Southern blot and microfingerprinting analysis of two DR7 haplotypes.
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Cell Line↗
Biomedical subjects
N Tanigaki
Publications and source records attributed to N Tanigaki.
Subset analysis of human class II molecules controlled by the DR2/Dw2 haplotype: two separate DR subsets carry the DR2 specificity.
Human class II molecules were isolated from cells of a DR2/Dw2-homozygous cell line, PGF. The three mutually exclusive subsets were separated by selective binding with monoclonal antibody MCS7 and alloantisera CCB921 and KY22. The specificity involved in the binding with alloantisera was identified to be a supertypic specificity associated with DR1, 2, and w9 for CCB921 and the DQw1 specificity known to be associated with DR1, 2, w6, and w10 for KY22. The MCS7 specificity appeared to be a cross-reactive specificity related to the DRw52-like specificity. On peptide mapping, the alpha chains of MCS7- and CCB921-reactive subsets were the same, showing the pattern characteristic of DR alpha chains, whereas the beta chains were very similar to, but distinguishable from, each other. These structural features conformed to those of DR or DR-like subsets. The KY22-reactive subset was distinctive in both alpha and beta chains from the above two subsets, and it displayed peptide patterns typical to DQw1-bearing Ia molecules. Interestingly, the MCS7- and CCB921-reactive subsets both carried the DR2 specificity, as indicated by their binding to alloantiserum Fe73/22 which was proven to be DR2-specific.
Alleles↗
Serological discrimination of DR4 haplotypes by radioimmunoassay.
Three new alloantigenic specificities of human major histocompatibility complex class II molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/Dw10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class II molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.
Antibody Specificity↗
Radioimmunoassay typing gives a more precise definition of the HLA association of type 1 (insulin-dependent) diabetes.
The problem of the HLA association of Type 1 (insulin-dependent) diabetes was re-examined by testing Class II antigenic specificities detectable by radioimmunoassay. Established (DRw53, DQw1, DQw2, DQw3) as well as newly described (DC5, DCalpha3) specificities were typed. The data obtained suggest that the association with DR3 and DR4 is secondary to that with DQ specificities in linkage disequilibrium with DR3 and DR4.
Diabetes Mellitus, Type 1↗
A radioimmunoassay typing study of non-DQw2-associated celiac disease.
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Adult↗
Serological and functional analysis of the epitope clusters on Leu3/T4 antigen.
Monoclonal antibodies (MoAbs) were generated against cells or cell membrane glycoproteins of a human T-cell line, HPB-ALL. Five, designated MCN 1, 3, 12, 19, and 29, were found to be specific to helper/inducer T-cells; they gave a positive membrane staining to approximately 48% of peripheral blood lymphocytes and 84% of thymocytes and these proportions did not change upon costaining with Leu 3a, a known anti-helper/inducer T-cell MoAb. Furthermore, their reaction pattern with a panel of human lymphoid cell lines was identical to that of Leu 3a. A reciprocal binding blocking test showed that the epitopes reactive with the MCN MoAbs are divided into three separate clusters. The MCN 3- and Leu 3a-reactive epitopes formed a cluster and they appeared to be the same epitope. This cluster was well separated from that represented by the MCN 1-reactive epitope. The MCN 12-, 19-, and 29-reactive epitopes could be assigned to a third cluster. MCN 12 and 19 were probably toward the same epitope. A sequential binding test indicated that the three epitope clusters reside on the molecule carrying Leu 3a-defined epitope, i.e., the Leu3/T4 antigen. On the functional analysis, MCN 3 gave a profound inhibitory effect on T-cell proliferative response to MHC class II antigens, whereas other MCN MoAbs did not show any modifying effect on the T-cell function.
Animals↗
A mouse monoclonal antibody against a polymorphic determinant in a defined subset of DR molecules.
The hybridoma technique was used to produce a mouse monoclonal antibody, designated as XI 21.4, which belongs to the IgG2a class. It is active in complement-dependent cytotoxicity and detects a B-cell antigenic determinant associated with DR1, DR4, DRw10, and, possibly, DRw9. Microfingerprinting of the immunoprecipitate from a homozygous DR4 cell line shows a typical alpha DR pattern and a beta pattern coinciding with that of DR4 molecules.
Animals↗
A new allodeterminant on HLA-DQ molecules carrying the DQw3 specificity.
