Broad Institute of MIT and Harvard, Cambridge, MA, United States.
Whitehead Institute for Biomedical Research, Cambridge, MA, United States.
Front Immunol. 2021 Nov 30;12:798235. doi: 10.3389/fimmu.2021.798235. eCollection 2021.
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 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.
虽然人类白细胞抗原 (HLA) 和 HLA 样蛋白构成了已知 NK 细胞受体配体的绝大多数，但 NK 细胞受体与非传统配体（尤其是碳水化合物抗原）的相互作用描述得较少。我们之前通过基于珠子的 HLA 筛选发现，杀伤细胞免疫球蛋白样受体 (KIR) 家族的先前孤儿成员 KIR3DS1 与 HLA-F 结合。在这项研究中，我们使用 Fc 融合构建体评估了 KIR3DS1 与细胞系的配体结合谱，并发现 KIR3DS1-Fc 与包括缺乏 HLA 的几种人类细胞系结合。为了鉴定这些非 HLA 配体，我们开发了一种基于磁珠富集的全基因组 CRISPR/Cas9 敲除筛选方法，并确定了参与肝素硫酸酯生物合成的酶是 KIR3DS1-Fc 与 K562 细胞结合的关键。KIR3DS1 与肝素硫酸酯之间的这种相互作用通过表面等离子体共振得到证实，并且从细胞表面去除肝素硫酸酯蛋白聚糖会消除 KIR3DS1-Fc 的结合。对其他 KIR-Fc 构建体的测试表明，含有 D0 结构域的 KIR 家族成员（KIR3DS1、KIR3DL1、KIR3DL2、KIR2DL4 和 KIR2DL5）与肝素硫酸酯结合，而不含 D0 结构域的成员（KIR2DL1、KIR2DL2、KIR2DL3 和 KIR2DS4）则没有。总的来说，这项研究表明使用全基因组 CRISPR/Cas9 敲除策略可以公正地鉴定 NK 细胞受体的非传统配体。此外，我们发现各种激活和抑制性 KIR 与肝素硫酸酯蛋白聚糖的以前未被认识到的结合可能在 NK 细胞受体信号传导和靶细胞识别中发挥作用。
