Target‐Induced Natural Killer Cell Loss as a Measure of NK Cell Responses

Hilary Warren1

1 Cancer Immunology Research Unit, The Canberra Hospital, Garran, Canberra, Australian Capital Territory, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.29
DOI:  10.1002/0471142735.im1429s101
Online Posting Date:  April, 2013
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Abstract

Natural killer (NK) cells are an important effector cell of innate immunity. Their interaction with susceptible target cells triggers NK cell cytotoxicity and the release of cytokines. Immunofluorescence flow cytometry–based assays are now the preferred methods for measuring NK cell responses. For these assays, assessment is made on NK cells (CD3–CD56+ CD16+) within the viable lymphocyte gate, defined by the parameters of size (FSC) and granularity (SSC). Accordingly, NK cells that have not dissociated from target cells at the time of measurement, or that have undergone target cell–induced apoptosis, are excluded from the viable lymphocyte gate and therefore from analysis. This unit describes a protocol for assessing NK cell function in response to various target cells (natural killing, antibody-dependent cell cytotoxicity, and NK cell alloreactivity) based on the loss of NK cells from the lymphocyte gate. This target-induced NK loss (TINKL) should provide a sensitive measure of NK cell responses in a clinical laboratory setting. Curr. Protoc. Immunol. 101:14.29.1-14.29.21. © 2013 by John Wiley & Sons, Inc.

Keywords: human NK cells; cytotoxicity; ADCC; Rituximab; NK cell alloreactivity

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Assessing NK Cell Responses in Unfractionated Peripheral Blood Mononuclear Cells
  • Alternate Protocol 1: Assessing NK Cell Killing of Autologous B Cells by Rituximab
  • Alternate Protocol 2: Assessing NK Cell Responses Using Purified NK Cells
  • Alternate Protocol 3: Assessing NK Cell Alloreactivity Using Purified NK Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Assessing NK Cell Responses in Unfractionated Peripheral Blood Mononuclear Cells

 Materials
  • Human PBMC: freshly isolated (unit 7.1) or from liquid nitrogen storage (see units 16.1 & 8.17)
  • RPMI/20% FBS: complete RPMI-1640 medium (appendix 2A) supplemented with 20% heat-inactivated fetal bovine serum (FBS)
  • K562 erythroleukemic cells (ATCC, cat. no. CCL-243)
  • RPMI/10% FBS: complete RPMI-1640 medium (appendix 2A) supplemented with 10% heat-inactivated fetal bovine serum (FBS)
  • Raji B lymphoblastoid cells (ATCC, cat. no. CCL-86)
  • RPMI/15% FBS: complete RPMI-1640 medium (appendix 2A) supplemented with 15% heat-inactivated fetal bovine serum (FBS)
  • Rituximab (Roche) anti-CD20 mAb, stock concentration 10 mg/ml: dilute an aliquot to a sub-stock concentration of 1 mg/ml in PBS containing 0.1% BSA, and filter sterilize (store at 4° to 8°C)
  • PBS/FBS/azide: phosphate-buffered saline (PBS; appendix 2A) containing 5% (v/v) fetal bovine serum (FBS) and 0.1% (w/v) sodium azide
  • FITC-conjugated anti-CD3 mAb (any manufacturer) and PE-conjugated anti-CD56 mAb (Miltenyi Biotec)
  • 2% paraformaldehyde in PBS (see unit 5.3 for preparation)
  • 24-well tissue culture plates
  • 10-ml conical centrifuge tubes
  • Refrigerated centrifuge with plate carrier
  • 96-well round-bottom plates
  • 5-ml flat-base screw-capped tubes (Sarstedt, cat. no 60.9921.532.PS)
  • 96-well V-bottom plates
  • 1.2-ml cluster tubes suitable for insertion into 4-ml tubes for the flow cytometer; cluster tubes are provided in racks at 96 per rack (Corning Costar Brand Cluster Tube System; Corning, cat. no. 4401; Fisher Scientific, cat. no. 07-200-319); cluster tubes are also available as bags of 960 (Corning, cat. no. 4410) to refill the racks
  • Flow cytometer (see Chapter 5)
  • Additional reagents and equipment for obtaining fresh PBMC from whole blood (see units 7.1 & 16.1), frozen storage and thawing of PBMC (see units 16.1 & 8.17), basic culture techniques including counting cells (appendix 3A), and flow cytometry (Chapter 5)

Alternate Protocol 1: Assessing NK Cell Killing of Autologous B Cells by Rituximab

 Additional Materials (also see Basic Protocol)
  • PE-conjugated anti-CD3 mAb, PE-conjugated anti-CD19 mAb, FITC-conjugated anti-CD56 mAb, and FITC-conjugated anti-CD94 mAb

Alternate Protocol 2: Assessing NK Cell Responses Using Purified NK Cells

 Additional Materials (also see Basic Protocol)
  • Whole blood or PBMC (fresh or frozen in liquid nitrogen) to be used for isolation of NK cells as described in unit 7.34
  • Red blood cells (optional; see step 1b below)
  • Counting beads for flow cytometry (e.g., Flow-Count Fluorospheres, Beckman Coulter)
  • Additional reagents and equipment for isolation of NK cells from whole blood using the RosetteSep method (unit 7.34)

