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Biomedical subjects

C K Hurley

Publications and source records attributed to C K Hurley.

At least 19 recordsLinked to original sources
PubMed1996

Molecular interactions between transfected human TCR, immunodominant myelin basic protein peptide 152-165, and HLA-DR13.

Chimeric TCR transfectants expressing human extracellular sequences and murine intracellular/transmembrane sequences were generated to analyze the trimolecular interaction between myelin basic protein (MBP) autoantigen, HLA, and a TCR isolated from a patient with multiple sclerosis. Chimeric transfectants responded to TCR activation by CD3- and TCRBV22S1-specific mAbs and by superantigen. Additionally, chimeric transfectants responded to autoantigen-specific activation with MBP 152-165 when presented by DR(alpha,beta1*1301) independent of the CD4 adhesion molecule. Transfectants did not respond to Ag presented by other HLA-DR molecules, including the closely related DR(alpha,beta1*1302). In peptide-binding studies with a panel of serial alanine-substituted MBP peptides, HLA contact residues necessary for anchoring MBP 152-165 to DR(alpha,beta1*1301) were also defined: 154 (F), 159 (R), and 162 (R). The chimeric TCR transfectant's differential response to a similar panel of MBP analogues defined residues that interact with the TCR: 153 (I), 155 (K), 156 (L), 160 (D), and 161 (S). Analysis of molecular interactions, such as those described in this work, may be central to developing new strategies for suppressing Ag-specific responses in human autoimmune disease.

Amino Acid Sequence↗
PubMed1996

Diverse usage of human T-cell receptor gene segments in HLA-DR1 allospecific T-cell clones.

T-cell recognition of alloantigen involves both the MHC molecule and its associated peptide ligand. To understand the relationship between the specificity of alloantigen recognition and the structure of TCR molecules, we have investigated TCR gene utilization by sequencing TCR genes from well-defined allospecific T-lymphocyte clones. Alloreactive TLC consisted of a panel of clones primed to recognize DR1-related alloantigens. Our sequencing results revealed extensively diverse, but nonrandom, usage of TCR AV and BV gene segments and essentially no conservation in CDR3 or junctional sequences. Such observations are consistent with allospecific TCR that interact with MHC molecules on a generic level while recognizing specific peptides. They also reduce potential enthusiasm for anti-TCR therapy in allograft rejection.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Cellular crossreactivity. Implications for solid organ transplantation matching.

This study evaluates the cellular crossreactivity among DR11, DR13, and DR8 molecules using TLC reagents generated in reciprocal priming combinations where the responder and stimulator cells express different microvariants of DR11. The large majority of T lymphocyte clones (TLC) derived from such stimulation detect not only the product of the specific DR11 allele expressed by the stimulator but also detect subsets of DR molecules that span serologic specificities. Thus, TLC generated in response to DR(alpha,beta1*1102) detect DR(alpha,beta1*1103) and products of specific DR13, DR8, DR2 and DR4 alleles. Whereas, TLC generated in response to DR(alpha,beta1*1104) detect DR(alpha,beta1*1101), DR(alpha,beta1*1103), and products encoded by specific DR8 and DR2 but not DR13 or DR4 alleles. Since DR11 microvariants cannot be identified serologically, this type of mismatch certainly occurs frequently between DR11 serologically matched donors and recipients. Particularly affected are populations, such as the African American population, that exhibit extensive HLA diversity and exhibit different frequencies of HLA alleles compared with those of the majority of serologically matched cadaveric donors. Rapid methods of DNA-based HLA typing now makes it feasible to utilize this methodology for allele level identificaiton of recipient and donor alleles. Based on the strength of the alloproliferative responses and on the recognition patterns of the TLC reported here, we suggest that retransplant patients might benefit by excluding subsequent donors expressing DR molecules that in vitro demonstrate strong cellular crossreactivity with DR molecules expressed by the previous donor(s) as well as those DR molecules shared with the previous donor(s). Since such a matching schema has the potential to improve retransplant allograft survival, particularly in patients from minority population groups, it should be evaluated clinically.

