Impaired ability of DCs to interact with and to prime naïve CD4+T cells. (A) Upper left panel shows a T-cell (green) 'engulfed' by a DC (red). Lower left panel shows a T-cell in contact with a DC but not 'engulfed'. Right panels show the same microscopic fields in transmitted light. (A-C) DCs were differentiated in vitro and pre-incubated with uninfected (DC+RBC) or P. yoelii-infected (DC+iRBC) erythrocytes, followed or not by incubation with LPS. DCs were loaded with OVA peptide 323–339 (black bars) or not (white bars) and incubated for 30 min with naïve DO11.10 T-cells labeled with CFSE (green). (D,E) CD11c+ DCs were isolated from P. yoelii-infected mice at different times of infection and incubated alone (white bars), with OVA peptide 323–339 (gray bars), or with LPS and OVA peptide 323–339 (black bars). DCs were incubated for 30 min with naïve DO11.10 T-cells labeled with CFSE (green). For microscopy analysis (A,B,D), DCs were fixed and actin was stained (red), for FACs analysis (C,E), DCs were labeled with cell tracker (red) before incubation with T-cells. (B,D) Number of 'engulfed' T-cells per 100 DCs analysed by microscopy. (C,E) Percentage of DC-T-cell conjugates analysed by FACS. (F,G) Up-regulation of CD69 in naïve CD4+ T-cells after a12 h incubation with OVA peptide 323–339-loaded unstimulated (white bars) or LPS-stimulated DCs (black bars). (F) DCs were differentiated in vitro and pre-incubated or not with uninfected or P. yoelii-infected erythrocytes. (G) DCs were isolated from infected mice at different times during blood-stage infection. Results are expressed as mean ± SD of triplicate samples. FACs analysis for (F) and (G) data is shown in Additional File 3.