Isolation and Functional Use of Human NKT Cells

Curr Protoc Immunol. 2017 Nov 1:119:14.11.1-14.11.20. doi: 10.1002/cpim.33.

Abstract

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 and some human NK marker+ T cells ('NKT') recognize the MHC class I-like CD1d protein and use an identical 'invariant' TCRα chain, which is generated in humans by precise Vα24 and Jα18 rearrangements with either no N-region diversity or subsequent trimming to identical or nearly identical amino acid sequence (hence, 'iNKT' cells). iNKT are mostly CD4+ or CD4-CD8- ('double negative'), although a few CD8+ iNKT can be found in some humans. 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. Basic Protocol 3 explains functional analysis of iNKT. 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. © 2017 by John Wiley & Sons, Inc.

Keywords: CD1d; NKT cells; human; iNKT cells; invariant T cell receptor; α-galactosylceramide.

MeSH terms

  • Animals
  • Antigens, CD1d / metabolism
  • CD4 Antigens / metabolism
  • CD8 Antigens / metabolism
  • Cell Culture Techniques*
  • Cell Proliferation
  • Cell Separation
  • Cells, Cultured
  • Cytotoxicity, Immunologic
  • Flow Cytometry
  • Humans
  • Mice
  • Natural Killer T-Cells / immunology
  • Natural Killer T-Cells / metabolism*
  • Receptors, Antigen, T-Cell / genetics*
  • Receptors, Antigen, T-Cell / metabolism

Substances

  • Antigens, CD1d
  • CD4 Antigens
  • CD8 Antigens
  • Receptors, Antigen, T-Cell
  • Valpha24 protein, human