would increase the number of cells to 96 144 x 10 9 Treg, and would thus

yield sufficient numbers of Treg.

Antigen-specific Treg immunotherapy has several advantages over polyclonal Treg therapy. If Treg specific for other targets present in the polyclonal Treg population dampen immune reactions that should not be suppressed, their infusion would increase the risk for opportunistic infections and tumor-growth. So using antigen- specific Treg would reduce the risk of non-specific immune suppression. Also, due to the low percentage of Treg within a polyclonal cell pool that is specific for a given antigen, large numbers of Treg need to be infused to efficiently suppress alloantigen reactivity. Using antigen-specific Treg reduces the number of Treg

7

needed for effective immunotherapy. In animal studies using antigen specific Treg, only 0.03 - 2 x 106 _{Treg are needed for successful immunotherapy [6;7;20;24;25;94].}

Extrapolation of these numbers to the human clinical setting indicates that 0.1 - 7 x 109 _{antigen-specific Treg would be needed. The efficient alloantigen-specific}

expansion protocol described in chapter 5 will help to provide sufficient numbers of Treg with direct alloantigen-specificity for transplantation settings. Other protocols yielding Treg with direct alloantigen-specificity [80;95], and protocols yielding Treg with indirect alloantigen-specificity [96;97] have been described. For autoimmunity, no expansion protocols for human autoantigen-specific Treg have been described to date. In an animal study, it has been shown that endogenous Treg from diabetic-prone mice can be expanded ex vivo using recombinant peptide stimulation [24]. A similar strategy can possibly be developed to obtain sufficient numbers of autoantigen-specific Treg for human patients.

Selection of Treg subsets according to additional criteria might further improve the efficacy of Treg immunotherapy. For example selection of Treg on the basis of trafficking receptor expression (as discussed above) might make Treg therapy more efficient and/or more specific. However, due to lack of experimental experience with this approach, no estimation of numbers of Treg needed in patients can be given.

TIMING Of Treg INfUsION

Treg immunotherapy might be effective for the induction of tolerance in transplantation, but also for dampening other unwanted immune responses, such as in autoimmune diseases. Transplantation is probably the best setting to gain the first experience with this type of therapy, as the time of initiation of the undesirable immune response is exactly known. In most animal models, Treg are administered before or around the time of transplantation. This approach seems to be effective, as it leads to prevention of alloantigen responses and promotes long-term tolerance [39]. When alloantigen-specific Treg are to be used, adoptive transfer around the time of transplantation requires foregoing knowledge of donor HlA-type and culture-time to select for alloantigen-specific Treg. This should be possible for planned transplantations such as stem cell transplantations and living donor solid organ transplantation. However, when solid organs derived from deceased donors are transplanted, there is obviously no prior knowledge of the donor characteristics and no time to select for alloantigen-specific Treg before to the transplantation. It is currently not known what would be more effective in this situation: polyclonal Treg around the time of transplantation, or alloantigen-specific Treg at a later point in time. In most animal models, Treg are given as a single infusion. It is not known whether a single infusion will be sufficient and optimal in clinical settings. Multiple infusions, with additional Treg administration at later stages after transplantation might be more effective to control ongoing responses. Also, a combination of specific subsets of Treg administered at different time points could hypothetically be very effective. For example, it could be beneficial to infuse Treg with direct antigen specificity at the time of transplantation to prevent acute rejection, followed by Treg with indirect antigen specificity at a later time point to prevent chronic rejection. Alternatively, it might be beneficial to infuse Treg with a lymphoid organ homing phenotype at the time of transplantation to limit

the induction of allograft rejection, followed by Treg with a target organ homing phenotype at a later time point to dampen local immune responses.

Clinical implementation of Treg based therapy will be highly facilitated if Treg can be stored prior to infusion, as this will allow a more flexible timing of Treg therapy. In chapter 6, we have shown that Treg survive cryopreservation, although suppressive capacity was decreased. fortunately, our experiments also show that Treg that are expanded in vitro either before or after cryopreservation retain normal suppressive capacity.

CONCLUDING REMARKs

Treg immunotherapy is a very promising approach to specifically dampen unwanted immune responses, such as alloantigen responses in transplantation and autoantigen responses in autoimmunity. Even though animal studies and small scale clinical trials have shown great potential, many important aspects of Treg biology and Treg immunotherapy have not been clarified yet. In this thesis, we described a number of studies addressing these issues, like Treg phenotype and function in secondary lymphoid organs and in a skin model, Treg expansion and selection of alloantigen-specific Treg, and Treg isolation in a clinical grade manner. slowly but surely, progress is made towards unravelling Treg behaviour in healthy states, as well as in inflammation in animal models and in humans. Tolerance biomarkers to monitor the success of Treg immunotherapy and to guide safe tapering of immunosuppressive drug treatment in patients with alloantigen or autoantigen tolerance have been described and should be validated in prospective trials [98]. In the future, all this knowledge can be used to optimize Treg immunotherapy and reduce immunosuppressive drug treatment in transplantation and autoimmunity. Although the isolation and selection of Treg for each patient will be costly and time-consuming, reaching the goal of inducing antigen-specific tolerance has the potential to greatly reduce patient morbidity and mortality by preventing transplant loss, graft-versus-host disease and autoimmune symptoms, without the increased incidence of infections and tumours associated with current immunosuppressive treatments.

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