The motif for peptide binding to the insulin-dependent diabetes mellitus-associated class II MHC molecule I-Ag7 validated by phage display library.

The MHC class II molecule I-Ag7 is essential for the development of insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse but the requirements for peptide binding to I-Ag7 are still controversial. We have now isolated I-Ag7-binding phage from a large phage display library encoding random nonamer peptides. Ninety peptide-encoding regions of phage eluted from I-Ag7 were sequenced and >75% of the corresponding synthetic peptides bound to I-Ag7. Peptide alignment led to the identification of position-specific anchor residues. Hydrophobic (V and P) and positively charged (K) residues were highly enriched at P6 and positively charged (R and K), aromatic (Y) or hydrophobic (L) residues at P9. In addition, small amino acid residues (G and A) were enriched at P7 and G at P8. The primary anchors at P6 and P9 defining the phage-derived motif were present in most high-affinity I-Ag7-binding peptides from IDDM candidate antigens but only in < or =25% of peptides that were low-affinity binders or failed to bind to I-Ag7. A comparison of these results with the proposed motifs for peptide binding to I-Ag7 validates the one we have previously described.

A peptide binding motif for I-Eg7, the MHC class II molecule that protects E alpha-transgenic nonobese diabetic mice from autoimmune diabetes.

The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due to a deletion in the E alpha gene promoter. E alpha-transgenic NOD mice express the E alpha E beta g7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4-13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and E alpha-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in E alpha-transgenic NOD mice.

Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices.

Most pockets in the human leukocyte antigen-group DR (HLA-DR) groove are shaped by clusters of polymorphic residues and, thus, have distinct chemical and size characteristics in different HLA-DR alleles. Each HLA-DR pocket can be characterized by "pocket profiles," a quantitative representation of the interaction of all natural amino acid residues with a given pocket. In this report we demonstrate that pocket profiles are nearly independent of the remaining HLA-DR cleft. A small database of profiles was sufficient to generate a large number of HLA-DR matrices, representing the majority of human HLA-DR peptide-binding specificity. These virtual matrices were incorporated in software (TEPITOPE) capable of predicting promiscuous HLA class II ligands. This software, in combination with DNA microarray technology, has provided a new tool for the generation of comprehensive databases of candidate promiscuous T-cell epitopes in human disease tissues. First, DNA microarrays are used to reveal genes that are specifically expressed or upregulated in disease tissues. Second, the prediction software enables the scanning of these genes for promiscuous HLA-DR binding sites. In an example, we demonstrate that starting from nearly 20,000 genes, a database of candidate colon cancer-specific and promiscuous T-cell epitopes could be fully populated within a matter of days. Our approach has implications for the development of epitope-based vaccines.

Identification of destabilizing residues in HLA class II-selected bacteriophage display libraries edited by HLA-DM.

HLA-DM (DM) functions as a peptide editor by catalyzing the release of class II-associated invariant chain peptides (CLIP) and other unstable peptides, thus supporting the formation of stable class II-peptide complexes for presentation. To investigate the general features that determine the DM susceptibility of HLA-DR1/peptide complexes, we generated a large DM-sensitive peptide repertoire from an M13 bacteriophage display library using a novel double selection protocol: we selected bacteriophage capable of binding to DR1 molecules and, subsequently, we enriched DR1-bound bacteriophage susceptible to elution by purified DM molecules. Sequence and mutational analyses of the DR1/DM double-selected peptides revealed that the amino acids Gly and Pro play a destabilizing role in the dissociation kinetics of DR1 ligands. This observation was confirmed also in natural peptide sequences such as CLIP 89-101, HA 307-319 and bovine collagen II (CII) 261-273. Our results demonstrate that DM susceptibility does not only depend on the number and nature of anchor residues, or the peptide length. Instead, less obvious sequence characteristics play a major role in the DM editing process and ultimately in the composition of peptide repertoires presented to T cells.

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules.

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires.

The role of HLA-DQ molecules in Ag presentation has, thus far, remained elusive. Here we report that two DQ allotypes, DQ7 (DQA1*0501/B1*0301) and DQ9 (DQA1*0201/B1*0303), are capable of binding peptide repertoires in complementarity with DR molecules. The results reflect fundamental differences in the binding modes of these two HLA class II isotypes, in that DQ7 and DQ9 but not DR molecules appear to have the capacity to bind peptide structures without type 1-like anchor residues. Consistent with this is our observation that none of the amino acid side chains of the class II-associated invariant chain peptides (CLIP) are required for association with DQ7 and DQ9, even though many of them are essential for CLIP-DR interaction. Together, these data reveal a functional complementarity of HLA-DR and -DQ molecules in Ag presentation.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice.

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association.

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

Precise prediction of major histocompatibility complex class II-peptide interaction based on peptide side chain scanning.

We 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 DRB1*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.