Cytokine-induced killer cell therapy for modulating regulatory T cells in patients with non-small cell lung cancer

Abstract

Previous studies have reported that regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self-tolerance, have a critical role in the regulation of the antitumor immune response. Targeting Tregs has the potential to augment cancer vaccine approaches. The current study therefore aimed to evaluate the role of cytokine-induced killer (CIK) cell infusion in modulating Tregs in patients with non-small cell lung cancer (NSCLC). A total of 15 patients with advanced NSCLC were treated by an infusion of CIK cells derived from autologous peripheral blood mononuclear cells (PBMCs). By using flow cytometry and liquid chip analysis, subsets of T cells and natural killer (NK) cells in peripheral blood, and plasma cytokine profiles in the treated patients, were analyzed at 2 and 4 weeks after CIK cell infusion. Cytotoxicity of PBMCs (n=15) and NK cells (n=6) isolated from NSCLC patients was evaluated before and after CIK cell therapy. Progression-free survival (PFS) and overall survival (OS) were also assessed. Analysis of the immune cell populations before and after treatment showed a significant increase in NK cells (P<0.05) concomitant with a significant decrease in Tregs (P<0.01) at 2 weeks post-infusion of CIK cells compared with the baseline. NK group 2D receptor (NKG2D) expression on NK cells was also significantly increased at 2 weeks post-infusion compared with the baseline (P<0.05). There was a positive correlation between NKG2D expression and the infusion number of CIK cells (P<0.05). When evaluated at 2 weeks after CIK cell therapy, the cytotoxicity of PBMCs and isolated NK cells was significantly increased compared with the baseline (P<0.01 and P<0.05). Correspondingly, plasma cytokine profiles showed significant enhancement of the following antitumor cytokines: Interferon (IFN)-γ (P<0.05), IFN-γ-inducible protein 10 (P<0.01), tumor necrosis factor-α (P<0.001), granulocyte-macrophage colony-stimulating factor (P<0.01), monocyte chemotactic protein-3 (P<0.01) and interleukin-21 (P<0.05) at 2 weeks post-infusion, compared with the baseline. At the same time, the expression of transforming growth factor-β1, which is primarily produced by Tregs, was significantly decreased compared with the baseline (P<0.05). Median PFS and OS in the CIK cell treatment group were significantly increased compared with the control group (PFS, 9.98 vs. 5.44 months, P=0.038; OS, 24.17 vs. 20.19 months, P=0.048). No severe side-effects were observed during the treatment period. In conclusion, CIK cell therapy was able to suppress Tregs and enhance the antitumor immunity of NK cells in advanced NSCLC patients. Therefore, CIK cell treatment may improve PFS and OS in patients with advanced NSCLC. CIK cell infusion may have therapeutic value for patients with advanced NSCLC, as a treatment that can be combined with chemotherapy and radiotherapy.

Keywords: cytokine-induced killer cells; non-small cell lung cancer; regulatory T cells.

Figure 1.

PBMC properties before and after CIK cell induction. (A) Total number of PBMCs, (B) number of CD3⁺CD56⁺ cells and (C) phenotypic analysis of PBMCs before and at day 21 after CIK cell induction (n=15). (D) Cytotoxicity of PBMCs against A549 target cells at E:T ratios of 10:1 and 20:1 before and at day 21 after CIK cell induction (n=15). Data are presented as the mean ± standard deviation. PBMC, peripheral blood mononuclear cell; CIK, cytokine-induced killer; E:T, effector-to-target.

Figure 2.

Dynamic phenotypic change of immunocytes in the peripheral blood of non-small cell lung cancer patients before and at 2 or 4 weeks after administration of CIK cells. (A) Percentage of Treg/CD4⁺ cells (n=15). (B) Percentage of NK cells (n=15). NK cell, natural killer cell; CIK, cytokine-induced killer; Treg, regulatory T cell.

Figure 3.

NKG2D expression on NK cells in peripheral blood before and at 2 or 4 weeks after CIK cell therapy. (A) Percentage of NK cells with NKG2D expression (n=15). (B) Relationship between the number of CIK cells infused and the percentage of NKG2D expression on NK cells (n=15). NK cell, natural killer cell; CIK, cytokine-induced killer; NKG2D, NK group 2D receptor.

Figure 4.

Cytotoxicity of PBMCs (n=15) and NK cells (n=6) against A549 target cells at E:T ratio of 10:1 before and at 2 or 4 weeks after cytokine-induced killer cell induction. Data are presented as the mean ± standard deviation. PBMCs, peripheral blood mononuclear cells; NK cell, natural killer cell; E:T, effector-to-target.

Figure 5.

Plasma cytokine profiles of non-small cell lung cancer patients before and at 2 or 4 weeks after cytokine-induced killer cell treatment. Antitumor cytokines (A) IFN-γ, (B) IP-10, (C) TNF-α, (D) GM-CSF, (E) MCP-3, (F) IL-21 and (G) TGF-β1 were evaluated (n=15). IFN-γ, interferon-γ; IP-10, IFN-γ-inducible protein 10; TNF-α, tumor necrosis factor-α; GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP-3, monocyte chemotactic protein-3; IL-21, interleukin-21; TGF-β1, transforming growth factor-β1; CIK, cytokine-induced killer.

Figure 6.

Progression-free survival and overall survival rates in non-small cell lung cancer patients who received routine chemoradiotherapy with or without CIK cell therapy. Survival rates were evaluated using Kaplan-Meier analysis with the log-rank test. (A) Progression-free survival (P=0.038). (B) Overall survival (P=0.048). CIK, cytokine-induced killer.