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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   

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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

  1. T25 Flasks (Corning, Falcon®, catalog numbers: 353108 )
  2. T75 Flasks (Corning, Falcon®, catalog numbers: 353136 )
  3. 15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
  4. 96 well plates (Corning, catalog number: 3799 )
  5. FACS tubes
  6. Tumor cell line (grown in flasks, in incubator at 37 °C and 5% CO2)
  7. RPMI 1640 (EuroClone, catalog number: ECM9106L )
  8. Fetal bovine serum (FBS) heat-inactivated for 1 h at 56 °C (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  9. Penicillin/streptomycin (EuroClone, catalog number: ECB3001D )
  10. L-glutamine (EuroClone, catalog number: ECB3000D )
  11. EDTA (Sigma-Aldrich, catalog number: E5134 )
  12. Phosphate buffered saline (PBS) (EuroClone, catalog number: ECB4004L )
  13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153-100G )
  14. Trypan-blue
  15. KIR-Fc fusion proteins
    1. Recombinant Human KIR2DL1/CD158a Fc chimera protein (R&D System, catalog number: 1844-KR-050 )
    2. Recombinant Human KIR2DL3/CD158b2 Fc chimera protein (R&D System, catalog number: 2014-KR-050 )
    3. Recombinant Human KIR3DL1 Fc chimera protein (R&D System, catalog number: 1225-KR-050 )
  16. Goat anti-Human Molecular ProbesTM secondary antibody, RPE conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: H10104 )
  17. Complete RPMI (see Recipes)
  18. 833 μM EDTA (see Recipes)
  19. FACS buffer (see Recipes)

Equipment

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

Software

  1. DIVA software version 6.1.3
  2. FlowJo software

Procedure

  1. Human tumor cells are cultured in fresh RPMI medium at a cell density ≥ 0.5-0.7 x 106 cells/ml in an incubator at 37 °C, 5% CO2. For each experiment are required 0.5 x 106 tumor cells.
  2. Wash adherent cells with PBS (5 ml) and detach cells by adding 833 μM EDTA (1 ml for T25 flask and 2 ml for T75 flask) for 3-5 min in an incubator at 37 °C, 5% CO2.
  3. Wash cells twice with PBS (2 x 5 ml in 15 ml tube) by centrifugation at 468 x g for 7 min and re-suspend them in cold FACS buffer.
  4. Count total viable cell number with Trypan-blue exclusion method.
  5. Seed cells in round-bottom 96 well plates at a cell density of 1 x 106 cells/well. Prepare 1 well for the control (without KIR-Fc fusion-proteins) and 1 well for each fusion protein to be tested.
  6. Spin cells at 832 x g for 2 min at 4 °C.
  7. Discard the supernatant and add 100 µl/well of cold FACS buffer containing single KIR-Fc fusion proteins (KIR2DL1-Fc, KIR2DL3-Fc, and KIR3DL1-Fc) diluted 1:5 and 100 μl of cold FACS buffer with none in control cells.
  8. Incubate for 1 h at 4 °C.
  9. Wash cells with 150 µl of cold FACS buffer and centrifuge at 832 x g at 4 °C.
  10. Discard the supernatant and re-suspend cells in 50 µl of cold FACS buffer containing Goat anti-Human secondary antibody, RPE conjugate antibody (diluted 1:100) and mix carefully avoiding formation of bubbles.
  11. Incubate for 25 min on ice in the dark.
  12. Wash cells with 150 µl of cold FACS buffer and centrifuge at 832 x g at 4 °C.
  13. Discard the supernatant and re-suspended cells in 150 µl of cold FACS buffer in FACS tubes and perform flow cytometric analysis. Viable human single cells will be selected by gating in forward scatter versus side scatter dot plot and the binding of KIR-Fc fusion proteins will be evaluated by mean fluorescence intensity (Figure 1).
  14. Acquire data using DIVA software and analyse using FlowJo software.


    Figure 1. KIR3DL1 binding to DAOY cells. Flow cytometric analysis of KIR3DL1-Fc fusion protein binding to DAOY cells (blue line). Isotype-matched negative control antibody is displayed as the red line.

