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

Abstract

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 the normal cell donor and for their ability to produce 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.

Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells

“…Clone E13-161.7 originally was raised against BALB/c mouse 'pre-T' cells (Aihara et al, 1986), whereas clone D7 was raised against the interleukin 2 (IL-2)-dependent mouse T-cell line CTL-L (Ortega et al, 1986). Although both antibodies react with Sca-1, they recognize distinct epitopes as D7 is unable to block binding of E13-161.7 (Bamezai and Rock, 1995).…”

Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators

“…The Ly-6A (Sca-1) antigen is useful for identifying stem cells from some strains of mice [22][23][24] and common lymphoid progenitors were isolated with this as a sort criterion. 6 We found nearly complete overlap between the "common lymphoid progenitor" and early pro-B cells on the basis of Sca-1 expression ( Figure 5).…”

“…[22][23][24] However, we found that early pro-B cells were not homogeneous with respect to Sca-1 density. There was a tendency for the c-kit Hi cells to have higher amounts of Sca-1 than did the c-kit Lo/Ϫ TdT ϩ cells ( Figure 5).…”

Transferrin receptor in T cell activation and transplantation

“…The R17 217.1.3 rat IgG2a anti-mouse TfR hybridoma, a gift from Dr. Jayne Lesley (Salk Institute) [22], and the isotype-matched control E13 161-7 rat IgG2a anti-mouse stem cell antigen-1 hybridoma (which fails to demonstrate complement-mediated cytotoxicity in isolated CBA/J splenocytes; American Type Culture Collection, Rockville, MD) [23] were grown in culture and purified with a protein G affinity column.…”