Joan C. Gorga

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding.

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present ‘processed antigens’ (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA‐DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent‐solubilized HLA‐DR1 and a papain‐cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA‐DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA‐DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.

Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

Immunoglobulin A supplementation abrogates bacterial translocation and preserves the architecture of the intestinal epithelium.

BACKGROUND Breast milk has been shown to prevent gut-origin infections in neonates through undefined mechanisms. Putative protective factors in breast milk include immunoglobulin (Ig)A, IgG, and lactoferrin. We examined their role in bacterial translocation in neonatal rabbits. METHODS IgA, IgG, and lactoferrin were isolated from rabbit breast milk through gel filtration and ion-exchange chromatography. Neonates were randomized to receive breast milk, formula alone, or formula supplemented with IgA, IgG, or lactoferrin. Quantitative cultures were performed on day 7 for bacterial translocation. Hematoxylin-eosin-stained sections of distal ileum were examined by light microscopy. Transmucosal bacterial passage was determined in vitro, and the ileal mucosal membranes were examined by confocal microscopy. RESULTS IgA supplementation abrogated bacterial translocation. IgG and lactoferrin had no significant effect. Neonates that received IgA or breast milk gained more weight than those in the other groups. IgA reduced transmucosal bacterial passage in vitro. In contrast to the normal-appearing distal ileum of neonates fed breast milk, intestinal epithelium from neonates that received formula or formula with IgG or IgA demonstrated prominent vacuoles by light microscopy. Those fed formula alone or formula with lactoferrin had slightly shortened villi. CONCLUSIONS IgA supplementation prevents bacterial translocation by enhancing gut mucosal barrier function.

Immunochemically purified DR antigens in liposomes stimulate xenogeneic cytolytic T cells in secondary in Vitro cultures

A monoclonal antibody directed against the human class II major histocompatibility antigen DR was generated. Use of this antibody, LB3.1, allowed isolation of large amounts of highly purified DR by immunoaffinity chromatography. The DR was reconstituted into liposomes and shown to stimulate secondary xenogeneic cytolytic T lymphocytes (CTL) specific for targets expressing DR antigens. DR digested with neuraminidase was equally as effective as native DR at stimulating CTL, while denatured DR and other purified membrane proteins were much less effective. The DR liposome-induced CTL lysed only target cells expressing class II antigens. Cytolysis of targets bearing class II antigens was blocked by DR-specific antisera.

Crystallization of HLA-DR antigens

The class II major histocompatibility antigens HLA-DR1, DR2, DR3, DR4, DR7 and DR8 were purified by immunoaffinity chromatography from homozygous human B lymphoblastoid cell lines. The purified, detergent-soluble molecules were cleaved with the protease papain to remove the hydrophobic transmembrane regions and cytoplasmic tails. Crystals were obtained for each of the papain-solubilized fragments. DR1 crystallized under a variety of different conditions, resulting in two different orthorhombic crystal forms, one of which diffracts as far as 3.5A. Crystals of DR2, DR3, DR4 and DR8 have the same unit cell dimensions as the DR1 crystals, and crystals of DR3 and DR4 have the same diffracting power as the DR1 crystals. The best DR7 crystals obtained thus far are hexagonal and diffract to only about 8A. Crystals of similar hexagonal form have also been observed for most of the other DR subsets.

Role of Ia antigens and interleukin 1 in T-cell proliferation to phytohemagglutinin☆

Highly purified human T cells were obtained by a four-step purification procedure which included: removal of plastic adherent cells, rosetting with sheep red cells, passage over nylon-wool columns, and treatment with mouse monoclonal antibodies to human Ia antigens and complement. The resulting T cells did not proliferate to phytohemagglutinin (PHA). Purified human interleukin 1 (IL-1) could not substitute for accessory cells in supporting a PHA response. Reconstitution with as little as 0.03% adherent cells resulted in a proliferative response to PHA. T-Cell proliferation to PHA was supported by monocytes, by Ia+ Epstein-Barr virus-transformed lymphoblastoid B-cells lines, and by Ia- cultured human dermal fibroblasts but not by Ia-containing liposomes. Addition of anti-Ia antibodies to monocyte-containing cultures did not inhibit the T-cell proliferative response to PHA. These results suggest that Ia antigen expression by accessory cells is neither necessary nor sufficient to support T-cell proliferation to PHA and that IL-1 is not sufficient to support the proliferation of T cells to PHA.

