PRODUCTION AND CHARACTERIZATION OF CYTO-TOXIC THY-1 ANTIBODY-SECRETING HYBRID CELL-LINES DETECTION OF T-CELL SUBSETS

Responses 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, F7D 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, F7D 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 F7 D 5 antibody behaved as a potent and absolute T cell‐depleting agent. This cell line and some anti‐Thy‐l.1 producing lines are available for research purposes. Copyright © 1979 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim