Noriko Steiner

The DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci

The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population, in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a DQw4 allelic product. This study characterizes the DR subregion of that haplotype. cDNA sequence analysis has revealed a DR beta gene which differs at several positions from previously described DR3 beta 1 genes. It is postulated that a gene-conversion-like event with a DRw52 beta gene as donor has generated some of these differences. The haplotype carries a DRw52a allele as defined by oligonucleotide hybridization studies. DNA restriction fragment analysis using a family and several unrelated individuals has allowed us to identify DR alpha and beta fragments associated with the DR3(w18),DQw4 haplotype. The most striking observation is that the DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci. Several genetic mechanisms including reciprocal recombination, gene conversion, and point mutation were involved in generating the differences between these haplotypes. Once established, the DR3(w18),DQw4 haplotype appears to be relatively stable in the population.

Allelic Variation in KIR2DL3 Generates a KIR2DL2-like Receptor with Increased Binding to its HLA-C Ligand

Although extensive homology exists between their extracellular domains, NK cell inhibitory receptors killer Ig-like receptor (KIR) 2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared with KIR2DL3*001, a low-affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 toward that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations.

Killer Cell Immunoglobulin-Like Receptors (KIR) Typing By DNA Sequencing

DNA sequencing is a powerful technique for identifying allelic variation within the natural killer cell immunoglobulin-like receptor genes. Because of the relatively large size of the KIR genes, each locus is amplified in two or more overlapping segments. Sanger sequencing of each gene from a preparation containing one or two alleles yields a sequence that is used to identify the alleles by comparison with a reference database.

Limited allelic diversity of stimulatory two-domain killer cell immunoglobulin-like receptors.

Genomic sequencing was used to characterize most of the coding regions of the five two-domain stimulatory killer cell immunoglobulin-like receptor (KIR) loci from 80 unrelated, primarily Caucasian, individuals. Specific loci were present in from 26% (KIR2DS3) to 98% (KIR2DS4) of individuals. The number of known alleles present varied from one (KIR2DS1, KIR2DS5) to five (KIR2DS4). The frequencies of loci and alleles were similar to observations made in populations of European and Asian ethnicities. New alleles were found at 2DS1 (*00202, *00302, *005, *006, *007) and 2DS4 (*008) loci.

HLA-B alleles associated with the B15 serologically defined antigens.

Cells expressing HLA molecules in the B15 family were identified by serologic typing in routine testing of volunteer donors of various ethnic backgrounds for a bone marrow registry. DNA sequencing was used to identify HLA-B15 alleles associated with each serologic type and to examine the diversity within the B15 antigen family. Alleles which appeared predominantly in each B15 serologic cluster included: B15 with no defined serologic subdivision (B*1501), B62 (B*1501), B63 (B*1516, B*1517), B75 (B*1502, B*1521), and B76/77 (B*1513). Other B*15 alleles were also found associated with the serotypes and some of these alleles (e.g., B*1501 and B*1516) were found in two or more serologic clusters illustrating the complexity of this family. The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype. Five new B*15 alleles (B*1530, B*1531, B*1533, B*1534, B*1535) and 5 other new HLA-B alleles (B*38022, B*3910, B*4010, B*51012, and B*5108) were also identified.

Extracellular domain alterations impact surface expression of stimulatory natural killer cell receptor KIR2DS5

In the human killer cell immunoglobulin-like receptors, KIR2DL2, and KIR2DL3, a triad of amino acids in the D1 domain interact to stabilize protein structure. Substitution of any one of these residues caused significant loss of cell surface expression. Although KIR2DS4 and KIR2DS5, two homologous receptors, differ for this triad, flow cytometry analysis of NK and T cell lines transfected with stimulatory KIR genes KIR2DS4 (allele *001) and KIR2DS5 (allele *002) demonstrated cell surface expression. For KIR2DS5, restoration of the triad sequence increased surface expression. Further studies of the receptor encoded by KIR2DS5*002 showed both mature and immature protein isoforms upon gel electrophoresis coupled with surface biotinylation or deglycosylation. In contrast, the KIR2DS5*001 allelic product was not expressed on the cell surface of either NK or T cells and exhibited only a single immature isoform upon gel electrophoresis. Site-directed mutagenesis demonstrated that absence of the KIR2DS5*001-encoded protein at the cell surface was imparted primarily by two amino acid polymorphisms in the D2 domain. Analysis using molecular dynamics simulations suggested that the substitution of a proline for a serine at residue 111 or the substitution of a serine for a phenylalanine at residue 164 caused destabilization of the domain structure and intracellular retention. A third polymorphism at residue 174 impacted the level of KIR2DS5 surface expression. This is the first description at a stimulatory KIR locus of the impact of specific amino acid variations on receptor maturation and the level of surface expression.

