ABSTRACT
The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.
Subject(s)
Cytotoxicity, Immunologic/drug effects , Killer Cells, Lymphokine-Activated/drug effects , Ornithine/pharmacology , T-Lymphocytes, Cytotoxic/drug effects , Animals , Antigens, Differentiation, T-Lymphocyte/analysis , Arginine/pharmacology , CD8 Antigens , Dose-Response Relationship, Drug , Immunity, Cellular/drug effects , Interleukin-2/pharmacology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred Strains , Putrescine/pharmacology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunologyABSTRACT
DL-alpha-Difluoromethylornithine (DFMO) is a specific inhibitor of the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC). DFMO (1 mM) added to C57BL/6 anti-DBA/2 murine mixed lymphocyte cultures (MLC) inhibited cytolytic T lymphocyte (CTL) activity on days 3 and 5 by 88% and 96%. Putrescine (PUT; 1 mM) and spermidine (SPD; 0.01 mM) reversed DFMO inhibition, indicating that DFMO inhibition was caused by ODC antagonism. T helper (Th) cell and accessory cell functions were not affected since DFMO did not inhibit MLC proliferation or lymphokine production. Furthermore, exogenous IL-1, IL-2, IL-4, interferon-gamma, or a rat Con A supernatant failed to abrogate DFMO inhibition. Inhibition was reversible within 48 h of removing cells from DFMO; moreover, subsequent development of DFMO-blocked CTL did not require CD4+ cells. Clonal expansion of CTL treated with 1 mM DFMO for three days in MLC, determined by subsequent analysis in limiting dilution microcultures, was only approx. 1 cell division less than control. These results indicate DFMO inhibition is exerted directly on the CTL, and that the process of differentiation was more affected by a reduction in polyamine biosynthesis than proliferation. This may be a useful model to the study stages and events of CTL development, and the roles played by polyamines in supporting these processes.
