(A) The production of IL-17 and IL-22 in cultures of PBMCs from healthy donors required priming with whole L. donovani parasites. Boosting was achieved using parasites or parasite extracts. Priming (on day 0; stimulation 1) and boost (on day 7; stimulation 2) were performed with opsonized (OpsLd) or non-opsonized (Ld) heat-killed L. donovani parasites or parasite extracts (Ld-ext). IL-17 and IL-22 were quantified by ELISA on days 7, 11, and 15. Two representative experiments (Exp 1 and Exp 2) from a series of 10 are shown. (B) IL-17 and IL-22 were produced by CD4⁺ T cells. CD4⁺ T cells were purified from the 12-day cultures of PBMCs primed and boosted with OpsLd. Purified CD4⁺ T cells were then stimulated for 48 hours with PMA plus ionomycin, concanavalin A (ConA), or phytohemagglutinin (PHA). IL-17 and IL-22 were quantified by ELISA. Three independent experiments with cells from different donors are shown. (C) Phenotypic characterization of IL-17⁺CD4⁺ T cells in cultures of normal PBMCs stimulated with OpsLd. Normal PBMCs were primed and boosted with OpsLd. Five days after the second stimulation, cells were stimulated with PMA plus ionomycin plus monensin for 5 hours. The x axis represents the fluorescence intensity after either CD4 labeling or IFN-γ labeling. The y axis corresponds to IL-17 labeling. Horizontal and vertical bars define the upper fluorescence intensity observed with labeled control cells. The numbers in each quadrant represent the percentage of cells in that quadrant relative to the total number of cells included in the analysis.