This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8+ T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.

No abstract

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324 -332, 1 FAPGNYPAL 9 ) to promote its folding in vitro of H-2D b . We found that H-2D b can be stabilized by the pentapeptide 5 NYPAL 9 , which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D b molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove. Major histocompatibility complex (MHC)5 class I molecules have evolved to present peptide epitopes of 8 -10 amino acids to cytotoxic T cells. Many MHC class I⅐peptide structures have now been solved by x-ray crystallography, and they all have a common tertiary structure (1). The structure consists of a polymorphic heavy chain (HC) and a nonpolymorphic light chain ␤2-microglobulin (␤2-m), noncovalently associated to form a molecule with two membrane proximal Ig-like domains (the ␣3 and ␤2-m domains) that support a membrane distal ␣1-␣2 "superdomain." The peptide-binding site is formed by a deep cleft between two ␣-helices in this superdomain. Antigenic peptides are always bound in the same orientation, with their N and C termini lying buried deep in pockets that define the ends of the peptide-binding groove (the A and F pockets, respectively). In addition, so-called "anchor residues" make allele specific interactions with polymorphic class I residues located deep inside the binding groove, in "specificitydetermining pockets."The peptide⅐MHC class I complex is formed in the endoplasmic reticulum (ER) and marks the end point of antigen processing (2). During antigen processing, proteins are unfolded and partially hydrolyzed in the cytoplasm, and the resulting polypeptides (of between 8 and 40 amino acids) are translocated across the ER membrane by the transporter associated with antigen processing. Once in the ER, some long peptide epitope precursors can undergo further trimming by the aminopeptidase ERAAP (3, 4) and are selected for assembly with newly synthesized MHC class I molecules that is dependent on their interaction with cofactor molecules such as calreticulin, tapasin, and ERp57. The process results in the preferential release from the ER of class I molecules presenting peptides that bind stably. Recent evidence suggests that this selection of high affinity peptides in vivo may occur by a mechanism that is more complex and controlled than simple competition between potential ligands for binding to class I in the ER (2) and may involve editing of the MHC-bound peptide repertoire in the early secretory pathway of antigen-presenting cells.It is not known whether the loading or editing of class I MHC peptide cargo...

Class I major histocompatibility complexes (MHC) are heterotrimeric structures comprising heavy chains (HC), ␤ 2 -microglobulin (␤ 2 -m), and short antigenic peptides of 8 -10 amino acids. These components assemble in the endoplasmic reticulum and are released to the cell surface only when a peptide of the appropriate length and sequence is incorporated into the structure. The binding of ␤ 2 -m and peptide to HC is cooperative, and there is indirect evidence that the formation of a stable heterotrimer from an unstable HC:␤ 2 -m heterodimer involves a peptide-induced conformational change in the HC. Such a conformational change could ensure both a strong interaction between the three components and also signal the release of stably assembled class I MHC molecules from the endoplasmic reticulum. A peptide-induced conformational change in HC has been demonstrated in cell lysates lacking ␤ 2 -m to which synthetic peptides were added. Many features of this conformational change suggest that it may be physiologically relevant. In an attempt to study the peptide-induced conformational change in detail we have expressed a soluble, truncated form of the mouse H-2D b HC that contains only the peptide binding domains of the class I molecule. We have shown that this peptidebinding "platform" is relatively stable in physiological buffers and undergoes a conformational change that is detectable with antibodies, in response to synthetic peptides. We also show that the structural features of peptides that induce this conformational change in the platform are the same as those required to observe the conformational change in full-length HC. In this respect, therefore, the HC ␣ 1 and ␣ 2 domains, which together form the peptide binding site of class I MHC, are able to act independently of the rest of the molecule.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.