Increased HLA class I and II diversity as 72 novel alleles are identified in volunteers for the National Marrow Donor Program Registry in 2010.

Seventy-two novel human leukocyte antigen (HLA) class I and class II alleles are described from volunteers for the 'Be The Match Registry®': 17 HLA-A alleles, 12 HLA-C alleles, 31 HLA-B alleles and 12 HLA-DRB1 alleles. Forty-six (≈ 64%) of the 72 novel alleles are single-nucleotide substitution variants when compared with their most homologous allele. Five of these single-nucleotide variants are silent substitutions and one creates a non-expressed allele (B*44:108N). The remaining novel alleles differ from their most similar allele by two to five nucleotide substitutions. One of the novel HLA-C alleles (C*07:150Q) is of questionable expression due to an insertion of 21 nucleotides starting at codon 143 that adds seven amino acids to exon 3. An inter-locus gene conversion may have created the novel allele HLA-A*23:31 that shares its codon differences with HLA-B*07:28. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 116 and 150), HLA-C (codon 114), HLA-B (codons 11, 21, 35, 42, 48, 73, 98 and 170) and HLA-DRB1 (codon 29) loci.

Novel HLA class I and II alleles identified during routine registry typing in 2010.

Twenty-one novel human leukocyte antigen (HLA) class I and class II alleles are described: seven HLA-A alleles, five HLA-C alleles, five HLA-B alleles and four HLA-DRB1 alleles. Seventeen (∼81%) of the 21 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants cause an amino acid substitution, while eight are silent substitutions. The remaining novel alleles differ from their most similar allele by two to three nucleotide substitutions. One novel HLA-C locus allele encodes an amino acid change at codon 10 that previously has not been reported to be polymorphic for this locus. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 24 and 156) and HLA-B (codons 40 and 115) loci.

Ninety-six novel HLA class I and II alleles identified in volunteers for the National Marrow Donor Program Registry in 2009.

Ninety-six novel human leukocyte antigen (HLA) class I and class II alleles are described from volunteers for the 'Be The Match Registry®': 15 HLA-A alleles, 11 HLA-C alleles, 36 HLA-B alleles and 34 HLA-DRB1 alleles. Sixty-eight (∼71%) of the 96 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Twenty-three of these single nucleotide variants are silent substitutions and one creates a non-expressed allele (B(*) 27:59N). The remaining novel alleles differ from their most similar allele by two to five nucleotide substitutions. One of the novel HLA-A alleles (A(*) 11:52Q) is of questionable expression because of a deletion of codon 11. Three of the novel alleles encode amino acid changes at codons 63 (HLA-C), 88 (HLA-B) and 79 (HLA-DRB1) which have not previously been reported to be polymorphic in these loci. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 115, 149 and 168), HLA-C (codon 90), HLA-B (codons 23, 44, 70, 120 and 153) and HLA-DRB1 (codon 88) loci.

Seventy-eight novel HLA class I and II alleles identified during routine registry typing in 2008 and 2009.

Seventy-eight novel human leukocyte antigen (HLA) class I and class II alleles are described: 19 HLA-A alleles, 30 HLA-C alleles, 21 HLA-B alleles, 7 HLA-DRB1 alleles and 1 HLA-DPB1 allele. Eight (∼10%) of the novel alleles were found in more than one individual and may be more common in the population. Sixty-two (∼80%) of the 78 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Twelve of these single nucleotide variants are silent substitutions and one creates a null allele (C*08:26N). One of the novel HLA-C alleles, C*03:58, is a hybrid that likely resulted from an intra-locus recombination. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Four of the novel HLA-C alleles encode amino acid changes at codons 41, 42, 55 or 200 which have not previously been reported to be polymorphic. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 55 and 120), HLA-C (codons 151, 153 and 176), HLA-B (codons 31 and 84) and HLA-DRB1 (codon 47) loci.

Sixty-five novel alleles at the HLA-A, -B, and -DRB1 loci identified from National Marrow Donor Program volunteer donors.

Sixty-five novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the 'Be The Match Registry': 29 HLA-A alleles, 24 HLA-B alleles, and 12 HLA-DRB1 alleles. Eight (∼12%) of the novel alleles were found in more than one individual and may be more common in the population. Forty (∼62%) of the 65 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Two of these single nucleotide variants are silent substitutions and one creates a null allele. The remaining novel alleles differ from their most similar allele by 2-10 nucleotide substitutions. Some of the novel alleles encode amino acid changes at codons not previously reported to be polymorphic, such as codons 23, 93, 129, and 155 in HLA-A alleles and codon 3 in HLA-B alleles.