Alleles↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Large-scale DRB and DQB1 oligonucleotide typing for the NMDP registry: progress report from year 2.

DNA typing of HLA class II alleles of the DRB1/3/4/5 and DQB1 loci using sequence-specific oligonucleotide probes and polymerase chain reaction amplified DNA has been used for the large-scale typing of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the second year of the project (10/1/939/30/94) show the typing continues to be highly accurate, specific, and reliable. The average percent of correctly classified HLA oligotypes (groups of alleles defined by a hybridization pattern with a panel of sequence-specific oligonucleotide probes) based on 9,244 DRB1 and 7,244 DQB1 assignments was 99.8% (range 99.4%100.0%) for DRB1/DRB3/DRB4/DRB5 and 99.8% (range 99.6%100.0%) for DQB1. This level of accuracy is particularly remarkable because the 4,636 DRB quality control samples were tested blindly and could not be distinguished from 57,580 donor samples tested at the same time by the laboratories.

Alleles↗
PubMed1996

Molecular and serological characterization of HLA-B71 in association with different class I haplotypes or in different ethnic groups.

The HLA-B70 antigen is among the most common antigens present in African Americans; however, monospecific serologic reagents defining B70 and its subtypes, B71 and B72, are rare. We have recently reported the molecular characterization of a B71 allele (B*1510) from an African American individual carrying the haplotype HLA-A30, Cw3, B71(w6). In order to better define the degree of polymorphism of molecules carrying the B71 serological specificity in the human population, we have used serology, cDNA sequencing, and PCR/SSOP typing to characterize B71 alleles from additional individuals from different ethnic populations and carrying different class I haplotypes. All carried either B*1510 or B*1518 alleles. Other HLA-B alleles isolated from these individuals (B*5001, B*4901, B*3501, B*3701) were identical to previously reported sequences except for a novel B41 allele (B*4102) identified in one Hispanic individual. This allele has concurrently been identified by Rufer and colleagues in Caucasian individuals. The B*4102 allele differs from B*4101 at codons 95 (Leu/Trp) and 97 (Ser/Arg). In addition, the B*4102 allele differs from B*4101 by two silent substitutions at codons 94 (ACC/ACT) and 99 (TAC/TAT). Since the polymorphic sequence present in B*4102 is also present in other HLA-B alleles (e.g.., B*2707, B*4002, B*0702), it may represent a gene conversion cassette. The allelic diversity at the class I loci and the scarcity of monospecific alloantisera support the importance of the application of molecular based methods to identify HLA class I alleles in matching unrelated donor/recipient pairs for bone marrow transplantation.

Alleles↗
PubMed1996

Novel HLA-B alleles, B*8201, B*3515 and B*5106, add to the complexity of serologic identification of HLA types.

Three class I alleles, B*8201, B*3515 and B*5106, have been described using DNA and cDNA sequencing. The B*8201 allele is most structurally related to B*5602, differing from it by 14 nucleotide substitutions resulting in 5 amino acid differences. The other two alleles, B*3515 and B*5106, differ from their most closely related HLA-B alleles by 2-3 nucleotide substitutions resulting in 1-2 amino acid substitutions, respectively. The majority of nucleotide substitutions marking these new alleles are observed in other HLA-B alleles suggesting that gene conversion and/or reciprocal recombination have created this diversity. All of the amino acid substitutions are predicted to alter the antigen binding site of the HLA-B molecule. The newly defined HLA-B allelic products were originally defined by their unusual serologic reactivity patterns. The B*8201 allelic product is serologically typed as a B "blank" or as a variant of B22 or B45. These patterns and the serologic reactivity of the other newly described allelic products are consistent with the protein sequence homology among specific HLA-B molecules. While serology remains a powerful tool for detecting HLA diversity, alleles generated by events resulting in the sharing of HLA sequence polymorphisms among alleles at a locus will continue to create complexity in the interpretation of typing results.

Alleles↗
PubMed1996

Typing the HLA-B locus by a nested primer approach and oligonucleotide hybridization.

