An Anti-MICA/B Antibody and IL-15 Rescue Altered NKG2D-Dependent NK Cell Responses in Hepatocellular Carcinoma

Simple Summary Primary liver cancer is one of the most common causes of cancer-related deaths worldwide. The present study shows that the cytokine IL-15 and a humanized antibody specific for a ligand recognized by a major activating receptor (NKG2D) expressed by a subset of lymphocytes named natural killer (NK) cells can restore dysfunctional NK cell responses in patients affected by the most common liver cancer hepatocellular carcinoma. This study emphasizes the importance of NK cells for immune responses to hepatocellular carcinoma and identifies the NKG2D activating receptor/ligand axis as a possible target for immunotherapeutic interventions. More importantly, building upon previously published studies, these findings provide a basis for the future treatment of tumors unresponsive to current therapies. Abstract Natural killer (NK) cells play a pivotal role in cancer immune surveillance, and activating the receptor/ligand interaction may contribute to control the development and evolution of hepatocellular carcinoma (HCC). We investigated the role of the natural killer group 2 member D (NKG2D) activating receptor and its ligand, the major histocompatibility complex class I chain-related protein A and B (MICA/B) in patients with cirrhosis and HCC subjected to surgical resection, patients with cirrhosis and no HCC, and healthy donors (HD). The NKG2D-mediated function was determined in peripheral blood (PB), in tumor-infiltrating lymphocytes (NK-TIL), and in matched surrounding liver tissue (NK-LIL). A group of patients treated with sorafenib because of clinically advanced HCC was also studied. A humanized anti-MICA/B monoclonal antibody (mAb) was used in in vitro experiments to examine NK cell-mediated antibody-dependent cellular cytotoxicity. Serum concentrations of soluble MICA/B were evaluated by ELISA. IL-15 stimulation increased NKG2D-dependent activity which, however, remained dysfunctional in PB NK cells from HCC patients, in line with the reduced NKG2D expression on NK cells. NK-TIL showed a lower degranulation ability than NK-LIL, which was restored by IL-15 stimulation. Moreover, in vitro IL-15 stimulation enhanced degranulation and interferon-γ production by PB NK from patients at month one of treatment with sorafenib. Anti-MICA/B mAb associated with IL-15 was able to induce PB NK cytotoxicity for primary HCC cells in HD and patients with HCC, who also showed NK-TIL degranulation for autologous primary HCC cells. Our findings highlight the key role of the NKG2D-MICA/B axis in the regulation of NK cell responses in HCC and provide evidence in support of a potentially important role of anti-MICA/B mAb and IL-15 stimulation in HCC immunotherapy.


Isolation of peripheral blood mononuclear cells and tissue-infiltrating lymphocytes
Peripheral blood mononuclear cells (PBMC) were isolated by Lympholyte (Cedarlane, Burlington, Canada) density gradient (1.0770) centrifugation following the manufacture's instruction. Briefly, whole blood was diluted with an equal volume of phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) and layered over Lympholyte. After centrifugation at 500 × g for 30 min at room temperature without the brake applied, the PBMC interface was carefully removed by pipetting and washed with PBS with 2% fetal calf serum (FCS, HyClone, GE Healthcare, South Logan, Utah, USA) by centrifugation at 400 × g for 10 min. PBMC were counted by excluding non-viable cells were identified by staining with trypan blue. PBMC were cryopreserved in liquid nitrogen in FCS containing 10 % dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored until required for downstream analyses.
Tissue samples were treated by mechanical and enzymatic dissociation with the human Tumor Dissociation Kit and by gentleMACS Dissociator (both from Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. The cell suspension was filtered in a 70µ m cell strainer (Miltenyi Biotec) and centrifuged twice at 50 × g for 2 min. The supernatant containing lymphocytes was processed for flow cytometry or cryopreserved in liquid nitrogen in FCS containing 10 % DMSO and stored until required for downstream analyses .The tumor cells were plated in culture as described below.
To establish in vitro primary HCC cell cultures, the cell pellet obtained after dissociation of resected tumor tissue was plated in tissue culture flasks (Corning, NY, USA) with DMEM supplemented with 10% FCS, 1% antibiotic antimycotic solution and 1% non-essential amino acids (Thermo Fisher Scientific) and used after low passages.
Expression of MICA/B was evaluated by labeling Huh 7.5 or HCC cells with anti-MICA/B PEconjugated (BioLegend, San Diego, CA, USA) or with control IgG isotype and examined by flow cytometry.

Functional assays
Redirecting assay, also named reverse antibody-dependent cellular cytotoxicity (rADCC) assay, was performed after cross-linking of activating receptors and FcγR+ P815 murine cell line. PBMC, TIL and LIL were incubated overnight with or without IL-15 (20 ng/ml, PeproTech EC, London, UK) and subsequently washed and incubated for 4 hours at 37°C with FcγR+ P815 murine target cells (E:T=1:1) in the presence of anti-NKG2D, -CD226 or -NKp30 specific mAbs (BioLegend and R&D System, Minneapolis, MN), anti-CD107a PE and the Protein Transport Inhibitor GolgiStop (both from BD Biosciences, San Diego, CA, USA). After washing, lymphocytes were stained for surface NK cell markers using anti-CD3 BV421™ (BD Biosciences) and anti-CD56 Pc5.5 (Beckman Coulter, Brea, CA, USA). Cells were fixed with BD Cytofix/Cytoperm and permeabilized with the BD Perm/Wash buffer (BD Biosciences) according to the manufacturer's instructions, in the presence of anti-IFNγ Alexa Fluor® 647 (BD Biosciences). LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit was used to determine cell viability. Data analysis was performed with the Kaluza 2.1 software (Beckman Coulter). Antibody-dependent cellular cytotoxicity (ADCC) was evaluated in several experimental conditions. When the Huh 7.5 or primary HCC cells were used as targets, 5x10 5 cells/well were seeded in 24-well plate in appropriate culture conditions with anti-CD107a PE and co-cultured for 4 hours with effector cells (E:T=5:1), in the presence of 10 g/ml control isotype (IgG) or human anti-MICA/B mAb (kindly provided by Innate Pharma, Marseille, France). The effectors were then harvested, washed and stained with anti-CD3 BV421™ and anti-CD56 Pc5.5. To evaluate target cell death, primary HCC cells were recovered and labeled with 7-Aminoactinomycin D (7-AAD, BD Biosciences), according to the manufacturer's instructions. When Daudi cells were used as target, the ADCC assay was performed in 96-round-bottom plates by co-culturing effector and target cells (E:T=1:1) for 4 hours at 37°C with anti-CD107a-PE antibody, with or without an anti-CD20 antibody (rituximab, 10 g/ml). At the end of culture, cells were harvested and labeled withLIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit, CD45 BV786™ (BD Biosciences), CD3 FITC (BD Biosciences) and CD56 Pc5.5.