Everolimus Enhances Immunomodulation of Alloreative T Cells by Multipotent Stromal Cells Due to Transforming Growth Factor - β Dependent Mechanisms

Abstract 2545

Steroid refractory Graft versus Host Disease (GvHD) is a serious complication following allogeneic haematopoietic stem cell transplantation (HCT). Recently, novel therapeutic strategies involving multipotent stromal cell (MSC) transfusions have shown promising clinical results. Nevertheless, little is known on the interaction between MSC and immunosuppressive agents currently used for GvHD treatment and prophylaxis. Here we investigated the effects of mammalian target of rapamycin (mTOR) and calcineurin inhibitors on MSC mediated allogeneic T cell suppression. Since both of these drugs inhibit protein translation and exert potent antiproliferative effects, we assumed that MSC immunomodulation would be abrogated following mTOR or calcineurin inhibitor treatment. In an experimental in vitro suppression assay human MSC were pre-incubated with either everolimus or cyclosporine at concentrations currently used in the clinical practice. After 3 hours, MSC were washed several times to remove any immunosuppressive drug in the supernatant and cultured with magnetically sorted allogeneic human CD4⁺ or CD8⁺ T cells at a ratio of 1 MSC to 10 T cells prior to T cell activation with anti-CD3/CD28 coated beads. Surprisingly, MSC treated with mTOR inhibitors exerted significantly enhanced suppression of allogeneic CD4⁺ T cell proliferation (76%+/−12%) as determined by thymidine incorporation in comparison to MSC pre-incubated with cyclosporine (59%+/−12%) or untreated MSC (39%+/−10%). Similar results were obtained when MSC were cultured with CD8⁺ T cells. High pressure liquid chromatography (HPLC) did confirm that no remaining immunosuppressive drug in the culture supernatant was responsible for this observation. Subsequently we investigated, whether regulatory T cells (Treg) expansion would account for this enhanced MSC mediated immunosuppression. When everolimus treated MSC were added to CD4⁺ T cells in the suppression assay, significantly more lymphocytes expressed a regulatory CD4⁺CD25^high FOXP3^high phenotype (22%) in short and long term cultures while cyclosporine pre-treatment of MSC induced a Treg population (10%) comparable to untreated MSC (7,3%). When neutralizing antibodies against transforming growth factor-beta (TGF-β) were added, lower numbers of Tregs were induced by mTOR treated MSC (15,7%), cyclosporine treated MSC (8,8%) and untreated MSC (7,3%). In addition, several reports proposed indoleamine 2,3-dioxygenase (IDO) as a potent mediator of MSC dependent immunosuppression, since IDO expression results in depletion of tryptophan that is essential for cell proliferation. In the presence of interferon-gamma (IFN-γ), MSC up-regulate IDO and exert enhanced suppression of alloreactive T cell proliferation. However, when MSC pre-treated with everolimus were stimulated with IFN-γ, IDO expression was reduced. In addition IFN-γ signalling abrogated Treg expansion induced by everolimus pre-treated MSC. Thus, combined effects of mTOR inhibitors and IFN-γ signalling reduced MSC mediated T cell suppression. Collectively, these data suggest that MSC pre-treated with mTOR inhibitors induce enhanced immunosuppressive capacity towards allogeneic T cells due to induction and expansion of Tregs in a, at least partially, TGF-β dependent way. In contrast, mTOR inhibitors and IFN-γ enhance MSC immunomodulation by independent mechanisms. However, when combined they antagonize each others effects. In conclusion, our results support the combined use of mTOR inhibitors and MSC for the treatment of steroid refractory GvHD. This combination may induce Treg expansion that can treat GvHD without limiting graft versus tumor effects. Additionally, determination of IFN-γ serum levels may predict the outcome of this combined therapy.