SummaryWe describe here a new method for predicting class II major histocompatibility complex-binding peptides, based on the preferences observed in a systematic series of peptide binding experiments where each position in a "minimal" peptide was replaced individually by every amino acid. The DRBI*0401 peptide binding preferences were determined and incorporated into a computer program that looks through sequences for potential epitopes and assigns each a score. These scores correlate well with previously determined T cell epitopes of foreign antigens and endogenous peptides from self proteins. Our findings hold implications for the design of subunit vaccines and in the identification of autoantigenic peptide regions within self proteins.M HC class II molecules are highly polymorphic proteins that bind peptides derived from processing of antigens and present them to T cells (1). Methods to predict regions in protein sequences capable of binding to human MHC class II molecules would be very valuable for many immunological applications. A quantitative prediction of MHC binding sites, however, requires the understanding of the rules governing peptide--MHC class II interaction. The use of M13 peptide display libraries to identify aUele-specific motifs (2-4), together with structural analyses of HLA-DRl-peptide complexes (5, 6) provided the basis to predict MHC class II-peptide interaction: (a) the identification of precisely spaced anchor positions in large HLA-DR-selected peptide pools and the x-ray crystallographic studies suggested an overall similar, sequence-independent peptide conformation (3, 5); (b) the fact that the anchor at position 1 (pl) 1 was found obligatory for high affinity binding allowed us to define frames in HLA-DR bound peptides (4); (c) the position of a particular side chain, with respect to the pl-frame,'determines whether a given amino acid behaves as anchor residue, inhibitory residue, or residue neutral for binding (4). This information led us to develop a new method for predicting class II MHC-binding peptides. The method is based on the hypothesis that peptide binding to HLA-DR correlates with the net result of all side chain effects in any given peptide, and that most side chain effects depend on their relative position to the pl anchor, rather than on the remaining peptide se- quence. Pl-anchored designer peptide libraries were thus used for scanning the relative peptide positions 2-9 for the effects of each amino acid side chain on binding to the human class II molecule DRBI*0401. When values, individually obtained by side chain scanning, were assigned to the amino acid residues of the selected protein region, the sum of these values correlated with the MHC-binding affinity of the respective peptide. The processing of the side chain scanning data into a software allowed a rapid and nearly perfect prediction of DRBI*0401 binding regions in any protein sequence. Materials and MethodsPreparation of HLA-DRBI*0401 Molecules. DRBI*0401 molecules were isolated from human lymphoblastoid cell...

SummaryTo investigate the development of HLA-DR-associated autoimmune diseases, we generated transgenic (Tg) mice with HLA-DRA-IEcx and HLA-DRBI*0401-IE[3 chimeric genes. The transgene-encoded proteins consisted of antigen-binding domains from HLA-DRA and HLA-DRBI*0401 molecules and the remaining domains from the IEd-ot and IEd-[3 chains. The chimeric molecules showed the same antigen-binding specificity as HLA-DRBI*0401 molecules, and were functional in presenting antigens to T cells. The Tg mice were backcrossed to MHC class II-deficient (IAl3-,IEoe-) mice to eliminate any effect of endogenous MHC class II genes on the development of autoimmune diseases. As expected, IA~x[3 or IEot[3 molecules were not expressed in Tg mice. Moreover, cell-surface expression of endogenous IE[3 associated with HLA-DRA-IEci was not detectable in several Tg mouse lines by flow cytometric analysis. The HLA-DRA-IEo~/HLA-DRBI*0401-IE[3 molecules rescued the development ofCD4 + T cells in MHC class II-deficient mice, but T cells expressing VI35, V1311, and VI312 were specifically deleted.Tg mice were immunized with peptides, myelin basic protein (MBP) 87-106 and proteolipid protein (PLP) [175][176][177][178][179][180][181][182][183][184][185][186][187][188][189][190][191][192], that are considered to be immunodominant epitopes in HLA-DR4 individuals. PLP175-192 provoked a strong proliferative response of lymph node T cells from Tg mice, and caused inflammatory lesions in white matter of the CNS and symptoms of experimental allergic encephalomyelitis (EAE). Immunization with MBP87-106 elicited a very weak proliferative T cell response and caused mild EAE. Non-Tg mice immunized with either PLP175-192 or MBP87-106 did not develop EAE. These results demonstrated that a human MHC class II binding site alone can confer susceptibility to an experimentally induced murine autoimmune disease.

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H. (1992) J. Biol. Chem. 267, 9988-9997). To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable. Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA. The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis. gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E. coli K-12 does not. Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.

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We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*11O1 molecules, by screening of an M13 peptide display library (420 million independent nonapeptides) for DRbinding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect ofanchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6-or 7-residuelong designer peptides is shown to result in a gradual increase of binding aMnity to the level of 13-residue-long hig-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elmination of other anchors results only in partial loss ofbinding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins.Each allelic form of the highly polymorphic major histocompatibility complex (MHC) molecules can bind a large array of different peptides to ensure T-cell-mediated immunity to the antigenic universe. Thus, provided that peptides bind to a given MHC molecule in similar or identical conformation (1-4), the sequencing of a sufficiently large pool of bound peptides should reveal the rules of MHC/peptide interaction.Recently, two different approaches have been described for the isolation and characterization of MHC-bound peptide pools. (i) Endogenously processed peptides were eluted from MHC molecules and sequenced in pool or individually. This approach resulted in binding motifs for class I MHC molecules (5-7). However, its application for MHC class II molecules proved to be difficult, because of the limited number of independent sequences identified and the high degree of length heterogeneity of bound peptides (8-11). (ii) Phage-expressed large (=20 million independent sequences) random peptide libraries were screened for MHC binding. Sequencing ofthe bound peptides has revealed allele-specific binding motifs for three different HLA-DR molecules (12, 13). Here, we report a further refinement of the phage library screening, which allowed us to identify additional DRB1*0401-binding anchors. We also demonstrate that the anchor residues in class II binding peptides are additive and position specific, and, in combination, enable short peptides to bind tightly to DR molecules. MATERIALS AND METHODSPreparation of HLA-DR Molecules. DR molecules isolated from cell lines BSM, PREISS (DRB1*0401), HOM-2, LG-2 (DRB1*0101), and SWEIG (DRB1*1101) wer...