Spleens were disaggregated through a 70 mm cell strainer into RPMI 1640. Following erythrocytic lysis (Sigma R7757), leucocytes were washed three times in RPMI 1640 and then cultured (37uC, 5% CO 2 ) on 96 well plates at 2610 6 cells/ml in RPMI 1640 supplemented with 24 mM Na HCO 3 , 10% heat inactivated foetal calf serum, 2 mM L-glutamine, 100 u/ml penicillin, 100 mg/ml streptomycin and 60 mM monothioglycerol. Triplicate sets of individual cultures (150 ml volume) were assigned to each experimental condition. Three cultures sets were stimulated with different TLR agonists: heat-killed endotoxin-free Listeria monocytogenes (TLR2 agonist) at 0.6610 8 cells/ml, or a 50:50 mix of Ultrapure Escherichia coli K12 lipopolysaccharide and Ultrapure Salmonella minnesota lipopolysaccharide (TLR4 agonists) at 3 mg/ml, or oligonucleotide ODN2006 (TLR9 agonist) at 6 mg/ml. All TLR agonist reagents utilized were tested for functional induction of the target TLR and for endotoxin contamination by the manufacturer (InvivoGen) and were used at concentrations previously determined to be optimal [18] . Further culture sets were stimulated with HbAg antigen at 50 mg/ml (pretreated with 500 U/ml polymyxin B for 4 h at 4uC) or left unstimulated. Culture supernatants and cells were harvested after 24 h for the unstimulated and TLR cultures and after 96 h for the HbAg cultures. Supernatants were immediately stored at 280uC and cells were placed overnight at 4uC in RNAlaterH (Ambion) and then stored at 280uC.