The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p

The heavy-chain gene-family usage and CDR3 length of polysaccharide-specific memory B cells.

<p>Sequences recovered from PS-specific MBCs were analyzed by IMGT/HighV-QUEST using the rhesus macaque immunoglobulin database. The heavy chain alleles of each B cell were generated by RT-PCR. (A) Heavy-chain gene-family usage at day 0 and 7 after immunization with 23vPS or 13vPnC. The number in the pie chart is the average number of variable sequences recovered from PS-specific MBCs. Pairwise statistical analysis was performed by Fisher’s Exact Test. (B) Distribution of heavy-chain gene-family usage for each PS serotype from PS-specific MBCs at 7 days following immunization with 13vPnC. The number in the pie chart is the average number of PS-specific MBCs against each antigen. Statistical analysis was performed by Fisher’s Exact Test. (C) The average CDR3 amino acid length of PS-specific MBCs at 0 and 7 days after immunization.</p

The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p

The fate of heat-killed and live <i>cpsII</i> parasites in host cells.

<p>C57BL/6 bone marrow derived macrophages were infected with <i>cpsII</i> parasites and examined using immunofluorescence assays (a). Bone marrow-derived macrophages activated with IFN-γ (100 U/ml) and LPS (0.1 ng/ml) were infected with freshly lysed or heat-killed parasites for 3 hours. Intracellular parasites were stained for host Irgb6 or LAMP1 recruitment in green. Parasites were stained with a mouse monoclonal antibody to GRA1 to identify the parasitophorous vacuole or rabbit polyclonal sera against GRA7 in red. IFN-γ and LPS activated bone marrow-derived macrophages were infected with freshly lysed <i>cpsII</i> parasites and fixed at 3, 24, 48 and 120 hours post-infection (b). Parasite vacuoles were identified with rabbit polyclonal sera to GRA7 (red) and host LAMP1 was identified with a rat monoclonal antibody. Scale bar = 10 µm. Electron micrograph images of infected macrophages treated with IFN-γ (50 units/ml) and LPS (10 ng/ml) or untreated at 2 hours post-infection (c). Parasites persist in intact vacuoles and do not display blebbing or disruption of the parasitophorous vacuole. Some <i>cpsII</i> parasites were found to exhibit non-productive cell division in IFN-γ and LPS- treated or untreated macrophages when examined 24 hours post-infection (d). Scale bars = 1.5 µm.</p

Composition of total cell populations, mCherry^+veViolet^−ve cell populations, and mCherry^+veViolet^+ve populations from the PECS of naïve and vaccinated mice.

<p>Mice were vaccinated with 10⁶ Violet-labeled, mCherry-expressing <i>cpsII</i> parasites intraperitoneally and sacrificed 18 hours post-vaccination. Cell type composition of total peritoneal cell populations in naïve and vaccinated mice, and the cell type composition of mCherry^+veViolet^−ve cells and mCherry^+veViolet^+ve cells in vaccinated mice were examined. Representative flow plots demonstrating infected cells and cells that have phagocytosed <i>T. gondii</i> for each major cell type present in the PECS are shown (a). The composition of the PECS in naïve mice and vaccinated mice, and the composition of infected cells (mCherry^+veViolet^+ve) and cells that have phagocytosed <i>T. gondii</i> (mCherry^+veViolet^−ve) are depicted (b). Percentages of macrophages and dendritic cells in the total peritoneal cell population in vaccinated mice are compared to the percentages of infected cells that are macrophages and dendritic cells (c). T/B/NK cells are identified by expression of CD3, CD19, or NK1.1. Dendritic cells were identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^HI,MHCII^HI. Monocytes and neutrophils were defined as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^LOW-INT,Gr-1^+ve. Macrophages were identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^LOW-INT,Gr-1^−ve,CD11b^INTorHI. *p<0.05; ***p<0.0005. AVG±STDEV. A paired, two-tailed student's t test was used to analyze the data in (c). Results shown are from one representative experiment. Similar results were obtained over the course of seven separate experiments.</p

Differences in pH sensitivity of two fluorescent markers can be used to distinguish parasites that have been phagocytosed from those that actively invade host cells.

<p>Fluorescence intensity of mCherry-expressing <i>cpsII</i> parasites labeled with CellTrace Violet and incubated overnight at varying pH in buffer solutions consisting of citric acid and disodium phosphate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004047#ppat.1004047-McIlvaine1" target="_blank">[93]</a> was measured by flow cytometry (a). Violet and mCherry fluorescence of immortalized murine bone marrow-derived macrophages exposed to Violet-labeled, mCherry-expressing <i>cpsII</i> parasites pre-treated with DMSO (top) or the irreversible inhibitor of invasion 4-p-bpb (bottom) 1 hour and 18 hours following exposure to parasites, measured by flow cytometry (b). Images of mCherry^+veViolet^+ve and mCherry^+veViolet^−ve bone marrow-derived macrophages 18 hours following exposure to Violet-labeled, mCherry-expressing <i>cpsII</i> parasites pre-treated with 4-p-bpb or DMSO (c). Violet and mCherry fluorescence of cells isolated from the PECS of mice 18 hours post-administration of 10⁶ DMSO-treated or 4-p-bpb-treated parasites (d). Cytospin analysis was performed on Violet^+ve cells isolated by FACS sorting, obtained from the PECS of a mouse 18 hours after vaccination with Violet-labeled <i>cpsII</i> parasites (e). Images of mCherry^+veViolet^+ve and mCherry^+veViolet^−ve cells isolated from the PECS of mice 18 hours post-administration of 10⁶ DMSO-treated or 4-p-bpb-treated Violet-labeled, mCherry-expressing <i>cpsII</i> parasites (f).</p

Active invasion is required for adaptive immune responses to <i>T. gondii</i>.

