Activation of Toll-like receptor 2 by a novel preparation of cell wall skeleton from Mycobacterium bovis BCG Tokyo (SMP-105) sufficiently enhances immune responses against tumors

Abstract

The cell wall skeleton of Mycobacterium bovis BCG has been investigated as an immunopotentiating adjuvant for immuno-therapy of malignant tumors via Toll-like receptor (TLR) 2 and TLR4. However, due to its high molecular weight, highly complicated lipoglycan structure, and complicated purification and isolation procedure, its exact structure-activity relationship has not been well established. We have newly isolated the cell wall skeleton from M. bovis BCG Tokyo (SMP-105) and examined the binding of SMP-105 with TLR. It was revealed that highly purified SMP-105 activates the nuclear factor-kB promoter in a TLR2-dependent manner, not a TLR4-dependent manner, using a reporter gene assay system. Peritoneal exudated cells of TLR2 and MyD88 knockout mice severely reduced the induction of tumor necrosis factor-alpha and interleukin-6 in the presence of SMP-105, whereas cells from TLR4 knockout mice produced similar levels of cytokines to wild-type mice. Dendritic cells and macrophages accumulated in the draining lymph nodes of treated mice. When mice were administered both SMP-105 and mitomycin C-inactivated Lewis lung carcinoma cells simultaneously, interferon-gamma-producing cells reacting to the tumor were increased distinctly in draining lymph nodes. When C57BL/6 mice, into which splenocytes from OT-I transgenic mice had been transferred, were administered with both SMP-105 and E.G7-OVA, OVA-specific cytotoxic T lymphocytes (CTL) increased markedly. Mice treated with SMP-105 and inactivated Lewis lung carcinoma cells suppressed the growth of implanted tumors. These results suggest that the activation of TLR2 by SMP-105 sufficiently enhanced immune responses, such as the number of interferon-gamma-producing cells and CTL, and prevented the growth of tumors without the contribution of TLR4.

Figure 1

Nuclear factor (NF)‐κB activation after SMP‐105 stimulation. (a) 293 kB/hTLR2 (), which expressed human Toll‐like receptor (TLR) 2 and the NF‐κB‐luciferase gene, and parental 293 kB (), which carried only the NF‐κB‐luciferase gene, were stimulated with SMP‐105 for 18 h. Luciferase activity was measured with luminescence. The mean ± SD of two separate measurements is shown. (b) HEK‐Blue4 was stimulated with 100 µg/mL (black bar) or 10 µg/mL (white bar) of the previous preparation of Mycobacterium bovis cell wall skeleton (BCG‐CWS), SMP‐105, or heat‐killed BCG Tokyo (hkBCG) for 18 h. Alkaline phosphatase activity was measured. Representative data from four experiments are shown. (c) 293 kB (white bar) or 293 kB/hTLR2 (black bar) was stimulated with Pam₃CSK₄ (0.3 µg/mL) or lipopolysaccharide (LPS) (0.5 µg/mL) for 18 h. Reporter gene induction was measured as luciferase activity. The mean ± SD of two separate measurements is shown. (d) HEK‐Blue4 was stimulated with Pam₃CSK₄ (10 µg/mL) or LPS (0.1 µg/mL) for 18 h. The induction of alkaline phosphatase activity was measured.

Figure 2

Interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α production by thioglycollate‐elicited peritoneal exudate cells (TG‐PEC) from wild‐type, Toll‐like receptor (TLR) 2, TLR4, and MyD88 knockout (KO) mice. TG‐PEC were cultured in the presence of 1 ng/mL recombinant mouse interferon‐γ with medium control (white bar) or 10 µg/mL of SMP‐105 (black bar) for 18 h. Culture supernatants were recovered and assayed for the production of (a) IL‐6 and (b) TNF‐α by enzyme‐linked immunosorbent assay. The mean ± SD of two mice is shown.

Figure 3

Induction of interferon (IFN)‐γ‐producing cells in vaccinated mice. (a) C57BL/6 mice were administered twice at 7‐day intervals with mitomycin C‐inactivated 3LL cells and SMP‐105. Spleen cells (spleen) and lymph node cells (LN) were collected 7 days after final administration, and were cultured with inactivated 3LL cells for 48 h. The amount of IFN‐γ in the culture supernatant was measured using enzyme‐linked immunosorbent assay. (b,c) C57BL/6 mice were administered into both hind footpads with mitomycin C‐inactivated 3LL cells and SMP‐105 (black bar) or vehicle (white bar). Single cell suspension was prepared from (b) popliteal lymph nodes or (c) spleens and was cultured in the absence or presence of mitomycin C‐inactivated 3LL cells for 24 h. The amount of IFN‐γ in the culture supernatant was measured using enzyme‐linked immunosorbent assay.

Figure 4

In vivo proliferation of OVA‐specific cytotoxic T lymphocytes (CTL). C57BL/6 mice, into which were adoptively transferred spleen cells of OT‐I transgenic mice, were administered with mitomycin C‐inactivated E.G7‐OVA cells alone (white bar), a mixture of cells and SMP‐105 (black bar), or CpG1826 ODN (gray bar). Popliteal lymph node cells were collected 4 or 7 days after administration and the number of OVA‐specific CTL was determined as CD8a⁺ OVA tetramer⁺ cells using a flow cytometer. The mean ± SD of two mice in each group on each day is shown.

Figure 5

Induction of antitumor activity by vaccination of inactivated tumor with SMP‐105. Wild‐type C57BL/6, Toll‐like receptor (TLR) 2 knockout (KO), TLR4 KO, and MyD88 KO mice were administered mitomycin C‐inactivated 3LL cells and SMP‐105 or vehicle four times at 7‐day intervals. Mice were challenged with live 3LL cells in the left flank 7 days after final administration and the growth of tumors was monitered using calipers. The mean ± SD of five mice is shown.