Isolation and Functional Use of Human NKT Cells

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.