Matthias Kalbus

Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans.

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.

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.

Ligand motif of the autoimmune disease-associated mouse MHC class II molecule H2-As

The MHC class II molecule H2‐As, expressed in the SJL mouse strain, is the principle restriction element of autoreactive CD4+ T cells mediating experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We deduced the H2‐As ligand motif from the analysis of naturally processed self peptides and from peptide binding studies. Major anchor residues were identified using various sets of substituted and truncated peptides, derived from natural peptide ligands and known H2‐As binders like myelin basic protein 81 – 99. The nine‐residue H2‐As core binding motif comprises an arrangement of anchors in relative positions P1, P4, P6, P7, and P9. The P1 pocket is relatively unspecific and the P6 pocket favors hydrophobic‐aliphatic side chains. The P1 pocket contributes little to peptide binding. Primary anchors were identified in P4, P7, and in particular in P9. The preferred anchor residues are Lys (P4), His / Arg (P7), and Pro (P9), respectively. Ala‐polysubstituted peptides containing only one of these dominant anchor residues still retain the capacity to bind to H2‐As. Thus, the presence of only one suitable anchor side chain in P4, P7, or P9 is sufficient for high‐affinity peptide binding, at least in the absence of negatively charged side chains nearby. The identified ligand motif facilitates the analysis of immunogenic peptides interacting with H2‐As and will allow a better prediction of pathogenetically relevant peptide antigens in the autoimmune mouse model.

New ligands for HLA DRB1*0301 by random selection of favourable amino acids ranked by competition studies with undecapeptide amide sublibraries

An efficient screening procedure for the identification of high affinity HLA class II ligands and their binding pattern has been established to characterize peptide specificities for the HLA allele DRB1*0301. The method is based on the screening of 209 synthetic undecapeptide amide sublibraries O/X10-NH2 representing collections of 19(10) individual peptides in a competition ELISA using HLA DRB1*0301 protein and the biotinylated natural ligand ApoB 2877-2894. Screening results represent the effect on competition induced by an individual amino acid residue in its sequence position of undecapeptide amides. Amino acids clustered as active in their position were randomly selected for the same position of a restricted set of 96 individual undecapeptide amides. This novel approach for the design of ligands was introduced to compensate for the inaccuracy induced by the translational invariance of amino acids in peptide libraries characterized by one defined amino acid. Translational invariance is facilitated by shifted docking of O/X10-NH2 libraries in the binding cleft and protrusion from the ends of the cleft. A second more directed deduced set of 24 peptides was obtained by combination of the most favourable residues in each position. All individual peptides were investigated in the competition assay. The most active HLA DRB1*0301 ligands were obtained by random selection of favourable amino acids and six of them showed improved affinity in comparison to the model ligand alpha AChR 310-325.

T-cell-reactivity against CNPase (a minor myelin component) in multiple sclerosis patients and normals

Introduction: A rather constant polyspecific antibody synthesis (e.g. against measles-, rubella-, herpes simplex-, zoster-virus) or autoantibody synthesis (e.g. anti ds-DNA) in brain is observed in patients with multiple sclerosis or autoimmune diseases with involvement of the central nervous system. Material and Methods: Antibody concentrations are enalysed in carebrosoinal fluid and serum and the intrathecal synthesis evaluated as increased Antibody-Index (1). Results: Immune modulating substances like cyclosporin A and corticosteroids influences the intrathecal virusspecific antibody synthesis. From a longitudinal study of these antibody concentrations in blood (daily blood analysis up to 50 days) from patients of the intensive care unit with and without • systemic inflammatory disease we find increasing concentrations of unspecific antibodies (s.g. against rubellavirus) in case of acute infections, e.g. pneumonia. The affinity of antibodies in acute and chronic diseases ere compared. Concluaion: The polyspecific immune response in CNS is sufficiently explained by an immune network memory as an emergent property of the complete system in the absence of antigen. tl) Rebec, H. emd Le, nge P; Cliff Ghem t99t;37:1 r53-t 160.