL-12 15 h after the transfer of DCs, and after 24 h, each mouse was infected with a lethal dose of P. yoelii YM. Additional doses of control immunoglobulin or anti-IL-12 were given after 3 and 6 d. This experiment was repeated twice. In all experiments, mice were monitored daily following infection and culled if required, but the hemoglobin and parasitemia levels were measured every 2–3 d. Error bars represent ± standard error of the mean. The mean hemoglobin level in 20 naïve mice is represented by a cross-symbol on the y-axis.
Figure 6.
Protection Is Mediated by IL-12 Secreted by Donor DCs
To confirm that IL-12 secreted by the donor DC-mediated protection, groups of six C57BL/6 and IL-12KO mice were infected with nonlethal P. yoelii 17XNL, total CD11c+ DCs were isolated from these infected mice after drug curing, and 1.5 × 107 DCs were transferred to groups naïve mice. After 24 h each mouse was infected with a lethal dose of P. yoelii YM. In all experiments, mice were monitored daily following infection and culled if required, but parasitemia levels were measured every 2–3 d. Error bars represent ± standard error of the mean. This experiment was repeated twice.