A system for intermediate level identification of the HLA-B locus alleles was devised. This system can be extended to identify individual alleles in any sample. The first step used primers which amplify all HLA-B alleles. This amplicon was subjected to SSOP hybridization to allow intermediate level typing of samples. In the second step, group-specific primers were utilized to obtain specific amplification of groups consisting of a few alleles. The oligotypes within each group were identified by the use of SSOP. The separation of groups of alleles by amplification allowed the use of a limited number of probes to identify oligotypes present in a sample. Additional probes can be added as new alleles are identified, increasing the flexibility of the system. HLA typing software was developed to determine the resolution of the system and to identify HLA oligotypes. PCR-SSOP methods are in wide use and have been extensively validated. The procedures reported here will be relatively easy to implement for large-scale DNA-based typing of the HLA-B locus.

Base Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Evolution in HLA-DRB1 and major histocompatibility complex class II haplotypes of Australian aborigines. Definition of a new DRB1 allele and distribution of DRB1 gene frequencies.

The distribution of HLA-DRB1 alleles was studied in Australian aborigines from different parts of Australia. There were significant differences in the frequencies of DRB1*0412, 1409, and 1410 between the Central Desert and Yuendumu populations and the previously reported Cape York and Kimberley aboriginal populations. A new DRB1 allele, DRB1*1414, present at low frequency in the Central Desert population, was identified. DRB1*1414 appears to be closely related to DRB1*1407 and is proposed to have arisen by intragenic recombination. A novel DR-DQ haplotype, DRB1*1402-DRB3*0101-DQA1*0501-DQB1*0402, was also identified. This haplotype may be ancestral to the DRB1*1409-DQB1*0402 haplotype present in these populations. The presence of alleles and haplotypes apparently confined to Australian aboriginal populations and differences in the distribution of these alleles in different populations suggests that evolution has occurred in the class II region in the period since colonization of Australia, an estimated 50,000 years ago.

Amino Acid Sequence↗
PubMed1995

Differences in peptide binding of DR11 and DR13 microvariants demonstrate the power of minor variation in generating DR functional diversity.

The influence of subtle HLA diversification on antigen binding was explored using murine L-cell transfectants expressing alleles in the DR11/DR13 family and a panel of peptides. The levels of binding among this family of DR microvariants were as diverse as the levels of binding among distantly related DR molecules. Even a single amino acid difference between allelic products had a profound effect on peptide binding. Specific amino acid substitutions, generated using site-directed mutagenesis to alter polymorphic residues at DR beta 32, 37, 57, 58, 67, 71, 86, demonstrated that a specific change within the context of a single DR molecule differed in its effect on the binding of specific peptides. In addition, a specific amino acid substitution had a differential effect on the binding level of a peptide to different DR molecules. Each polymorphic amino acid appeared to play a role in the binding of some peptide. Studies using the amino-terminal portion of the invariant chain CLIP peptide suggested that this peptide may offer varying degrees of competition in the binding of the cellular peptide pool in cells expressing different DR molecules. Finally, the results obtained with two strain-specific peptides from an immunodominant region of a malarial parasite show differential binding to two DR13 molecules, suggesting that immune pressure may promote parasite diversity. A dynamic interaction may exist between pathogens and the immune system shaping the HLA profile in a population. Thus even subtle diversification of the HLA molecules, possibly pathogen driven, can have a substantial effect on peptide binding and immune recognition.

Alleles↗
PubMed1995

A description of a new DR allele, DRB1*1113.

We have discovered a previously unpublished HLA-DRB1 allele, observed in a patient (SB), his mother, and one sibling. The undefined allele gave sporadic positive reactions with sera in the DR52-associated group. SSOPH analysis utilizing both generic and group specific primers and probes also gave ambiguous results. SB typed clearly as a DRB1*0301 (paternal allele) but the DNA from SB also bound probes specific for DRB1*14 and DRB1*11. Sequencing revealed that the undefined allele was similar to a DRB1*14 allele with a segment of sequence found in DRB1*11 alleles. The patient was MLC reactive with donors who express DRB1*0301, *1401 and *0301, *11 and was nonreactive solely to DRB1*0301 (Dw3) homozygous typing cells.

Alleles↗
PubMed1995

Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319.

Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1*1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.

Alleles↗