Flow cytometry for natural killer T cells: multi-parameter methods for multifunctional cells

Vα24-Jα18 invariant natural killer T cells (iNKT) are an evolutionary conserved sub-lineage of T cells with effector-memory phenotype that often express an NK cell surface antigen CD161 and is characterized by reactivity to self-glycolipids and α-galactosylceramide (αGalCer) that are presented by monomorphic HLA class-I-like molecule CD1d. Upon antigen recognition, iNKT cells can rapidly produce multiple cytokines and chemokines and regulate development of Th-2 and Th-1 immune responses. Potential importance of iNKT cells has been demonstrated in several animal models of infection diseases, autoimmunity, and cancer. Multi-parameter flow cytometry has been the main tool to study human and murine iNKT cells. Analysis of human iNKT cells is particularly demanding since their frequency among peripheral blood T cells is relatively low ranging from less than 0.01% to 1%, with a mean of about 0.1%. Herein, we discuss flow cytometry applications that are utilized for iNKT cell identification and enumeration, subset characterization, detection of intracellular cytokines, quantitative analysis of multiple secreted molecules, cell-mediated cytotoxicity, and migration.