The la subset that reacts with alloantiserum HON known to possess a strong anti-DRw53 activity was isolated from a 125I-labeled Ia preparation obtained from cells of RPMI 8057 cell line (DR1,4) and was found on peptide mapping to be lacking in the pattern characteristic of DR-like molecules carrying the DRw53 specificity and to display the structural features of DQ molecules, particularly those carrying the DQw3 specificity. Distribution analysis on a panel of selected la-positive cell lines indicated that the specificity involved is associated only with DR4 and DRw9, differing from the known DRw53 pattern (DR4, 7, and w9) and also from the known DQw3 pattern (DR4 and 5). Reciprocal sequential binding experiments demonstrated that the HON-defined specificity resides along with DQw3 specificity on the same molecules. Thus, HON alloantiserum possesses two different antibody activities; one directed to DRw53 specificity and another directed to a new DR4- and w9-associated DQ specificity.
Epitopes↗
DR5-associated DC molecules carry three different allospecificities.
Antiserum 8w670 which had been classified as anti-DR5 in the Workshop analysis was found by the direct binding test to react with about 40% of Ia molecules in an Ia preparation from LG38 cells (DR5,5) that was deleted of DR molecules and DR-like molecules we call BR and enriched in DC molecules by pretreatment with a rabbit antiserum raised against alpha-subunits of DR and BR molecules. The specificity involved in the binding reaction was shown by the binding inhibition assay to be present in 100% of DR5-positive cases (53 out of 53) and 8w13-positive cases (2 out of 2) and in 23% of DR4-positive cases (4 out of 17). This association pattern did not correspond to any of the known supertypic specificities including the DC beta 4 and DC alpha 3, both of which had been found on DR5-associated DC molecules. Yet sequential binding analysis revealed that the 8w670-defined specificity was indeed present on the same molecules carrying DC beta 4 and DC alpha 3 specificities. This specificity was designated DC5.
Antibody Specificity↗
An IgM pan-T monoclonal antibody that blocks E-rosette formation.
An IgM monoclonal antibody directed against the T-cell E-rosette receptor was obtained by preparing a hybridoma against cells of a human T-cell leukemia line, HPB-ALL. The antibody, named EDN-34B1, reacts exclusively with cells of T-cell lineage, including those of three T-cell leukemia lines. It does not react with cells of B-cell lines, normal B-cells, or monocytes. EDN-34B1 recognizes a single polypeptide of approximately 50,000 molecular weight, is capable of blocking E-rosette formation, and its binding to the target cell can be specifically blocked by monoclonal antibodies 9.6 and OKT-11, both anti-E-receptor antibodies. EDN-34B1 is 100% cytotoxic against normal thymocytes and peripheral T-cells, thus enabling the routine removal of such cells from a mixed lymphocyte population, and kills a higher percentage of normal T-lymphocytes than Ab 9.6, even though immunofluorescence studies have shown that both antibodies bind to the same fraction of PBLs. This phenomenon may be due to the IgM nature of EDN-34B1. Addition of EDN-34B1 and complement to PBLs totally abrogates T-cell functions such as mitogen (PHA) stimulation and response in MLR, while addition of EDN-34B1 alone only affects MLR.
Antibodies, Monoclonal↗
Serologically detectable polymorphism of the HLA-DC alpha-subunit.
Two 8th Workshop alloantisera, 8W1062 and 8W614, which were classified respectively as anti-DR5 and anti-DR3, were found to react with an Ia preparation from LG38 cells (DR5,5) that was depleted of DR and BR molecules and enriched in DC molecules by pretreatment with a monoclonal antibody and an antiserum against alpha-subunits of DR and BR molecules. The reaction of both alloantisera was inhibited by DR3-positive, DR5-positive, and DRW13-positive individuals. This pattern does not correspond to any of the hitherto reported supertypic specificities. Both antisera were shown to bind to the alpha-subunit isolated from the DC-enriched preparation but not to the beta-subunit. The specificity has been named DC alpha 3. Family studies show the segregation according to the HLA haplotype patterns. This demonstrates by formal genetics criteria the HLA linkage of the locus controlling the DC alpha-subunit.
Binding, Competitive↗
Three Ia species with different structures and alloantigenic determinants in an HLA-homozygous cell line.
