Characterization of Thymic Development of Natural Killer T Cell Subsets by Multiparameter Flow Cytometry

Natural killer T (NKT) cells are a subset of αβ T cells that recognize lipid antigens presented by the nonclassical MHC molecule CD1d. Although numerically small, these cells have been shown to play an important role in the regulation of multiple immune responses, including microbial infection, autoimmunity, and cancer. Even in the steady state, cytokine production by NKT cells influences the basal status and function of other immune cells, including dendritic cells and CD8 T cells. To fully understand their biology and harness them in the clinic, it is imperative to dissect the molecular mechanisms involved in the acquisition of their functionality. Unlike conventional αβ T cells, NKT cells acquire their effector function during development in the thymus. At this time, precursors commit to one of three functionally different effector lineages: NKT1, NKT2, and NKT17. These subsets are characterized by the secretion of different cytokines upon antigenic stimulation and by the expression of the master transcription factors Tbet, promyelocytic leukemia zinc finger (PLZF), and retinoic orphan receptor γ t (RORγt). Here we describe a multicolor flow cytometry protocol to identify NKT cell subsets and interrogate the progression of NKT precursors through their development in the thymus.