Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3.

Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined. By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser desorption ionization mass spectrometry, and Edman microsequencing), we were able to isolate and identify a panel of HLA-DR4/2 (HLA-DRB1*0401/0201/DRB5*0101)-associated self-peptides from the melanoma cell line FM3. Among ubiquitously HLA-DR-associated peptides such as peptides from the class II-associated invariant chain peptide region of the invariant chain, HLA-class I, the transferrin receptor, and the IFN-gamma receptor, we identified several potential tumor-associated antigens stemming from the MHC class I-restricted tumor antigen gp100, the Ca2(+)-binding protein annexin II, and proteins from the hsp70 family. Chinese hamster ovary cells cotransfected with HLA-DRA, DRB1*0401, and CD80 genes were shown specifically to prime T lymphocytes from HLA-DRB1*0401 donors to the annexin II and gp100 peptides. These results demonstrate that MHC class II molecules expressed by melanoma cells potentially present a variety of novel antigens to the immune system, some of which could be exploited for immunotherapy.

Identification of Dll1 (Delta1) target genes during mouse embryogenesis using differential expression profiling.

The Notch signaling pathway has pleiotropic functions during mammalian embryogenesis. It is required for the patterning and differentiation of the presomitic and somitic paraxial mesoderm and of the neural tube. We used DNA-chip expression profiling and 2D-gel electrophoresis combined with peptide mass fingerprinting to identify genes and proteins differentially regulated in E10.5 Dll1 (delta-like 1, Delta1) mutant embryos. The differential expression profiling approach identified 47 regulated transcripts and 40 differentially expressed proteins. The majority of these genes has until now not been associated with Notch signaling. Subsequent whole-mount in situ hybridization confirmed that a subset of the identified transcripts has restricted and distinct patterns of expression in E10.5 mouse embryos. For most genes these expression patterns were affected in the presomitic mesoderm, in differentiating somites of Dll1 mutant embryos and in the neural tube and cells differentiating from it. Similar effects were observed in embryos homozygous for the Headturner (Htu) and pudgy (pu) mutations, which are alleles of the Notch ligands Jag1 and Dll3. The regulated expression of a subset of the proteins was validated by immunoblots. Remarkably six of the proteins down-regulated in Dll1 mutant embryos are proteasome subunits. The large set of regulated genes identified in this differential expression profiling approach is an important resource for further functional studies.

A 16mer peptide of the human autoantigen calreticulin is a most prominent HLA-DR4Dw4-associated self-peptide.

The human Ca(2+)-binding (storage) protein calreticulin, located in the lumen of the endoplasmic reticulum, is proposed to play a role as autoantigen: anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis (calreticulin shows a high sequence homology to the Onchocerca volvulus antigen RAL-1). Here we present sequencing data of a HLA-DR4Dw4-associated calreticulin peptide fragment, Cal(295-310), purified from a DR4Dw4 self-peptide pool. Cal(295-310) proved to be one of three commonest self-peptides associated with DR4Dw4 molecules that were isolated from the EBV-transformed B-cell line BSM (DR4Dw4, DRw53). We tested the binding of Cal(295-309) and the analogous RAL-1 peptide to HLA-DR molecules: Cal(295-309) exhibited specific binding characteristics for DR4Dw4. Binding assays using self-peptide analogues with replaced amino acids led us to a DR4Dw4-binding motif with anchor residues at relative positions 1 and 6. The sequencing data suggest that calreticulin is a frequently processed intracellular protein. The abundance of calreticulin makes the presentation of different calreticulin peptides associated with HLA-D molecules likely to occur, supporting the immunologic relevance of this molecule.

Characterization of peptides bound to extracellular and intracellular HLA-DR1 molecules.