Three distinct molecular subsets with different structures and alloantigenic determinants were identified in human Ia antigens from cells of an HLA-Dw7 homozygous cell line. The subsets carried DR7 specificity, BR4X7 supertypic specificity and MB2 supertypic specificity, respectively, and were immunospecifically separated by the use of operationally monospecific alloantisera. These specificities showed HLA-linked segregation in families and they were distributed in the population according to different but partially overlapping patterns. On peptide mapping analysis, the three subsets showed marked differences in the beta-chains. The alpha-chains of DR7 and BR4X7 subsets were very similar to each other, whereas the alpha-chains of MB2 subset were distinctive from those of DR7 and BR4X7. These data indicate the presence of a minimum of three HLA-linked loci; DR locus, a locus that encodes BR4X7, and a locus that encodes MB2, and substantiate the three-loci concept for the genetic control of human I antigens.
Amino Acid Sequence↗
The distribution of DR5, MT2, and MB3 specificities on human Ia subsets.
Human Ia molecules were isolated from cells of LG-38, an HLA-homozygous lymphoid cell line of DR5 specificity. Three Ia subsets could be distinguished and separated by using specific alloantisera and a monoclonal antibody with polymorphic reactivity. These subsets carried the specificities DR5 and MT2, MT2 alone and MB3 alone. The structure of the three molecular species was analyzed by microfingerprinting. The subset carrying only MT2 was similar, in both the component alpha and beta chains, to the major subset carrying DR5 and MT2, whereas the subset carrying MB3 was distinct in both chains from the other two subsets. These data are compatible with our previous findings obtained for the products of two Ia loci closely linked to the DR locus and provisionally called DC and BR; they also support the conclusion that the subset carrying only MT2 is an allelic product of the BR locus, whereas the MB3 subset is an allelic product of the DC locus. MT2 appears to be a shared specificity of DR and BR loci products.
Antibodies, Monoclonal↗
Evidence that celiac disease is primarily associated with a DC locus allelic specificity.
Sixty patients with celiac disease were typed by radioimmunoassay for the specificities HLA-DR3, HLA-DR7, and for an allelic specificity, DC3, of the HLA-associated DC locus. We found that celiac disease is primarily associated with the DC determinant. The previously described associations with DR3, DR7, B8, B13, and A1 can be explained by decreasing degrees of linkage disequilibrium with DC3.
Adolescent↗
Influence of ionic strength on the antibody binding of MHC products.
Ionic strength has a strong influence on the binding of human Ia and HLA(A,B,C) molecules by the corresponding antibodies. A control system, BSA-anti-BSA, was influenced only minimally. The effect on the binding of anti-beta 2-microglobulin was very small when isolated chains were used, but more pronounced when using whole HLA(A,B,C) molecules. An inverse relationship exists between degree of inhibition and antibody concentration.
Antigen-Antibody Reactions↗
Microfingerprinting analysis of human Ia molecules favours a three loci model.
Alpha subunits from DC1 Ia molecules, when compared with DR alpha subunits, are shown to possess distinctive features revealed by differences in microfingerprinting patterns after peptic digestion. Alpha chains from BR4X7 molecules differ from DC1 alpha chains and are more similar to DR alpha chains. Since DC1 and BR4X7 beta chains (which carry the HLA-controlled alloantigenic determinants) associate with different alpha subunits, it is considered unlikely that they are controlled by alleles at the same locus. The proposed model implies the existence of three tightly linked HLA loci controlling the beta subunits of DR, DC and BR molecules respectively.
Alleles↗
A current view of the human Ia system based on serological and immunochemical analysis.
We have approached the analysis of the human Ia system by localizing antigenic determinants on subsets of Class II molecules. For this purpose two classes of reagents have been used: alloantisera and mouse anti human monoclonal antibodies (moabs). We summarize here the most recent data obtained by these two approaches.
Alleles↗
Human Ia molecules carrying DC1 or BR4X7 determinants are not homologous to murine I-E molecules.
An anti-I-Ek alloantiserum was shown to react with purified human Ia preparation. All Ia preparations tested were actively bound irrespective of their HLA-DR phenotypes. However, from a quantitative point of view, DR7 molecules were significantly less reactive. No reaction was observed with isolated Ia subunits. Only molecules carrying DR determinants, but not those carrying either DC1 or BR4X7 determinants, were bound.
Animals↗