Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

Highlights

• CIK therapy recruits MDSCs in tumor tissues through inflammatory cytokines.

• MDSCs inhibit CIK tumor lytic function in an ARG1 and iNOS dependent manner.

• Tadalafil, an FDA approved PDE5 inhibitor, suppressed the number and function of MDSCs.

• Tadalafil enhances antitumor efficacy of CIK cells in in vivo murine HCC models.

• Human MDSCs inhibit human CIK cell cytotoxicity.

Background & Aims

Cytokine-induced killer (CIK) cell-based immunotherapy is effective as an adjuvant therapy in early stage hepatocellular carcinoma (HCC) but lacks efficacy in advanced HCC. We aimed to investigate immune suppressor mechanisms in HCC, focusing on the role of myeloid-derived suppressor cells (MDSCs) in response to CIK therapy.

Methods

MDSCs were quantified by flow cytometry and quantitative real-time PCR. Cytokines were detected by cytokine array. A lactate dehydrogenase cytotoxicity assay was performed in the presence or absence of MDSCs to study CIK function against HCC cells in vitro. An FDA-approved PDE5 inhibitor, tadalafil, was used to target MDSCs in vitro and in vivo. Two different murine HCC cell lines were tested in subcutaneous and orthotopic tumor models in C57BL/6 and BALB/c mice. The antitumor effects of human CIKs and MDSCs were also tested in vitro.

Results

Adoptive cell transfer of CIKs into tumor-bearing mice induced inflammatory mediators (e.g., CX3CL1, IL-13) in the tumor microenvironment and an increase of tumor-infiltrating MDSCs, leading to impaired antitumor activity in 2 different HCC models. MDSCs efficiently suppressed the cytotoxic activity of CIKs in vitro. In contrast, treatment with a PDE5 inhibitor reversed the MDSC suppressor function via ARG1 and iNOS blockade and systemic treatment with a PDE5 inhibitor prevented MDSC accumulation in the tumor microenvironment upon CIK cell therapy and increased its antitumor efficacy. Similar results were observed when human CIKs were tested in vitro in the presence of CD14⁺HLA-DR^−/low MDSCs. Treatment of MDSCs with a PDE5 inhibitor suppressed MDSC suppressor function and enhanced CIK activity against human HCC cell lines in vitro.

Conclusion

Our results suggest that targeting MDSCs is an efficient strategy to enhance the antitumor efficacy of CIKs for the treatment of patients with HCC.

Lay summary

Cytokine-induced killer cells are a mixture of immune cells given to eliminate cancer cells. However, not all patients respond to this treatment. Herein, we show in 2 different liver cancer models that myeloid-derived suppressor cells are increased in response to cytokine-induced killer cell therapy. Targeting these myeloid-derived suppressor cells may provide an additional therapeutic benefit alongside cytokine-induced killer cell therapy.

Introduction

Hepatocellular carcinoma (HCC) is a common and fatal cancer, with an increasing incidence worldwide.¹ Though many curative therapies have been developed, the overall response to these therapies is inadequate and the long-term prognosis of patients with HCC remains poor because of its high recurrence rates.² A lot of data have shown that tumor progression is correlated with the accumulation of myeloid-derived suppressor cells (MDSCs) which induce local and possibly systemic immunosuppression.³ Moreover, a greater prevalence of MDSCs has been correlated with early recurrence and was shown to be a predictor of poor prognosis in patients with HCC who underwent curative resection,⁴ radiotherapy,⁵ and hepatic arterial infusion chemotherapy.⁶ MDSCs have been shown to suppress CD8⁺[7], [8], [9] and CD4⁺ T¹⁰ cells as well as natural killer (NK)¹¹ cells through diverse direct or indirect mechanisms.¹²

Cytokine-induced killer (CIK) cells are a mixed cell population of effector cells with diverse T cell receptor specificities that also possess non-major histocompatibility complex-restricted cytotoxic activity against tumor cells. CIK cells, which comprise cytotoxic T cells, NK cells, and NK-like T cells that express both NK- and T-cell markers are expanded ex vivo using recombinant IFN-γ, IL-2 and anti-CD3.¹³ CIK cell-based immunotherapies have been widely studied and used in the treatment of patients with cancer, including HCC.¹⁴ Currently, 90 registered clinical trials are listed on the ClinicalTrials.gov website (http://www.clinicaltrials.gov) when the following keywords are used in the search: cytokine-induced killer cells or CIK.¹⁵

A recent study determined that adjuvant immunotherapy using CIK cells appeared to reduce the recurrence of HCC and to improve overall survival.¹⁶ Although adjuvant CIK cell-based immunotherapy is a promising treatment option for early stage HCC, it lacks efficacy in advanced HCC.[17], [18], [19] We hypothesized that CIK cells could trigger a counter-regulatory immunosuppressive mechanism through recruitment of MDSCs that might hinder CIK cell antitumor activity.

We show that adoptive CIK cell therapy leads to an accumulation of MDSCs in the tumor microenvironment, which in turn suppress CIK function. We demonstrate that a PDE5 inhibitor can not only suppress MDSCs accumulation and function, but also enhance CIK cell-based therapy in murine HCC tumor models. Finally, our murine data were corroborated by human in vitro data using human CIK and tumor cells as well as MDSCs treated with a PDE5 inhibitor.