Novel TLR7 agonist stimulates activity of CIK/NK immunological effector cells to enhance antitumor cytotoxicity

Abstract

Toll-like receptor (TLR) 7/8 agonists have been applied in combination with chemo-, radio- or immunotherapy for lymphoma, and used as topical drugs for the treatment of viral skin lesions and skin tumors. In the present study, the role of an adenine analog, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine [termed Gao Dong (GD)], a novel TLR7 agonist, in the activation of cytokine-induced killer/natural killer (CIK/NK) cells was determined. The results of the present study indicated that GD was able to activate CIK/NK cells. The proportion of GD-induced CD3⁺CD56⁺ CIK and CD3^-CD56⁺ NK cells was ~4% higher respectively compared with the control. Notably, combination therapy with CIK/NK cells stimulated by GD, markedly suppressed the proliferation of the chronic myelogenous leukemia K562 cell line. Following GD treatment, the cytotoxicity improved by ~25 and 21% when the effector/target ratio was 20:1 and 10:1, respectively. The results of the present study suggested a novel protocol for CIK/NK cell proliferation and revealed that GD may serve as a potent innate and adaptive immunomodulator in immunocyte culture.

Keywords: Toll-like receptor 7; combination therapy; cytokine-induced killer cells; cytotoxicity; natural killer cells.

Figure 1.

Molecular structure of GD, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine.

Figure 2.

Cell proliferation following three distinct treatments of peripheral blood mononuclear cells derived from three human donors. *P<0.05, vs. ‘none’. GD, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine; IFN-γ, interferon-γ.

Figure 3.

(A-F) Proportions of CIK, NK and T cells on days 0, 7 and 15 of culture. *P<0.05, vs. ‘none’. GD, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine; IFN-γ, interferon-γ; CIK, cytokine-induced killer; NK, natural killer.

Figure 4.

Phenotype of CIK, NK and T-cells in group ‘+GD’ on days 0, 7 and 15 of culture. Results are representative of three experiments. CIK, cytokine-induced killer; NK, natural killer; GD, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine; FITC, fluorescein isothiocyanate; CD, cluster of differentiation; APC, allophycocyanin; PE, phycoerythrin; PerCP, peridinin chlorophyll protein complex.

Figure 5.

Cytotoxicity of CIK/NK cells towards the K562 cell line. *P<0.05, vs. ‘none’. GD, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine; IFN-γ, interferon-γ; CIK, cytokine-induced killer; NK, natural killer; E:T, effector/target ratio.