Identification of Rare Thymic NKT Cell Precursors by Multiparameter Flow Cytometry

Mouse invariant natural killer T (NKT) cells are a subset of T lymphocytes which have been shown to play a significant role in innate and adaptive immune responses. Features of innate responses are attributed to these cells because they can be stimulated simultaneously with the same ligand to produce quickly and in large amount cytokines without prior immunization. Because these characteristics could be exploited for clinical applications, NKT cells have attracted considerable interest. Many studies have investigated the molecular mechanisms through which they are selected and differentiate. These studies are based on developmental models that serve as a scaffold to understand the specific roles played by various factors and to identify checkpoints during cellular development. Analysis of NKT cell precursors at the HSA^high stage, stage 0, can reveal potential selection defects, whereas analysis of NKT cells at the HSA^low stage can shed light on defects in the maturation/differentiation of the different NKT cell subsets (NKT1, 2, and 17). Unlike HSA^low NKT cell subsets, HSA^high NKT cell precursors are not accurately identified by flow cytometry because of their extreme rarity. Here, we describe an NKT cell enrichment strategy to identify unambiguously NKT cell precursors at the HSA^high stage that can be used to assess their distribution and characteristics by multicolor flow cytometry.