Figure 1. A putative iNKT cell ontogenetic pathway. Early steps, CD4 and CD8 double negative (DN)-1–4 through immature CD8 single positive (ISP8) stages, of thymocyte development are common to both iNKT lymphocyte and conventional T cell lineages. The ontogenetic programming of the unique features of iNKT cell function occurs at the CD4 and CD8 double-positive (DP) stage; it begins with the rearrangement of the V14J18 TCR chain and after its interaction with the positively selecting ligand, CD1d-self lipid complex. Stage-specific iNKT cell markers (grey boxes) and lineage specific differentiation signals are indicated. Several iNKT cell markers, e.g. CD4, CD44, CD122 (IL-2R), CD127 (IL-7R) and CD161 (NK1.1) indicated in grey boxes as well as CD5, CD24 (heat-shock antigen), DX5 and Ly49A/D and C/I (not shown), are developmentally regulated. IL-7 and IL-15 are cytokines, which utilize specific (IL-7R and IL-15R, respectively) and the shared common chain (c) receptor proteins that mediate intercellular communications. IL-15 also uses IL-2R that it shares with IL-2 for intercellular communications. Csf-2 (granulocyte–macrophage colony-stimulating factor) uniquely signals functional differentiation of iNKT cells; its absence during ontogeny results in iNKT cells that are incompetent for cytokine secretion. CD1d and pre-T cell receptor (TCR)- (pT) are structural proteins, while J18 segment and C FG-loop are structural parts of the TCR essential for positive selection of iNKT cells. Fyn and Lck are Src (cellular protein homologous to the Rous sarcoma virus oncogene) kinases (protein phosphorylation enzymes) essential for transmitting TCR signals from the plasma membrane to inside of the cell. Fyn also transmits signals relayed from SLAM (signalling lymphocyte activation molecule) through the adapter protein SAP (SLAM-associated protein). Protein kinase C (PKC)- processes TCR signalling and activates nuclear factor-B (NF-B), which is a transcription factor. Other transcription factors such as Ets-1, MEF, Nur77 and T-bet are also essential for iNKT cell ontogeny.

Figure 2. Natural iNKT cell antigens and glycolipid analogues. iNKT cell antigens include Sphingomonas cell wall-derived glycolipids, which contain 1¢-1-O-linked galacturonic acid (or glucuronic acid; not shown), that resemble the marine sponge Agelas-derived glycolipid -galactosylceramide (GalCer). OCH, C20:2 and -C-GalCer are GalCer-based altered lipid ligands that elicit distinct cytokine responses from iNKT cells. Borrelia burgdorferi cell wall-derived -monogalactosyl-diacylglycerol (MGalD) is a recently identified glycolipid antigen that weakly elicits IFN- response from iNKT cells. Isoglobotrihexosylceramide (iGb3) is a recently identified endogenous self antigen. The triangle and the box indicate differences in the glycerol and sphingosine backbone. The anomeric bonds as well as differences in hexose atoms are indicated in red.

Figure 3. Two distinct strategies for iNKT cell activation by microbes. Microbes containing TLR ligands such as LPS activate iNKT cells by inducing IL-12 production by DC, which amplifies weak responses elicited upon the recognition of CD1d bound with self-glycolipids by the iNKT cell receptor. Isoglobotrihexosylceramide (iGb3) has been suggested to function as an endogenous glycolipid for stimulating iNKT cell responses. The generation of iGb3 requires the action of -hexosaminidase B (HexB) and the binding of iGb3 to CD1d requires Sap B (left). Some microbes such as Sphingomonas capsulata, which belong to the class of -Proteobacteria, synthesize -anomeric glycolipids (see Figure 2) for their cell walls. These glycolipids, when presented by CD1d, activate iNKT cells directly, without the need for endogenous iNKT cell antigens. As with the loading of CD1d with -galactosylceramide, the loading of the bacterial glycolipids such as -glucuronosylceramide and -galacturonosylceramide may require lysosomal lipid transfer proteins G_M2 activator (G_M2A) and/or saposins (Sap) (right).

Figure 4. The assembly of CD1d with iNKT cell antigen and its evasion. (a) Infection of M and DC delivers microbes to the CD1d-containing lysosomes. Differential interference contract (DIC) picture of M observed under a light microscope showing the gross cellular outline; the prominent structure within this cell is its nucleus. Cellular organelles and their contents are observed by confocal fluorescence microscopy. In the micrographs shown, the lysosome is stained red (1) because it is marked with a fluorescent dye, Lyso-tracker; the microbe, in this case Borrelia burgdorferi, stained with the fluorescent dye PKH-II, is seen green (2); CD1d, which is detected with a specific monoclonal antibody 1B1 tagged with the fluorescent dye allophycocyanin, is stained blue. B. burgdorferi bacteria colocalize with lysosomes and, hence, they appear yellow in the merged picture 1+2. Similarly, the regions of the cell where lysosomes and CD1d colocalize appear pink in the merged picture 1+3, while the regions where CD1d and microbe colocalize appear light blue in the merged picture 2+3. The regions where lysosomes, B. burgdorferi and CD1d colocalize appear white in the merged picture 1+2+3. (b) CD1d assembly with lipids begins within the rough endoplasmic reticulum (ER) with the assistance of several chaperones. Partially folded -chain–2m complex is then thought to bind ER-resident lipids with the assistance of lipid transfer proteins (LTP) such as microsomal triglyceride transfer protein (MTP), a protein that facilitates the assembly of apolipoprotein B. Upon complete assembly, the CD1d–lipid complexes egress from the ER and negotiate the secretory pathway to the plasma membrane. By virtue of late endosome/lysosome targeting motif (tyrosine-glutamine-glycine-valine-leucine and tyrosine-glutamine-aspartate-isoleucine-arginine in human and mouse CD1d, respectively) within the cytoplasmic tail of CD1d, it recycles through the MHC class II enriched compartment (MIIC). During its time in the MIIC (late endosomes/lysosomes), CD1d exchanges its ER-loaded lipids for antigenic glycolipids that activate iNKT cells in vivo. The extraction of bound lipids from CD1d and the loading of antigenic glycolipids are facilitated by lysosomal LTP such as Saposins (Sap), G_M2 activator (G_M2A), and Niemann-Pick C-2 (not shown), which are essential for the enzymatic catabolism of glycolipids. Locations where viruses and their products block CD1d trafficking are indicated. The modulator of immune recognition (MIR)-1 and MIR-2 proteins of Kaposi sarcoma-associated herpesvirus (KHSV) are ubiquitin ligases that ubiquitinylate the cytoplasmic tail of CD1d, which triggers endocytosis of surface CD1d thereby reducing cell surface CD1d expression. The Nef (negative regulatory factor) protein of Human immunodeficiency virus (HIV-1), which causes acquired immunodeficiency syndrome, also reduces CD1d expression perhaps by increased endocytosis of cell surface CD1d molecules coupled with inhibition of CD1d transport to the cell surface. Similarly, in Herpes simplex virus (HSV)-1-infected cells, CD1d molecules accumulate in the MIIC, owing to a defect in recycling CD1d molecules back from endosomal compartments to the cell surface.