A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors

Klara Klein,Tim Wang,Tae-Eun Kim,Haley L Dugan,Wilfredo F Garcia-Beltran,Stephanie Jost,Marcus Altfeld,Angelique Hölzemer

doi:10.3389/fimmu.2021.798235

Abstract

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.

Highlights

NK-cell function is dictated by germline-encoded activating and inhibitory receptors that interact with a plethora of ligands expressed on target cells [1]
Using a soluble construct of KIR3DS1, an activating NK-cell receptor previously shown by our group to bind human leukocyte antigen (HLA)-F [20], we found that enzymes involved in the biosynthesis of heparan sulfate were critical for generating KIR3DS1 ligands on the cell surface of HLA-deficient cells (K562 cells)
Using a genome-wide CRISPR/Cas9 knock-out screen, we identified heparan sulfate proteoglycans as ligands for the activating NK-cell receptor KIR3DS1 on K562 cells

Summary

Introduction

NK-cell function is dictated by germline-encoded activating and inhibitory receptors that interact with a plethora of ligands expressed on target cells [1]. Major classes of NK-cell receptors are [1] natural cytotoxicity receptors (NCRs), which are comprised of NKp30, NKp44, and NKp46, [2] killer-cell lectinlike receptors (KLRs), which include NKG2A, NKG2C, and NKG2D, and [3] killer-cell immunoglobulin-like receptors (KIRs), the most polymorphic family of NK-cell receptors [2]. These receptors allow NK cells to discriminate between healthy “self” and a variety of pathological cell states [3]. Heparan sulfate has been referred to as “the most informationdense biopolymers found in nature” [16]

Methods

Results

Conclusion