Diversity of alleles encoding HLA-B40: relative frequencies in United States populations and description of five novel alleles

The frequency of each B*40 allele was determined by DNA sequencing in four major United States populations: Caucasians, African Americans, Asians/Pacific Islanders, and Hispanics. Thirty-two individuals from each ethnic group, who were previously described serologically as B40, B60, or B61, were randomly selected out of a pool of 82,979 unrelated individuals for allele characterization. Out of nine different B*40 alleles identified in this study, B*4001 and B*4002 were the two most frequent B*40 alleles in all the population groups. B*4001 was the primary B*40 allele seen in Caucasians (83%) and African Americans (76%), while B*4002 was found in the majority of Hispanics (62%). The distributions of both alleles were comparable in the Asian/Pacific Islander population. These two alleles were the only B*40 alleles detected in Caucasians while four to five additional B*40 alleles were seen in the other population groups. The other B*40 alleles detected in this study included: B*4003 and B*4010 in Asian/Pacific Islanders; B*4012 and B*4016 in African Americans; and B*4004, B*4006, and B*4027 in Hispanics. Analysis revealed significant differences between Hispanics and all other groups as well as between African Americans and Asian/Pacific Islanders. This report also describes five novel B*40 alleles: B*4019, B*4020, B*4024, B*4027, and B*4028.

Investigation of killer cell immunoglobulin‐like receptor gene diversity in KIR3DL1 and KIR3DS1 in a transplant population

Several overlapping amplicons were used to obtain the sequence of genomic DNA covering most of the coding regions of KIR3DL1 and KIR3DS1 from a family and 77 bone marrow transplant patients and their unrelated donors. Alleles 3DL1*00101 and *002 were most frequently observed in addition to 12 other known 3DL1 alleles. A single 3DS1 allele, 3DS1*01301, was identified in the 31 of 32 individuals carrying this gene. Two new alleles, 3DL1*01702 and 3DS1*058, were characterized. Three samples appeared to carry the duplicated killer cell immunoglobulin-like receptor (KIR) haplotype observed in other studies based on the presence of 3DS1 and two 3DL1 alleles. Additionally, one sample appeared to carry a novel KIR haplotype containing one 3DL1 and two 3DS1 alleles.

Allelic variation of killer cell immunoglobulin-like receptor 2DS5 impacts glycosylation altering cell surface expression levels

Natural killer cell stimulatory receptor gene, KIR2DS5, is polymorphic. While KIR2DS5*002 is most frequently observed, other alleles have also been found. The proteins encoded by these alleles (KIR2DS5*002-*009) are expressed at varying levels on the surface of NKL and Jurkat transfectants. Gel electrophoresis of all allelic products showed two isoforms which differ in the extent of maturation of N-linked glycosylation. These isoforms differed in intensity and molecular weight among the allelic products. Site-directed mutagenesis was used to identify polymorphic variation at residues 123 and 157 as key in altering glycosylation and levels of surface expression.

Promoter variants of KIR2DL5 add to diversity and may impact gene expression

Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encompassing the putative proximal promoter and the coding region was used to identify KIR2DL5 alleles from 77 unrelated Caucasian individuals. PCR and sequencing were used to link each new allele to its neighboring KIR locus to identify 2DL5A or 2DL5B loci. Allele 2DL5A*001 was found in 24 of the 37 2DL5 positive individuals; 2DL5B*0020101 and 2DL5A*0050101 were also observed. Two new alleles, 2DL5B*008 and 2DL5B*009, contained substitutions altering the amino acid sequence of the leader and transmembrane region, respectively. Two other novel alleles, 2DL5B*0020102 and 2DL5A*0050102, contained alterations of the 5′ upstream region, bringing the number of unique promoter sequences to six. Promoter activity of the alleles was compared using luciferase reporter assays. Our results support those recently published, in which the promoter of 2DL5B*0020101 was shown to be more active in vitro compared to 2DL5A*001, and also provide additional information about the transcriptional activity of the promoters of the newly characterized alleles related to two altered transcription factor binding sites.