Production and characterization of cytotoxic Thy‐1 antibody‐secreting hybrid cell lines Detection of T cell subsets

Abstract

Of London, London UniversityResponses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells.For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7 D 5 , which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons.Serologically, F 7 D 5 Thy-1.2 antibody was found to behave as a conventional Thy-1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F 7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1producing lines are available for research purposes. + Supported by a Fellowship of the MRC of Canada.

Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.

“…Spleen cells for fusion were prepared 4 days later. Cell fusion was performed as described by Lake et al (1979). The plasmacytoma cell line Sp2lO-AG 14 (Sp2lO) (Shulman et al, 1978)was used for fusion.…”

Semliki Forest Virus-induced, Immune-mediated Demyelination: Adoptive Transfer Studies and Viral Persistence in Nude Mice

“…T lymphocytes were removed from preparations of spleen cells using a cytotoxic monoclonal anti-Thy 1.2 antibody (F7D5; Lake et al, 1979). This was a kind gift of Professor N. A. Mitchison (University College, London, U.K.) but can also be obtained from Olac Ltd (Bicester, U.K.).…”

Subpopulation of T Cells Sensitive to Natural Thymocytotoxic Autoantibody (NTA) of New Zealand Mice: I. Distinct Cytotoxic Sensitivity of Functional T Cell Subsets to NTA and Anti‐Thy‐1 Antibodies

“…Monoclonal anti-Thy-1.2 antibody was purchased from Olac Ltd., Blackthorn Bicester, U.K. (Clone F7D5, IgM class). This clone was established originally by Lake et al (16). An NTA-positive NZB mouse serum pool was obtained from the blood of 12-month-old NZB mice.…”