Population of the HLA ligand database.

We have established an HLA ligand database to provide scientists and clinicians with access to Major Histocompatibility Complex (MHC) class I and II motif and ligand data. The HLA Ligand Database is available on the world wide web at http://hlaligand.ouhsc.edu and contains ligands that have been published in peer-reviewed journals. HLA peptide datasets prove useful in several areas: ligands are important as targets for various immune responses while algorithms built upon ligand datasets allow identification of new peptides without time-consuming experimental procedures. A review of the HLA class I ligands in the database identifies strengths and deficiencies in the database and, therefore, the utility of the dataset for identifying new peptides. For instance, 212 HLA-A phenotypes exist of which 23 have a motif determined and 43 have peptides characterized. In terms of number of ligands, HLA-A*0201 has 258 characterized ligands, A*1101 has 25 peptides, while the remaining two-thirds of the HLA-A phenotypes have less than 10 associated peptide sequences. Characterization of ligands and motifs remains roughly the same at the HLA-B locus while the peptides of the HLA-C locus tend to be less characterized. These data show that 74% of HLA class I molecules do not have ligands represented in the database and thus algorithms based on the dataset could not predict ligands for a majority of the US population. Building upon this dataset and knowledge of HLA allelic frequencies, it is possible to plan a systematic expansion of the HLA class I ligand database to better identify ligands useful throughout the population.

Non-conservative substitutions distinguish previously uncharacterized HLA-A molecules.

The extent of class I HLA polymorphism is not yet realized, and to provide a glimpse of the HLA-A polymorphism which remains undetected, we have analyzed approximately 3,700 National Marrow Donor Program (NMDP) Donor/Recipient Pair Retrospective Study Samples with HLA-A DNA sequence-based typing (SBT). Seventeen new HLA-A alleles were detected, with a total of 19 nucleotide substitutions distinguishing these new alleles from their closest HLA-A relatives. Nearly all of the new alleles differ by single nucleotide substitutions; a majority of these substitutions can be explained by gene conversion events but 6 alleles likely originated by point mutation. Fifteen of the 19 nucleotide substitutions translate into amino acid differences in the molecule. Structurally, the inferred amino acid alterations were non-conservative in terms of chemical property, and most substitutions were positioned in 1 or more of the specificity pockets which determine peptide binding. Although these new alleles were identified in a primarily Caucasian sample population, 9 of the 17 new HLA-A alleles were found in samples of non-Caucasoid origin. A new allele detection rate of 1 in approximately 200 individuals in our data set would, therefore, be higher in a non-Caucasoid sample population. In summary, the single nucleotide substitutions that distinguish undetected HLA-A alleles translate into functionally distinct HLA-A molecules. Further studies of the role of HLA-A in transplantation, in disease association, and in evolution must therefore accommodate the discovery of new alleles differing by single nucleotides.

C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells.

Purification of specific class I molecules prior to peptide ligand characterization is complicated by the presence of multiple class I proteins in most cell lines. Immortalized B, T, and tumor cell lines typically express endogenous HLA-A, -B, and -C; and most individuals from which the cell lines are derived are heterozygous at these loci. Antibodies specific for a particular HLA molecule may be used for purification, but allele-specific antibodies can be biased by ligands occupying the peptide-binding groove. Through the use of C-terminal tagging, we have developed a method of soluble HLA production such that downstream purification does not skew the peptide analysis of the examined molecule. Comparison of peptides eluted from HLA class I molecules with and without C-terminal tags demonstrates that addition of a tag does not abrogate the peptide binding specificity of the original molecule. Both pooled Edman sequencing and mass spectrometric sequencing identified no substantial differences in peptides bound by untailed, 6-HIS-tailed, and FLAG-tailed class I molecules, demonstrating that the peptide specificity of a given molecule is not distorted by either tag. This production methodology bypasses problems with isolation of specific molecules and permits ligand mapping and epitope discovery in a variety of pathogen-infected and tumor cell lines.

Alpha-2 domain polymorphism and HLA class I peptide loading.

Diversity within the class I HLA antigen binding groove is positioned to moderate the presentation of peptide ligands. Polymorphism is widely dispersed about the peptide binding groove, and unravelling the functional significance of a given polymorphism requires comparative analysis of peptides presented by class I subtypes differing at the position(s) in question. Previous studies have demonstrated that not all class I polymorphisms act equally, and to determine the impact of substitutions specifically located in the alpha2 domain, peptides purified from B*1501, B*1512, B*1510, and B*1518 were examined by pooled Edman sequencing and comparative mass spectrometric analysis. Molecule B*1512 differs from B*1501 at residues 166 (Glu to Asp) and 167 (Trp to Gly) of the alpha2 domain. The pooled motif and ion mass ligand maps for B*1512 tightly matched those of B*1501, demonstrating that the 166/167 polymorphism between B*1501 and B*1512 has little impact upon ligand presentation. Although the 166/167 polymorphism minimally affects peptide binding preferences, this polymorphism makes B*1512 and B*1501 quite distinct by serology. We then compared the B70 molecules B*1510 and B*1518. The two are almost indistinguishable by serology and differ only by an alpha2 polymorphism at 116. Comparative peptide mapping shows that a Tyr to Ser polymorphism at 116 drastically changes the ligands bound by B*1510 and B*1518; no overlaps could be found. Polymorphisms in alpha2 therefore vary from subtle to extreme in the manner by which they moderate ligand presentation, and serologic crossreactivity did not reflect the ligands presented by these B15 subtypes.

Sequence-based typing provides a new look at HLA-C diversity.

Although extensive HLA-A and HLA-B polymorphism is evident, the true diversity of HLA-C has remained hidden due to poor resolution of HLA-C Ags. To better understand the polymorphic nature of HLA-C molecules, 1823 samples from the National Marrow Donor Program research repository in North America have been typed by DNA sequencing and interpreted in terms of HLA-C diversification. Results show that HLA-Cw*0701 was the most common allele with a frequency of 16%, whereas 28% of the alleles typed as Cw12-18 (serologic blanks). The frequency of homozygotes was 9.8% as compared with previous studies of 18% for sequence-specific primers and 50% for serology. Most startling was the frequency at which new alleles were detected; 19 new HLA-C alleles were detected, representing a rate of approximately 1 in 100 samples typed. These new HLA-C alleles result from 29 nucleotide substitutions of which 4 are silent, such that coding substitutions concentrated about the Ag-binding groove predominate. Polymorphism at the HLA-C locus therefore resembles that at the HLA-A and HLA-B loci more than previously believed, indicating that antigenic stress is driving HLA-C evolution. However, sequence conservation in the alpha-helix of the first domain and a clustering of unique amino acids around the B pocket indicate that HLA-C alleles respond to antigenic pressures differently than HLA-A and HLA-B. Finally, because the samples characterized were predominantly from Caucasians, we hypothesize that HLA-C polymorphism will equal or exceed that of the HLA-A and -B loci as DNA sequence-based typing is extended to include more non-Caucasian individuals.