<p><i>cpsII</i>-OVA parasites were heat-killed, treated with the invasion inhibitor 4-p-bpb or left untreated and administered to mice intraperitoneally. Tetramer-specific and total CD4⁺ (a) and CD8⁺ (b) T cell responses were measured from cells isolated from the spleen and lymph nodes (pooled) 10 days post-vaccination. Flow plots are gated on Foxp3^−ve CD4⁺ T cells (a, top) or CD4⁺ T cells (a, bottom) and the population examined at the bottom of A was enriched for tetramer^+ve cells. Flow plots in B are gated on CD8⁺ T cells. *p<0.05; **p<0.005. AVG±SE.</p

Infected cells are sufficient to induce CD4⁺ and CD8⁺ T cell responses.

<p>Bone marrow-derived dendritic cells were cultured overnight with Violet-labeled, mCherry-expressing <i>cpsII</i> parasites. The following day, dendritic cells were sorted into mCherry^+veViolet^+ve (infected) and mCherry^+veViolet^−ve (uninfected) populations and 10⁴ dendritic cells from each population were administered to mice. In parallel, dendritic cells that had phagocytosed <i>T. gondii</i> were obtained by sorting on bone marrow-derived dendritic cells that were incubated with invasion-blocked Violet-labeled, mCherry-expressing <i>cpsII</i> parasites. 10 days later, mice were sacrificed and CD4⁺ (a) and CD8⁺ (b) T cell responses in the peritoneal cavity and spleen were analyzed. Populations shown depicting CD4⁺-tetramer binding are enriched for the tetramer^+ve population and these cells were isolated from the spleen (a, bottom). All other cell populations shown were harvested from the peritoneal cavity, although similar trends were apparent when splenocytes were examined. Flow plots are gated on Foxp3^−ve CD4⁺ T cells (a, top), CD4⁺ T cells (a, bottom) or CD8⁺ T cells (b). Parasite burdens from the PECS of mice transferred infected dendritic cells or dendritic cells that have phagocytosed <i>T. gondii</i> 5 days post-challenge with 10³ tachyzoites of a highly virulent, replicating strain of <i>T. gondii</i>, administered 6 weeks following vaccination with 10⁴ infected or uninfected dendritic cells, analyzed by flow cytometry (c). Significance in (c) was determined using a Mann-Whitney U-test. *p<0.05; **p<0.005. AVG±SE.</p

Dendritic cells are required for optimal CD4⁺ and CD8⁺ T cell responses.

<p>CD11c-DTR mice were administered diphtheria toxin 1 day prior to <i>cpsII</i>-OVA vaccination. At the time of vaccination, some mice were sacrificed to determine the efficiency of depletion. Percentages and numbers of dendritic cells from the spleen are shown. FACS plots are gated on CD3⁻,CD19⁻,NK1.1⁻ cells (a). Eight days following vaccination, mice were sacrificed and total and tetramer-specific CD4⁺ and CD8⁺ T cell responses were analyzed. Total CD4⁺ T cell responses from the spleens are shown (b, top). Tetramer-specific CD4⁺ T cell responses from pooled lymph nodes and splenocytes were determined in a separate experiment (b, bottom). Flow plots are gated on CD4⁺ T cells (b), and the population examined was magnetically enriched for the tetramer^+ve population (b, bottom). Total and OVA-specific CD8⁺ T cell responses from the PECS are depicted (c), and flow plots are gated on CD8+ T cells. Significant differences in tetramer and total CD8⁺ T cell responses between WT and CD11c-DTR mice were also apparent in the spleen. *p<0.05; **p<0.005. AVG±SE.</p

Activation status of mCherry^+veViolet^−ve and mCherry^+veViolet^+ve macrophages and dendritic cells.

<p>Mice were administered parasites as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004047#ppat-1004047-g003" target="_blank">Figure 3</a>. At 18 hours post-vaccination, expression of the antigen presentation molecules MHCI (a) and MHCII (b) and expression of the costimulatory molecules CD86 (c) and CD40 (d) on CD11b^HI macrophages and dendritic cells was determined by flow cytometry. Macrophages are identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^−ve,Gr-1^−ve,CD11b^HI cells. Dendritic cells are identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^HI,MCHII^HI. Confidence intervals were determined using the Bonferroni correction method. *p<0.017; **p<0.0017; ***p<0.00017. AVG±SE. Paired, two-tailed student's t tests were used to compare expression levels of molecules on populations within <i>cpsII</i>-vaccinated mice.</p