Exogenous antigens are internalized by antigen-processing cells and processed within vesicular compartments to produce antigenic peptides that bind to newly synthesized MHC II molecules. These MHC class II peptide complexes are displayed at the plasma membrane and stimulate specific CD4+ T cells. In the present study, we established a method to isolate intracellular MHC molecules in a preparative scale (2-3 mg HLA-DR1) from endosomal compartments by Percoll density-gradient centrifugation. Peptides associated with HLA-DR1 in these intracellular fractions were released, purified by microbore HPLC, characterized by sequencing, and compared with the amino acid composition of peptides derived from MHC class II molecules obtained by solubilization of the plasma membrane. The binding affinity of these MHC fractions was analyzed by our highly sensitive binding assay using different DR1-restricted IM and Ii peptides. The results indicate that (a) intracellular MHC molecules show higher peptide-binding capacity, (b) peptides that are about 18-25 amino acids long need only a core region of 11 amino acids for binding, (c) specific positions of the peptides are important for DR1 binding, (d) most of the naturally processed peptides show a proline at position 2 or 3 that may represent a stop signal for trimming, and (e) Ii peptides are very abundant in DR1 peptide pools derived from intracellular compartments.

T cell reactivity to DR*0401- and DQ*0302-binding peptides of the putative autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is thought to be an autoimmune disease mediated by T lymphocytes recognizing critical islet cell antigens. Recently, the tyrosine phosphatase like protein IA-2 was suggested as a putative autoantigen in type 1 diabetes since autoantibodies are detected in sera of diabetic patients and prediabetic subjects. Similarly, T cell responses of peripheral blood lymphocytes of type 1 diabetic patients to this protein have been described. Only very few data is available about immunodominant epitopes of IA-2 recognized by T cells. We have studied T cell responses in type 1 diabetic patients and age and partly HLA matched controls to IA-2 peptides designed to bind HLA risk alleles of IDDM as DR*0401 and DQ*0302. Both diabetic patients and controls responded to IA-2ic and some of the peptides. Three peptides of the C-terminal region of IA-2 were recognised by T cells of a fraction of diabetic patients but at least two of these peptides triggered also T cell responses in DR*0401/DQ*0302-matched controls. Most peptides bound to different HLA alleles ("promiscous binders"). The identification of autoantigenic epitopes may offer clues to related sequences e.g. of viral origin what relates to models of diabetes pathogenesis ("molecular mimicry"). Secondly, the design of antigen- or even epitope-specific immune intervention strategies aiming at tolerization of disease specific T cells in type 1 diabetes may profit from the knowledge of immunodominant T cell epitopes of a putative autoantigen.

[Comparative gas chromatographic studies of heroin samples. Results of a pilot project in West Germany in 1982]. / Vergleichende gaschromatographische Untersuchungen von Heroinproben. Ergebnisse eines Pilotprojektes in der Bundesrepublik Deutschland im Jahr 1982.

From 1.4. -30.10.1982 all heroin samples weighing more than 5 g seized in the States of Baden-Württemberg, Hesse and by the Bundeskriminalamt were analyzed by capillary gas chromatography during a pilot project. For comparison of samples ratios of concentrations of heroin (including its decomposition products 0(6)-acetylmorphine and morphine) and its natural by-products of synthesis acetylcodeine, papaverine, and narcotine (noscapine) were determined. The application of these parameters and further qualitative and quantitative criteria for heroin comparison for investigative and legal purposes are discussed.

Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues.

A major advantage of the mouse model lies in the increasing information on its genome, transcriptome, and proteome, as well as in the availability of a fast growing number of targeted and induced mutant alleles. However, data from comparative transcriptome and proteome analyses in this model organism are very limited. We use DNA chip-based RNA expression profiling and 2D gel electrophoresis, combined with peptide mass fingerprinting of liver and kidney, to explore the feasibility of such comprehensive gene expression analyses. Although protein analyses mostly identify known metabolic enzymes and structural proteins, transcriptome analyses reveal the differential expression of functionally diverse and not yet described genes. The comparative analysis suggests correlation between transcriptional and translational expression for the majority of genes. Significant exceptions from this correlation confirm the complementarities of both approaches. Based on RNA expression data from the 200 most differentially expressed genes, we identify chromosomal colocalization of known, as well as not yet described, gene clusters. The determination of 29 such clusters may suggest that coexpression of colocalizing genes is probably rather common.

