Isolation and Functional Use of Human NKT Cells

Mark A. Exley1, Brian Wilson2, Steven P. Balk1

1 Cancer Biology Program, Hematology‐Oncology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, 2 Diabetes Center of Excellence, University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.11
DOI:  10.1002/0471142735.im1411s90
Online Posting Date:  August, 2010
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This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term iNKT derives from the fact that a large fraction of murine NKT cells recognize the MHC class I‐like CD1d protein, are CD4+ or CD4‐CD8‐ (double negative), and use an identical “invariant” TCRα chain, which is generated by precise Vα14 and Jα281 (now renamed Jα18) rearrangements with either no N‐region diversity or subsequent trimming to nearly identical amino‐acid sequence (hence, ‘iNKT’). Basic Protocol 1 and Alternate Protocol 1 use multi‐color FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high‐speed FACS sorting of iNKT cells. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A Support Protocol for secondary stimulation and rapid expansion of iNKT cells is also included. Basic Protocol 3 explains functional analysis of iNKT. Curr. Protoc. Immunol. 90:14.11.1‐14.11.17. © 2010 by John Wiley & Sons, Inc.

Keywords: NKT cells; human; CD1d; α‐galactosylceramide; invariant T cell receptor

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Table of Contents

  • Introduction
  • Identification and Quantitation of Invariant NKT Cells
  • Basic Protocol 1: Identification of Invariant NKT Cells by Flow Cytometry Using Anti‐Vα24 and Anti‐Vβ11
  • Alternate Protocol 1: Flow Cytometry with Vα24 and Anti‐Invariant 6B11 mAbs or CD1d Tetramer
  • Isolation and Expansion of Invariant NKT Cells
  • Basic Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by Immunomagnetic Beads Followed by Selective Expansion with α–GalCer
  • Alternate Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by FACS Followed by Selective Expansion with α‐GalCer
  • Alternate Protocol 3: Generation of Invariant NKT Cell Clones
  • Support Protocol 1: Secondary Stimulation of Invariant NKT Cell Lines and Clones
  • Basic Protocol 3: CD1d‐Specific Functional Assays for Invariant NKT Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Identification of Invariant NKT Cells by Flow Cytometry Using Anti‐Vα24 and Anti‐Vβ11

  • Isolated PBMC from healthy human donors (unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Flow cytometry buffer (FC buffer): PBS ( appendix 2A) with 1% human serum, 1% FBS, 0.1% sodium azide
  • Conjugated anti‐Vα24 mAb (clone C15B2, PE or FITC conjugates, Coulter Immunotech)
  • Conjugated anti‐Vβ11 mAb (clone C21D2, Coulter PE or FITC conjugates)
  • Conjugated isotype‐matched control mAbs (Coulter, Pharmingen, eBioSciences)
  • Optional lineage markers for inclusion (e.g., CD3), exclusion (e.g., CD19), and sub‐typing (see CD4 etc. below)
  • PBS with 4% paraformaldehyde: heat only in a functional fume hood to 80°C with a stirring bar to dissolve and then cool; divide into aliquots in the hood before removal for individual use; aliquots can be stored indefinitely at −20°C
  • Heparinized tubes
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for isolating human PBMCs (unit 7.1)

Alternate Protocol 1: Flow Cytometry with Vα24 and Anti‐Invariant 6B11 mAbs or CD1d Tetramer