Description of novel class I alleles encountered during routine registry typing in 2007.

Twenty-six novel human leukocyte antigen (HLA) class I alleles are described: 3 HLA-A alleles, 19 HLA-B alleles and 4 HLA-C alleles. Only one of the novel alleles (HLA-B*0753) was found in multiple individuals and likely is not uncommon in the population. Nineteen (approximately 70%) of the 26 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Four of these single nucleotide variants are silent substitutions, and one creates a null allele. The remaining novel alleles differ from their most similar allele by two to six nucleotide substitutions. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-B lows (codons 30, 67 and 72), while HLA-Cw*0347 encodes an amino acid change at a position not previously reported to be polymorphic for this locus.

TNF, LTA and TGFB1 genotype distributions among acute graft-vs-host disease subsets after HLA-matched unrelated hematopoietic stem cell transplantation: a pilot study.

Cytokine single nucleotide polymorphisms and consequent production levels have been associated with acute graft-vs-host disease (aGVHD) development. The aim of this pilot study was to determine whether polymorphisms in tumor necrosis factor (TNF), lymphotoxin alpha (LTA) and transforming growth factor beta 1 (TGFB1) showed any association with aGVHD severity. Novel alleles and polymorphisms were identified for each cytokine locus. Genotype distributions were examined in 38 recipient-donor pairs (all chronic myelogenous leukemia in the first chronic phase) with either low-grade (grades 0-I) or high-grade (grades III-IV) aGVHD. Although no significant differences were found, some trends were noted in genotype distributions among aGVHD-grade groups. Power calculations determined that substantially more pairs would be required to show significant associations in distributions among aGVHD-grade groups.

Characterization of 104 novel alleles at the HLA-A, -B, and -DRB1 loci from National Marrow Donor Program volunteer donors.

One hundred and four novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the National Marrow Donor Program: 37 HLA-A alleles, 37 HLA-B alleles, and 30 HLA-DRB1 alleles. Seventeen ( approximately 16%) of the novel alleles were found in multiple individuals and likely are relatively common in the population. Seventy-two ( approximately 69%) of the 104 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants are silent substitutions and three create null alleles. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Some of the novel alleles encode amino acid changes at positions not previously reported to be polymorphic, such as codons 6 and 11 in HLA-A alleles and codons 5, 105, and 141 in HLA-B alleles. Interestingly, one of the novel HLA-DRB1 alleles (*1471) has a change that is not the typical glycine/valine dimorphism at codon 86, which plays a key role in peptide binding to DR molecules. This is only the second DRB1 allele described that encodes an amino acid other than glycine or valine at this position.

Characterization of new HLA-B and -DRB1 alleles from Singapore.

Six novel alleles, three human leukocyte antigen (HLA)-B and three HLA-DRB1 alleles, are described. Five of the variants are single nucleotide substitutions from their most homologous allele, of which three result in amino acid changes (B*3572, *9509 and DRB1*1157) and two are silent substitutions (B*370103 and DRB1*150204). DRB1*0462 differs by three nucleotide substitutions that alter two amino acids.

Novel alleles at the HLA-DRB1 and -DQB1 loci.

Twelve novel human leukocyte antigen class II alleles are described; eight DRB1 alleles and four DQB1 alleles. Nine of the variants are single nucleotide substitutions from their most homologous allele, of which six result in amino acid changes (DRB1*0459, *1156 and *1522; DQB1*0205, *0320 and *0321) and three are silent substitutions (DRB1*030105 and *040304, and DQB1*030104). The remaining alleles (DRB1*0906, *1464 and *1468) differ from their most similar alleles by two to three nucleotide substitutions which alter one to two amino acids.

Twenty-three novel alleles increase diversity at the HLA-C locus.

Twenty-three novel HLA-C alleles are described. Nine of the new alleles are single-nucleotide substitutions from their most homologous allele of which seven result in amino acid changes (Cw*0327, *0508, *0514, *0613, *0735, *0739 and *1517) and two are silent substitutions (Cw*030305 and *070107). The remaining 14 alleles (Cw*0113, *0207, *0212, *0216, *0318, *0411, *0417, *0512, *0722, *0733N, *1216, *1218, *1515 and *1607) differ from their most similar alleles by 2-4 nucleotide substitutions that result in 1-3 amino acid differences.

Expanding diversity at the HLA-B locus: 28 novel HLA-B alleles.

