Biomedical subjects

H Kalbacher

Publications and source records attributed to H Kalbacher.

At least 37 records · Page 2Linked to original sources

PubMed2000

Enhanced T cell responsiveness to citrulline-containing myelin basic protein in multiple sclerosis patients.

Myelin basic protein (MBP), a candidate autoantigen in multiple sclerosis (MS), exists in different isoforms and charge isomers generated by differential splicing of exons and by a combination of posttranslational modifications, respectively. These various isoforms and charge isomers of MBP vary in abundance and most likely serve different functions during myelinogenesis and remyelination. The least cationic among the charge isomers of MBP is citrullinated and is referred to as MBP-C8. MBP-C8 is relatively increased in the population of MBP isomers in more developmentally immature myelin and in MS brain tissue. In a previous study, we found that MBP-C8-reactive T cells could be detected in CD4+ T cell lines (TCL) generated with MBP from both MS patients and normal controls. Here, we examined the frequency and peptide specificity of MBP-C8-specific TCL generated with MBP-C8 in MS patients and controls. Ten subjects grouped in five sets, each an MS patient and a control, were studied. In all cases, the MS patient had either a higher overall number of MBP-C8-responding lines, responded with greater sensitivity to the MBP-C8 antigen or both. Few lines responded to the MBP-C8 peptides but, if they did, they appeared to be specific to the carboxyl-half of the MBP-C8 molecule. Given the large amounts of citrullinated MBP in MS brain tissue, a preferential T cell response to MBP-C8 may be involved in the induction and perpetuation of this disease. Multiple Sclerosis (2000) 6 220 - 225

Adult↗

PubMed1999

NMR structure and functional characteristics of the hydrophilic N terminus of the potassium channel beta-subunit Kvbeta1.1.

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming alpha-subunit or certain auxiliary beta-subunits. Upon coexpression, Kvbeta1.1 was found to endow non-inactivating members of the Kv1alpha family with fast inactivation via its unique N terminus. Here we investigated structure and functional properties of the Kvbeta1.1 N terminus (amino acids 1-62, betaN-(1-62)) using NMR spectroscopy and patch clamp recordings. betaN-(1-62) showed all hallmarks of N-type inactivation: it inactivated non-inactivating Kv1.1 channels when applied to the cytoplasmic side as a synthetic peptide, and its interaction with the alpha-subunit was competed with tetraethylammonium and displayed an affinity in the lower micromolar range. In aequous and physiological salt solution, betaN-(1-62) showed no well defined three-dimensional structure, it rather existed in a fast equilibrium of multiple weakly structured states. These structural and functional properties of betaN-(1-62) closely resemble those of the "unstructured" ID from Shaker B, but differ markedly from those of the compactly folded ID of the Kv3.4 alpha-subunit.

Amino Acid Sequence↗

PubMed1999

A rapid method to separate endosomes from lysosomal contents using differential centrifugation and hypotonic lysis of lysosomes.

Here we describe a fast and efficient subcellular fractionation procedure that permits lysosomes to be separated from endosomes. Differential centrifugation is used to isolate a subcellular fraction containing both endosomes and lysosomes. Because lysosomes are sensitive to osmotic stress, hypotonic conditions destroy them, whereas endosomes, which are osmotically insensitive, stay intact. We demonstrate that hypotonic lysis of an endosome-lysosome-pool releases 85% of the lysosomes into the supernatant as measured by the activity of the lysosomal marker enzyme N-acetyl-beta-D-glucosaminidase (beta-AGA). The endosomal fraction is thoroughly characterised using a variety of subcellular markers. After pulsing cells with fluorescein isothiocyanate labelled transferrin (FITC-Tf), only about 12% of the marker is released under hypotonic conditions. A typical fractionation procedure takes about 1-2 h from initial cell homogenisation. The fractionation gives a pure lysosomal fraction (fraction L) containing high activities of lysosomal enzymes and an endosomal fraction (fraction E) reflecting different stages of endosomes.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1999

Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides.

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.

Alanine↗

PubMed1999

Differential behavioral effects of TFF peptides: injections of synthetic TFF3 into the rat amygdala.

TFF peptides (formerly named P-domain peptides or trefoil factors) are also released from the brain as well as being secreted typically by mucin producing cells. The amygdala, besides the hypothalamus, represents a defined neuronal locality of TFF3 synthesis. In a passive avoidance test synthetic TFF3/monomer or 0.9% sodium chloride (control) was injected bilaterally into the basolateral nucleus of the amygdala of male rats either immediately (consolidation test) or 23 h after a footshock (retrieval test). Application of a low TFF3 dose (2 x 6 pg) decreased avoidance latency in a time dependent manner. In contrast, a high dose (2 x 60 pg) increased avoidance latency. Maximal effects of TFF3 were observed about 24 h after the injection. This bidirectional effect was also observed using the elevated plus-maze test. The locomotor activity on the open arms was significantly increased 24 h after a low dose injection of TFF3 into the amygdala. In contrast, a high-dose injection significantly decreased the activity on the open arms. The results of both tests can be explained by an anxiolytic effect at a low dose and an anxiogenic effect at a high dose of synthetic TFF3/monomer.