  • Conjugated 6B11 anti‐invariant TCR mAb (Pharmingen, eBioSciences, Miltenyi Biotech.).
  • Lineage markers for inclusion (e.g., CD3), exclusion (e.g., CD19), or sub‐typing (see CD4 etc. below)
  • Stable α–GalCer analogue, PBS‐57‐loaded CD1d tetramer (NIH Tetramer Core Facility; conjugated to appropriate chromophore such as APC.
  • Un‐loaded CD1d tetramer control conjugate (NIH Tetramer Core Facility)
NOTE: As with MHC tetramers (unit 17.3), assembled CD1d tetramers have finite shelf life and need to be tested and replaced periodically. Alternatively, investigators may obtain MHC tetramers from the NIH Tetramer Facility ( as monomers with indefinite shelf‐life when stored deep‐frozen, and which can be assembled with fluorescent streptavidin from individual aliquots periodically. Alternatively, CD1d dimers are commercially available for loading with ligands like α–GalCer. These have the distinction of lower avidity than tetramers (maximally binding trivalently), and so can discriminate between exact consensus iNKT TCR and related sequences, which also bind 6B11 with lower avidity, but to which CD1d tetramers bind nearly equally. Hence, choice can be based on need to include as many as possible or all α–GalCer‐reactive TCR (which include a small population of non‐Vα24+ T cells) or greater selectivity for truly invariant Vα24Jα18 TCR, in which case 6B11 can identify all (with lower avidity/MFI for nonconsensus rearrangements) and CD1d dimers can identify optimal consensus sequences.This protocol is identical to the above dual staining, substituting the conjugated Vβ11 mAb (as iNKT cells can be Vβ11‐negative) with 6B11. This is why 6B11 and Vβ11 may not be an ideal combination. For CD1d tetramer staining, a second label is similarly required for inclusion of true T cells and/or exclusion of B cells and other contributors to background. Usually CD3 is used rather than Vα24, but these can be combined with or without CD19 to exclude B cells. In this case, the negative control is unloaded CD1d tetramer.

Basic Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by Immunomagnetic Beads Followed by Selective Expansion with α–GalCer

  • Isolated PBMC from healthy human donors (unit 7.1)
  • Wash buffer: PBS with 2 mM EDTA (PBS/EDTA)
  • FcR‐blocking reagent (Human IgG; Miltenyi Biotech, cat. no. 130‐059‐901), optional
  • Unconjugated anti‐Vα24 mAb (Coulter)
  • Unconjugated or PE‐conjugated 6B11 anti‐invariant TCRα mAb (eBioSciences, Pharmingen)
  • Binding buffer: PBS with 2 mM EDTA and 2% human serum
  • Goat anti‐mouse IgG or anti‐PE microbeads (Miltenyi Biotech)
  • T cell medium (see recipe)
  • DMSO freezing mixture: 90% FBS/10% DMSO
  • α–GalCer (
  • IL‐2
  • MS columns (for up to 108 starting cells; Miltenyi Biotech, cat. no. 130‐042‐201)
  • LS columns (for up to 108 starting cells; Miltenyi Biotech, cat. no. 130‐042‐401)
  • Magnetic separation device
  • γ‐irradiator
  • Round‐bottomed 96‐well tissue culture plates
  • Additional reagents and equipment for isolating human PBMCs (unit 7.1)
NOTE: An alternative to all reagents above is the direct 6B11‐conjugated microbeads/iNKT kit now available (Miltenyi).

Alternate Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by FACS Followed by Selective Expansion with α‐GalCer

  • FACS buffer: PBS with 1% human serum and 1% FBS (no azide)
  • FACS machine (e.g., MoFlo, Cytomation)

Alternate Protocol 3: Generation of Invariant NKT Cell Clones

  • Phosphate‐buffered serum (PBS; appendix 2A) with 10% human serum
  • T cell cloning medium: RPMI‐1640, 10% autologous human serum, 2 µg/ml PHA‐P, 20 U/ml IL‐2, and 20 U/ml IL‐7 (Genzyme)

Support Protocol 1: Secondary Stimulation of Invariant NKT Cell Lines and Clones

  • Isolated PBMC from healthy human donors (unit 7.1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • B lymphoblastoid cell line (JY or C1R)
  • Expansion medium: RPMI‐1640 with 10% AB‐negative human serum, 10 mM HEPES, and gentamicin (FBS may be used in place of human serum, but lots of the human and bovine sera should be tested for their ability to stimulate these cells)
  • Mitogenic anti‐CD3 [clones UCHT1 (IgG1) from Ancell or OKT3 have been used with success]
  • Recombinant IL‐2 and IL‐7 (Genzyme)
  • γ‐irradiator
  • 25‐cm2 flasks
  • Microscope
  • Additional reagents and equipment for isolating human PBMC (unit 7.1)

Basic Protocol 3: CD1d‐Specific Functional Assays for Invariant NKT Cells

  • Isolated iNKT cells (see protocol 3, protocol 4, or protocol 5)
  • T cell medium (TCM; see recipe) containing 10 U/ml IL‐2
  • Human CD1d‐specific mAb (clone 42.1; Pharmingen)
  • α‐GalCer‐pulsed APCs, optional
  • Cytokine ELISA kits (Endogen, Pharmingen, or other)
  • 96‐well, flat‐bottom plates
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