Recipes

  1. Complete RPMI
    440 ml RPMI 1640
    50 ml FBS
    5 ml penicillin-streptomycin
    5 ml L-glutamine
  2. 833 µM EDTA
    Dissolve 7.3 g EDTA in 50 ml sterile water, adjust pH to 8.0
    Add 83.3 µl of this solution into 50 ml sterile PBS
    Filter sterilize through a 20 µm filter
    Store at 4 °C
  3. FACS buffer
    50 ml PBS
    1 g BSA

Acknowledgments

This protocol was adapted from Cifaldi et al. (2015). We acknowledge funding from Italian Ministry of Health (Rome, Italy) grant and the special project 5 x 1,000 Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy) grant.

References

  1. Cifaldi, L., Romania, P., Falco, M., Lorenzi, S., Meazza, R., Petrini, S., Andreani, M., Pende, D., Locatelli, F. and Fruci, D. (2015). ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors. Cancer Res 75(5): 824-834.
  2. Lanier, L. L. (2005). NK cell recognition. Annu Rev Immunol 23: 225-274.
  3. Raulet, D. H., Vance, R. E. and McMahon, C. W. (2001). Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 19: 291-330.

简介

自然杀伤(NK)细胞在针对病毒和肿瘤细胞的先天和适应性免疫应答中发挥关键作用。它们的功能依赖于通过结合靶细胞上表达的配体的受体在激活和抑制信号之间的动态平衡。不存在与其配体的抑制性受体接合和通过活化受体与特异性配体相互作用传递的激活信号的存在导致NK细胞活化(Lanier,2005; Raulet等人,2001)。因此,针对抑制性和激活受体表达的配体的平衡决定了NK细胞是否将被激活以杀死靶细胞。该协议允许给NK细胞上的任何给定受体分配精确的配体特异性。因此,如果肿瘤细胞表达配体,则该方案将允许评价其与特异性受体的相互作用。特别地,通过与HLA I类重链残基和结合肽的氨基酸残基的直接接触,杀伤细胞免疫球蛋白(Ig)样受体(KIR)识别它们的配体(HLA I类分子)。该方案将允许测试氨基酸取代或其他突变对KIR与HLA I型结合的影响。我们使用该方案描述ERAP1(MHC I类抗原加工的关键组分)在调节NK细胞中的作用通过控制抑制性受体的接合来控制细胞功能(Cifaldi等人,2015)。

关键字:自然杀伤细胞, 杀伤细胞免疫球蛋白(Ig)-样受体(KIR), HLA I类, KIR风行蛋白, 肿瘤细胞

材料和试剂

  1. T25烧瓶(Coring,Falcon ,目录号:353108)
  2. T75烧瓶(Coring,Falcon ®,目录号:353136)
  3. 15ml离心管(Coring,Falcon ,目录号:352096)
  4. 96孔板(Coring,目录号:3799)
  5. FACS管
  6. 肿瘤细胞系(在烧瓶中,在37℃和5%CO 2培养箱中生长)
  7. RPMI 1640(EuroClone,目录号:ECM9106L)
  8. 在56℃热灭活1小时的胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  9. 青霉素/链霉素(EuroClone,目录号:ECB3001D)
  10. L-谷氨酸(EuroClone,目录号:ECB3000D)
  11. EDTA(Sigma-Aldrich,目录号:E5134)
  12. 磷酸盐缓冲盐水(PBS)(EuroClone,目录号:ECB4004L)
  13. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153-100G)
  14. 台盼蓝
  15. KIR-Fc融合蛋白质
    1. 重组人KIR2DL1/CD158a Fc嵌合体蛋白(R& D系统,目录号:1844-KR-050)
    2. 重组人KIR2DL3/CD158b2 Fc嵌合蛋白(R& D系统,目录号:2014-KR-050)
    3. 重组人KIR3DL1 Fc嵌合体蛋白(R& D System,目录号:1225-KR-050)
  16. 山羊抗人类分子探针二抗,RPE偶联物(Thermo Fisher Scientific,Invitrogen,目录号:H10104)
  17. 完成RPMI(参见配方)
  18. 833μMEDTA(参见配方)
  19. FACS缓冲区(参见配方)

