Identification of Human Memory‐Like NK Cells

Elena I. Kovalenko1, Maria A. Streltsova1, Leonid M. Kanevskiy1, Sophia A. Erokhina1, William G. Telford2

1 Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russian Federation, 2 National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.50
DOI:  10.1002/cpcy.13
Online Posting Date:  January, 2017
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Our understanding of NK biology is increased dramatically, a product of improved flow‐cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory‐like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically. Here we describe an approach for identification and analysis of adaptive NK cells in human peripheral blood. CD57‐positive cells with high expression of activating‐receptor NKG2C, increased expression of KIR receptors, lack of co‐expression with inhibitory receptor NKG2A, and decreased expression of activating receptor NCR3 (NKp30) all characterize this cell type. The flow‐cytometric method described below can identify this NK cell subset on a relatively simple flow cytometer. © 2017 by John Wiley & Sons, Inc.

Keywords: memory‐like NK cells; adaptive NK cells; NK cell repertoire; differentiation; CD57; NKG2A; NKG2C

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Table of Contents

  • Introduction
  • Significance Statement
  • Basic Protocol 1: Analysis of Memory‐Like NK Cells Associated with HCMV Infection Circulating in Human Blood
  • Support Protocol 1: Identification of HCMV Infection‐Associated Adaptive NK Cell Subset in NK Cells Ex Vivo‐Purified by Magnetic Separation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Analysis of Memory‐Like NK Cells Associated with HCMV Infection Circulating in Human Blood

  • Lymphocyte separation medium 1.077 (Lonza, cat. no. 17‐829E)
  • Human blood collected in heparin‐ or EDTA‐containing tubes
  • Phosphate‐buffered saline, pH 7.2 (PBS; Fisher Scientific, cat. no. 20‐012‐027)
  • Staining solution (see recipe)
  • Ice
  • Monoclonal antibodies:
    • Anti‐human CD3‐PE‐Cy7 (Beckman Coulter, clone UCHT1, cat. no. 6607100)
    • Anti‐human CD56‐APC [Beckman Coulter, clone N901 (HLDA6), cat. no. IM2474]
    • Anti‐human NKG2C‐AlexaFluor (AF) 488 (R&D Systems, clone 134591, cat. no. FAB138G‐100)
    • Anti‐human NKG2A‐PE (R&D Systems, clone no. 131411, cat. no. FAB1059P‐100)
    • Anti‐human KIR2DL2/DL3 (CD158b)‐PE (Miltenyi Biotech, clone DX27, cat. no. 130‐092‐618)
    • Anti‐human CD57‐PE (eBioscience, clone TB01, cat. no. 12‐0577‐42)
    • Anti‐human NKp30‐PE (eBioscience, clone AF29‐4D12, cat. no. 12‐3379‐42)
    • Anti‐human HLA‐DR‐FITC (Beckman Coulter, clone B8.12.2, cat. no. IM0463U)
  • 50‐ml conical centrifuge tubes (Fisher Scientific, cat. no. 14‐432‐22)
  • Centrifuge
  • Automatic pipets
  • Cell counter or hemacytometer
  • 5‐ml polystyrene round‐bottom tubes (Fisher Scientific, cat. no. 352008)
  • Multi‐laser flow cytometer with appropriate filters for collection of fluorescence emission
NOTE: For this protocol two 9‐ml tubes with blood should be enough. For the protocol 2Support Protocol (including NK cells separation) minimum 36 ml (four 9‐ml tubes) of blood should be taken.

Support Protocol 1: Identification of HCMV Infection‐Associated Adaptive NK Cell Subset in NK Cells Ex Vivo‐Purified by Magnetic Separation

  Additional Materials (also see protocol 1Basic Protocol)
  • Separation buffer (see recipe)
  • NK Cell Isolation Kit, human (MiltenyiBiotec, cat. no. 130‐092‐657) containing:
    • NK Cell Biotin‐Antibody Cocktail
    • NK Cell MicroBead Cocktail
  • 15‐ml conical centrifuge tubes (Fisher Scientific, cat. no. 14‐959‐53A)
  • 4°C incubator
  • MACS LD Columns (MiltenyiBiotec, cat. no. 130‐042‐901)
  • MidiMACS Separator (MiltenyiBiotec, cat. no. 130‐042‐302)
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Literature Cited

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