Functional capacities of CD11b^highLy6C⁺ cells from the spleens of mice infected with P chabaudi. (A) FACS analysis of CD11b^highLy6C⁺ spleen cells from C57Bl/6 mice. Expression of iNOS and ROI in CD11b^highLy6C⁺ monocytes from the spleens of uninfected and day 8–infected C57Bl/6 mice. Cells were stained with antibodies against CD11c, Ly6C, CD11b, CD19, CD3, Ter119 and gated as described in Figure 1A. Live cells were gated on forward and side scatter and CD19, CD3, Ter119-positive cells were excluded. The fluorescence profiles of gated CD11b^highLy6C⁺ cells from uninfected (left) and day 8–infected mice (middle) labeled with Ab to iNOS (black line) and the isotype control Ab (shaded histogram) are indicated. (Right) Fluorescence is shown from DHR 123 taken up by gated CD11b^highLy6C⁺ cells from uninfected (stippled lines) and infected (solid lines) mice as indicator of ROI activity. (B) Expression of cytokine mRNA in CD11b^highLy6C⁺ spleen cells from uninfected and day 8–infected C57Bl/6 mice. CD11b^highLy6C⁺ monocytes and CD11c^high DCs were sorted to greater than 95% purity, and quantitative polymerase chain reaction was performed as described in “Quantitative reverse transcription–polymerase chain reaction.” (Primer sequences are shown in supplemental Table 1). The data are shown as fold increase relative to ubiquitin. These values shown are the means and SEMs of 3 independent experiments, each using total RNA from a pool of 3 mice. P values were derived from the Student t test. (C) Ability of CD11b^highLy6C⁺ splenic cells to phagocytose CFSE-labeled P chabaudi–iRBCs in vitro. Dual parameter contour plots and histogram of C57Bl/6 spleen cells isolated at day 8 of a P chabaudi infection. Cells were enriched with the use of CD11b MACS beads as described and stained with antibodies against CD11c, Ly6C, CD11b, CD19, CD3, CD8, and Ter119 after incubation with 100:1 iRBCs as described. (Left) Cells were gated for live cells on forward and side scatter, and CD19, CD3, Ter119, CD8 lineage-positive and 7AAD⁺ cells were excluded. (Middle) Contour plot of CD11b and Ly6C profiles of lineage-negative cells. (Right) Histogram of CFSE on gated CD11b^highLy6C⁺ cells incubated with CFSE-labeled iRBCs (solid lines) and without iRBCs (shaded histogram). iRBCs were prepared as described in “Methods.” (D) Ability of CD11b^highLy6C⁺ spleen cells to phagocytose GFP-expressing P chabaudi parasites in vivo during an infection. Histogram shows GFP expression in CD11b^highLy6C⁺ cells from splenocytes of C57Bl/6 mice 8 days after infection with 10⁵ GFP–P chabaudi (solid lines) or WT P chabaudi (shaded histogram). Cells were stained with antibodies against CD11c, Ly6C, CD11b, CD19, CD3, Ter119 and gated as described in Figure 1A. Live cells were gated on forward and side scatter. and CD19, CD3, Ter119-positive cells were excluded. Each experiment was performed with at least 3 mice per group and repeated 3 times. Numbers show the percentage of GFP⁺ cells within the regions. (E) The proportion of CD11b⁺Ly6C⁺ splenocytes that contain GFP 15 minutes after intravenous injection of 10⁹ GFP P chabaudi parasites into day 13–infected mice. Splenocytes were stained with antibodies against CD11c, Ly6C, CD11b, CD19, CD3, Ter119, and 7AAD as described in Figure 1. (Left) GFP expression on FSC and SSC gated cells from GFP–P chabaudi–injected (solid lines) and uninjected (shaded histograms) mice. (Right) CD11b and Ly6C expression on GFP⁺ cells. Similar gating strategies were used as described above. Number shows the percentage of cells within the region. Experiment was performed with 3 mice per group.