设备

  1. 离心机,使用最大加速和减速(Eppendorf,型号:5810R)
  2. 将培养箱(5%CO 2,37℃)(Forma TM Supter-Cult TM CO 2 Sub培养箱 - Thermo Fisher Scientific)
  3. 流式细胞仪,配有2个激光器(8个检测器),通过使用DIVA软件版本6.1.3(BD,型号:FACSCanto II)与PC接口,

软件

  1. DIVA软件版本6.1.3
  2. FlowJo软件

程序

  1. 将人肿瘤细胞在37℃,5%CO 2的培养箱中在新鲜RPMI培养基中以细胞密度≥0.5-0.7×10 6个细胞/ml培养。 对于每个实验需要0.5×10 6个肿瘤细胞
  2. 用PBS(5ml)洗涤贴壁细胞并通过加入833μMEDTA(1ml的T25烧瓶和2ml的T75烧瓶)在37℃,5%CO 2的培养箱中3-5分钟 。
  3. 通过在468×g离心7分钟,用PBS(2×5ml,在15ml管中)洗涤细胞两次,并将它们重悬在冷的FACS缓冲液中。
  4. 用台盼蓝排除法计数总活细胞数
  5. 在圆底96孔板中以1×10 6个细胞/孔的细胞密度接种细胞。 准备1个孔用于对照(无KIR-Fc融合蛋白),1个孔用于待测试的每个融合蛋白。
  6. 在4℃下以832×10 9 /分钟的速度旋转细胞2分钟。
  7. 弃去上清液,加入100μl/孔的冷FACS缓冲液,其含有以1:5稀释的单一KIR-Fc融合蛋白(KIR2DL1-Fc,KIR2DL3-Fc和KIR3DL1-Fc)和100μl冷FACS缓冲液, 。
  8. 在4℃孵育1小时。
  9. 用150μl冷FACS缓冲液洗涤细胞,并在4℃下以832×g离心。
  10. 弃去上清液并将细胞重悬于50μl含有山羊抗人二抗,RPE偶联物抗体(1:100稀释)的冷FACS缓冲液中,小心混合以避免形成气泡。
  11. 在冰上在黑暗中孵育25分钟。
  12. 用150μl冷FACS缓冲液洗涤细胞,并在4℃下以832×g离心。
  13. 丢弃上清液和重悬浮的细胞在150μlFACS管中的冷FACS缓冲液,并执行流式细胞术分析。通过在前向散射对侧向散射点图中选通来选择存活的人单个细胞,并通过平均荧光强度评估KIR-Fc融合蛋白的结合(图1)。
  14. 使用DIVA软件获取数据,并使用FlowJo软件进行分析

    图1.KIR3DL1结合DAOY细胞。 KIR3DL1-Fc融合蛋白与DAOY细胞结合的流式细胞术分析(蓝线)。同种型匹配的阴性对照抗体显示为红色线。

食谱

  1. 完成RPMI
    440ml RPMI 1640
    50ml FBS
    5ml青霉素 - 链霉素 5ml L-谷氨酰胺
  2. 833μMEDTA
    将7.3g EDTA溶于50ml无菌水中,调节pH至8.0 将83.3μl的该溶液加入到50ml无菌PBS中 通过20μm过滤器过滤灭菌
    存储在4°C
  3. FACS缓冲区
    50 ml PBS
    1 g BSA

致谢

此协议改编自Cifaldi等人。 (2015)。 我们承认意大利卫生部(意大利罗马)的资金和5 x 1,000意大利意大利安哥拉(意大利米兰)的特别项目。

参考文献

  1. Cifaldi,L.,Romania,P.,Falco,M.,Lorenzi,S.,Meazza,R.,Petrini,S.,Andreani,M.,Pende,D.,Locatelli,F.and Fruci, 2015)。  ERAP1调节自然杀伤细胞功能,通过控制 抑制性受体的结合。 Cancer Res 75(5):824-834
  2. Lanier,LL(2005)。  NK细胞识别。/a> Annu Rev Immunol 23:225-274
  3. Raulet,DH,Vance,RE和McMahon,CW(2001)。  自然杀伤细胞受体库的调节。 Annu Rev Immunol 19:291-330。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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.
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