P. chabaudi infection causes reduced expansion of B cells and failure of CD4+ T-cell migration through modulation of DCs. (a) Uninfected (squares) or P. chabaudi-infected (circles) BALB/c mice received OVA-specific CD4+ T cells and HEL-specific B cells 12 days after infection and were immunized with OVA-HEL/LPS (filled symbols) 24 h later. Controls remained unimmunized (open symbols). Results shown are the absolute number of HEL-specific B cells in the spleen (left) and lymph nodes (LN, right) and represent the mean of 3 mice per group ±1 s.d. and are representative of 3 similar experiments (*p ≤ 0.05, #p ≤ 0.005 uninfected and immunized versus P. chabaudi-infected immunized). (b) Five days after immunization, spleens from the mice described in (a) were snap frozen and prepared for immunohistochemistry. Sections were stained using biotinylated-KJ1.26 followed by Streptavidin-AlexaFluor 647 to detect OVA-specific T cells (red) and B220-FITC to identify B-cell areas (green). Images shown are representative of 3 mice per group from 2 similar experiments. (c) Sections stained as in (b) were analyzed by laser-scanning cytometry. Results are expressed as mean proportion of OVA-specific T cells per unit area in the regions indicated. The number of OVA-specific T cells contained in identically sized regions of follicle and periarteriolar lymphoid sheath (PALS) was calculated and expressed as a proportion of total KJ1.26+ cells in the section to avoid bias due to the difference in expansion between uninfected and infected spleens. Results represent triplicate readings of 3 mice per group ±1 s.d. (*p ≤ 0.05 uninfected and immunized versus P. chabaudi-infected and immunized). (d) Bone-marrow-derived DCs were cultured with P. chabaudi pRBCs (circles) or RBCs (squares) for 18 h before pulsing with 5 mg/ml OVA (filled symbols). Controls remained unpulsed (open symbols). OVA-specific CD4+ T cells were then added at a ratio of 1:1 and cultured for 72 h in vitro. T cells were isolated, washed and transferred into uninfected recipients immunized 48 h before transfer to synchronize the immune response. Clonal expansion was then assessed as described above. Results show the mean proportion of CD4+KJ1.26+ T cells and represent the mean of 3 mice per group ±1 s.d. (*p ≤ 0.05, RBC-cultured, OVA-pulsed DCs versus pRBC-cultured, OVA-pulsed DC). (e) DCs were purified from spleens of uninfected or P. chabaudi-infected mice and pulsed with 5 mg/ml OVA for 2 h. Cells were then harvested, washed and 5 × 105 DCs transferred into uninfected BALB/c mice along with CFSE-labeled OVA-specific T cells. The level of CFSE in OVA-specific CD4+ cells was analyzed 5 days later, as described in Figure 8.