Andreas Ziegler

Analysis by Sequential Immunoprecipitations of the Specificities of the Monoclonal Antibodies TÜ22, 34, 35, 36, 37, 39, 43, 58 and YD 1/63.HLK Directed Against Human HLA Class II Antigens

The monoclonal antibodies (MOABs) TU22, TU34, TU35, TU36, TU37, TU39, TU43, TU58 and YD1/63.HLK were used to identify subpopulations of class II antigens encoded by the human major histocompatibility complex. Since all MOABs reacted with B lymphocytes of HLA-DR1-8 homozygous as well as all heterozygous cells tested, they recognize monomorphic determinants, with the possible exception of TU58 and YD1/63.HLK which do not fix complement. As shown by radioactive binding assays and immunoprecipitations of labeled chains, 3 MOABs reacted strongly and 3 others weakly with isolated beta-chains, and the former also bound alpha-chains, albeit very weakly. Immunoprecipitations with the MOABs from 125I-labeled KR3598 cells (Dw5, DR5, MT2, MB3 homozygous, SB2, SB4) demonstrated that at least 4 different subpopulations of class II antigens were present in the lysate. Possibilities to reconcile these biochemical data with the reactivity of the MOABs with HLA mutant cell lines and with functional as well as tissue distribution studies are discussed.

Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class I and III regions by field inversion gel electrophoresis

Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class field inversion gel electrophoresis 11HlllUllO-genetics The human major histocompatibility (HLA) complex is located on the short arm of chromosome 6 in the 6p21.31-6p21.33 region (Spring et al. 1985, Ziegler et al. 1985a). The physical length of the entire HLA complex is unknown so far, but our estimate based on the separation of DNA fragments containing HLA genes by pulsed-field gel electrophoresis (Schwartz and Cantor 1984, Carle and Olson 1984) indicates that it encompasses at least 2500 kb pairs (Ragoussis et al. 1986). This estimate has recently been confirmed (Lawrance et al. 1987). Apart from the highly polymorphic class I and II loci, the HLA complex contains the genes for several complement components and 21-steroid hydroxylase (class III region) (Lamm and Olaisen 1985). In addition, the loci for tumor necrosis factor (TNFA) as well as lymphotoxin (TNFB) are also near or even within the HLA region (Nedwin et al. 1985). Spies and co-workers (1986) demonstrated that the TNF genes map either centromeric to HLA-DP or telomeric to the class I1 region, although apparently not in the vicinity of any known class I or III genes. The recent demonstration that the TNF loci are situated 70 kb upstream of the H-2D gene in the BALB/c mouse between the class III and class I regions (Mfiller et al. 1987a, b) suggested an analogous location in man, because the genetic organization of the major histocompatibility complexes (MHC) of both species is very similar. To clarify the position of the TNFA and TNFB genes on the HLA map, we have assigned TNFA to large DNA restriction fragments separated by field inversion gel electrophoresis (FIGE) (Carle et al. 1986), which hybridize with either class 1II-or class I-specific probes as well. These results prove that the TNFA locus is localized between the HLA class III region and the HLA-B locus. To avoid interpretative difficulties which might arise from haplotype-specific restriction fragment length poly-morphisms, mutant human cell lines with monosomy 6 or HLA hemizygosity were employed. All mutants were derived from BJAB-B95.8.6 lymphoma cells with the HLA haplotypes A1, Cw4,B35 and A2, C-,B13 (Spring et al. 1985). Mutant BM 19.7 is a monosomy 6 mutant cell line retaining the A2 haplotype (Ziegler et al. 1985b). BM 28.7 also exhibits monosomy 6, but with loss of the chromosome bearing the A2 haplotype (Ragoussis et al. …

Refinement of HLA gene mapping with induced B-cell line mutants

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned “wild-type” cells were phenotyped HLA-A1, A2, B 13, 1335, Bw4, Bw6, Cw4, DR5, DRw52, DQwl, DQw3, DPw2, DPw4, GLO1*1, PGM3*2-1, and ME1*0 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TÜ101) and Bw4 (TÜ48, TÜ109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, 835, Bw6, Cw4, DR5. DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class 11 antigens. One group retained DQw1 and DPw2, another was DQw1−, DPw2+, and a third was DQw1−, DPw2−. Karyotyping of the “wild-type” line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and -B loci directly to the distal part of the 6p2l.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6.

Immunophenotyping of T-lymphoblastic lymphoma/leukemia: correlation with normal T-cell maturation

Twenty-nine cases of T-lymphoblastic lymphoma/leukemia were classified with conventional morphologic methods and the aid of monoclonal antibodies. All cases were investigated with a sensitive immunohistochemical method, using a panel of 22 monoclonal antibodies. In addition, normal thymus glands in the 22nd and 36th weeks of gestation were studied. Eight different groups of T-lymphoblastic lymphomas/leukemias could be distinguished, each of which showed a characteristic marker constellation. The results indicate that a complete detection of all thymic and prethymic lymphomas and leukemias is possible. By comparison with the phenotypic pattern of normal peripheral T-lymphocytes and their thymic precursors, the groups could be arranged in a sequence that resembles normal T-cell maturation, monoclonal antibodies.

