Limiting dilution analysis of induced unresponsiveness to trinitrophenyl: increased suppression of cytotoxic T precursor cells at unchanged frequencies.

In this paper we address the problem of tolerance in the immune system. We are discussing results of experiments that we designed to distinguish between the two major alternative hypotheses that have been invoked for immunological tolerance: clonal deletion/anergy versus suppression. These alternative hypotheses make essentially different predictions with respect to the frequencies of lymphocytes that react with antigens to which the immune system is tolerant: clonal deletion models predict reduced frequencies, whereas in suppression models precursors of effector cells are postulated to occur at frequencies similar to the nontolerant situation. We investigate these questions by limiting dilution analyses of cytotoxic T cell precursors (CTLP), using two different ways to activate them into functional cytotoxic cells (CTL): polyclonally by Concanavalin A (ConA) and specifically by antigen. Tolerance should be apparent in either protocol since neither antigen nor ConA usually activates self-reactive cytotoxic cells. Moreover, in addition to providing information on precursor frequencies, the multihit results usually obtained in limiting dilution experiments of ConA activated T cells allow the determination of quantitative and qualitative parameters of suppression. In addition, clonal anergy situations could be expected to become apparent by a differential sensitivity of precursors to activation with ConA and with antigen. As experimental system we chose the induced unresponsiveness of mice to trinitrophenyl (TNP) achieved by intravenous injection of reactive trinitrobenzenesulfonic acid (TNBS) and measured in a primary cytotoxic response to TNP-coupled syngeneic cells (TNP-SC). An equally specific but perhaps not identical form of unresponsiveness is induced by coupling the responder cell population with TNBS in vitro. Although we do not propose that this model ideally reflects all aspects of self tolerance, we think that in this particular system of induced unresponsiveness the antigen becomes associated with the surfaces of a large proportion of the cells of the body. Therefore, tolerance to widely expressed cell surface determinants might be quite adequately "simulated" by this experimental unresponsiveness. In our limiting dilution experiments we can distinguish between frequent and infrequent cytotoxic precursors which correspond to virgin and memory cells, respectively. For the frequent CTLP the situation is quite clear: they occur at essentially equal frequencies in normal and tolerant mice, as demonstrated by their reactivity to antigen at low cell densities. However, they are under the influence of a suppressive activity which is strongly increased when compared to that of normal mice.(ABSTRACT TRUNCATED AT 400 WORDS)