The chicken B-complex in commercial pure lines: comparison of polymorphisms defined by serotyping and DNA analysis.
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Animals↗
Biomedical subjects
M G Tilanus
Publications and source records attributed to M G Tilanus.
Biochemical polymorphism of B-G and B-F antigens of the chicken major histocompatibility complex.
To refine and improve the definition of the polymorphism of the chicken B-complex at the level of expressed products we used a biochemical approach, including SDS-PAGE and/or IEF. In chickens this approach appeared as successful as in several other species. Chickens of a commercial pure White Leghorn line were typed for B-F (class I) and B-G (class IV) major histocompatibility (B) complex antigens by two methods. Serotyping was performed by direct haemagglutination using alloantisera raised against erythrocytes. Biochemical analysis involved immunoprecipitations from radiolabelled cells using monoclonal antibodies recognizing monomorphic determinants of B-F (F21-2) or B-G (18-6G2) followed by analysis on SDS-PAGE and/or IEF. Specific banding patterns of B-G proteins were observed by 1D-IEF. Moreover, separation on molecular weight of B-G proteins also enabled a precise identification of these B-G types. Within the White Leghorn line there were no discrepancies between serotypes and typing patterns for B-G by either IEF or SDS-PAGE. Analysis of B-F immunoprecipitates enabled us to identify B-F polymorphism separately of B-G. Identical B-F patterns were found with two different B-G types, indicating that in these chickens recombinations between B-F and B-G have occurred.
Animals↗
Polymorphism and complexity of HLA-DR: evidence for intra-HLA-DR region crossing-over events.
HLA-DR molecules were isolated from HLA-DR3, -5, and -w6 positive homozygous B-cell lines by immunoprecipitation with monoclonal antibodies and analyzed by gel electrophoretic techniques. DNA isolated from the same cell lines was digested with the restriction enzyme Taq I and hybridized with a DR beta full-length cDNA probe. We demonstrated that certain DR beta I alleles are found in combination with different DR beta III alleles as defined by Southern blotting, protein chemistry, a functional assay using purified protein derivative-specific T-cell lines, and, in one case, also alloreactive T-cell reagents. Our results indicate that within the family of HLA-DRw52-associated haplotypes DR beta chain genes may have been transferred from one haplotype to another. The implications of these findings are discussed.
Alleles↗
Correlations between polymorphisms at the DNA and at the protein level of DRw52 haplotypes, revealed with a variety of techniques.
LB-Q1 and LB-Q4 are two subtypes of DRw52, defined by proliferative T-cell clones. These subtypes represent a polymorphism of the DR beta III gene. Similar subtypes of DRw52 can be defined by oligonucleotide typing, serology and RFLP analysis. In the present study we compared these typing techniques on a panel of 22 HLA-D homozygous, DRw52-positive typing cells. All typing techniques correlated very well. Three subtypes of DRw52 could be identified. Our results show that typing for cellularly defined structures can be done with a variety of noncellular techniques. This observation has important implications for matching in unrelated bone marrow transplantation and for disease association studies.
DNA↗
Molecular localization of LB-Q1, a DRw52-like T-cell recognition epitope and identification at the genomic level of associated shared hybridizing fragments.
In this paper we report on the molecular localization of LB-Q1, a supertypic HLA class II determinant which we previously identified by the use of proliferative T cells. The population distribution shows that each of the DRw52 associated specificities DR3, DR5, and DRw6 may occur with and without LB-Q1. DNA from nine DR3, six DR5, and 14 DRw6 homozygous B-cell lines were digested with the enzymes TaqI, EcoRI, and PvuII. Using a DR beta cDNA probe, shared hybridizing fragments were observed that correlate completely with the presence or absence of LB-Q1. T-cell recognition of LB-Q1 can be blocked with a monoclonal antibody (7.3.19.1) which in some haplotypes selectively reacts with the DR beta III chains, but cannot be blocked with a monoclonal antibody (I-LR2) reacting in those same haplotypes exclusively with DR beta I chains. Therefore, LB-Q1 maps to the DR beta III molecule. These data suggest the occurrence of relatively frequent previous recombinations between the two DR beta chain genes present in DRw52 haplotypes.
Antibodies, Monoclonal↗
HLA class II DNA analysis by RFLP reveals novel class II polymorphism.
