SummaryCD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating α galactosylceramide (αGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for αGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing αGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

By identifying the lipid LPC as an endogenous antigen, recognized by the invariant subset of human NKT cells, this study establishes a novel link between these immunoregulatory cells and an inflammatory lipid mediator.

V␣14i natural killer T (NKT)-cell function has been implicated in a number of disease conditions. The molecular events that drive V␣14i NKT-cell development remain elusive. We recently showed that T-bet is required for the terminal maturation of these cells. Here we identify some of the genetic targets of T-bet during V␣14i NKT-cell lineage development. Microarray gene-expression analyses on developing V␣14i NKT cells were performed and provide a molecular framework to study these maturation events. In vitro ectopic expression of T-bet in immature V␣14i NKT cells, which do not yet express T-bet, was sufficient to promote V␣14i NKT-cell maturation, driving the expression of multiple genes, including those that participate in migration, survival, and effector func- IntroductionNatural killer T (NKT) cells are a unique lymphocyte lineage that coexpresses a rearranged T-cell receptor (TCR) in association with the CD3 complex as well as several receptors first identified on bona fide NK cells, such as NK1.1, the interleukin-2 (IL-2)/15R␤ chain (CD122) and various Ly49 molecules. 1 Most NKT cells in the mouse express a semi-invariant TCR composed of a V␣14-J␣18 rearrangement that preferentially associates with either V␤8, V␤7, or V␤2, 2 and are positively selected by CD1d, a nonclassical major histocompatibility (MHC) class I molecule. We refer to this CD1d reactive subgroup as V␣14 invariant (V␣14i) NKT cells to distinguish them from other populations of NKT cells that have been defined. 3,4 The early and potent cytokine secretion by V␣14i NKT cells following TCR stimulation may provide an important link between the innate and adaptive immune systems. 3,5 Consistent with this, V␣14i NKT cells appear to be important for responses to tumors, infectious agents, the maintenance of self tolerance, and the prevention of autoimmunity. 1,3 Recent progress has been made in identifying V␣14i NKT-cell developmental intermediates. [6][7][8][9] V␣14i NKT cells develop from CD4 ϩ CD8 ϩ double-positive (DP) V␣14-J␣18 TCR ϩ thymocyte precursors. 6,[10][11][12] Following positive selection by CD1d molecules, V␣14i NKT cells undergo a maturation process defined by the sequential up-regulation of diverse markers and the late fate specification of the NKT-cell lineage. 6-9 NKT-cell precursors can be identified on the basis of TCR specificity in the thymus of young mice using the CD1d tetramer. These cells are phenotypically indistinguishable from conventional mature lymphocytes, being HSA low , CD44 low , and NK1.1 Ϫ . 7 This phenotype is defined as stage 1. 13 Ontogeny and transfer studies defined a developmental sequence from CD44 low NK1.1 -(stage 1) to CD44 high NK1.1 -(stage 2). At this latter stage, V␣14i NKT cells are still considered immature but can leave the thymus and colonize peripheral organs. 7,8 A final maturation step that occurs either in the thymus or in the peripheral organs is accompanied by the expression of NK1.1, Ly49 receptors and CD122 (stage 3). 7,8,14 Interestingly, these 3 developmental intermediates each ha...