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The role of antigen-presenting cells (APC) in the induction of antigen-specific unresponsiveness was examined, using two functionally distinct murine macrophage hybridomas, #59 and #63 cells. Derivatized with the hapten (dinitrofluorobenzene; DNFB), #59 cells induced contact hypersensitivity (CH) in mice. Hapten-derivatized #63 cells failed to induce CH. Instead, they prevented recipients from acquiring CH when exposed subsequently to a sensitizing dose of the hapten. Similarly, hapten-derivatized #59 cells, pretreated in vitro with transforming growth factor-beta2 (TGF-beta2) lost their capacity to evoke CH, and induced tolerance. Hapten-derivatized #63 cells and TGF-beta2-treated #59 cells eliminated CH in mice sensitized to hapten. Reverse transcription-polymerase chain reaction analysis of mRNAs for various accessory molecules important in T-cell activation revealed that #63 and TGF-beta2-treated #59 cells differed only in their expression of tumour necrosis factor-alpha (TNF-alpha) mRNA. The latter expressed higher levels of TNF-alpha mRNA than did untreated #59 cells. As a consequence, #63 and TGF-beta2-treated #59 cells, both of which induce tolerance, secrete TNF-alpha protein unlike untreated #59 cells, which do not induce tolerance to hapten. Since neutralizing anti-TNF-alpha antibodies abrogated the tolerogenic potential of #63 cells in vivo, we conclude that TGF-beta2 equips hapten-bearing APC with the capacity to evoke systemic immune deviation in which CH is selectively silenced. We speculate that one effect of TGF-beta2 is to cause APC to up-regulate TNF-alpha production. In turn, this cytokine biases the functional property of responding hapten-specific T cells in a direction that not only interferes with acquisition, but suppresses induction of CH.

Cyclosporin A (CsA) is reported to inhibit programmed cell death. We confirmed this by using T-cell hybridomas which are inducible to programmed cell death by activation with immobilized anti-CD3 antibody or with anti-Thy 1.2 antibody. Cell death and DNA fragmentation, characteristic features of programmed cell death, were almost completely blocked by CsA or FK506. To investigate whether CsA inhibits only the cell death through the signals via the TCR/CD3 complex or all of the programmed cell death induced by various reagents, we further established CD4+8+ thymic lymphomas which result in programmed cell death after activation with calcium ionophore, dexamethasone, cyclic AMP or anti-CD3 antibody. It was revealed that CsA could block only the cell death mediated by the TCR/CD3 complex. For the clarification of the site of action of CsA, Ca2+ influx and endocytosis of receptors after stimulation with anti-CD3 antibody were monitored in the presence of CsA, and no significant effects of CsA were observed. Furthermore, prevention of cell death was examined by adding CsA at various periods of time after initiation of culture. CsA was found to exert its effect even when added after 4 h of cultivation, and the kinetic pattern of suppression was similar to that of the suppressive effect on IL-2 production. These observations indicate that in the events of programmed cell death, the major site of action of CsA will not be the inhibition of the immediate membrane events after activation of the TCR/CD3 complex but rather the interference in the function of molecules that transmit signals between membrane events and the activation of genes in the nucleus.