Indeed, their immunologically “innate” status needs to be questioned if NK cells are incapable of independently responding to PfRBC in the absence of “adaptive” T cells. However, the insights presented do provide encouraging implications for malaria vaccine development, since they suggest that by inducing classical memory PI3K Inhibitor Library T-cell responses vaccination will simultaneously achieve enhanced NK responses “into the bargain”. Protocols for and clinical course of stringently controlled experimental human malaria infections at our centre have been described in detail earlier 12, 13. Briefly, after providing written informed consent, five healthy malaria-naïve Dutch volunteers were infected with malaria by exposure

to the bites of five P. falciparum-infected mosquitoes and followed-up closely for symptoms and signs of malaria. As soon as a standard microscopic thick smear of peripheral blood became positive for malaria parasites, volunteers were treated with a standard curative regimen of the anti-malarial drug artemether–lumefantrine. The study

was approved by the Institutional Review Board of the Radboud University Nijmegen Medical Centre (CMO 2006/207). Preparations of mature parasitized RBC (PfRBC) and mock-cultured uninfected erythrocytes (uRBC) were obtained by routine methods as described previously 12 and cryopreserved at 150×106/mL in 15% glycerol/PBS in aliquots for use in stimulation assays. Cryopreserved PfRBC form almost as strong a stimulus as fresh PfRBC and have identical stimulatory characteristics Opaganib research buy (Supporting Information Fig. 2). Their use in large experiments has logistical advantages, in addition to reducing confounding due

to inter-batch variation. One single large batch of cryopreserved PfRBC was used for the entire follow-up study described above. Venous whole blood was collected into citrated CPT vacutainers (Becton and Dickinson, Basel, Switzerland) prior to challenge (day C−1), during blood-stage malaria infection (day C+9), 3 wk after treatment (day C+35) and again 20 wk after challenge (day C+140). PBMC were obtained by density gradient centrifugation, washed 3× in cold PBS, enumerated, frozen down in 10% DMSO/FBS and stored in liquid nitrogen. Immediately prior to use, cells were thawed, washed twice in RPMI and resuspended in complete culture medium (RPMI 1640 containing check 2 mM glutamine, 1 mM pyruvate, 50 μg/mL gentamycine and 10% v/v human A+ serum, Sanquin, Nijmegen) for a final concentration of 2.5×106/mL. PBMC were transferred into 96-well round-bottom plates and were stimulated in duplo wells with either 5×106/mL cryopreserved PfRBC or uRBC. PBMC were stimulated for 24 h at 37°C/5%CO2; 4 h prior to cell harvest, 100 μL/well supernatant was collected and stored at −80°C for subsequent cytokine measurement and replaced with 100 μL/well fresh culture medium containing brefeldin A (Sigma) for a final concentration of 10 μg/mL.