Alternate Protocol 3: Assessing NK Cell Alloreactivity Using Purified NK Cells

 Additional Materials (also see Basic Protocol and Alternate Protocol 2)
  • Phytohemagglutinin (PHA; also see unit 7.10)
  • DMEM/10% FBS (see recipe)
  • Human recombinant IL-2 (rIL-2; any manufacturer)
  • Cy5-conjugated anti-CD158a, anti-CD158b, and anti-CD158e1 (any manufacturer)
  • Biotinylated anti-CD158a, anti-CD158b, and anti-CD158e1 (any manufacturer; optional; see step 17 below)
  • Streptavidin-Cy5 (optional; see step 17 below)
  • PE-conjugated anti-CD158a, anti-CD158b, anti-CD158e1, anti-NKG2A, anti-CD85J (any manufacturer)
  • FITC-conjugated anti-CD3 (any manufacturer)
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Figures

  •  FigureFigure 14.29.1 Analysis of PBMC showing gating of lymphocytes (A) and of NK cells (CD3– CD56+) and T cells (CD3+) within the lymphocyte gate (B).
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  •  FigureFigure 14.29.2 Triplicate cultures were set up containing 5 × 104 PBMC (control) alone (A) or with Rituximab (ADCC) (B). After 6 hr, the cultures were harvested and then analyzed by immunofluorescence staining and flow cytometry. NK cells were defined as CD3– CD56+ CD94+ (15.7%). B cells (16.8%) and T cells (65.3%) were identified with anti-CD19-PE mAb and anti-CD3-PE mAb, respectively. NK cells and B cells were quantified relative to T cells as an internal control (C). The 100% control values corresponded to 0.2524 ± 0.0024 (NK/T ratio) and 0.2835 + 0.0068 (B/T ratio).
    View Image
  •  FigureFigure 14.29.3 Analysis of NK cells showing gating of NK cells and counting beads. Single beads and doublets are indicated.
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  •  FigureFigure 14.29.4 Analysis of single KIR+ NK cells. Triplicate cultures were set up containing 2 × 104 NK cells with 2 × 104 activated T cells as target cells. After 6 hr, the cultures were harvested and stained with mAb to identify NK cell subsets expressing a single KIR in the absence of other KIR and in the absence of CD94/NKG2A and CD85J. The plot of FSC/SSC allows determination of the number of counting beads (A). The plot of FSC/anti-CD3-FITC allows NK cells to be gated away from the activated T cell targets (B). The plot of CD158b, CD158e1, NKG2A and CD85J (PE-stained) on the y axis with CD158a-Cy5 on the x axis identifies the single CD158a KIR+ population (C). The plot of CD158a, CD158b, NKG2A, and CD85J (PE-stained) on the y axis with CD158e1-Cy5 on the x axis identifies the single CD158e1 KIR+ population (D).
    View Image
  •  FigureFigure 14.29.5 Comparison of methods for assessing NK cell responses to target cells, by the TINKL assay using the Basic Protocol (% NK loss), by the 51Cr-release cytotoxicity assay (% specific lysis), and by the CD107a assay (% CD107a+).
    View Image

Videos

Literature Cited

Literature Cited
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    Brunner, K.T., Mauel, J., Cerottini, J.C., and Chapuis, B. 1968. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr-labelled allogeneic target cells in vitro: Inhibition by isoantibody and by drugs. Immunology 14:181-196.
    De Santis, D., Foley, B.A., John, E., Christiansen, F.T., and Witt, C.S. 2010. Rapid, flow cytometric assay for NK alloreactivity reveals exceptions to rules governing alloreactivity. Biol. Blood Marrow Transplant. 16:179-191.
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    Goda, R., Bachanova, V., Gleason, M., McCullar, V., Yun, G.H., Cooley, S., Verneris, M.R., McGlave, P.B., and Miller, J.S. 2010. Natural killer cell killing of acute myelogenous leukemia and acute lymphoblastic leukemia blasts by killer cell immunoglobulin-like receptor-negative natural killer cells after NKG2A and LIR-1 blockade. Biol. Blood Marrow Transplant. 16:612-621.
    Horowitz, A., Behrens, 1R.H., Okell, L., Fooks, A.R., and Riley, E.M. 2010. NK cells as effectors of acquired immune responses: Effector CD4+ T cell-dependent activation of NK cells following vaccination. J. Immunol. 185:2808-2818.
    Jewett, A. and Bonavida, B. 1996. Target-induced inactivation and cell death by apoptosis in a subset of human NK cells. J. Immunol. 156:907-915.
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    Warren, H.S. 2011. Target-induced natural killer cell loss as a measure of NK cell responses. J. Immunol. Methods 370:86-92.
    Warren, H.S. and Rana, P.M. 2003. An economical adaptation of the RosetteSep™ procedure for NK cell enrichment from whole blood, and its use with liquid nitrogen stored peripheral blood mononuclear cells. J. Immunol. Methods 280:135-138.
    Warren, H.S., Kinnear, B.F., Phillips, J.H., and Lanier, L.L. 1995. Production of IL-5 by human NK cells and regulation of IL-5 secretion by IL-4, IL-10, and IL-12. J. Immunol. 154:5144-5152.
    Warren, H.S., Kinnear, B.F., Kastelein, R.L., and Lanier, L.L. 1996. Analysis of the costimulatory role of IL-2 and IL-15 in initiating proliferation of resting (CD56dim) human NK cells. J. Immunol. 156:3254-3259.
    Yamauchi, A., Taga, K., Mostowski, H.S., and Bloom, E.T. 1996. Target cell-induced apoptosis of interleukin-2-activated human natural killer cells: Roles of cell surface molecules and intracellular events. Blood 87:5127-5135.
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