Mice were injected i.p. with 10⁶ infected erythrocytes. At days 0 (naive mice) and 10 post-injection, DC subsets were sorted by FACS. DC subsets were then co-cultured at various ratios with naive CD4 T cells isolated from DO11.10 mice, with or without OVA peptide. After 80 hours of co-culture, the culture media was assayed for cytokine content using the BD Cytometric Bead Array. Error bars represent standard deviation within co-cultures of cells sorted from 3 individual mice (*, P < 0.05; **, P<0.01; ***, P < 0.001 when comparing CD11c^hi co-cultures to CD11c^loCD45RB^hi co-cultures by Student's t-test).

Mice were injected i.v. with 15μg LPS (white squares) or vehicle (PBS, black circles). (A) At the indicated days post-injection, splenocytes from groups of 3 mice were analyzed for the two subpopulations, expressed as the proportion of CD11c⁺ cells (DCs). (B) Each subset was also assayed for expression of costimulatory molecules, expressed as mean fluorescence intensity (MFI). Error bars represent standard deviation within groups of at least 3 mice (*, P < 0.05; ***, P < 0.001 when comparing LPS to PBS by two-way ANOVA). (C) FACS plots showing expression of CD40 on DCs from mice that had received vehicle (grey shaded histogram) or LPS (empty histogram with heavy line). An isotype-matched control was also analyzed (thin grey line).

Mice were injected i.p. with 10⁶ control or infected erythrocytes. At the indicated days post-injection, spleen CD11c⁺ DC subsets were analyzed for their expression of the costimulatory molecules. (A) CD40, CD80 and CD86 expression on DCs from control (RBC, black circles) or infected (iRBC, white squares) mice, expressed as mean fluorescence intensity (MFI). Error bars represent standard deviation within groups of 5 mice (*, P < 0.05; **, P<0.01; ***, P < 0.001 when comparing iRBC to RBC by twoway ANOVA). (B) FACS plots showing expression of CD40 on DCs from control (grey shaded histogram) and infected (empty histogram with heavy line) mice. An isotype-matched control was also analyzed (thin grey line). Parasitaemia were similar to those indicated in Figure 1. The results are representative of two independent experiments.

Mice were injected i.v. with 1000, 100 or 10 sporozoites (spz, black and grey bars) or the vehicle RPMI (white bars). (A) At the indicated days post-injection, splenocytes from groups of 3 mice were analyzed for the two subpopulations, expressed as the proportion of CD11c⁺ cells (DCs). Error bars represent standard deviation within groups of 3 mice (***, P < 0.001 when comparing iRBC to RBC by one-way ANOVA). (B) Parasitaemia were counted via Giemsa-stained slides (white symbols) and are overlaid here with the parasitemia curves from infections started with 10⁶ and 10⁵ infected erythrocytes from Figure 5B for comparison (grey symbols). The differences between the doses of 10⁶ and 10⁵ infected erythrocytes on day 3 become apparent because of the logarithmic scale used. The graph is normalized to account for the 2 days of development required by sporozoites before starting blood-stage infections. Parasitemia counts below the level of detection are plotted below the black dashed line.

(A-C) Mice were injected i.p. with 10⁶ control (RBC, black circles) or 10⁶, 10⁵, 10⁴, or 10³ infected (white symbols) erythrocytes. (A) At the indicated days post-injection, splenocytes from groups of 3 mice were analyzed for the two subpopulations, expressed as the proportion of CD11c⁺ cells (DCs). (B) Parasitaemia were quantified by Giemsa-stained slides. (C) The expression of costimulatory molecules for the splenic DC subsets from (A), expressed as mean fluorescence intensity (MFI). The results are representative of two similar independent experiments. (D-E) Mice were injected i.p. with 10⁶ control or infected erythrocytes. At days 0, 1 or 2 post-injection, splenocytes from groups of 3 mice were analyzed for the two subpopulations. (D) Subpopulations from control (RBC, black circles) or infected (iRBC, white squares) mice are expressed as the proportion of CD11c⁺ cells (DCs). Error bars represent standard deviation within groups of at least 3 mice (no significant difference when comparing iRBC to RBC by two-way ANOVA). (E) FACS plots showing expression of CD40 on DCs from control (grey shaded histogram) and infected (empty histogram with heavy line) mice. An isotype-matched control was also analyzed (thin grey line).

(A-B, E-H) BALB/c mice were injected i.p. with 10⁶ control (RBC, black circles) or P. yoelii-infected (iRBC, white squares) erythrocytes. Splenocytes from groups of 4 mice were stained with antibodies to CD11c and CD45RB. (A) Two subpopulations, R1 (CD11c^hi) and R2 (CD11c^loCD45RB^hi) were gated for analysis by flow cytometry. The FACS plots are representative of splenocytes from day 0 and day 10 post-infection. (B) At the indicated days post-injection, splenocytes were analyzed for the two subsets, expressed as the proportion of CD11c⁺ cells (DCs). (C-D) C57BL/6 mice were injected i.p. with 10⁶ P. berghei ANKA-infected erythrocytes (C). BALB/c mice were injected i.p. with 10⁶ P. yoelii YM-infected erythrocytes (D). Splenocytes were harvested on the days indicated and stained as in (A). The values on the plots represent the proportion of the CD11c⁺ gate that each subset comprises. (E) On the days indicated post-injection, two subpopulations, CD11c^hi and CD11c^loCD45RB^hi were gated, and analyzed for their expression of mPDCA-1 (heavy line) by flow cytometry. A staining control was also analyzed (grey line). (F-G) At the indicated days post-injection, the number of total spleen cells (F) and the absolute number of each DC subset (G) were quantified. (H) Parasitaemia were quantified by Giemsa-stained slides. The results are representative of three independent experiments. Error bars represent standard deviation within groups of 4 mice (*, P < 0.05; ***, P < 0.001 when comparing iRBC to RBC by two-way ANOVA).

Mice were injected i.p. with 10⁶ infected erythrocytes. At days 0 (naive mice) and 10 post-injection, DC subsets were sorted by FACS. DC subsets were then co-cultured with naive CD4 T cells isolated from DO11.10 mice in the presence of OVA peptide. (A) After 4 days of co-culture, T cell proliferation was determined using the incorporation of [H³]-thymidine for the final 16 hours of culture as a readout. Error bars represent standard deviation within co-cultures of cells sorted from 3 individual mice. (B-C) After 24 hours or 7 days of co-culture with DCs sorted from infected (B) or naive (C) mice, T cells were collected and assayed via flow cytometry for markers of T cell activation. FACS histograms show the expression of the surface marker when T cells are co-cultured with CD11c^hi DCs (thick line) or CD11c^loCD45RB^hi DCs (thin line). Stainings with isotype control antibodies are shown in the grey shaded histograms. Each FACS plot is representative of one out of three individual mice. (D) After 7 days of co-culture with sorted DCs from day 0 (naïve) or day 10-infected mice, cells were restimulated with PMA and ionomycin for 5 hours. CD4 T cells were stained intracellularly for cytokine expression. Isotype controls for the cytokine-specific antibodies are shown as FACS histograms, as detailed in B. Each FACS plot is representative of one out of three DC-T cell co-cultures, and are representative of two independent experiments.