Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids.

Stephan Klöss,Nicole Chambron,Lubomir Arseniev,Ruth Esser,Ulrike Köhl,Elke Pogge Von Strandmann,Michael A Morgan,Sandra Weil,Oliver Seitz,Joachim Koch,Tanja Gardlowski

doi:10.3389/fimmu.2015.00543

Abstract

Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-β1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 μg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor surveillance in relapsed HNSCC patients could be reversed by the re-establishment of ADCC-mediated effector cell activity, thus supporting NK cell-based immunotherapy in combination with antineoplastic monoclonal mAbs.

Highlights

Natural killer cells are lymphoid effector cells important for the innate immune response against virally infected and malignant cells [1, 2]
To analyze the NK cell-mediated killing activity in presence and absence of cetuximab, we developed a matched effector-target cell system based on a flow cytometry (FCM)-based cytotoxicity assay
Freshly isolated patient NK cells revealed low to moderate expression levels of the natural cytotoxicity receptors (NCRs), NKp30, NKp44, NKp46, and NKG2D compared to higher frequencies of IL-2 stimulated NK cells from healthy controls (HCs)

Summary

Introduction

Natural killer cells are lymphoid effector cells important for the innate immune response against virally infected and malignant cells [1, 2]. Tumor-infiltrating NK cells can trigger stimulating interactions via “cell cross-talk” with dendritic cells (DCs), possibly facilitating tumor antigen presentation and induction of tumor antigen-directed T-cell responses This demonstrates the constitutive role of NK cells as mediators between the innate and acquired immune systems [4,5,6]. Elevated levels of soluble forms of these NKG2D ligands (sMICA and sMICB), generated by matrix metalloproteinase (MMP)-dependent proteolytic cleavage (“shedding”) were detected in plasma/serum of cancer patients [13] These soluble NKG2D ligands are responsible for systemic reduction of NKG2D expression on the surface of various circulating blood lymphocytes, especially cytotoxic NK cells, NK-like T (NKT) cells, and CD8+ αβ+- and γδ+-T cells. These immune modulating effects resulted in decreased tumor surveillance by attenuated recognition and elimination of malignant cancer cells [14,15,16]

Results

Discussion

Conclusion