Splenic CD11b^highLy6C⁺cells are poor stimulators of CD4 T cells and express less MHC class II and costimulatory molecules than CD11c^high cells during a P chabaudi infection. (A) Representative examples showing surface expression of MHC class II, CD86, and CD40 on CD11c^high (gray line) and CD11b^highLy6C⁺ (black line) on spleen cells obtained from BALB/c mice infected for 8 days with P chabaudi. CD11b^highLy6C⁺ cells were gated as shown in Figure 1. The gray-shaded area represents the staining pattern of the cells incubated with an isotype-control antibody. Each experiment was performed with at least 3 mice per time point and repeated 3 times. (B) Proliferation and T-cell–cytokine production by malaria-specific TCR Tg T cells. (Left) Proliferation of MSP1-specific transgenic CD4 T cells (B5) after coculture with 5 × 10⁴ purified CD11b^highLy6C⁺ (■) and CD11c^high (□) subpopulations of splenocytes obtained from mice infected for 8 days with P chabaudi. CD11b^highLy6C⁺ cells were cultured in the presence () and absence of 1μM of specific peptide. T-cell proliferation was determined by the incorporation of ³H-thymidine after 4 days. The values shown represent the means and SEMs of the mean of triplicate cultures of a representative experiment of 3 performed. Proliferation of APCs alone is shown. (Middle and right) IFNγ, IL-4, and IL-10 produced in the supernatants of B5 transgenic CD4 T cells cultured with CD11c^high (□) and CD11b^highLy6C⁺ in the absence (■) and presence of 1μM peptide () cells purified from spleens of P chabaudi–infected mice 8 days after infection. After 6 days of coculture, T cells were transferred into plates coated with anti-CD3 Ab and cultured for a further 48 hours. Cytokines were measured in the culture supernatant by enzyme-linked immunoabsorbent assay. The bars and error bars represent the means and SEMs of triplicate cultures of a representative experiment of 3 performed. (C) IL-2 production by MSP1-specific CD4 T-cell hybridoma, B5, after coculture with purified CD11b^highLy6C⁺ (■) and CD11c^high (○) subpopulations of splenocytes obtained from mice infected for 8 days with P chabaudi. CD11b^highLy6C⁺ cells were also cultured in the presence (▾) of 1μM specific peptide. The amount of IL-2 in the culture supernatant was determined after 24 hours using the CTLL2 proliferation assay. Each symbol is the mean and SEM of triplicate samples of 1 representative experiment of 3 performed. SEM less than 10% of the mean is not shown.