Determination of frequency of T cells expressing the T cell-specific serine proteinase 1 (TSP-1) reveals two types of L3T4+ T lymphocytes.

Abstract

TSP-1 is a murine T cell-specific serine proteinase which is exclusively expressed in activated but not in resting T lymphocytes. Among T lymphocyte clones tested so far the enzyme was found to be associated with all Ly-2+ but only with a fraction of L3T4+ lines. Here we have applied a limiting dilution system to determine the frequency of precursor cells of resting L3T4+ and Ly-2+ lymphocytes which can be induced in vitro by antigen/lectin to express TSP-1. T cell subsets were either positively enriched by flow cytofluorometry cell sorting or by negative selection using monoclonal antibodies and complement. Following stimulation of lymphocytes in vitro, individual microwells were tested for growth by visual examination and for the TSP-1 protein/enzyme by analyzing cell lysates using either a specific rabbit anti-TSP-1 antiserum and/or the chromogenic model peptide substrate H-D-Pro-Phe-Arg-p-nitroanilide. Moreover, a large panel of L3T4+ and Ly-2+ T lymphocyte clones generated from primary cultures were similarly investigated. In some cell cultures the presence of TSP-1 was also tested on the mRNA level using a TSP-1-specific oligonucleotide probe. The data show that the majority, if not all, of antigen/lectin-induced-Ly-2+ T cells expressed TSP-1. In contrast, only 12%-27% of the growing lectin or antigen-reactive L3T4+ lymphocytes were positive for the enzyme. Studies performed in parallel with L3T4+ and Ly-2+ lymphocyte populations sensitized in bulk culture showed that under these conditions similar levels of TSP-1-specific mRNA and enzyme activity are detected in both subsets. The finding of primary L3T4+ T lymphocyte clones with distinct patterns of TSP-1 production provides evidence for the existence of two types of L3T4+ effector cells with different functional capacities. The data also suggest a cooperation between distinct L3T4+ lymphocytes for induction of optimal TSP-1 activity in L3T4+ T cells.