Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.

The immune system preserves and makes use of autoreactive lymphocytes with specialized functions. Here we showed that one of these populations, CD8alphaalpha(+)TCRalphabeta(+) intestinal intraepithelial lymphocytes (IELs), arose from a unique subset of double-positive thymocytes. This subset of cells was precommitted to preferentially give rise to CD8alphaalpha(+)TCRalphabeta(+) IELs, but they required exposure to self-agonist peptides. The agonist-selected TCRalphabeta(+) thymocytes are CD4 and CD8 double-negative, and their final maturation, including the induction of CD8alphaalpha expression, appeared to occur only after thymus export in the IL-15-rich environment of the gut. These developmental steps, including precommitment of immature thymocytes, TCR-mediated agonist selection, and postthymic differentiation promoted by cytokines, define a unique pathway for the generation of CD8alphaalpha(+)TCRalphabeta(+) IEL.

The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist ␣-galactosyl ceramide (␣-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, ␣-galacturonosyl-ceramides from Sphingomonas wittichii, although structurally similar to ␣-GalCer, have significant differences in the sugar head group as well as the ceramide portion. The Sphingomonas glycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-␥ secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with ␣-GalCer. Because ␣-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.

Certain glycolipid Ags for Vα14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, α-galactosylceramide. Although the CD1d-exposed portions of OCH and α-galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-γ, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.

CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.

Natural killer T (NKT) cells provide an innate-type immune response upon T cell receptor interaction withCD1d-presented antigens. We demonstrate through equilibrium tetramer binding and antigen presentation assays with V␣14i-positive NKT cell hybridomas that the Sphingomonas glycolipid ␣-galacturonosyl ceramide (GalA-GSL) is a NKT cell agonist that is significantly weaker than ␣-galactosylceramide (␣-GalCer), the most potent known NKT agonist. For GalA-GSL, a shorter fatty acyl chain, an absence of the 4-OH on the sphingosine tail and a 6 -COOH group on the galactose moiety account for its observed antigenic potency. We further determined the crystal structure of mCD1d in complex with GalA-GSL at 1.8-Å resolution. The overall binding mode of GalA-GSL to mCD1d is similar to that of the short-chain ␣-GalCer ligand PBS-25, but its sphinganine chain is more deeply inserted into the F pocket due to alternate hydrogen-bonding interactions between the sphinganine 3-OH with Asp-80. Subsequently, a slight lateral shift (>1 Å) of the galacturonosyl head group is noted at the CD1 surface compared with the galactose of ␣-GalCer. Because the relatively short C 14 fatty acid of GalA-GSL does not fully occupy the A pocket, a spacer lipid is found that stabilizes this pocket. The lipid spacer was identified by GC͞MS as a mixture of saturated and monounsaturated palmitic acid (C 16). Comparison of available crystal structures of ␣-anomeric glycosphingolipids now sheds light on the structural basis of their differential antigenic potency and has led to the design and synthesis of NKT cell agonists with enhanced cell-based stimulatory activities compared with ␣-GalCer.adjuvant ͉ glycolipid T he CD1 family of antigen-presenting glycoproteins mediates T cell responses through the presentation of self and foreign lipids, glycolipids, lipopeptides, or amphipathic small molecules to T cell receptors (TCR) (1-10). In humans, the various CD1 isoforms are categorized into group I (CD1a, CD1b, CD1c, and CD1e) and group II (CD1d) based on sequence similarity (11). Through the binding and presentation of endogenous and exogenous lipid antigens to TCRs, the CD1 pathway is reminiscent of peptide presentation by MHC class I and class II molecules.The group II isotype, CD1d, presents antigens to a unique population of T lymphocytes termed natural killer T (NKT) cells (4), which express an invariant V␣14i TCR in mice or V␣24i TCR in humans, in addition to NK 1.1 and other receptors typical of NK cells (12). These cells are sometimes called V␣14i or V␣24i NKT cells to distinguish them from other T lymphocytes that can express NK1.1. The first defined and most potent V␣14i NKT cell agonist, ␣-galactosylceramide (␣-GalCer), was originally isolated from a marine sponge and subsequently optimized by medicinal chemistry (13). When bound to CD1d, ␣-GalCer strongly activates V␣14i NKT cells, causing a rapid release of T helper type 1 and 2 cytokines. Although ␣-GalCer has been of great value and use in exploration of NKT cell biology, its unusual origin ...

Mouse natural killer T (NKT) cells with an invariant Vα14-Jα18 rearrangement (Vα14 invariant [Vα14i] NKT cells) are either CD4+CD8− or CD4−CD8−. Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor α rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8+ Vα14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by Vα14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of Vα14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of Vα14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing Vα14i NKT cells.

The phenotype and development of T cells from transgenic mice expressing a T cell receptor with specificity for insulin presented by the MHC class Ib molecule Qa-1(b) was investigated. Peripheral T cells from the transgenic mice express CD8 and, after activation, kill Qa-1(b)-positive lymphoid target cells in the presence of soluble insulin. Thymic selection requires expression of Qa-1(b) but not the dominant Qa-1-associated peptide, Qdm. In contrast to conventional T cells, selection is at least as efficient when the selecting ligand is expressed only on hematopoietic lineage cells as compared to expression on epithelial cells in the thymus. Our findings suggest that there is a dedicated population of Qa-1-restricted T cells that are selected by interaction with Qa-1 and that the cellular requirements for selection may differ from conventional T cells.