Bricf Detlnitive Rcport T200 Cell Surface Glycoprotein of the Mouse Polymorphism Defined by the Ly-5 System of Alloantigens

The Ly-5 alloantigen, originally believed to be restricted to thymocytes and thymus-derived (T) lymphocytes (1), has recently been shown to be present on most murine hematopoietic cells, including prothymocytes and pluripotent stem cells (2). Immu-noprecipitation studies by Michaelson et al. (3) demonstrated that Ly-5 alloantisera react with high molecular weight surface proteins of thymocytes and spleen cells. Several components with apparent molecular weights that ranged from 175,000 to 220,000 could be resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The 220,000-mol wt species was found only in spleen cells and appeared to be derived from bone marrow-derived (B) cells. The tissue distribution and biochemical properties of the Ly-5 alloantigen are very similar to the d'istribution and properties of the T200 glycoprotein, a major cell surface glycoprotein of murine hematopoietic cells that is a dominant antigen in xenoimmunization (4-6). We report here that Ly-5 alloantisera define a polymorphism of the T200 glycoprotein. Materials and Methods Cell Lines. The cell lines used in these studies were the murine T cell lymphoma cell line, BW5147, and two mutant cell lines, BW5147 (T200-) and BW5147PHA'~ 1.8, that were derived from BW5147 cells. Further details of the mutant cell lines are given in Results. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% horse serum. Immunological Reagents and Procedures. Ly-5.1 and Ly-5.2 alloantisera were prepared as described previously (2). Monoclonal T200 antibody was obtained from I3/2.3 hybridoma cells (6), and monoclonal Thy-1 antibody was obtained from the hybridoma T24/31.7 (7). For trace antibody binding assays, monoclonal antibodies were obtained from culture supernates of the appropriate hybridoma cells. For immunoprecipitation studies, a 50% ammonium sulphate fraction of ascitic fluid from tumor-bearing mice was the source of T200 antibody (8). Trace antibody binding assays were performed as previously described (6). Immunoprecipitation studies were carried out with 0.15 M NaCI-0.01 M phosphate buffer (pH 7.2) that contained 1% Nonidet P-40 (Shell Chemical Co., New York), 1% sodium deoxycholate, and 0.1% SDS to solubilize cells. Antibody-antigen complexes were isolated from cell lysates by adsorption to fixed Staphylococcus aureus. These procedures were as previously reported (8), and give low backgrounds of nonspecific binding. For precipitation of Ly-5 antigen, 5/LI of alloantiserum was used, and antibody-antigen complexes were bound directly to S. aureus. Biochemical Procedures. Cells were labeled by the glucose oxidase modification of the lacto-peroxidase technique (9). Immunoprecipitates were analyzed on 7.5% polyacrylamide gels with a discontinuous buffer …