(A) To assess the role of CD4 T cells in protracted protection, A/J mice immunized with 10³ (AS) prbc in control ODN or CpG-ODN were depleted of CD4 T cells 12 weeks after immunization. Mice were challenged i.v. with 10⁵ homologous (AS) prbc immediately after depletion (left panels, effector) or 21 days after depletion to allow for naive/helper CD4 T cells to replenish (right panels, helper). Parasitemias were monitored. Results are representative of 3 separate experiments and data for individual mice are shown. ^†Animals that succumbed to infection. (B) To assess for cytokine secretion in memory CD4 T cells, spleen cells from A/J mice immunized with 10³ (AS) prbc in CpG-ODN were purified 12 weeks after immunization, incubated for 96 hours in the presence of homologous (AS) prbc, and cells stained for intracellular IFN-γ and IL-2. Numbers represent percentages of cytokine⁺ CD4 T cells. (C) To identify the specificity of CD4 T cell memory subsets, IFN-γ secretion and expression of lymph node homing receptor (CD62L) were assessed by harvesting spleen cells from A/J mice immunized with 10³ (AS) prbc in CpG-ODN at 12 weeks after immunization. Cells were incubated for 96 hours in the presence of homologous (AS) prbc and stained. Numbers represent percentages of IFN-γ⁺ CD62L^loCD4 T cells. (D) To determine the existence of memory populations, spleen cells from A/J mice immunized with 10³ (AS) prbc in CpG-ODN were FACS-sorted into CM (CD44^hiCD62L^hi) and EM (CD44^hiCD62L^lo) populations directly ex vivo. CM and EM cells were then incubated for 96 hours in the presence of homologous (AS) prbc and stained for intracellular cytokine (IFN-γ) and proliferation (BrDU). Numbers represent percentages of CD4 T cells in corresponding quadrants. Results are representative of 3 independent experiments performed.