Antisera were produced in rabbits and monkeys by immunization with either HSB (T cell) or SB (B cell) lymphoblastoid cell lines. With a C-dependent microcytotoxicity assay, appropriately absorbed antisera to HSB were strongly reactive with human thymocytes, T lymphoblastoid cell lines, and some acute lymphoblastic leukemia cells (>90% lysis). Approximately 75% of normal peripheral blood lymphocytes (PBL) were lysed by the antisera. Reactivity was at background level when monocytes and cells from patients with chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and most acute myelocytic leukemia (AML) and acute myelomonocytic leukemia (AMML) were tested against the antisera. Further absorption of anti-HSB sera with CML cells or platelets did not remove serologic reactivity, whereas absorption with thymus, PBL, or concanavalin A- (Con A) stimulated PBL removed reactivity against all cells tested. Antisera produced to SB and absorbed with HSB were strongly reactive with B cell lines, CLL cells, tonsil, adenoid, enriched B cell preparations from normal PBL, and cells from most AML and AMML patients. Additional absorption of anti-SB sera with thymus, CML cells, or platelets did not remove reactivity. Anti-SB sera that were additionally absorbed with CLL cells were negative against all cells tested.
Sodium deoxycholate- (DOC) solubilized membrane preparations from a variety of cells were labeled with 125I and subjected to radioimmunoprecipitation (RIP) and polyacrylamide gel electrophoresis (PAGE) techniques. Unlabeled DOC-solubilized membrane antigens of HSB were able to inhibit the cytotoxic reaction of antisera to HSB with PBL and thymocytes. The reactivity of SB antisera with CLL target cells was not affected by the solubilized HSB antigens. Unlabeled DOC solubilized membrane preparations from HSB, PBL, and thymocytes were able to block the RIP activity of anti-HSB sera.
One major peak of radioactivity (m.w. approximately 150,000 daltons) was consistently observed during PAGE of reduced immunoprecipitates of either monkey or rabbit anti-HSB sera and labeled surface antigens from HSB, PBL, or thymus. Treatment of the immunoprecipitates with additional 2-mercaptoethanol or reduction with dithiothreitol failed to alter the observed m.w. of the antigen. No significant peaks were observed in PAGE when the same antigens were precipitated with either normal sera of antisera to SB.
PAGE of reduced immunoprecipitates of absorbed anti-SB sera and labeled SB membranes revealed two peaks of radioactivity, corresponding to m.w. of 38,000 and 30,000 daltons. When labeled membrane preparations from SB, K562, or CLL cells were precipitated with absorbed antisera to HSB, no significant peaks were observed.
This work was supported by Grant AM 08054 from the National Institute of Arthritis, Metabolism and Digestive Diseases, Grant CA 08975 from the National Cancer Institute to Duke University, and by Grant FR 00165 from the Division of Research Resources to Emory University.