Allospecific Regulatory Effects of Sirolimus and Tacrolimus in the Human Mixed Lymphocyte Reaction

Background. Tacrolimus (TAC) and sirolimus (SRL), two commonly used immunosuppressive agents, have demonstrated contrasting immunoregulatory effects. We recently described factors affecting the generation of allospecific CD4(+)CD25(High) forkhead/winged helix transcription factor P3 (FOXP3(+)) T-regulatory (Treg) cells in mixed lymphocyte reaction (Treg MLR) and now report additional findings on the effects of TAC and SRL. Methods. TAC, SRL, or media without agents were added separately to MLRs using human leukocyte antigen two DR-matched and -mismatched healthy volunteers and prekidney transplant donor/recipient pairs. Concentrations correlated with subtherapeutic and therapeutic blood levels. Stimulation indices of H-3-TDR uptake, cell proliferation, and the generation of carboxy-fluorescein diacetate succinimidyl ester (CFSE) labeled CD4(+)CD25(High)FOXP3(+) cells by flow cytometry were initially compared. Each group of (non-CFSE labeled) MLR-generated cells were then added as third components to CFSE-labeled responding cells in freshly prepared primary MLRs, to determine allospecific and nonspecific inhibitory and Treg recruitment effects. Results. TAC inhibited stimulation indices and CD4(+)CD25(High)FOXP3(+) cell generation in both human leukocyte antigen DR-matched and -mismatched pairs, particularly at therapeutic levels (>= 5 ng/mL). SRL had an equivalent effect in matched pairs but was associated with a significantly higher %generation of CD4(+)CD25(High)FOXP3(+) cells than TAC. SRL-MLR-generated Tregs added as third components allospecifically inhibited MLR proliferation and recruited additional CFSE-labeled autologous Tregs compared with addition of TAC- or media-MLR-generated Tregs. Conclusions. Calcineurin and mammalian target of rapamycin inhibitors have disparate effects on allospecific Treg generation using the Treg MLR. This assay can thereby be helpful in assessing allospecific regulatory effects of diverse immunosuppressive agents.