The action of antibodies and their fragments on leukaemic lymphocytes

Glennie, Martin John (1980) The action of antibodies and their fragments on leukaemic lymphocytes. University of Southampton, Doctoral Thesis.

Abstract

Some metabolic effects of antibody reacting with the surfaces of leukaemic lymphocytes have been investigated. The lymphocytes concerned are of B type and belong to the L2C line, which is passaged in vivo in strain 2 guinea pigs. Most of the work has involved antibody reacting with the surface immunoglobulin (Ig) of these cells in vitro. The work is divided into two main sections: the effects of antibody on production of surface Ig, and the resistance of the cells to lysis by anti-Ig plus complement. In both cases major effects were dependent on the surface Ig being cross-linked by bivalent antibody. In the first Section a radiolabelling assay was used to demonstrate that IS and the histocompatibility antigens Ia(2,4) and B.1 were all turning over at the cell surface with a half life of between 4-5 hours. Cross-linking the surface Ig with anti-Ig at 37°C was found to suppress its delivery to the surface membrane during subsequent culture. This suppression manifested as a considerable delay in re-expression of surface Ig compared with that observed after its removal by papain. Bivalent F(ab')2 fragments from anti-Ig also suppressed Ig delivery, while Fab'Y and Fab/c fragments did not, thus confirming the need for cross-linking by antibody. The inhibitory effects of anti-Ig appear specific, since throughout these experiments the production and turnover of both B.1 and Is were completely unaffected. Some preliminary studies showed that anti-Ig may act via a cyclic nucleotide: the specific inhibitory effect of anti-Ig could be mimicked by cholera toxin which like anti-Ig causes a surge in intracellular cyclic AMP. In the second Section the monovalent antibody fragment Fab/c, consisting of one Fab arm attached to the Fe via an intact heavy chain, was prepared by partial proteolysis of rabbit IgG. The fragment was used to investigate the role of antibody in the induction of 'antigenic modulation' - a process whereby antibody treated cells at 37°C are rendered resistant to lysis upon subsequent exposure to antibody plus complement. Thus L2C cells pre-treated with anti-Ig at 37°C for 15 min or longer underwent no lysis on addition of complement, although parallel batches of cells maintained at 0°C during the pro-treatment were susceptible to lysis. In contrast when Fab/c fragments of anti-Ig replaced the antibody in these experiments they demonstrated firstly the ability to invoke complete complementmediated lysis, and secondly the failure to modulate at 37°C. These results are consistent with antigenic modulation requiring the cross-linking of surface antigens by antibody. Fluorescent labelling studies confirmed the monovalentnature of Fab/c by showing that it resembled Fab'Y fragments in remaining dispersed on the surface after incubation with metabolically active cells at 37°C. The ability of monovalent antibody to induce L C cell lysis without antigenic modulation or reduced Ig delivery is discussed with regard to improving the therapeutic value of antibody directed against the idiotypic determinants on the surface 19 of B lymphomas.

This record has no associated files available for download.

Contributors

Download statistics