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization.

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.

Self-peptides from four HLA-DR alleles share hydrophobic anchor residues near the NH2-terminal including proline as a stop signal for trimming.

Naturally processed MHC class II-associated peptides proved to be heterogeneous in size, varying from 13 to 25 amino acids. Truncation variants suggested sequence motifs that afford the amino termini to be shifted for obtaining an alignment: a 9- to 11-residue core region that is bordered by primary anchor residues is surrounded by extra sequences of variable lengths and hitherto unknown functions. Herein we present bulk sequencing analyses of self-peptides from four HLA-DR alleles and HLA-DQw7 clearly showing that the length of most of the NH2-terminal preanchor sequence is limited to 1 to 3 residues. Most strikingly, proline is the dominant residue reappearing at positions 2 and 3 in any allele. Proline revealed to function as a stop signal for NH2-terminal trimming as well as a secondary anchor: crude cytosolic and endosomal peptide fractions could be processed by aminopeptidases in vitro, whereupon DR1 binding peptides with increased affinity were generated. In addition, aminopeptidase treatment of DR1: self-peptide complexes implied that proline together with sterical constraints of the MHC molecule do protect the peptides' NH2-termini from further processing, whereas their COOH-termini were accessible to cathepsin B processing. Finally, bulk sequencing profiles contained signals from further putative anchor residues clustering in the NH2-terminal region:tyrosine, phenylalanine, leucine, isoleucine, and valine are enriched at positions 2 to 4 in DR1, DR5, and DR6, however, at positions 4 to 6 in DR3. Isotype-specificity is demonstrated by DQw7 displaying glutamine and asparagine at position 2. Obviously, the degenerate occurrence of aromatic or aliphatic side chains close to the NH2-terminal guarantees for essential interactions with a hydrophobic pocket of the investigated DR molecules. Most probably, this pocket is located in the nonpolymorphic DR alpha-chain rationalizing previous findings of promiscuous peptide binding to different DR alleles.

Defensins are dominant HLA-DR-associated self-peptides from CD34(-) peripheral blood mononuclear cells of different tumor patients (plasmacytoma, chronic myeloid leukemia).

The HLA-DR-associated peptides from peripheral blood mononuclear cells of 2 patients with plasmacytoma and 1 with chronic myeloid leukemia were isolated, identified, and compared. Several were identified as derivatives of the defensin family. Defensins (or human neutrophil peptides [HNP]) are antimicrobial, cationic peptides of 29 to 35 amino acids in length and are the major constituents of the azurophilic granules of human neutrophils. Using peripheral blood cells from leukapheresis, containing about 90% of polymorphonuclear cells, we could identify HNP-1, -2, and -4 and propeptides of up to 49 amino acids in length, eluted from HLA class II molecules. Binding of isolated and synthetic defensin peptides to various HLA-DR alleles using an in vitro binding/competition assay based on size exclusion chromatography revealed that defensin may bind into the peptide-binding groove. In a T-cell competition assay, defensins were able to reduce the proliferation of an HLA-DR-restricted T-cell line after preincubation of stimulating cells (CHO-DRB1*0401 transfectants) with defensin. Therefore, binding of defensins might prevent T-cell recognition of HLA class II molecules expressed on different blood precursor cells (all of which are "nonprofessional" antigen-presenting cells) by blocking the HLA peptide-binding groove or, alternatively, might protect defensin-expressing cells from self-destruction. (Blood. 2000;95:2890-2896)

Identification of wheat flour allergens by means of 2-dimensional immunoblotting.

