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IMMUNOsUPPREssIVE DRUGs

The induction of tolerance crucially depends on reduction and control of the alloantigen reactive Tconv pool [172;173]. While Treg based immunotherapy aims to achieve this goal, immunosuppressive drugs can certainly help in this process. Therefore, introduction of Treg based therapy into the human clinical setting will likely take place under the cover of concurrent treatment with immunosuppressive drugs with known efficacy to reduce the alloantigen reactive Tconv pool. Many immunosuppressive drugs that affect Tconv also have the potential to influence Treg, so it is crucial to select drugs that efficiently reduce the number or function of alloantigen-specific Tconv cells but allow Treg function and/or iTreg induction as much as possible. Calcineurin inhibitors such as cyclosporine and tacrolimus decrease T-cell receptor mediated Il-2 production and efficiently reduce the expansion of Tconv [174-176]. Caution is warranted if these drugs are to be used in combination with Treg based therapy, as although Treg suppressor function is maintained by calcineurin inhibitor treatment [177;178], Treg proliferation is severely reduced and induction of iTreg is abrogated [179]. Rapamycin is an immunosuppressive drug that is widely used in transplantation settings. This drug inhibits T-cell cycle progression by binding to mTOR [180]. Rapamycin is promising for combination with Treg based therapies, as it inhibits proliferation of Tconv more potently than that of nTreg [181;182] and it also allows nTreg suppressive function [177;182]. This is reflected in the finding that patients treated with rapamycin have higher percentages of Treg in peripheral blood as compared to cyclosporine treated patients [183;184]. Rapamycin also seems to promote the induction of TGF-β mediated iTreg [179]. Interestingly, in an animal model of transplantation, the administration of rapamycin combined with recombinant Il-10 leads to tolerance by in vivo induction of Tr1 cells [185].

Antibody therapy aimed at reducing the pool of Tconv after transplantation is being used in the clinical setting for decades. In recent years, it has been reported that some antibodies not only cause a reduction of Tconv numbers, but also mediate iTreg conversion. This has been shown for OKT3 (anti-CD3 monoclonal antibody) [186], anti-thymocyte-globulin (polyclonal antibody cocktail) [187;188], and alemtuzumab (anti-CD52, present on T-cells, B-cells, monocytes, and granulocytes) [189;190]. The monoclonal anti-CD25 antibodies daclizumab and basiliximab, also used in the clinic, are aimed at inhibition of CD25pos _{activated Tconv, but obviously}

also affect CD25high _{nTreg. Indeed, nTreg populations are transiently undetectable}

in peripheral blood after daclizumab treatment [191]. The function of human nTreg is not affected by daclizumab and basiliximab in vitro [191;192], and a single dose allows the formation of Treg with indirect alloantigen-specificity in vivo in humans [23]. However, in an animal model of stem cell transplantation, tolerance induction was abrogated if daclizumab was administered at the time of transplantation [93]. The experimental drug malononitrilamide inhibits cell cycle progression and suppresses human Tconv while inducing iTreg conversion in vitro [193]. The novel immunosuppressive drug fingolimod interferes with cell trafficking between lymphoid organs and blood. Treatment of animals with fingolimod shifts the Treg to Tconv ratio towards the Treg side in lymphoid tissues as well as at inflammatory sites and enhances the suppressor activity of the Treg pool [194;195].

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Taken together, these studies strongly indicate that for successful implementation of tolerance inducing strategies in the clinical setting, the choice of immunosuppressive drugs may be crucial.

fUTURE PERsPECTIVE

Recently, the first clinical trials on infusion of ex vivo selected nTreg and ex vivo generated iTreg have been initiated in patients receiving stem cell transplantations. An overview of these studies is provided in recent review papers [196;197]. Several important issues should be clarified by these frontline trials. First, a cautious estimation of the clinical potential of Treg therapy may be obtained. Furthermore, data regarding the safety of Treg infusion in human patients will become available. Issues that require special attention are the occurrence of non-specific suppression and the detrimental effects of co-infused Tconv. The results of these first trials will have a great impact on the further development of Treg infusion therapy in the human transplantation setting.

For the near future, efforts should be made to find biomarkers to monitor the efficacy and safety of Treg therapy after transplantation. Up to now, evaluation of the efficacy of therapy is largely based on clinical outcome. A genuine assessment of the presence of tolerance after organ transplantation would require complete withdrawal of immunosuppressive drug treatment, which is not an attractive experiment for the graft recipient. When an accurate marker of tolerance would be available this could guide safe tapering of immunosuppressive drug treatment in patients with alloantigen-tolerance, and maintenance or intensification of treatment in patients in which the tolerance induction treatment has failed. Recent studies have described biomarkers that are associated with the presence of operational allograft tolerance [198;199]. However, there is no test available yet that can reliably identify patients in whom non-specific immunosuppression can be tapered successfully.

We expect that in future years, the technical difficulties regarding GMP Treg isolation will be largely resolved, which will lead to an increase in the number of clinical trials on Treg infusion after transplantation. Tracking of infused cells will provide information about the in vivo homing and stability of Treg, which is also crucial for optimal timing of Treg transfusion. An important issue regarding the infusion of Treg is that the techniques required for the custom-made cell preparations for each recipient are very laborious and expensive. Therefore, in vivo interventions resulting in increases of Treg number and function form attractive adjuncts or even alternatives. We expect that the current strategies will be fine-tuned for use in the human clinic and that therapies aimed to enhance the Treg mediated suppression of donor-specific immune responses at the expansion of Treg facilitation in vivo will significantly contribute to the establishment of transplantation tolerance.

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