All Stem Cells Japan

Abstract
Natural killer T (NKT) cells are a distinct T-cell sublineage, originally named because of their coexpression of an alphabeta T cell antigen receptor (TCR) characteristic of T lymphocytes, and NK1.1, a C-type lectin expressed by natural killer (NK) cells. NKT cells use their TCR to recognize glycolipids bound to or presented by CD1d. Until recently, most studies used the synthetic glycolipid alpha-galactosylceramide (αGalCer) to activate these lymphocytes, and very little was known about the natural antigens recognized by NKT cells. Given the pivotal role played by the NKT cells in many immune responses, including antimicrobial responses, tumor rejection, and the development of autoimmune diseases, the identification of the natural antigens recognized by these cells, and analogs that may alter their cytokine production, are goals of primary importance. This chapter discusses methods that can be used to assess the potency of potential glycolipid antigens for this unique population of T lymphocytes, including methods for in vitro NKT cell activation and expansion, in vivo activation, and measurement of their avidity for different antigens.

Overview
Natural killer T cells are named after their coexpression of an αβ T cell antigen receptor (TCR) along with surface receptors typical for NK cells, such as NK1.1 in mice (Kronenberg and Gapin, 2002). For several reasons, however, this classification has become inadequate. First, only three commonly used inbred mouse strains, C57BL/6, NZB, and SJL, express an allelic form of NK1.1 that can be recognized by the available NK1.1 monoclonal antibody (mAb). Second, in addition to CD1d reactive cells, some conventional T lymphocytes up regulate NK1.1/CD161 on activation. Moreover, some CD1d-dependent ‘NKT’ cells do not express NK1.1(Godfrey et al., 2004).
A newer nomenclature, therefore, was established on the basis of the expression of an invariant α chain TCR rearrangement. Contrary to conventional T cells that express highly diverse antigen receptors, most mouse NKT cells express an invariant Vα14-Jαβ18 TCR α rearrangement coex-pressed with a limited number of β chains (Vβ8.2, Vβ7, or Vβ2). These cells, therefore, are sometimes referred to as Vα14 invariant (Vα14i) NKT cells. The homologous population in human expresses an invariant Vα24-Jα18 rearrangement paired with Vβ11 (Dellabona et al., 1994; Porcelli et al., 1993); these are the orthologs of mouse Vα14 and Vβ11. We refer to the human NKT cell population as Vα24α NKT cells, and collectively to these subpopulations in mice and humans as iNKT cells.
On activation, iNKT cells very rapidly produce large amounts of different cytokines, including IFN# and ILα4, suggesting they could have a potent immunoregulatory function. This has been confirmed in numerous studies demonstrating the pivotal roles played by iNKT cells in many different types of immune responses and cancer (Smyth et al., 2002; Van Kaer, 2004).
Conventional T lymphocytes respond to peptides presented by the polymorphic class I and class II antigen α presenting molecules. iNKT cells, by contrast, are activated on recognition of glycolipids presented by CD1d, a nonclassical or nonpolymorphic antigen α presenting molecule. CD1d is distantly related to both the class I and class II molecules, and like class I molecules, it consists of a heavy chain noncovalently associated with β2α microglobulin (Porcelli, 1995). When bound to CD1d, the lipid tails of glycolipid are buried in the hydrophobic CD1d groove, leaving predominantly the carbohydrate portion available for TCR recognition together with CD1d (Zajonc et al., 2005). αGalCer, a synthetic glycosphingolipid, originally extracted from a marine sponge in a screen for compounds that could prevent tumor metastases to the liver of mice (Kobayashi et al., 1995), was the first antigen described that activates iNKT cells (Kawano et al., 1997). αGalCer was not thought to be the natural antigen for iNKT cells, however, because of its unusual α linkage of the 10 carbon of the sugar to the 1 carbon of the sphingosine base, whereas in nearly all other glycosphingolipids the bond connecting the sugar to the lipid is in the β anomeric form. Recent publications, however, showed that Sphingomonas, a common environmental bacteria, have α linked glycosphingolipids, and these compounds are able to bind to CD1d and activate both mouse and human iNKT cells (Kinjo et al., 2005; Mattner et al., 2005; Sriram et al., 2005). iNKT cells are weakly self α-reactive for CD1dþ antigen α presenting cells, and isoglobotrihexosyl ceramide (iGb3) was identified recently as an autoantigen likely to be required for the development of these lymphocytes (Zhou et al., 2004).
Methods used to identify, expand, and assay Vα14i NKT cells, and methods to test the potency of different glycolipid antigens.

Source & See More : http://www.ncbi.nlm.nih.gov/pubmed/17132506