Dolly B. Ness

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk− cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.

Genetic control of IgM responses to (T,G)-A — L

The primary antibody response to aqueous immunization with a low molecular-weight lot of (T,G)-A — L (#420) was studied in congenic pairs of inbred mouse strains. Two new genetic controls were identified, both of which quantitatively regulate the production of IgM anti-(T,G)-A — L antibody. Testing of F1 and F2 progeny demonstrated that one of these genes is linked to the major histocompatibility (H-2) complex, and that high response is dominant over low response. Whether this gene is identical toIr-1A is not yet known. The other gene, designatedIg-TGALM, is linked to the immunoglobulin heavy-chain allotype locus (Ig-1) and is expressed in a genedose dependent manner. Following secondary challenge with (T,G)-A — L 420, quantitative differences in IgG antibody response were observed inIr-1A high-responder congenics differing only at theIg-1 locus. Breeding studies, however, failed to demonstrate any linkage between this locus and the quantitative control of IgG anti-(T,G)-A — L antibody. These data demonstrate thatH-2-linked immune response genes can regulate IgM as well as IgG antibody responses, that genetic control of the IgM response to (T,G)-A — L is linked toIg-1, and that bothH-2-linked andIg-1-linked genes may simultaneously affect an IgM antibody response to the same antigen.

New polymorphisms of HLA-B27 and other B locus antigens detected by RFLP using a locus-specific probe

Genomic DNA from 46 B27+ ankylosing spondylitis, Reiters syndrome, or normal individuals was digested with Taq I and probed, in Southern blots, with the HLA-B locus specific probe, EI7. Four restriction fragment length polymorphisms (RFLP), 2.5, 3.4, 3.8 and 4.0 or 8.0 kb, were observed for the B27 gene. In Caucasians, one of the B27 variants (2.5 kb) was more frequent in normals and almost never appeared in patients, suggesting a trend that is not yet statistically significant. In the course of defining the B27 polymorphisms, three and two RFLP, respectively, were also found for the B18 and B44 genes.

Adjuvant effect of lps on in vivo antibody response. Genetic defect in c3h/hej mice.

Among several inbred strains of mice studied, only C3H/HeJ failed to demonstrate LPS enhancement of antibody response to the synthetic polypeptide (T,G)-A-L. LPS antibody enhancement was designated the LPS/adjv(+) trait and results of testing F1 and backcross animals were consistent with the postulate that the LPS/adjv(+) trait may be determined by a single autosomal dominant gene.

ST-16: A new Bw16 subtype present in Mexican-Americans

Using a large battery of Bw16, w38, and w39 antisera, a new variant of Bw16 has been identified in four unrelated Mexican-American families. The serologic pattern obtained is distinct from that for Bw38, Bw39, and 8W57 antigens. Absorption studies confirm the existence of this new Bw16 subtype which we term ST-16. ST-16 is Bw6-associated, with antigen frequency estimated to be 2.5% in Mexican-Americans.

HLA Class I Gene Typing with an HLA-B-Specific DNA Probe

HLA class I antigens and genes are of interest as markers of disease susceptibility and targets of cytolytic T lymphocytes [1, 2]. In addition to the HLA-A, B, and C loci, human analogs of the murine class I Qa and T1 loci also probably exist [3, 4]. The well-known serological polymorphism of the class I antigens forms the basis of a substantial part of this workshop; however, early studies using recombinant DNA techniques such as Southern blotting to detect restriction fragment length polymorphisms (RFLPs) suggest that even greater complexity may be found at the level of the genes themselves, with estimates of more than 20 class I loci [3]. Identification of differences at the gene level has been difficult because their great homology results in strong cross-reactivity of cloned class I gene probes with all members of this multigene family. A strategy was therefore devised to prepare a DNA probe that would be HLA-B locus specific and thus permit clear-cut characterization for RFLP of that locus. We report here the preparation and use of such a probe with preliminary results demonstrating the first intragenic DNA restriction endonuclease site specific for an HLA allele.

A simplified new method for HLA-DR typing using the TM1 monoclonal antibody

A new modification of an HLA-DR typing technique is described which makes DR typing as rapid and simple as routine HLA-A,B,C typing. In this new method, designated the TM1 technique, carboxyfluoresceindiacetate labeled peripheral blood lymphocytes are added directly to DR typing trays. The T cells are then lysed by addition of TM1, a pan-T cytotoxic IgM monoclonal antibody, and residual B-cell reactivity with cytotoxic DR alloantibodies is read as in routine fluorochromasia microlymphocytotoxicity. HLA-DR typing by the TM1 technique compares favorably to typing by methods using B cells enriched by sheep red blood cell rosetting or by Degalan bead columns. The TM1 technique also works well with cells that have been cryopreserved as well as with cells that have been separated from whole blood drawn as much as 3 days earlier. Finally, because TM1 is so effective in lysing normal T lymphocytes, this antibody may prove useful in functional in vitro and in vivo studies requiring T-cell depletion.

Strong linkage disequilibria among HLA-Bw50, Bfs1, and HLA-DR3/DR7, and mapping of the Bf locus

Based on genotypic and phenotypic studies we have found strong linkage disequilibria in Caucasians among the genes HLA-Bw50, BfS1, and HLA-DR3 and/or -DR7. The relative disequilibria, which are among the highest described in man, are delta r (BfS1, DR7) = 0.51, delta r (Bw50, BfS1, DR7) = 0.36, delta r (Bw50, DR3 or 7) = 0.72, delta r (BfS1, DR3 or 7) = 0.91, delta r (Bw50, BfS1, DR3 or 7) = 0.73. The previously described high delta r (Bw50, BfS1) and delta r (Bw50, DR7) have also been confirmed. A B parallel DR crossover family is also presented that, together with previously reported recombinant families, confirms that the Bf locus resides between HLA-B and HLA-DR. These data suggest the existence of a supergene complex of Bw50, BfS1, DR3/7 (or MB2), and hypotheses to account for the observed disequilibria are discussed.