Isolation and functional use of human NKT cells

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. © 2010 by John Wiley & Sons, Inc.