Use of the HLA-DR antigens incorporated into liposomes to generate HLA-DR specific cytotoxic T lymphocytes.

Publisher Summary This chapter reviews that in order to elucidate the molecular mechanisms involved in the induction of cytolytic T lymphocytes (CTLs) in the immune response, it is helpful to be able to separate the antigens expressed on the cell surface, and to test their individual abilities to act as stimulators of and targets for the action of CTLs. It discusses that human histocompatibility antigens at various stages of purification have been used to generate a CTL response. Isolated lymphoblastoid cell membranes and the dialyzed, detergent-soluble fraction from isolated membranes are both effective in eliciting CTLs. The chapter also explores that recently; HLA-DR antigens (DR) have been purified by immune affinity chromatography and used to generate CTLs. The use of purified DR to generate DR-specific CTLs reduces the chances that minor contaminants of the preparation, such as class I antigens, lentil lectin, or cytoskeletal proteins will affect the results. Purified antigen also facilitates analysis of both the requirements for generating CTLs and their specificities.

Human class II MHC molecule HLA-DR1: X-ray structure determined from three crystal forms.

The three-dimensional structure of the extracellular region of a 60 kDa class II major histocompatibility glycoprotein, HLA-DR1, was determined to 3.3 A by X-ray crystallography using three crystal forms, each containing two molecules per asymmetric unit. Phases were initially determined to 4.2 A using two crystal forms both containing DR1 from human lymphocytes complexed with a mixture of endogenous peptides. One of these crystal forms also contained a 28 kDa superantigen, Staphylococcus aureus enterotoxin B (SEB), bound to each DR1 molecule. Single-isomorphous replacement phasing followed by iterative two- and fourfold non-crystallographic real-space averaging between the two crystal forms resulted in 4.2 A resolution electron-density maps from which the paths of the polypeptides could be traced. Cryocrystallography and synchrotron radiation were then used to extend the resolution to 3.3 A for the two lymphocyte-derived crystal forms and for a third crystal form grown from DR1 produced in insect cells and complexed in vitro with a specific antigenic peptide. Iterative sixfold non-crystallographic real-space averaging resulted in an electron- density map into which 340 of 371 residues could be fit unambiguously. Crystal contacts and the existence of a parallel dimer of the DR1 alphabeta heterodimer in the three crystal forms are discussed.

Purification of Complexes between Peptide Antigens and Class II Major Histocompatibility Complex Antigens Using Biotinylated Peptides

The Major Histocompatability Antigens (MHC) are cell surface glycoproteins which are involved in the regulation of the immune response to foreign antigens. The MHC proteins bind peptides generated by cellular processing of native proteins (Babbitt et al., 1985; Buus et al., 1986; Watts & McConnell, 1986; Chen & Parham, 1989), and the peptide-MHC complexes are presented on cell surfaces to T lymphocytes (Schwartz, 1985). Class I MHC proteins maintain surveillance over proteins which are found in the cell cytoplasm, such as newly synthesized viral proteins in an infected cell (Morrison et al., 1987; Moore et al., 1988). The foreign peptide-MHC complexes are recognized by cytotoxic T lymphocytes, which respond by lysing the infected cell.

Comparison of the Secondary Structures of Human Class I and Class II MHC Antigens by Ftir and CD Spectroscopy

Considerable evidence exists that the structures of class I and class II histocompatibility antigens are similar. Most of the evidence (summarized in Kappes and Strominger, 1988 and Brown et al., 1988) is based on sequence homologies and similarities in domain structure at both the protein and DNA levels. In addition, some T cells that are specific for either class I or class II molecules use the same receptor (Rupp et al, 1985; Marrack and Kappler, 1986). However, the secondary structures of purified class I and class II antigens have not been directly compared.