Amygdala↗

PubMed1999

Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.

Fast N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and occurs by a 'ball-and-chain'-type mechanism. In this mechanism an N-terminal protein domain (inactivation gate) occludes the pore from the cytoplasmic side. In Kv3.4 channels, inactivation is not fixed but is dynamically regulated by protein phosphorylation. Phosphorylation of several identified serine residues on the inactivation gate leads to reduction or removal of fast inactivation. Here, we investigate the structure-function basis of this phospho-regulation with nuclear magnetic resonance (NMR) spectroscopy and patch-clamp recordings using synthetic inactivation domains (ID). The dephosphorylated ID exhibited compact structure and displayed high-affinity binding to its receptor. Phosphorylation of serine residues in the N- or C-terminal half of the ID resulted in a loss of overall structural stability. However, depending on the residue(s) phosphorylated, distinct structural elements remained stable. These structural changes correlate with the distinct changes in binding and unbinding kinetics underlying the reduced inactivation potency of phosphorylated IDs.

Animals↗

PubMed1999

Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif.

A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells.

Alleles↗

PubMed1999

T cell reactivity to DR0401- and DQ0302-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.

Adolescent↗

PubMed1998

Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

Antigens, Neoplasm↗

PubMed1998

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.

Animals↗

PubMed1998

Analysis of T-cell reactive regions and HLA-DR4 binding motifs on the latex allergen Hev b 1 (rubber elongation factor).

BACKGROUND: The rubber elongation factor in Hevea rubber (Hev b 1) is one of the important latex-allergenic proteins inducing the production of specific IgE antibodies in latex-exposed subjects. In addition, Hev b 1 induces lymphocyte proliferation responses. A study about detailed T-cell epitope analysis of major latex allergens has not been published yet. OBJECTIVE: The aim of this study was to define the T-cell reactive regions on the latex allergen Hev b 1. METHODS: Nine overlapping peptides with 19 or 17 amino acid lengths representing the complete sequence of Hev b 1 were used for T-cell epitope mapping. Peripheral blood mononuclear cells (PBMCs) of latex-sensitized patients and healthy subjects were isolated and stimulated with the synthetic peptides to determine the specific proliferation response. The examined patients were sensitized to latex by occupational exposure (n=14) and suffered from rhinitis, conjunctivitis, contact urticaria and/or asthma. Two control groups of non-sensitized subjects were studied, one occupationally exposed to latex (n=4), the other one not exposed to latex (n=4). RESULTS: Positive proliferation response induced by one or more peptides was detected in 10 of the latex-sensitized patients and in two of the latex-exposed non-sensitized subjects. Each patient exhibited an individual epitope reactivity pattern. The whole protein was found to be immunogenic, but the intensity of lymphocyte responses induced by the peptides was different. The majority of the patients' PBMCs (more than 66%) responded to the peptides 31-49 and 91-109. Using the peptide selection of Stille's algorithms, three epitopes were predicted covering region 38-74 and two others covering region 82-104; the two peptides 31-49 and 91-109 are part of these regions. A strong HLA-DR4Dw4 (DRB1*0401)-binding motif according to published DR4w4 binding motifs was predicted in the region of 102-110 including peptide 91-109 to which the majority of tested HLA-DR4-positive patients responded. CONCLUSIONS: Our data indicate that the whole 14 kDa protein Hev b 1 is immunogenic having regions that demonstrate individual and varying PBMC stimulation. Experimental data of T-cell reactive regions based on PBMC-stimulation complement the information on T-cell epitope prediction. In addition, the Hev b 1 molecule contains a HLA-DR4Dw4 (DRB1*0401)-binding motif.

Adult↗

PubMed1998

Identification of HLA-DR and -DQ alleles conferring susceptibility to pollen allergy and pollen associated food allergy.

