2.6. Lymphoproliferation assay

PBMCs from day 0, 42, and 70 were assessed for P. falciparum derived peptide-specific activation induced proliferation. PBMCs were washed and cultured overnight. They were then suspended in RPMI media without any supplements at a cell density of 10×10⁶ live cells/ml and stained with 5 μM of CFDA SE (Molecular Probes, Eugene, OR). Following staining, cells were washed and suspended in HR10 at a cell density of 2×10⁶ cells/ml and plated into 96 well tissue culture plates (Corning, Corning, NY) at cell density of 2×10⁵ cells/well. Cells were stimulated either with individual P. falciparum derived HLA-DR-specific peptides (10 μg/ml), pan-DR epitope, PADRE (10 μg/ml), candida antigen (Greer Lab, 10 μg/ml) or the superantigen SEB (Toxin Technology, Sarasota, FL, 10 ng/ml). Peptides included in this analysis are outlined in Supplemental Table 5. Unstimulated cells or those stimulated with DMSO alone served as negative controls. Plates were incubated in 37°C, 5% CO₂ incubator for 6 days followed by staining with an antibody cocktail consisting of anti-human CD3-Alexa Fluor 700 (BD Biosciences), CD4-eFluor450 (eBioscience, San Diego, CA) and CD8-APC cy7 (BD Biosciences). Data was acquired on BD LSR-II flow cytometer and analyzed using Flowjo software (Treestar, Ashland, OR). The percentage of CD3+CD4+ and CD3+CD8+ cells in the proliferating gate were calculated.