Modulation of TLR9 on anti-tumor immune responses of peripheral blood mononuclear cells from patients with non-small-cell lung cancer

OBJECTIVE: To assess the modulation of TLR9 on anti-tumor immune responses in peripheral blood mononuclear cells (PBMC) from patients with non-small-cell lung cancer (NSCLC). METHODS: PBMCs were isolated from 36 NSCLC patients. Lung cancer cells were isolated from these patients and further enriched. PBMCs were cultured in RPMI-1640 medium (blank control group), and medium with cytosine guanine oligodeoxynucleotide (CpG ODN, an TLR9 agonist) or control ODN for 72 h; and then flow cytometry was used to examine the expression of CD69 antigen on the surface of CD3 cells, [3H]-thymidine incorporation method was used to examine the cell proliferation, and the IFN-alpha level in the supernatant was measured. Another PBMCs were cultured in medium with interleukin (IL)-1 and then CpG ODN, control ODN, and CpG ODN + chloroquine or inhibitory ODN were added respectively for 24-48 h. Then the IFN-alpha in the supernatant was measured. Subsets were assessed by flow cytometry and the expression of TLR9-mRNA in freshly isolated PBMC was detected by RT-PCR. The production of interferon (IFN)-alpha in the PBMCs was measured by ELISA. The proliferation of the PBMCs was determined by [3H]-thymidine incorporation. The PBMCs co-cultured with CpG ODN and autologous lung tumor cells treated with mitomycin C were used as effector cells, and K562 cells and autologous tumor cells were used as target cells flow cytometry was used to detect the capacity of PBMCs to kill autologous lung tumor cells and K562 cells. Meanwhile we investigated the intracellular expression of IFN-gamma and IL-4 in CD8+ T. RESULTS: The expression level of TLR9 of the PBMCs from patients was not significantly different from that of the PBMCs from the healthy donors. The proportion of CD69 antigen expressing CD3+ T cells of the CpG ODN group was (39.5 +/- 8.9)%, significantly higher than those of the blank control group [(8.8 +/- 1.2)%, t = 40.30, P = 0.00] ands control ODN group [(10.6 +/- 1.0)%, t = 41.85, P = 0.00]. Examination with beta liquid scintillation counter showed that the cpm value of the CpG ODN group was (1.61 +/- 0.20) x 10(4), significantly higher than those of the blank control group [(0.27 +/- 0.14) x 10(4), t = 20.43, P = 0.00] and control ODN group [(0.34 +/- 0.13) x 10(4), t =20.20, P = 0.00]. Chloroquine and inhibitory ODN dose-dependently inhibited the IFN-alpha levels in the supernatant. The CD4 + T/CD8 + T of the CpG ODN group was (3.44 +/- 0.20), significantly higher than those of the control ODN group (1.73 +/- 0.27, t = 19.85, P = 0.00) and blank control group (1.69 +/- 0.13, t = 29.32, P = 0.00). The IFN-gamma positive CD8 + T cells of the CpG ODN group was (18.5 +/- 4.2)%, significantly higher than those of the control ODN group [(4.2 +/- 1.0)%, t = 24.12, P = 0.00] and blank control group [(3.1 +/- 1.2)%, t = 25.1, P = 0.00]. There was no significant differences in the proportion of IL-4 positive CD8 + T cells among different groups. When the E/T was 40:1 the killing capacity of PBMCs against the K562 cells was (19.5 +/- 1.0), significantly higher than those of the control ODN group (7.9 +/- 1.1, t = 19.9, P = 0.00) and blank control group (5.1 +/- 1.6, t = 21.9, P = 0.00), and the killing capacity of PBMCs against the autologous lung tumor cells was (29.8 +/- 2.1), significantly higher than those of the control ODN group (8.1 +/- 0.9, t = 36.9, P = 0.00) and blank control group (5.7 +/- 1.6, t = 35.7, P = 0.00). CONCLUSION: TLR9 signal takes part in the immunomodulation of PBMCs. The activation of TLR9 results in enhanced anti-tumor response in the PBMCs against autologous lung cancer cells and K562 cells.