Polymorphism of the HLA-D/DR region has been defined by serologic and cellular methods. Additionally, protein and DNA analyses not only confirmed and refined the definition of the established polymorphisms but also revealed further polymorphisms for which no serologic or cellular correlate are known (yet). To study these in more detail, we analyzed the banding patterns obtained from Southern blot hybridizations with DR beta and DQ alpha cDNA probes. Specific fragments reflecting already defined polymorphisms could be identified. A refined HLA-D/DR definition based upon the presence of DNA subtypes could be introduced. Fragments have also been identified that are associated with the DR/Dw specificities. Moreover, individuals with different DR types may also share fragments in hybridization assays. These shared hybridizing fragments (SHFs) are, for instance, found in individuals typed as DR4, DR5, DR7, and DRw8. In total, 20 SHFs were found using two restriction enzymes and the DR beta and DQ alpha cDNA probes. Some of these SHFs correlate with antigenic determinants defined by broad reacting alloantisera, such as DRw52, but for 14 of these SHFs, no serologic equivalent has been found so far. Thus, SHFs reflect a conservation at the DNA level of the HLA class II region, which suggests that the polymorphic class II genes may be more conserved than previously thought. The possible biologic implications of conserved sequences in the HLA class II genes will be discussed.
Cell Line↗
Polymorphisms within the HLA-DRw6 haplotype. III. DQ alpha and DQ beta polymorphism associated with HLA-D.
We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQ alpha and DQ beta polymorphism. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQ alpha and beta chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.
Antibodies, Monoclonal↗
The HLA-Dw HAG specificity is defined by DR beta cDNA hybridization as a hybrid haplotype carrying DR5 and DRw6 determinants.
The serological and cellular class II definition of HTC9W1802 HAG was difficult to establish. RFLP obtained with a DR beta cDNA probe indicated that HAG could be a hybrid haplotype composed of fragments characteristic for DR5 and DRw6 specificities.
DNA↗
Two alleles detected for HLA-DZ alpha on the basis of a polymorphic restriction site in the third exon.
The restriction enzyme ApaI has been used to define Restriction Fragment Length Polymorphism (RFLP) within the DZ alpha gene. Digestion of genomic DNA of 96 individuals with ApaI reveals the existence of two different patterns. Both homozygous and heterozygous individuals were detected. Hardy-Weinberg analysis indicates that DZ alpha is part of a di-allelic system with allele frequencies of 50%. DZ alpha polymorphism in the population studied does not correlate with serologically determined class I and HLA-DR, DQ and cellular DP typing data. A limited family study demonstrates that DZ alpha polymorphism segregates with HLA-class II phenotypes.
Alleles↗
RFLP of the HLA-DQ alpha region: a diallelic DX alpha polymorphism, not linked to DR and DQ specificities.
HLA-DQ alpha DNA polymorphism was studied by Southern blot analysis in a panel of 117 individuals, consisting of 56 randomly selected and 61 HLA-DR homozygous individuals. Hybridizing fragments representing DQ alpha genes correlate with DR specificities owing to linkage disequilibrium between DQ and DR. Two fragments representing DX alpha genes were identified with the restriction enzymes PvuII and TaqI and a DQ alpha cDNA probe. The two fragments of PvuII-DQ alpha hybridization (a 7200 and a 7000 basepair fragment) and the two of TaqI-DQ alpha (2200 and 1900 basepairs) have an identical distribution in the panel, and reveal gene frequencies of 49.1% and 50.9%; they behave as alleles of the putative DX alpha locus. The panel study shows that the DX alpha fragments are not linked with the DR or DQ specificities but segregate along with HLA as shown in family studies.
Alleles↗
HLA-DO polymorphism associated with resistance to type I diabetes detected with monoclonal antibodies, isoelectric point differences, and restriction fragment length polymorphism.
A new HLA-DQ-related genetic system with two alleles, 2B3 and TA10, defined serologically by MAbs and alloantisera, showed an almost perfect correlation with charge differences on DQ beta molecules, as well as with two polymorphic DNA fragments hybridizing with a DQ beta probe and various restriction enzymes on a panel of 14 DR4+ homozygous typing cells. It was therefore concluded that the serologically defined alleles 2B3 and TA10 are coded by the DQ beta gene and situated on the HLA-DQ beta chain. This 2B3/TA10 polymorphism is independent of HLA-D and segregates with HLA in families. The TA10 allele appears to be a new marker for resistance to type I diabetes, which is independent from the known resistance marker DR2, whereas no association was observed between this DQ beta polymorphism and rheumatoid arthritis.
