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Detection and Characterization of Human Leukemia-Associated Antigens on Leukemic Cell Lines and Thymocytes

II. Isolation of Human Thymus-Leukemia-Associated Antigens from the Cell Surface and Partial Characterization¹

From the Department of Immunology Research, Roswell Park Memorial Institute, ² Buffalo, New York 14263

Abstract

Common human thymus-leukemia-associated (HTL) antigens were identified for seven leukemic T cell lines and several thymocyte preparations derived from different donors by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The leukemic T cell lines tested were MOLT-4, CCRF-CEM, RPMI 8402, CCRF-HSB-2, JM, HPB-ALL, and HPB-MLT. The leukemic T cells and normal thymocytes along with normal peripheral blood lymphocytes (PBL) and other control cells were labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination and then lysed with Renex 30, a nonionic detergent, in the presence of protease inhibitors Trasylol and phenylmethanesulfonyl fluoride. The supernatant of the cell lysate was subjected to various treatments to minimize the occurrence of nonspecific precipitation of radio-activity in the subsequent analyses. The treated supernatant was subjected to immunoprecipitation by adding the G fraction of rabbit antiserum rendered specific to HTL antigens (Abs-RMOLT) followed by the subsequent addition of a slight excess of goat antiserum to the Fc fragment of rabbit IgG. The resulting immunoprecipitate was reduced with dithiothreitol and analyzed by SDS-PAGE. Two different types of human thymocytes were identified and designated types I and II. Type II thymocytes showed three HTL antigenic components with approximate sizes of 150,000, 120,000, and 63,000 daltons, whereas type I thymocytes showed only the 63,000 dalton component. The seven leukemic T cell lines studied were similar to type II thymocytes in the SDS-PAGE pattern of HTL antigens rather than to type I thymocytes, although there were some differences in the pattern among the different cell lines. The HTL antigens were not detected on PBL, on normal B cell lines, on the leukemia cells of a patient with chronic lymphocytic leukemia, and on the leukemia cells of a patient with acute myelogenous leukemia. Control experiments were carried out with normal rabbit serum or with Abs-RMOLT absorbed with thymocytes in place of Abs-RMOLT.

Footnotes

¹ This work was supported by Grant CA 19304, awarded by the National Cancer Institute, Department of Health, Education and Welfare.

² A unit of the New York State Department of Health.