Low-dose immunosuppression by cyclosporine operating via antigen-presenting dendritic cells

The inhibitory effect of cyclosporine on the stimulation of lymphocytes was tested in 20 microliters hanging drop cultures. Cells were stimulated by concanavalin A, or by allogeneic dendritic cells in a mixed leukocyte culture (MLC). Significant inhibition of lymphocyte proliferation was obtained with low concentrations of CsA (10(-9)-10(-7) micrograms/ml) in addition to the well-documented, dose-dependent inhibition at concentrations greater than or equal to 10(-1) micrograms/ml. Intermediate doses (10(-6)-10(-2) micrograms/ml) caused little inhibition. To establish whether the CsA was acting on the antigen-presenting cells or on the responding T cells, each population was pulsed with CsA before adding to the MLC. Higher doses of CsA were required to cause inhibition using this short-term pretreatment rather than continuous presence of CsA; however, when DC were pulsed with CsA, biphasic effects of the drug were seen. The time of DC pretreatment with 1 microgram/ml CsA was varied, and inhibition was seen after 1-3 hr and 16-24 hr pretreatment but not at intermediate time points. Similarly, a biphasic dose-response developed when DC-pretreatment time was fixed (2 hr) and drug concentration was varied. By contrast, on pulsing responder lymphocytes with CsA there was a biphasic effect in some experiments, but more often a single dose-dependent inhibition occurred, particularly when DC were removed from the responder cells. Major dose-dependent inhibitory effects of CsA may therefore act on both antigen-presenting DC and on lymphocytes, but this work reveals an additional low-dose inhibition that can operate via DC.