BACKGROUND: Allergenic crossreactivity of pollen and foods due to the antigeneic similarity of oligopeptides is a well established clinical phenomenon. OBJECTIVE: To determine the immunopathological relevance of antigen presentation, we analysed the HLA class-II genotype of patients with either pollen allergy or pollen associated food allergy. METHODS: One hundred and twenty patients with pollen allergy and 80 patients with pollen associated food allergy were evaluated by skin- prick tests, RAST, and HLA class-II genotyping. The control population comprised 4251 healthy blood and bone marrow donors. RESULTS: Monovalent pollen allergy was observed in 57% (n=68) of patients with pollinosis (57x grass pollen, 11x birch pollen), but only in 15% (n=12) of patients with food allergy (9x grass pollen, 3x birch pollen). Hazelnut (71%), almond (65%), walnut (44%) and apple (41%) were the most common food allergens and frequently associated with birch pollen allergy. Grass pollen allergy was associated with an increased frequency of HLA-DQB1*0301 (RR=2.3; EF=0.4; P=0.0016) when compared with the control population. HLA-DRB *08 conferred a sixfold higher risk for peanut allergy (EF=0.3; P=0.0013) and -DRB1*12 a 13-fold higher risk for carrot allergy (EF=0.3; P<0.000001). The differences on allele frequencies detected among patients with food allergies diminished or turned statistically insignificant when their genotypes were directly compared to those of patients with the corresponding pollen allergies. This was found in the case of birch pollen associated hazel nut allergy for the extended haplotype HLA-DRB1*01, -DQA1*0101, -DQB1*0501 as well as in grass pollen associated peanut allergy for HLA-DRB1*08 (from RR=6, P=0.0013 to insignificant) and in birch pollen associated carrot allergy for HLA-DRB1*12 (from RR=13, P < 0.000001 to insignificant). CONCLUSION: We were able to identify HLA class-II alleles associated with some allergies thus indicating that these alleles might confer susceptibility to the respective allergens. Similarities at the level of the HLA class-II genotype parallel the empirical finding of distinct cross-reactivity patterns thus complementing investigations of IgE specificities. Our observations provide evidence for the major importance of antigen presentation on the manifestation of distinct crossreactivity patterns.

Alleles↗

PubMed1998

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.

Adult↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Abnormal thymocyte development and generation of autoreactive T cells in mixed and cortical thymomas.

To gain insight into the pathogenesis of thymoma-associated myasthenia gravis, thymocyte maturation was investigated in mixed and cortical thymomas by three-color flow cytometry. Although we detected cells at all recognizable stages, we noted an unusual increased percentage of early CD4+/CD3- thymocytes--especially in mixed thymoma--and a pronounced decreased percentage of mature CD4+/CD3+ cells in cortical thymomas as well. The percentage of CD3+/CD69+ cells that arose after positive selection was reduced in both thymoma subtypes compared with control thymuses, which suggests differences in the rate or efficiency of positive selection particularly in mixed thymomas. Mature T cells in 10 of 11 thymomas were not activated in situ as shown by the absence of CD25 expression. After stimulation with recombinant human acetylcholine receptor alpha-subunit fragments, thymocytes from 8 of 11 thymomas of both subtypes proliferated more strongly than those from controls, regardless of whether the donors were myasthenic. Responses of residual thymus cells to tetanus toxoid correlated well with those of autologous blood T cells, whereas those from the thymomas clearly did not--implying minimal colonization of thymomas by mature recirculating T cells. In conclusion, our results show that cortical and mixed thymomas exhibited differences in thymocyte maturation. Nevertheless, both thymoma subtypes seem to contribute to the pathogenesis of paraneoplastic myasthenia gravis by generating naive but potentially autoaggressive T cells; in some thymomas, these cells may then be actively immunized inside the tumor.

Autoantigens↗

PubMed1997

The epitope recognized by a monoclonal antibody in the myelin-associated protein CNP.

The epitope recognized by a monoclonal antibody (MAb-46-1) directed against the myelin-associated protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase; EC 3.1.4.37) from several species was characterized. MAb-46-1 can be employed for immunoprecipitation, immunostaining in Western blots and in immunohistochemistry. Short peptides derived from the human CNP1 peptide sequence were synthesized and used in enzyme linked immunosorbent assays to test the reactivity of MAb-46-1. Coarse screening experiments enabled us to localize the epitope recognized by MAb-46-1 to the amino acid residues 9 to 19 close to the N-terminus. Further investigations using shorter peptides comprising this part of the protein allowed us to identify a 9 amino acid residue long peptide (amino acids 11 to 19: ELQFPFLQD) which represents the minimal epitope recognized by MAb-46-1, probably through a 3-dimensional structure and less likely a straight linear peptide. The epitope seems to be stabilized also by the attached amino acids 7 to 10 (KDKP). The peptide sequence 9-19 is conserved in all CNP sequences described so far. Thus, MAb-46-1 might be of general usefulness for further studies of the not yet identified function of the myelin-associated protein CNP.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1997

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.

Animals↗

PubMed1997

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy.

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induced IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine epsilon-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.

Carrier Proteins↗