BACKGROUND: Wheat flour proteins are allergens for 60% to 70% of bakers with workplace-related respiratory symptoms. OBJECTIVE: The aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized bakers and to identify the most frequently recognized allergens. METHODS: Water/salt-soluble wheat flour proteins from the cultivar Bussard were separated by using 2-dimensional gel electrophoresis with immobilized pH gradients. IgE-reactive proteins were identified by means of immunoblotting with sera of 10 subjects with baker's asthma. Mass spectrometric fingerprinting was used to identify the proteins most frequently recognized by IgE. RESULTS: The IgE immunoblots obtained with 10 different sera exhibited a remarkable heterogeneity. Each patient showed an individual IgE-binding pattern with 4 to 50 different allergen spots. Altogether, more than 100 IgE-binding protein spots were detected. Nine of the predominant IgE-binding protein spots were identified by using mass spectrometric fingerprinting. The obtained masses matched 2 different isoforms of glycerinaldehyde-3-phosphate dehydrogenase from Hordeum vulgare, triosephosphate isomerase from H vulgare, and serpin, a serine proteinase inhibitor from Triticum aestivum. CONCLUSIONS: The results show a great interindividual variation of IgE-binding patterns of wheat flour proteins in baker's asthma. The clinical relevance of the identified 4 new allergens will be further investigated in the near future.

BCR/ABL leukemia oncogene fusion peptides selectively bind to certain HLA-DR alleles and can be recognized by T cells found at low frequency in the repertoire of normal donors.

Chronic myelogenous leukemia (CML) is characterized by the t(9;22) translocation that results in chimeric genes encoding bcr/abl fusion proteins. Junction-spanning sequences represent unique tumor-specific moieties that might be exploited therapeutically. We investigate here the binding of synthetic bcr/abl peptides to various HLA-DR alleles and their recognition by T cells from normal donors and CML patients. A 23-mer b3/a2 peptide bound very strongly to isolated HLA-DRB1*1101 (Dw5) and relatively strongly to DRB1*0301 (Dw3) and DRB1*0402 (Dw10) molecules, as estimated using a competition assay. It failed to bind to several other DR alleles, including three different DR4 alleles. In contrast, a 23-mer b2/a2 peptide bound only to the DRB1*0301 (Dw3) allele. Peripheral blood mononuclear cells from normal donors were sensitized in vitro against the b3/a2 peptide. After four repetitive stimulations, T cells responding to the peptide were found at low frequency in 5 of the 11 donors tested. Three of the five were HLA-DR11+, and all three of the DR11+ donors tested were found to respond. T cells recognizing bcr/abl peptides were not identified in any of the CML patients studied, regardless of HLA type. Finally, even peptide-reactive T-cell lines from normal donors were not stimulated by native CML cells in the absence of exogenous peptide. These results show the presence of low-frequency major histocompatability complex class II-restricted bcr/abl-responses in the normal T-cell repertoire of donors with certain HLA types, but suggest that unmodified tumor cells cannot be recognized by such peptide-sensitized T cells.

New ligands binding to the human leukocyte antigen class II molecule DRB1*0101 based on the activity pattern of an undecapeptide library.

Major histocompatibility complex (MHC) class II molecules present peptide antigens to CD(4+)-T cells. These heterogeneous peptides are derived from internalized exogenous proteins or from endogenous membrane proteins that are processed by the antigen-presenting cell. Peptides are bound to the MHC class II molecules in an extended conformation and extend out of the binding groove. The aim of this study was to estimate the influence of every amino acid in all the possible undecapeptide amides (2.048 x 10(14) individuals) on the binding to human MHC-DRB1*0101 molecules (HLA-DR1) and to identify new peptide ligands. 220 undecapeptide sublibraries, O/X10, each composed of ten degenerate positions and one defined position, were screened for binding to isolated HLA-DR1. Competition of the sublibraries with a fluorescence-labeled peptide ligand allowed definition of the amino acids favourable or unfavourable for DR1-binding at every sequence position. From the activity pattern of the undecapeptide library, 54 individual peptides were deduced (27 potential hits and 27 potential falls) and prepared by chemical synthesis. As anticipated, 27 positive and 27 negative results were obtained from the competition experiments. The 27 peptides that bind obey the rules for the HLA-DR1-binding motif. The synthetic peptide library approach proved to be valuable for the design of synthetic MHC class II ligands and thus can be considered as a basis for drug design in immunotherapy.

Tumour-specific MHC-class-II-restricted responses after in vitro sensitization to synthetic peptides corresponding to gp100 and Annexin II eluted from melanoma cells.