Twenty-eight novel human leukocyte antigen-B alleles are described: one B*07 (*0745), two B*08 (*0826 and *0831), one B*27 (*2731), four B*35 (*350106, *350402, *3566, and *3568), two B*37 (*370102 and *3711), two B*38 (*380102 and *3813), two B*39 (*3935 and *3939), one B*41 (*4108), one B*42 (*4209), two B*44 (*4444 and *4448), two B*48 (*480302 and *4815), one B*51 (*5140), one B*55 (*5523), one B*56 (*5617), two B*57 (*570103 and *5710), and three B*15 (*9505, *9510, and *9519). Sixteen of the variants are single-nucleotide substitutions from their most homologous allele, of which 10 result in amino acid changes (B*0745, *0831, *3813, *3935, *3939, *4815, *9505, *9509, *9510, and *9519) and 6 are silent substitutions. The remaining alleles (B*0826, *2731, *3566, *3568, *3711, *4108, *4209, *4444, *4448, *5140, *5523, *5617, and *5710) differ from their most similar alleles by two to seven nucleotides substitutions, altering from one to five amino acids.

Thirty-two novel HLA-A alleles identified during intermediate resolution testing.

Thirty-two novel human leukocyte antigen-A alleles are described: four A*01 (*0110, *0112, *0113 and *0117), four A*02 (*0263, *0280, *0292 and *9201), two A*03 (*0316 and *0325), four A*11 (*111502, *1117, *1122 and *1123), five A*24 (*2441, *2450, *2455, *2456 and *2457), one A*26 (*2627), two A*29 (*2909 and *2914), two A*30 (*3013 and *3016), one A*32 (A*3213), two A*34 (*3407 and *3408) and five A*68 (*6828, *6829, *6830, *6831 and *6834). Seventeen of the variants are single-nucleotide substitutions from their most homologous allele, which results in amino acid changes (A*0117, *0263, *0292, *0316, *0325, *1122, *1123, *2455, *2456, *2457, *2627, *2909, *3016, *3407, *6828, *6831 and *6834), and only one is silent substitution (A*111502). The remaining alleles differ from their most similar alleles by two to six nucleotide substitutions.

Novel polymorphisms and the definition of promoter 'alleles' of the tumor necrosis factor and lymphotoxin alpha loci: inclusion in HLA haplotypes.

Tumor necrosis factor (TNF) and lymphotoxin alpha (LTA) influence a variety of cellular responses and play a complex role in the immune response. Several single nucleotide polymorphisms (SNPs) have been reported in these major histocompatibility complex (MHC)-linked loci; however, a comprehensive examination of polymorphisms in the promoter regions of TNF and LTA has not been carried out and was undertaken here. Seven novel SNPs in LTA were identified by sequence analysis of 69 samples. Eight novel TNF alleles and 16 novel LTA alleles were designated. The TNF alleles clustered into two closely related groups, while the LTA alleles clustered into three distinct groups using phylogenetic and percentage difference analyses. A total of 52 unique TNF-LTA-HLA haplotypes are reported. There appear to be some associations between TNF/LTA alleles and HLA haplotypes, but not with specific HLA alleles. The majority of the SNPs appear to be randomly associated within and between the two loci except for the LTA SNPs at -293, +81 and +369. These observations may provide an explanation for the oftentimes contradictory results of studies associating individual cytokine gene SNPs with expression level phenotypes, HLA and disease.

Specific recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells.

The CD94/NKG2 receptors expressed by subpopulations of NK cells and T cells have been implicated as receptors for a broad range of both classical and nonclassical HLA class I molecules. To examine the ligand specificity of CD94/NKG2 proteins, a soluble heterodimeric form of the receptor was produced and used in direct binding studies with cells expressing defined HLA class I/peptide complexes. We confirm that CD94/NKG2A specifically interacts with HLA-E and demonstrate that this interaction is dependent on the association of HLA-E with peptide. Moreover, no interaction between CD94/NKG2A and classical HLA class I molecules was observed, as assayed by direct binding of the soluble receptor or by functional assays using CD94/NKG2A+ NK cells. The role of the peptide associated with HLA-E in the interaction between HLA-E and CD94/NKG2A was also assessed. All class I leader sequence peptides tested bound to HLA-E and were recognized by CD94/NKG2A. However, amino acid variations in class I leader sequences affected the stability of HLA-E. Additionally, not all HLA-E/peptide complexes examined were recognized by CD94/NKG2A. Thus CD94/NKG2A recognition of HLA-E is controlled by peptide at two levels; first, peptide must stabilize HLA-E and promote cell surface expression, and second, the HLA-E/peptide complex must form the ligand for CD94/NKG2A.

NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor.

