Secreted Ligands of the NK Cell Receptor NKp30: B7-H6 Is in Contrast to BAG6 Only Marginally Released via Extracellular Vesicles.

Viviane Ponath,Nathalie Hoffmann,Elke Pogge Von Strandmann,Bilal Alashkar Alhamwe,Leonie Bergmann,Christina Mäder,Christian Preußer

doi:10.3390/ijms22042189

Viviane Ponath, Nathalie Hoffmann + Show 5 more

Open Access

https://doi.org/10.3390/ijms22042189

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Abstract

NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses. Ligands for NKp30, including BAG6 and B7-H6, are upregulated in virus-infected and tumor cells but rarely detectable on healthy cells. These ligands are released by tumor cells as part of the cellular secretome and interfere with NK cell activity. BAG6 is secreted via the exosomal pathway, and BAG6-positive extracellular vesicles (EV-BAG6) trigger NK cell cytotoxicity and cytokine release, whereas the soluble protein diminishes NK cell activity. However, the extracellular format and activity of B7-H6 remain elusive. Here, we used HEK293 as a model cell line to produce recombinant ligands and to study their impact on NK cell activity. Using this system, we demonstrate that soluble B7-H6 (sB7-H6), like soluble BAG6 (sBAG6), inhibits NK cell-mediated target cell killing. This was associated with a diminished cell surface expression of NKG2D and NCRs (NKp30, NKp40, and NKp46). Strikingly, a reduced NKp30 mRNA expression was observed exclusively in response to sBAG6. Of note, B7-H6 was marginally released in association with EVs, and EVs collected from B7-H6 expressing cells did not stimulate NK cell-mediated killing. The molecular analysis of EVs on a single EV level using nano flow cytometry (NanoFCM) revealed a similar distribution of vesicle-associated tetraspanins within EVs purified from wildtype, BAG6, or B7-H6 overexpressing cells. NKp30 is a promising therapeutic target to overcome NK cell immune evasion in cancer patients, and it is important to unravel how extracellular NKp30 ligands inhibit NK cell functions.

Highlights

Clinical and experimental data in different model systems and tumor entities demonstrate the important role of natural killer (NK) cells in the immunological control of tumors [1,2,3]
NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses
We demonstrate that soluble B7-H6, like soluble BAG6, inhibits NK cell-mediated target cell killing

Summary

Introduction

Clinical and experimental data in different model systems and tumor entities demonstrate the important role of natural killer (NK) cells in the immunological control of tumors [1,2,3]. Ligands for activating NK cell receptors are released by tumor cells and part of the cellular secretome. These extracellular ligands may interfere with NK cell regulation and antitumor immune responses, representing an important immune escape mechanism. In contrast to NKG2D ligands, the role and activity of NKp30 ligands BAG6 and B7-H6, either cell-surface expressed or present in the cellular secretome, are not well understood. Some of the patients in this study had high serum levels of the soluble NKp30 ligand B7-H6, which were associated with reduced NK cell activity and continued to be associated with chemoresistance and survival. We purified vesicles as well as recombinant soluble ligands from cells overexpressing BAG6 and B7-H6, respectively, to characterize the different ligand natures and analyze their impact on NK cell activity

Results and Discussion

Western Blot Analysis

NK Cell Receptor Surface Marker Expression

Quantitative RT-PCR