In a search for potentially tumour-specific MHC-class-II-restricted antigens, the immunogenicity of endogenous peptides that had been eluted from HLA-DR molecules of the human melanoma cell line FM3 (HLA-DRB1*02x, DRB1*0401) was tested in vitro. Two 16-mers representing gp100 positions 44-59, and annexin II positions 208-223 bound well to isolated DRB1*0401 molecules and are discussed here. HLA-DR-matched normal donors' T cells were cultured with peptide-pulsed artificial antigen-presenting cells (CHO cells cotransfected with genes for HLA-DRB1*0401 and CD80 and coexpressing high levels of both human molecules). Specific sensitization was achieved against both peptides, as measured in assays of autocrine proliferation and interleukin-2 secretion. Moreover, responses to native autologous melanoma cells but not to autologous B cells were also observed. In view of the expression of fas by the activated T cells and of fas ligand by the melanoma cells, blockade of potential fas/ fas-ligand interactions was undertaken using monoclonal antibodies (mAb). The antagonistic fas-specific mAb M3, but not the fas agonist M33, caused a markedly enhanced T cell response to FM3 cells. These results demonstrate that synthetic peptide antigens are able to sensitize T cells in vitro for effective MHC-class-II-restricted recognition of melanoma cells.

Human monoclonal antibody with T-cell-like specificity recognizes MHC class I self-peptide presented by HLA-DR1 on activated cells.

Alloreactive T cells recognize peptides presented in the binding groove of major histocompatibility complex molecules (MHCs), whereas B cells mainly recognize the MHCs independent of bound peptides. Here, we demonstrate that the human B-cell repertoire comprises B cells which can be stimulated during pregnancy to produce antibodies reacting with MHCs in a way similar to T cells. The human monoclonal antibody UL-5A1 recognizes DR1(DRA/DRB1*0101) molecules on lymphoblastoid cell lines only if they co-express HLA-A2 or if they have been loaded with HLA-A2-derived peptides. The effect of the HLA-A2 peptide 105-117 on UL-5A1 reactivity was specific, time and dose-dependent. Reactivity increased when naturally processed peptides were removed from DR1 molecules before the HLA-A2 peptide 105-117 was loaded. UL-5A1 reacted specifically with cells that had been activated. The results imply a role of activation of cells in peptide processing and/or loading.

Glutamic acid decarboxylase T lymphocyte responses associated with susceptibility or resistance to type I diabetes: analysis in disease discordant human twins, non-obese diabetic mice and HLA-DQ transgenic mice.

Glutamic acid decarboxylase (GAD65) has been implicated as a targeted self antigen in the immune destruction of pancreatic beta cells. T cell responses to GAD65 peptides have been detected in both patients with type I diabetes and in the non-obese diabetic (NOD) mouse. To establish which GAD65 epitopes are important in the immunopathogenesis of disease we initially compared T cell responses to GAD65 epitopes in conditions of disease susceptibility and protection. T cell responses to GAD65 peptides were measured in monozygotic twin pairs selected on the basis of disease discordance and T cell recognition of immunogenic regions of GAD65. Peptides of interest were then used to immunize susceptible NOD mice and H2-E transgenic NOD mice which are protected from diabetes. A differential response to the epitope GAD65 521-535 discriminated diabetic from non-diabetic human twins as well as susceptible from protected mice. This epitope as well as GAD 505-519 induces T cell responses despite binding the type I diabetes associated HLA-DQA1*0301/DQB1*0302 product with low affinity. Since DQ-restricted T cell responses are difficult to study in humans, HLA-DQ8 transgenic mice were then used: GAD epitopes 521-535 and 505-519 induced responses in DQ8 transgenic mice and T cell lines were established. Long-term T cell lines against GAD 505-519 were HLA-DQ restricted, and responded to peptide with a strong IFN-gamma and IL-10 response. The findings implicate GAD 521-535 as a possible target peptide in pathogenesis and are compatible with a model whereby self-reactive T cells specific for low-affinity peptide-MHC complexes may escape thymic negative selection.