Regulatory CD4+CD25+Foxp3+ T cells (Tregs) play an important role in the induction of allospecific tolerance. Yet, the sole transfer of Tregs from the natural repertoire was not capable of inducing allotolerance so far. In addition, while polyspecific Tregs are sufficient to prevent graft versus host disease after stem cell transplantation, they are largely inefficient in preventing unwanted immune responses in autoimmunity and transplantation. Instead antigen-specific Tregs will be needed under such non-lymphopenic conditions. Finally the stability of the Treg phenotype has recently been questioned. Therefore, translation of adoptive Tregs into clinics will need stable, antigen-specific Tregs in sufficient numbers. Alloantigen-specific Tregs were generated by stimulating naïve recipient CD4+ T cells with irradiated donor splenocytes: CD25+CD69+ allospecific recipient CD4+ T cells were isolated and transduced with Foxp3. These transduced Tregs phenotypically resembled natural Tregs and could be expanded up to 18-fold in vitro while maintaining a stable Treg phenotype. Furthermore the transduced cells showed suppressive capacity in vitro as well as in vivo by preventing acute allograft rejection in a lymphopenic and for the first time in a non lymphopenic and strongly immunogenic skin transplantation model with full MHC mismatch (C57BL6→BALB/c). Long term tolerance could be achieved in both models. Interestingly alloantigen-specific Tregs with direct and indirect antigen recognition were far more efficient in this model than their polyspecific counterparts. For the first time we could demonstrate long-term persistence and local accumulation as major mechanisms of Treg mediated allotolerance. In fact tolerance could be transferred by Tregs of the tolerated graft. Infectious tolerance and deletional tolerance of alloreactive T cells did not contribute significantly to tolerance identifying the transferred Tregs as major mediators of long-term tolerance. In addition for maintenance of tolerance the local presence of alloantigen-specific Tregs was sufficient and presence of these cells in secondary lymphoid compartments was not needed. Foxp3 transduced Tregs exhibited a stable phenotype even under lymphopenic and inflammatory conditions. Furthermore adoptive Treg therapy was safe and no malignant transformation was observed. Constant expression from the integrated Foxp3 locus was needed, as the endogenous Foxp3 locus remained methylated despite expression of endogenous Foxp3. The results prove that large numbers of stable alloantigen-specific Tregs can be generated from a polyclonal repertoire of naïve T cells. This is the first time that allotolerance was achieved in a non lymphopenic transplant model using skin grafts in an immunogenic strain combination. Therefore, antigen-specific Tregs might have a huge therapeutic potential after solid organ transplantation.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Fragestellung: In vitro expandierte, antigenspezifische CD4+CD25+ regulatorische T Zellen (Tregs) können die Autoimmunantwort im Typ I Diabetes Modell der Nonobese Diabetic Maus (NOD) unterdrücken. Es ist jedoch fraglich, ob ausreichende Mengen antigenspezifischer Tregs mit hochaffinen T Zellrezeptoren aus einem polyklonalen T Zellrepertoire generiert werden können.

Beta-cell specific autoreactive T cells can be found in patients with type I diabetes (T1D) and in healthy controls. They are usually controlled by a network of regulatory mechanisms including CD4+CD25+Foxp3+ regulatory T cells (Tregs). It was suspected that defects in Treg number and activity are causally related to the development of T1D. Although there are hints that this concept might be true, it is neither proven in animal models nor in patients with T1D. However, increasing the number of Tregs by adoptive transfer can be used to prevent and treat even established T1D. It was demonstrated that Tregs recognizing beta-cell antigens are far more efficient in treating the disease than polyspecific Tregs. The use of beta-cell specific Tregs is also leading to a tissue specific immunotolerance without perturbing the general immunocompetence. Two sources for beta-cell specific Tregs are currently employed: First natural Tregs specific for beta-cells are expanded IN VITRO and reinfused into diabetic animals. Second naïve or activated T cells specific for beta-cell antigens are IN VITRO converted to Tregs by genetic manipulation or by specific cytokine combinations. Both approaches were successful in treating even established diabetes in animal models. Before such therapies can be used in patients safety measures regarding the fate and the effects of the transferred Tregs have to be studied. Besides this ethical considerations are important in regard to what risks we should take to treat a disease in young patients which can otherwise be treated medically. In the meantime the concept of Tregs for therapy of T1D is supported by successful clinical attempts to induce these cells IN VIVO by administration of monoclonal antibodies against CD3. If subsequent studies show that Tregs represent a safe and efficient source for therapy, they could become an important weapon in the fight against immune mediated pathology.

Pilat, N1; Baranyi, U1; Klaus, C1; Jaeckel, E2; Mpofu, N2; Muehlbacher, F1; Wekerle, T1 Author Information