Antibodies, Monoclonal↗
Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals.
In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR beta, -DQ alpha, and -DQ beta cDNA probes in Southern blot analysis. Three subtypes are found using the DR beta cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, Dw10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ alpha cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ beta detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ alpha and DQ beta cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ alpha probe, probably due to DX polymorphism. DR beta RFLP divides the LB14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.
Cell Line↗
An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses.
HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.
Collodion↗
Polymorphisms within the HLA-DRw6 haplotype. I. Restriction fragment length variation and its correlation with serology.
The Dw6/DRw6 complex, one of the MHC class II specificities that can be defined by cellular techniques and by serology, probably has one or more immunoregulatory functions. To obtain information on the molecular structure of the DRw6 region, we studied several DRw6 homozygous cell lines, of which three were of consanguineous origin. DNA-DNA hybridization comprised the use of seven restriction enzymes in combination with three DR beta cDNA probes. The obtained results were compared with similar analyses of an HLA homozygous cell panel, expressing DR1-w8 specificities. This comparison indicated that in DRw6 homozygous individuals the coding potential for DR beta chains resembles closely that of all other DR specificities, thus identifying DRw6 as a regular DR region. In addition, we found a restriction fragment length pattern unique for DRw6, indicating the possibility to type for DRw6 by DNA-DNA hybridization. Comparisons within the DRw6 cell panel revealed the occurrence of several HLA class II DNA subtypes. These subdivisions partly correlated with serologically obtained reaction patterns. No correlation, however, could be observed between the different DNA subtypes and cellular reaction patterns as obtained by MLC and T cell cytotoxicity.
Antigen-Antibody Reactions↗
The effect of serologically defined major histocompatibility complex haplotypes on Marek's disease resistance in commercially bred White Leghorn chickens.
In commercial pure white leghorn lines, A, B, and C, the effects on resistance against a virulent strain of Marek's disease virus were assessed for B19 and B21 haplotypes of the chicken major histocompatibility complex. B haplotypes were identified by direct hemagglutination using alloantisera raised against erythrocyte antigens. In homozygous B21 female chicks from lines A and B, mortality upon challenge with virus was 16% and 9%, respectively; in B19 chicks, mortality was 42% and 60%, respectively. Intermediate mortality was observed in heterozygous B19/B21 birds. When line A and B hens were crossed with B15/B15 or B5/B19 cocks from line C, differences between B19 and B21 were significant only in the progeny from B5/B19 sires. Therefore, it was concluded that selection for major histocompatibility complex-associated disease resistance markers may be useful only when B haplotypes complement each other in commercial line crosses and when interactions with genetic background do not severely obscure the differential haplotype effects, as are observed within pure lines.
Animals↗
Cytotoxic T lymphocytes infiltrating the human cardiac allograft show a restriction in T-cell receptor V beta gene usage: a study on serial biopsy and blood specimens.
T lymphocytes were propagated in vitro from endomyocardial biopsy specimens that were obtained weekly from four patients during the first 2 to 3 months after heart transplantation. The culture was performed in the presence of recombinant interleukin-2 and interleukin-4, with or without mitogen, in which especially CD8+ donor-specific cytotoxic T cells expanded. These cells, presumably reflecting an in vivo activated population, could even be cultured from biopsy specimens without histopathologic signs of rejection. A preferential expression of T cell receptor V beta gene families was found in these T-cell lines. This finding is in contrast with the heterogenous expression in peripheral blood T cells of the same patient. The restriction in V beta gene family expression was substantiated in the evaluation of clones obtained from two cell lines. Among 17 donor-specific cytotoxic T-cell clones derived from one cell line, only four V beta gene families were expressed. All five clones from the other cell line used the V beta 8 family. Some clones expressing a distinct V beta gene family used the same V-D-J junction sequence, indicative of their origin from the same precursor. With the use of oligonucleotide probes complementary to clone-specific V-D-J junction sequences, four of five clones were detected not only in the parent T-cell line but also in T-cell lines derived from biopsy specimens with rejection reactions taken 1 week earlier and 2 weeks later and in blood cells taken before and 0.5, 3, and 6 months after transplantation; these clones were not detected in blood cells harvested 12 months after transplantation. This study showed a restricted usage of the V beta gene families by activated donor-specific cytotoxic T lymphocytes in the heart transplant.
Antigens, CD↗