An attempt to produce “pre-T” cell hybridomas and to identify their antigens

With the aim of identifying some of the stages in the development of pre-T cells (cells of the T cell lineage before they enter the thymus), we have produced a large number of hybridomas by the fusion of BALB/c bone marrow cells, bone marrow cells from BALB/c-nu/nu mice, BALB/c fetal liver cells and BALB/c fetal thymocytes with the AKR thymoma BW5147. The hybridomas were selected for the expression of the Thy-1.2 antigen of th3 normal celldonor and for their ability t9produce interleukin 2 (IL 2) upon co-culture with irradiated normal spleen cells. A set of these hybridomas is described in this communication. The hybridomas were then used to immunize rats and to generate monoclonal antibody-producing B cell hybridomas. Most, if not all, of the immunizing hybridomas were derived from pre-T cells as evidenced by the fact that they produce IL 2, and express some of the T cell markers (the Thy-1.2, Ly-1, Ly-2 or L3T4 antigens). The monoclonal antibodies were tested on a panel of pre-T cell hybridomas and on normal cells obtained from spleen, lymph nodes, thymus and bone marrow. The testing was carried out by the microcytotoxicity assay and flow cytometric analysis. Three groups of antibodies could be distinguished. Some antibodies were broadly reactive, being positive with virtually all the clones in the pre-T cell panel and with a substantial fraction of normal lymphoid cells. The identity of the antigens detected by these antibodies remains unknown but they do not seem to correspond to any of the known cell surface markers. Other antibodies reacted only with some of the pre-T cell clones and did not react at all with normal lymphoid cells obtained from adult animals. Finally, other antibodies still reacted only with a minor subpopulation of thymocytes or of thymocytes and bone marrow cells, as well as some of the pre-T cell clones; they did not react with spleen and lymph node cells. These antibodies might be specific for cells in the prethymic phase of the T cell differentiation pathway. They should prove useful for the identification of pre-T cell markers and hence for the isolation of pre-T cells and their functional analysis.