Assignment of the TCP1 locus to the long arm of human chromosome 6 by in situ hybridization

TCP1, the human homolog of the Tcp-1 locus in the mouse, which is part of the murine t complex and codes for an abundant testicular germ-cell protein, has been mapped within the human genome by in situ hybridization. Using a cDNA probe for TCP1, pB1.4 hum, we assigned TCP1 to human chromosome region 6q23----qter, with the most likely localization being 6q25----q27.

Mapping of HLA genes using pulsed-field gradient electrophoresis.

The technique of pulsed‐field gradient electrophoresis (PFGE) allows the determination of gene linkage relationships since DNA fragments up to 2 Mb can be separated. PFGE was employed to study linkage of class I, II and III genes belonging to the human major histocompatibility (HLA) complex. The results establish that the class II DOβ and DZα genes are linked with the DP Subregion, centromeric to the DQ/DX‐DR‐C4 chromosomal segment, and allow us to estimate the minimal length of the entire HLA complex.

Monosomy 6 in a human lymphoma line induced by selection with a monoclonal antibody.

The human Epstein Barr Virus-superinfected B lymphoma cell line BJAB-B95.8.6 was mutagenized by gamma irradiation, and HLA mutants were selected with the HLA-Bw6-specific monoclonal antibody SFR8-B6. One of the mutants obtained, BM19, had lost one of the chromosomes 6 present in the wild type cells. Electrophoretic analysis of phosphoglucomutase isozyme PGM3 and erythrocyte glyoxalase 1 from both cells supports this conclusion. The HLA antigens expressed on BM19 were HLA-A2, B13, Bw4, C-, DR2 (questionable), DRw52 (weak) and DQw1. This constitutes one of the haplotypes of the wild type cells, the other (lost from BM19 cells) being HLA-A1, B35, Bw6, Cw4, DR5, DRw52 (strong) and DQw3. Possibilities to employ BM19 cells for the analysis of the major histocompatibility complex and other chromosome 6-encoded genes as well as their products are discussed.

Do monoclonal antibodies Tü15 and Tü67 detect heterogeneity of human transferrin receptor molecules

The possible molecular heterogeneity of human transferrin receptors was analyzed using two murine monoclonal antibodies. Tü 15 and Tü67. Both reagents precipitated from lysates of 125I‐labeled HL‐60 cells a major component of 88 kDa which could be identified as the transferrin receptor by comparison with the proteins detected by monoclonal antibody OKT9. Although sequential immunoprecipitations appeared to demonstrate molecular heterogeneity of transferrin receptors, since the Tü15‐reactive species were fully included in the Tü67‐positive population, but not vice versa, the possible association of Tü15‐reactive molecules with transferrin receptor is also discussed.

Identification of human spermatozoa antigens using monoclonal antibodies and the alkaline phosphatase anti-alkaline phosphatase-technique.

Summary: Both cytoplasmic and surface‐membrane antigens of human spermatozoa were detected by means of monoclonal antibodies (MoAbs) and of the alkaline phosphatase anti‐alkaline phosphatase‐ (APAAP‐) technique. Several advantages of this technique for the identification of sperm could be demonstrated. The labeling of cytocentrifuge preparations from 16 ejaculates proved the presence of glycosphingolipids, nuclear and mitochondrial antigens of spermatozoa. However, there were no HLA‐molecules and other leukocyte antigens on sperm cells.

Towards a Physical Map of the HLA Complex

The human major histocompatibility (HLA) complex is located on the short arm of chromosome 6 in the 6p21.31→6p21.33 region (1,2). There are three clusters of genes, the HLA class I, II, and III regions. Whereas the class III loci are only moderately polymorphic (see (3) for review), the class I and II genes which encode cell surface glycoproteins show an extreme degree of polymorphism. There are a minimum of 17 class I loci (4) and at least 15 genes for class II alpha and beta chains (5). In addition, we (6) and others (7) have recently demonstrated that also the loci for tumor necrosis factor (TNFA) and lymphotoxin (TNFB) are part of the HLA complex [see also Ragoussis et al., this volume]. A precise knowledge of the exact arrangement of HLA genes and the physical distances involved is essential for an understanding of several important properties of the HLA system, among them the association of haplotypes with diseases (8) and the phenomenon of linkage disequilibrium between alleles of different loci (9). Towards this end, we have assigned individual HLA genes to large DNA restriction fragments separated by pulsed field gel electrophoresis or related techniques. This allowed us to construct a molecular map of the HLA complex spanning at least 4000 kilobasepairs (kb).