Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.
HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.
The human B cell-specific alloantigen which is closely linked genetically to HLA contains two non-covalently associated, sialogycoprotein subunits of molecular weight (MW) 29,000 (p29), and 34,000 (p34). Although p29 and p34 have different amino-terminal sequences, their tyrosine peptide maps indicate considerable similarity in other portions of their polypeptide chains. Thus the genes for their proteins may have evolved by duplication of a common ancestral gene. Another lymphocyte cell surface protein of MW 16,000 (p16) has also been characterised. Both p16 and p44 (the heavy chain of HLA-A,B antigens) have been compared with p29 and p34.
The HLA-linkedB cell alloantigen (p29,34) is composed of two subunits of 29,000 (p29) and 34,000 (p34) molecular weight. The partially purified HLA-linkedB cell alloantigen was purified by a final step of preparativesodiumdodecylsulfate-gelelectrophoresis. An antiserum was prepared against p29,34 which specifically lysed B lymphocytes. In sodiumdodecyl sulfate at 21 degrees, p29 and p34 remained noncovalently associated and retained immunologic activity; subunit dissociation at higher temperatures correlated with loss of immunologic activity. Although the pI values of p29 and p34 are 6.1 and 5.2, respectively, the subunits co-electrofocus under nondenaturing conditions. In addition, cross-linking studies showed the B cell antigen has a (p29)1(p34)1 subunit structure.
From these data, a model was prepared which summarizes schematically our present knowledge of the structure and orientation of the HL-A antigenic molecule in the lymphocyte membrane (Fig. 3). It seems likely that the heavy chain spans the membrane, with the hydrophobic region inserted in the membrane and the hydrophilic C-terminus inside the cell. This C-terminal region bears one (possible two) SH residue which has the potential for forming interchain disulfides. Whether or not these are actually formed physiologically remains an interesting question. There is the attractive possibility that whatever the physiological functions of HL-A antigens are, structurally these molecules provide the potential for signaling from outside the cell to inside the cell because they span the membrane. It is even conceivable that this function might be expressed via the opening and closing of disulfide bridges.
The structure of monomeric and disulfide-bonded dimericforms of HLA antigens has been studied. Detergent-solubleHLA antigen heavy chains contain one or two easily reduced sulfhydryl groups not found in papain-solubilized HLA antigens, as demonstrated by amino acid analysis (Springer, T. A., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2481-2485, and Terhorst, C., Parham, P., Mann, D.L., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 910-914) and by labeling with iodo[3H]acetate. Dimer formation occurred during purification, since it was prevented by pretreatment of membranes containing HLA antigen with iodoacetamide. Cross-linking studies showed that the non-disulfide-bonded form of HLA antigens contains one subunit each of the Mr = 44,000 heavy chain and the Mr = 12,000 light chain (beta2-microglobulin).
Purified, detergent-solubleHLAantigens (p44,12) are composed of a glycoprotein of molecular weight 44,000 (p44) and a peptide of molecular weight 12,000 (p12), beta2-microglobulin. Upon digestion with papain, p44,12 is converted to p39,12, then to p34,12, which retains antigenic activity. The NH2-terminal amino acid sequences of p34 and p44 are identical. p44, p39, and p34 were purified, and comparison of their amino acid compositions showed that the COOH-terminal peptide removed by the first papain cleavage is hydrophilic and contains cysteine that can be alkylated after mild reduction. The penultimate COOH-terminal peptide removed by the second papain cleavage is hydrophobic, and presumably anchors HLAantigens to the membrane. This correlates with the observation that p44,12 and p39,12 bind detergent, while p34,12 does not. The orientation and integration of HLAantigensin the lymphocyte membrane were thus defined, and the structure suggests that HLAantigens span the plasma membrane.
A number of immunological methods have been employed to show that the smallsubunit of HL-Aantigens, isolated either after papain treatment or after solubilization with detergent, is identical to beta(2)-microglobulin, a protein previously isolated from human urine and shown to be homologous in structure to constant region domains of immunoglobulins. Moreover, quantitative data indicate virtually total identity between the smallsubunit of HL-Aantigens and beta(2)-microglobulin. Studies of the turnover of labeled HL-Aantigens from the lymphocyte surface indicate that the two subunits turn over at similar rates, although only the smallsubunit could be detected in the culture medium. The significance of these observations is discussed.
HL-Aantigensolubilized with the non-ionic detergent, Brij 99, has been purified to about 50% of homogeneity from a cultured human lymphoblast line. It consists of two nonidentical subunits of 44,000 and 12,000 molecular weight (MW). Upon papain proteolysis the 44,000 MW peptide is converted by at least two cleavages to a 34,000 MW peptide, but the 12,000 MW peptide appears to be unchanged. Concomitantly, the apparent molecular weight in gel filtration chromatography under nondenaturing conditions in the presence of Brij 99 is reduced from 460,000 to 45,000. HL-A molecules produced by direct papain proteolysis of membranes and bypapain treatment of purified detergent-soluble HL-A are identical.