CD94 is a C-type lectin expressed by natural killer (NK) cells and a subset of T cells. Blocking studies using anti-CD94 mAbs have suggested that it is a receptor for human leukocyte antigen class I molecules. CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s). This report shows that NKG2A, a 43-kD protein, is covalently associated with CD94 on the surface of NK cells. Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94. Analysis of NK cell clones showed that NKG2A was expressed in all NK cell clones whose CD16-dependent killing was inhibited by cross-linking CD94. The induction of an inhibitory signal is consistent with the presence of two immunoreceptor tyrosine-based inhibitory motifs (V/LXYXXL) on the cytoplasmic domain of NKG2A. Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.

The impact of DR3 microvariation on peptide binding: the combinations of specific DR beta residues critical to binding differ for different peptides.

HLA-DR molecules are a group of highly polymorphic glycoprotein heterodimers that present peptide antigens to T lymphocytes for immune surveillance. To assess the significance of limited polymorphism on the functional differentiation of DR molecules, the binding of several immunogenic peptides to the DR3 microvariants [DR(alpha, beta 1*0302) and DR(alpha, beta 1*0301)] and to mutants of these DR3 molecules was examined. This analysis has shown that each residue (DR beta 26, DR beta 28, DR beta 47, and DR beta 86), which differentiates these two DR3 molecules, contributes to their functional distinction and that the relative contribution of each residue varies for different peptide/DR3 complexes. For example, DR beta 28 and DR beta 86 controlled the mycobacterium tuberculosis 65-kD heat shock protein peptides 3-13 and 4-15 (HSP) binding specificity to DR (alpha, beta 1*0301). [HSP does not bind to DR(alpha, beta 1*0302)], whereas DR beta 26, DR beta 28, and DR beta 86 controlled the influenza hemagglutinin peptide 306-318 (HA) binding specificity to DR(alpha, beta 1*0302). [HA does not bind to DR(alpha, beta 1*0301).] In comparison, DR beta 86 alone controlled the binding level difference of sperm whale myoglobin peptide 132-151 (SWM) and of myelin basic protein peptide 152-170 (MBP) [both bind to DR(alpha, beta 1*0301) at levels five times greater than to DR(alpha, beta 1*0302)] to the DR3 molecules. Although not critical, additional DR beta residues influenced the binding level of individual peptides of each of the DR3 molecules and, again, the combinations of these residues differed for different peptide/DR3 complexes. These data showed that individual DR residues vary in their relative contribution to the interaction between a specific DR molecule and different peptides and that limited polymorphism can create substantial differences in the peptide binding profiles among DR molecules.

The relative importance of individual DR binding motif positions as defined by peptide anchor analysis of influenza hemagglutinin peptide 306-318 and human myelin basic protein peptide 152-165 binding to several DR molecules: definition of a common extended DR binding motif.

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306-318 and human myelin basic protein (MBP) 152-165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(alpha, beta 1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(alpha, beta 1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(alpha, beta 1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (alpha, beta 1 *0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.

Microvariation creates significant functional differences in the DR3 molecules.

Two DR3 molecules differ by four amino acids whose side chains point into the DR antigen-binding groove. To begin to assess the role of microvariation on DR3 function, DRB1*0302 residues were replaced with DRB1*0301 residues at beta-chain positions 26, 47, 86, and 47 plus 86. Murine fibroblast cell lines expressing DR(alpha, beta 1*0301), DR(alpha, beta 1*0302), and the four mutant 0302 molecules were examined for alloproliferative DR(alpha, beta 1*0302)-specific TLC stimulation and peptide binding. Changing position 26 had the most profound effect on T-cell recognition (seven of nine TLCs did not respond). Two TLCs did not respond to the mutant 0302V86 molecule and four TLCs that did respond to this mutant lost responsiveness when positions 47 and 86 were mutated together. These data suggest that each of these variant residues, including position 47, influence T-cell recognition. Surprisingly, none of the mutations had an effect on the absolute binding of HA 307-319 (DR[alpha, beta 1*0302] specific) and HSP 3-13 (DR[alpha, beta 1*0301] specific); however, the mutant 0302 molecules changed at position 86 (glycine to valine) consistently bound HA 307-319 at significantly higher levels than DR(alpha, beta 1*0302). These data for position 86 are in contrast to other DR molecules and indicate that peptide contact residues for a specific DR molecule cannot be predicted based on binding results obtained with other DR molecules. These data suggest that each of these variant groove residues, although not accessible to the TCR, contribute to the significant functional differences between the DR3 microvariants through subtle influences on the DR3-peptide complex.