Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments.

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.

Efficient purification of unique antibodies using peptide affinity-matrix columns.

Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85-99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells. Investigations of the fine specificity of the ER6.1 peptide demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER6.1 peptide.

Chimeric beta2 microglobulin/CD3zeta polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8(+) T cells.

CD8(+) T cells are key mediators of transplant rejection and graft-versus-host disease and contribute to the pathogenesis of autoimmune diseases. We tested whether TCR ligands can be converted into T cell activation receptors, redirecting genetically modified T cells at pathogenic CD8(+) T cells. For this purpose we exploited the ability of the non-polymorphic beta(2) microglobulin light chain to pair with all MHC class I heavy chains. In this report we describe the design and expression in a T cell hybridoma of two modalities of beta(2) microglobulin polypeptides, fused with the transmembrane and intracellular portion of CD3zeta chain. In the absence of a particular antigenic peptide, the chimeric product associates with different endogenous MHC class I heavy chains and triggers T cell activation upon heavy chain cross-linking. When an antigenic peptide is covalently attached to the N-terminus of the chimeric polypeptide, transfectants express high level of surface peptide-class I complexes and respond to antibodies and target T cells in a peptide-specific manner. Our results provide the basis for a universal genetic approach aimed at antigen-specific immunotargeting of pathogenic CD8(+) T cells.

Non-randomness in Shine-Dalgarno regions: links to gene characteristics.

A probabilistic approach to the study of the Shine-Dalgarno region was used to identify the most non-random positions based on parsing of genomes in four species: Escherichia coli, Bacillus subtilis, the AT-rich Clostridium perfringens, and the GC-rich Streptomyces coelicolor. The compositional non-randomness shows a clear peak centered around 9-11 nucleotides upstream of the start codon. This peak was in all species associated with guanine as the most abundant nucleotide, flanked by guanine in the closest proximity and adenines farther away (cytosine in case of S. coelicolor). Using contingency tables, the nucleotides in the Shine-Dalgarno region were shown to have a strong association to the choice of start codons. We also show that gene characteristics such as length, aromaticity, and lipophilicity are related to the nucleotide at this peak position upstream of the start codon.