**4. Strategies to reduce the burden of immunosuppression and future directions**

Strategies to limit the impact of chronic immunosuppression include protocols of drug minimization towards individualization of organ-specific immunosuppression *regimens*, development of new non-nephrotoxic agents and trials of tolerance induction. Drug minimi‐ zation *regimens* are being explored in select patient populations to improve the safety of current immunosuppression protocols while preserving their efficacy. This strategy is based on the concept that, over time, the risk of rejection decreases and, at the same time, the cumulative risk for toxicity increases. Studies have concentrated on corticosteroids minimization and calcineurin-inhibitors minimization (review in [66]). Careful patient selection and close monitoring of graft function are mandatory steps for a successful conduct of a drug minimi‐ zation attempt in order to avoid rejection and graft loss.

At present we still lack reliable methods to identify transplant recipients who can be weaned of immunosuppression, although a number of candidate assays have been proposed to identify operationally tolerant patients. Among them, transcriptional profiling with either microarray or real-time PCR is currently a promising approach [67]. Peripheral and intra-graft expression markers of immune activation are used as tools to guide patient selection and monitor the progress of drug minimization trials [68]. In renal transplantation, non-invasive urine bio‐ markers have been described by measuring mRNA of inflammatory cytokines [69]. In addition, studies on urine proteomics allowed to identify different causes of graft dysfunction [70]. These non-invasive tools with or without protocol allograft biopsy offer the opportunity to monitor patients enrolled in trials of drug minimization.

In recent years, advances in immunosuppression that target specific pathways of the alloim‐ mune response have been developed (review in [71]). In particular, new medications targeting the processes related to ischemia-reperfusion injury are currently under evaluation [72]. The ischemic insult to the allograft associated with the procurement and implantation processes contributes to trigger the immune activation of the recipient via the release of immunologically active substances known as damage-associated molecular patterns (DAMPs) [73]. In addition, new agents are being developed acting on the cellular and humoral mechanisms of the adaptive immune response. These include antibodies and fusion proteins interfering with Tcell-mediated activation via LFA-1/ICAM-1, CD2/LFA-3, CD40/CD154, and CD28/B7.1 and B7.2 interactions [74]. Furthermore, intracellular targets involved in T- and B-cell activation pathways are being evaluated, including protein kinase C inhibitors, Janus-associated kinase (JAK) inhibitors, and proteasome inhibitors. Several new medications demonstrate promise in inhibiting donor-directed humoral immunity by targeting B-cell-activating factor (BAFF) and complement activation pathways. Finally, other strategies are targeting the "memory" component of the T-cell repertoire [75] or the regulatory component [76].

Currently, transplant recipients are bound to lifelong immunosuppression. However, there have been reports of rare instances of "tolerance", defined as the maintenance of allograft function without immunosuppression. Although several definitions of tolerance have been proposed ("complete tolerance", "prope" tolerance, "operational" tolerance and others) and consensus is still lacking on the underlying mechanisms involved in tolerance, indeed there are patients who either intentionally or accidentally fail to reject the allograft and maintain allograft function while under minimal or no immunosuppression. As an example, in 1993 a series was reported of 11 liver transplant recipients maintaining normal liver function following the discontinuation of all immunosuppressive drugs as a consequence of either noncompliance or lymphoproliferative disorders [77]. Unfortunately, due to the heterogeneity of the human immune response, it has been so far prohibitively difficult to replicate these results on a larger number of patients and to establish tolerance in the clinical setting. The individualization of immunosuppression, identification of biomarkers of tolerance and of rejection and real-time monitoring of post-transplant immune responses may facilitate induction of lasting tolerance in humans [78,79] The advancement of many high-throughput 'omic techniques such as genomics, proteomics and metabolomics has allowed to identify potential mechanisms of specific graft injury and to develop novel biomarkers for acute rejection, chronic rejection and operational tolerance [80,81]. Finally, the pharmacogenomics of organ transplantation has emerged recently as a complement to the immunogenetic information that has accumulated over the past decade [82]. Polymorphism studies focus on genes that interact across the group of immunosuppressive drugs (cyclosporin, tacrolimus, sirolimus and corticosteroids) such as CYP3A5, ABCB1, IMPDH1 and IMPDH2, and cytokines and growth factors. Although not routinely used in the clinic, it is expected that in the near future clinical pharmacogenomics techniques will become additional tools in the management of organ transplant patients.
