Thomas Halder

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.

The Role of Endogenous Peptides in the Direct Pathway of Alloreactivity to Human MHC Class II Molecules Expressed on CHO Cells

Allograft rejection is caused by the recognition of major histocompatibility complex {MHC) molecules by recipient T cells via two different pathways. The indirect pathway of alloreactivity requires the presentation of MHC peptides derived from the graft by the autologous antigen-presenting cells of the host. The direct pathway, on the other hand, requires the recognition of foreign MHC molecules on graft cells, apparently in an MHC-unrestricted fashion. It is believed that antigen receptors on T cells recognize foreign MHC molecules because of molecular mimicry (i.e. foreign MHC determinants and self MHC/foreign peptide complexes are recognized by virtue of cross-reactivity), and that the endogenous peptides bound by the MHC contribute to the generation of the epitope recognized {Lechler et al. 1990). A great deal of circumstantial evidence in favour of an important role for peplides in alloreactivity has been presented, ranging from the tissue specificity of ailorecognition to the finding that stimulation by MHCpositive antigen-processing mutants is impaired {Sayegh et al. 1994). However, despite the general acceptance of this hypothesis for class I-directed alloreactivity (Heath et al. 1989, Rotzschke et al. 1991), there are very few published examples