NKT Cell Responses to Glycolipid Activation

NKT cells are a distinct lineage of T lymphocytes that are usually identified by the co-expression of the semi-invariant CD1d-restricted alphabeta TCR and the NK1.1 allelic marker of NK lineage receptors in the C57BL/6 mice and related strains. NKT cells can be subdivided based on CD4/CD8 expression and on tissue of origin. NKT cells express significantly the TCR gene products Valpha24 JalphaQ in humans, the homolog of mouse Valpha14 Jalpha18, paired with Vbeta11, the homolog of mouse Vbeta8.2. NKT cells are most frequent in liver (up to 30% of T cells in mice and approximately 4% of hepatic T cells in human), bone marrow, and thymus and represent a smaller proportion of T cells in other tissues including spleen, lymph nodes, blood, and lung. NKT cells recognize a broad array of glycolipids in the context of CD1d presentation, and many studies have characterized a cascade of functions following in vitro and in vivo stimulation by alpha-GalCer, including production of high levels of immune-regulatory cytokines and bystander activation of several cell types including NK, B, T, and dendritic cells. Both in vitro and in vivo methods have been developed for the study of NKT responses to glycolipid presentation by CD1d. In practice, CD1d-glycolipid-loaded tetramers would most reliably identify these cells. In vitro, splenocytes can be used to monitor cytokine release as this population contains all the cells necessary for sequestering, loading onto CD1d molecules, and presentation of glycolipids to NKT cells. Another system involves the use of NKT cell hybridoma and CD1d coated onto plastic plates to measure responses limited to NKT cells more precisely. In vivo, responses are typically measured by injecting the glycolipid into mice and monitoring plasma cytokine levels or DC maturation in the spleen. This chapter describes methods that can be used to identify NKT cells and to asses in vitro and in vivo their activation and expansion.