Everolimus Failure to Counteract the Cytotoxic Response Mediated by CD8+ T and NK Cells in Heart-Transplant Patients

Abstract

The negative effects of immunosuppression after heart transplantation (HT) are hard to avoid, but they can be partially overcome using new drugs such as the everolimus (EVL) mTOR (mammalian target of rapamycin) inhibitor. Initial studies have shown that EVL administration in combination with low-doses of calcineurin inhibitors and corticosteroids is effective in reducing the acute rejection and cytomegalovirus infection, two of the main causes of the development of cardiac allograft vasculopathy (CAV), and induce the expansion of regulatory T cells (Tregs). However, the impact of EVL introduction after HT on the in vivo immune response remains poorly understood. Here, we analyzed the changes in the immune phenotype on peripheral blood samples of 56 HT recipients who were treated with mycophenolate mofetil (MMF) or switched from MMF to EVL, and low-dose tacrolimus. We studied two cohorts; a “late cohort” of recipients of more than 1 year post-HT, who were converted from MMF to EVL (EVL group, n=20) following clinical criteria or maintained on MMF (MMF group, n=20) and an “early cohort” (n=16), who were switched from MMF to EVL at 3 months post-HT. Blood samples were taken at a single time one year post-EVL initiation and at different post-transplant times (before EVL initiation 1, 3 and 9 months after EVL conversion) in the early cohort. Immune phenotype was analyzed by flow cytometry and DNA methylation profiles in immune genes by pyrosequencing. Our results showed that EVL conversion from MMF induced the expansion of cytotoxic NK (CD56dim CD16+) and effector-memory (CD45RA- CCR7-) CD8+ T populations, showing significantly higher levels of expression of cytotoxic molecules (granzyme, perforin and IFN-γ) and greater degranulation ability under activation. Treatment with EVL modifies the NK cell receptors repertoire increasing the activation state and cytolytic activity of these cells. These changes are already evident at 1 month after EVL initiation and gradually increase during the first year post-transplant. Moreover, only one month after EVL initiation, HT patients showed a significant expansion of Tregs and decrease of B cells. We observed that these phenotypic alterations were associated with changes in the DNA methylation patterns of key immune genes (IFNG, PRF, FASL and PD1). Thus, quantification of the methylation levels in these genes could help to predict the alterations in the immune phenotype and its functionality after EVL conversion. In short, we observed that both early and late EVL conversion from MMF induces changes in the immune system which could condition the post-transplant immune response. The consequences of EVL initiation after HT can be considered as a double-sided coin; on the one hand induces the Tregs expansion and reduces the B cells number contributing to the immune tolerance, but at the same time failure to blockage the cytolytic ability mediated by CD8+ T and NK cells. According to that, EVL conversion as a general strategy after HT must be taken with caution.