Inhibitory Receptor Crosslinking Quantitatively Dampens Calcium Flux Induced by Activating Receptor Triggering in NK Cells

Sridharan Ganesan,Petter Höglund

doi:10.3389/fimmu.2018.03173

Sridharan Ganesan, Petter Höglund

Open Access

https://doi.org/10.3389/fimmu.2018.03173

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Abstract

Natural killer (NK) cell function is regulated by a balance between activating and inhibitory receptors, but the details of this receptor interplay are not extensively understood. We developed a flow cytometry-based assay system in which Ca2+ flux downstream of antibody-mediated activating receptor triggering was studied in the presence or absence of inhibitory receptor co-crosslinking. We show that the inhibitory influence on activating receptor-induced Ca2+ flux is quantitatively regulated, both on murine and human NK cells. Furthermore, both activating and inhibitory receptors operate in an additive way, suggesting that a fine-tuned balance between activating and inhibitory receptors regulate proximal NK cell signaling. We also demonstrate that murine NK cell expression of H2Dd lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2Dd and Ly49A reduce the signaling capacity of Ly49A in this setting. Finally, we show that priming of NK cells by IL-15 rapidly augments Ca2+ flux after activating receptor signaling without attenuating the potential of inhibitory receptors to reduce Ca2+ flux. Our data shed new light on NK cell inhibition and raises new questions for further studies.

Highlights

Natural Killer (NK) cells are innate lymphocytes involved in the immune defense against viral infections and transformed cells [1], and they exert control over adaptive immune responses [2, 3]
To gain further insight into this process, we established an in vitro system based on co-crosslinking of activating and inhibitory NK cell receptors on mouse and human NK cells, followed by a FACSbased assay for Ca2+ flux in real time (Figure S1). We reasoned that this setup would allow us to investigate if inhibitory receptor triggering quantitatively downregulates NK cell activation, or if inhibition would operate in a threshold mode
When NK1.1 was co-crosslinked with either Ly49A or Ly49G2, we found that IL-15 priming induced Ca2+ flux in the Ly49A+ and Ly49G2+ NK cell subsets (Figures 4A,B, compare red and black lines), and that the efficiency of the inhibitory receptor to shut off NK1.1-triggered Ca2+ release cell subset expressing Ly49A and the red line represents Ly49A-negative NK cells in the same sample. (B) Same setup as in A but on NK cells from B6 mice. (C) Mean fluorescence intensity (MFI) of Ly49A staining at various dilutions of anti-Ly49A on NK cells derived from B6 or H2Dd mice

Summary

Introduction

Natural Killer (NK) cells are innate lymphocytes involved in the immune defense against viral infections and transformed cells [1], and they exert control over adaptive immune responses [2, 3]. In contrast to T cells, each of which possess a unique T cell receptor generated by somatic gene recombination, NK cells express several distinct types of germlineencoded activating receptors that are differentially expressed, signal differently and bind unique ligands on target cells [9]. A similar diversity can be found for inhibitory receptors for MHC class I. In mice these belong to the lectin-like Ly49 receptor family, whereas in humans they constitute immunoglobulin superfamily killer immunoglobulinlike receptors (KIR) [10]. Both mice and humans express NKG2A, a lectin-like

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