Progress in defining the nature of T cell receptors for antigen has been slow and many conflicting results have been reported (reviewed in reference 1). Recently more success has been achieved by developing monoclonal antibodies, directed against T cell clones, that show unique specificities for the clones (2-4). In this study we have utilized a similar approach and have screened monoclonal antibodies made against cells from a human T cell leukemia for idiotypelike specificity. Two such monoclonal antibodies were found that reacted with the same membrane molecule; one showed absolute specificity for the immunizing cells, the other also reacted with a small population of normal T cells. Materials and MethodsLeukemic cells were obtained from a male from the Dominican Republic. He presented with typical clinical and laboratory findings of Sezary Syndrome. Peripheral leukocyte counts ranged from 30,000 to 97,000 with -~85% Sezary cells. The patient was leukopheresed and leukemic cells obtained by Ficoll-Hypaque separation. Surface markers on the leukemic cells were: sheep erythrocyte rosette +, OKT3 +, OKT4 +, OKT8 -, Ia -, and surface immunoglobulin -. The leukemic cells were cryopreserved until use.Cryopreserved cells were thawed and 2 x 107 cells in alum were injected intraperitoneally into BALB/c mice (The Jackson Laboratories, Bar Harbor, ME). 3 wk later the mice were boosted with an equal number of cells, and splenic leukocytes were obtained for fusion 3 d later. Spleen cells were fused with SP 2/0 cells by the technique of Kennett et al. (5). Supernatants from viable hybridoma cultures were screened by standard indirect immunofluorescence technique by the use of fluorescein-conjugated Fab~ fragments of goat anti-mouse IgG antiserum (Tago Inc., Burlingame, CA) and a Cytofluorograf 30-H (Ortho Diagnostic Systems Inc., Westwood, MA). Results obtained by flow cytometry were correlated by visual immunofluorescence. Hybridomas of interest were cloned in soft agar. Cloned cells were injected into pristane-primed BALB/c mice and the monoclonal antibody was isolated from the ascites by ammonium sulfate precipitation and ion exchange chromatography.Normal peripheral blood mononuclear cells were subjected to rosetting with neuraminidase-treated sheep erythrocytes. Rosetting and nonrosetting populations plus granulocytes, erythrocytes, and platelets were used to screen the reactivity of the cloned hybridomas. Human T cell lines (MOLT 4, CEM-T, Jurkat, KE 37, 1301, 3639, and HUT 102), B cell lines (8866, Cess, and Josh 7), and myeloid cell lines (K 562, HL 60, and U 937) were also evaluated by flow cytometry. T cell blasts were generated by stimulation with concanavalin A at 10 #g/ml (Sigma Chemical Co., St. Louis, MO), phytohemagglutinin 1:100, or pokeweed mitogen 1:100 (Gibco Laboratories, Grand Island, NY) and stained with the monoclonal antibodies. Samples of peripheral blood from patients with various T cell malignancies were also analyzed by indirect immunofluorescence. The malignancies included two samples of b...

Two different human T cell leukemias were compared, using antiidiotype-like murine monoclonal antibodies. In each case these antibodies immunoprecipitated disulfide-linked heterodimer molecules from their respective leukemic cells. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the two idiotype-bearing molecules a major difference in molecular weight was observed, which could be attributed to a similar difference in size of the heavily iodinated chain of either heterodimer. The lightly iodinated chains of both molecules co-migrated at 43 Kd, but appeared to have different isoelectric points on two-dimensional gel analysis. The possibility that these two different heterodimers correspond to different classes of the putative T cell receptor for antigen is discussed. Assays of proliferation of the leukemic cells using Sepharose-bound antiidiotype-like monoclonal antibody showed that one of the leukemic cell types proliferated readily in response to its antiidiotypic antibody. This proliferation was not associated with measurable production of IL-2 and appeared to be a direct effect of the antiidiotypic antibody, which may mimic antigen in its interaction with the T cell receptor for antigen. The other leukemic cell type did not respond to Sepharose-bound antiidiotypic antibody and was generally unresponsive to lymphokines and mitogens. It is possible that the two leukemic cell types represent different stages of T cell differentiation.