TLR8 and TLR9 conferred the stimulatory activity of plasma of NSCLC patients. (A) HEK cells transfected with TLR3, TLR4, TLR8 and TLR9 were incubated with 20% plasma from NSCLC patients before treatment for 6 h, and then assayed for the expression of IL-8 using quantitative PCR. Negative controls were HEK transfectants stimulated with plasma from normal adult volunteers. Positive controls were HEK transfectants stimulated with TLR agonists. (B) TLR8 and TLR9-expressing HEK cells were co-transfected with CD32-expression vector and then stimulated with 20% plasma from NSCLC patients before treatment for 6 h. *P < 0.001. (C and D) PBMCs derived from normal volunteers were transfected with the indicated TLR shRNA respectively for 24 h, and then cultured with 20% plasma derived from NSCLC patients before treatment for 6 h. The efficacy of TLR shRNA was evaluated by detecting their relative mRNA expression. *P < 0.01. (E) HEK cells transfected with the indicated TLR with or without CD32 were incubated with the isolated IC from NSCLC plasma before treatment for 6 h. *P < 0.001. Data points represented individual patients. Each bar represented the means (±SD) from 5 NSCLC patients.