The nonstimulated, the interleukin (IL)-2, or TLR-activated LMCs were added to triplicate wells at an effector to target cell ratio of 20:1 in a total volume of 200 μL of complete RPMI medium. The IL-2-stimulated effector LMCs used for the assay were stimulated for 3 days with IL-2 (100 units/mL)
and the TLR-activated LMC comprised of a series of cell cultures incubated with a single or mixture of TLR ligands each at a predetermined optimal concentration of 2-10 μg/mL of the appropriate TLR-L prior to their addition to the target cells. The TLR ligands used included TLR2 ligand (lipoteichoic acid, LTA: TLR2-L), TLR3 ligand (polyinosine-polycytidylic acid, poly (I:C): TLR3-L), TLR4 ligand (lipopolysaccharide, LPS: TLR4-L), TLR5 ligand (Flagellin: TLR5-L), TLR7/8 ligand (CL097: TLR7/8-L), TLR9 Tamoxifen in vitro ligand type A (ODN2216, CpG type A: TLR9-LA), www.selleckchem.com/products/Decitabine.html and TLR9 ligand type B(ODN2006, CpG type B: TLR9-LB). The combination of TLR ligands used for activation of LMC included (1) TLR2-L + the ligands for either TLR3, 4, 5, 7/8, 9-LA, or 9-LB; (2) TLR3-L + the ligands for either TLR4, 5, 7/8, 9-LA, or 9-LB; (3) TLR4-L + ligands for either TLR5, 7/8, 9-LA, or 9-LB; (4) the TLR5-L + the ligands for either 7/8, 9-LA, or 9-LB; (5) TLR7/8-L + the ligands of either 9-LA to TLR9-LB; (6) TLR9-LA + TLR9-LB. The TLR ligands were purchased from Invitrogen (San Diego, CA). Controls consisted of triplicate
wells containing target cells cultured in media alone and target cells that were incubated with 10% Triton X-100 to determine spontaneous and maximal 51Cr release, respectively. Following incubation of the cocultures of the effector with target cells for 8 hours, 100 μL of supernatant fluid was collected from each well and counted and the percentage of specific 51Cr release calculated as (cpm of experimental release − cpm of spontaneous release) / (cpm of maximal
release − cpm of spontaneous release) Thiamet G × 100). Experiments using the combination of TLR3-L and TLR4-L were performed on aliquots of samples at least three times from each of the patients. As further controls, polymyxin B and chloroquine were used as specific inhibitors of LPS and poly I:C, respectively, for assays involving TLR4 and TLR3-induced activation. Although polymyxin B was added at the time of TLR4 activation, chloroquine was added 2 hours prior to the activation of the TLR3 pathway for the cytotoxicity assay. Hepatic Mo, T cells, and NK cells were isolated from LMC following in vitro activation with TLR3-L and TLR4-L for 3 days. Subsequently, highly enriched populations of Mo, T cells, NK cells, and LMC depleted of Mo, T cells, and NK cells were assessed for their cytotoxic activity against autologous BEC at an effector-to-target cell ratio of 5:1. Thence enriched populations of NK cells and LMC were stimulated with several combinations of TLR3-L and TLR4-L in the presence of a variety of supernatant fluids prepared as described above.