Immunoregulatory effects of everolimus on in vitro alloimmune responses

Everolimus (EVL) is a novel mTOR-inhibitor similar to sirolimus (SRL) that is used in organ transplant recipients, often in combination with tacrolimus (TAC) or mycophenolate (MPA). The current study aims to determine its effects on regulatory T cells. Increasing concentrations of EVL, MPA and TAC alone or in combination were added to MLRs of healthy volunteers. Lymphoproliferation by ³H-TdR incorporation and the percentage of newly generated CD4⁺CD127^-CD25⁺FOXP3⁺ (total Treg) and CD4⁺CD127^-CD25^HighFOXP3⁺ (natural Treg) in CFSE labeled responder cells were assessed by flow cytometry. In comparison to medium controls, EVL and other agents dose-dependently inhibited ³H-TdR incorporation in HLA-2DR-matched and HLA-mismatched MLRs (n = 3-10). However, EVL significantly amplified newly generated total and natural Tregs in CFSE labeled responder cells (p<0.05) at all concentrations, while MPA and SRL did this only at sub-therapeutic concentrations and inhibited at therapeutic levels. In contrast, TAC inhibited newly generated Tregs at all concentrations. When tested in combination with TAC, EVL failed to reverse TAC inhibition of Treg generation. Combinations of EVL and low concentrations of MPA inhibited proliferation and amplified Treg generation in an additive manner when compared to medium controls or each drug tested alone (p<0.05). The relative tolerogenic effect from high to low was EVL > SRL> MPA > TAC. If the results from these in vitro studies are extrapolated to clinical transplantation, it would suggest EVL plus low concentrations of MPA may be the most tolerogenic combination.