杀伤细胞免疫球蛋白样受体结合肿瘤细胞实验

Loredana Cifaldi; Franco Locatelli; Doriana Fruci

doi:10.21769/BioProtoc.1931

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Peer-reviewed

Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells

杀伤细胞免疫球蛋白样受体结合肿瘤细胞实验

LC Loredana Cifaldi

FL Franco Locatelli

Doriana Fruci email

发布: 2016年09月20日第6卷第18期 DOI: 10.21769/BioProtoc.1931 浏览次数: 7212

评审: HongLok LungBenoit ChassaingAnonymous reviewer(s)

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Cover of Cancer Research, featuring study using the protocol.

Mar 2015

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Abstract

Natural killer (NK) cells play key roles in innate and adaptive immune responses against virus and tumor cells. Their function relies on the dynamic balance between activating and inhibiting signals through receptors that bind ligands expressed on target cells. The absence of inhibitory receptor engagement with their ligands and the presence of activating signals transmitted by activating receptors interacting with specific ligands, leads to NK cell activation (Lanier, 2005; Raulet et al., 2001). Thus, the balance of the ligands expressed for inhibitory and activating receptors determines whether NK cells will become activated to kill the target cells. This protocol allows to assign a precise ligand specificity to any given receptor on NK cells. Thus, if a tumor cell expresses the ligand, this protocol will allow to evaluate its interaction with the specific receptor. In particular, killer cell immunoglobulin (Ig)-like receptors (KIR) recognize their ligands (HLA class I molecules) through the direct contact with HLA class I heavy chain residues and amino acid residues of the bound peptide. This protocol will allow to test the effect of amino acid substitutions or other mutations on the binding of KIR to HLA class I. We used this protocol to depict the role of ERAP1, a key component of the MHC class I antigen processing, in regulating NK cell function by controlling the engagement of inhibitory receptors (Cifaldi et al., 2015).

Keywords: NK cells (自然杀伤细胞)

Killer cell immunoglobulin (Ig)-like receptors (KIR) (杀伤细胞免疫球蛋白（Ig）-样受体（KIR）)

HLA class I (HLA I类)

KIR funsion protein (KIR风行蛋白)

Tumor cells (肿瘤细胞)

Materials and Reagents

T25 Flasks (Corning, Falcon^®, catalog numbers: 353108 )
T75 Flasks (Corning, Falcon^®, catalog numbers: 353136 )
15 ml centrifuge tubes (Corning, Falcon^®, catalog number: 352096 )
96 well plates (Corning, catalog number: 3799 )
FACS tubes
Tumor cell line (grown in flasks, in incubator at 37 °C and 5% CO₂)
RPMI 1640 (EuroClone, catalog number: ECM9106L )
Fetal bovine serum (FBS) heat-inactivated for 1 h at 56 °C (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270-106 )
Penicillin/streptomycin (EuroClone, catalog number: ECB3001D )
L-glutamine (EuroClone, catalog number: ECB3000D )
EDTA (Sigma-Aldrich, catalog number: E5134 )
Phosphate buffered saline (PBS) (EuroClone, catalog number: ECB4004L )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
Trypan-blue
KIR-Fc fusion proteins

Recombinant Human KIR2DL1/CD158a Fc chimera protein (R&D System, catalog number: 1844-KR-050 )
Recombinant Human KIR2DL3/CD158b2 Fc chimera protein (R&D System, catalog number: 2014-KR-050 )
Recombinant Human KIR3DL1 Fc chimera protein (R&D System, catalog number: 1225-KR-050 )

Goat anti-Human Molecular Probes^TM secondary antibody, RPE conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: H10104 )
Complete RPMI (see Recipes)
833 μM EDTA (see Recipes)
FACS buffer (see Recipes)

Equipment

Centrifuge, used with maximal acceleration and deceleration (Eppendorf, model: 5810R )
Incubator (5% CO₂, 37 °C) (Forma^TM Steri-Cult^TM CO₂ Incubator-Thermo Fisher Scientific)
Flow cytometer, equipped with 2 lasers (8 detectors), interfaced with PC by using DIVA Software version 6.1.3 (BD, model: FACSCanto II )

Software

DIVA software version 6.1.3
FlowJo software

Procedure

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如何引用

Cifaldi, L., Locatelli, F. and Fruci, D. (2016). Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells. Bio-protocol 6(18): e1931. DOI: 10.21769/BioProtoc.1931.