Infected red blood cells from the blood of infected mice (parasitemia, 30–50%) were purified (> 95%) by centrifugation in 74% Percoll density gradient as described previously [21]. MHC II+CD11chiCD3−CD19−, MHC

II+CD11c−CD3−CD19−IgM+, and MHC II+CD11c−CD3−CD19−IgM− cells were purified by Opaganib price cell sorting as described. Cells (1 × 105) were cultured in the presence of iRBC or RBC (4 × 106) in a final volume of 200 µL for 16 hr and the concentrations of cytokines in the supernatant determined by a sandwich ELISA as described previously [22]. OT-II mice were immunized i.p. with OVA (200 µg) in complete Freund’s adjuvant. After 5 days, CD4+ T cells were prepared from the spleens of OT-II mice using a CD4+ T cell isolation kit (Milteny Biotech, CD4+ T cells; 87.5%) and labeled with 15 µM CFSE (Invitrogen, Carlsbad, CA, USA) for 10 min. MHC II+CD11chiCD3−CD19− DCs and MHC II+CD11c−CD3−CD19− cells were prepared by cell sorting and pulsed with OVA323–339 (10 µg/mL) or with OVA (1 mg/mL) for 3 hr. OT-II (1 × 105) and MHC II+CD3−CD19− cells (1 × 104) were cocultured for 3 days and cell divisions assessed on the basis of diminution of CFSE dye using a FACS Canto II. The supernatant was collected after 2 days of culture to measure the concentrations of IL-2. ELISA was performed as described previously [22]. The statistical significance

of differences was determined by two-sided Student’s t-test using SRT1720 chemical structure GraphPad PRISM 5 software. P values less than 0.05 were considered significant. After excluding T and B cells with CD3 and CD19 markers, MHC class II+ cells were examined using spleen cells from B6 mice infected with P. yoelii. Splenic CD3−CD19− cells were medroxyprogesterone stained for CD11c and MHC II, and MHC II+ cells divided into three subpopulations based on the degree of CD11c expression, namely CD11chi, CD11cint and CD11c− cells (Fig. 1a). In MHC II+CD3−CD19− cells, the degree of MHC II expression was greater in CD11chi cells (MFI: uninfected, 6199; infection day 8, 3279) than in CD11cint (MFI: uninfected, 2884; day 8, 2219) or CD11c− (MFI: uninfected, 2638; day 8, 1295) cells.

MHC II+CD11chiCD3−CD19− cells included conventional DCs and constituted the major population of MHC II+ cells prior to infection. MHC II+CD11cintCD3−CD19− cells included plasmacytoid DCs and regulatory DCs [7]. After day 5 post-infection, the numbers of both MHC II+CD11chiCD3−CD19− and MHC II+CD11cintCD3−CD19− cells decreased steadily (Fig. 1b). In contrast, the number of MHC II+CD11c−CD3−CD19− cells increased until day 9 post-infection in parallel with the degree of parasitemia (Fig. 1c). However, the number of these cells decreased after day 11 post-infection despite continuing increase in parasitemia. These MHC II+CD11c−CD3−CD19− cells were next stained for CD138, a plasma cell marker, and Igs (Fig. 2a).