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A: Immunity/Tolerance

Research Area A is developing concepts to modulate immune mechanisms at the xenograft-host interface. A1 will test the hypotheses that: i) SLA class-I deficiency with additional downregulation of SLA class-II and/or transgenic expression of hPD-L1 will result in tissues with particularly low immunogenicity; and ii) clinically suitable donor-recipient combinations can be defined by SLA-HLA matching. A2 will develop strategies of local immunomodulation delivered either by recombinant adeno-associated viral vectors (rAAV), micro-RNAs, small hairpin (sh) RNAs, or by genetic modification of the donor animal to support long-term survival of cardiac xenografts. A3 and A5 will synergistically address mechanisms of the instant blood-mediated inflammatory reaction (IBMIR) induced by intraportal islet cell transplantation and test strategies to overcome them in unique ‘humanised’ mouse models. A4 will characterise the functional peculiarities of xenoreactive regulatory T cells (Treg) and establish protocols for Treg-mediated tolerance induction, e.g. by expression of xenospecific chimeric antigen receptors (CARs).

To overcome the major immunological barriers to xenotransplantation, strategies have to be developed that ensure long-term engraftment of the transplanted tissue or even achieve tolerance. Although probably difficult in the near term for vascularised xenografts, it is a realistic option for cellular xeno-transplants such as pancreatic islets. Mesenchymal stem cells are known to have an immunomodulatory effect when co-transferred with donor cells, and this should be applicable to xeno-islets. Furthermore, donor-reactive regulatory T cells (Treg) have been shown to facilitate tolerance induction in allograft settings. Such cell-based immunomodulatory therapies provide opportunities to control donor rejection of xenografts. However, there are clear differences between allo- and xenografts. For example, Treg function in alloimmunity mainly depends on cell contact mechanisms, while in xenografts immunomodulatory cytokines play a major role. The endothelium is a major target for immune responses to vascularised grafts. Thus, in addition to strategies applying Treg for immunomodulation, modification of the endothelium could provide graft protection. In this context, expression of immunomodulatory genes in endothelial cells, the use of mesenchymal stem cells to improve vascular function; and partial substitution of graft endothelial lining with host-derived endothelial cells might facilitate adaptation to the host immune system. New concepts for immunomodulation/tolerance induction will be developed by projects A2 and A4.

An important prerequisite for further development of clinical xenotransplantation is the availability of monitoring platforms particularly adapted to assess xenogeneic immune responses in the recipient and the status of the xenograft. One important result expected from monitoring data is the definition of biomarker profiles that serve as rapid surrogate markers to identify patients at high risk of graft rejection versus those where it may be possible to decrease immune suppressive medications Sawitzki 2009) Projects A1 and A4 include work packages that aim to define the individual risk profile of patients receiving xenografts. Work in this area will be crucial to organ and cell transplantation during research and development stages and in pre-clinical and clinical trials. The scientific topics addressed by projects of group A are closely connected to project groups B (Novel transgenic strategies) and C (Preclinical and clinical xenotransplantation).

Genetic variability in immunologically relevant molecules plays an important role in the regulation of an individual’s immune response. R. Schwinzer/J. Hundrieser (project A1) will test the hypothesis that variability in molecules encoded by the major histocompatibility complex (MHC) and the natural killer gene complex (NKC) determines the intensity and/or quality of the individual recipient’s immune response to a cellular xenograft. Congenic and recombinant rat strains expressing defined MHC and NKC haplotypes will be used as recipients for different porcine cell grafts (e.g. islets, endothelial cells). In vivo and in vitro studies will be performed to characterise the immune response patterns of cells from different rat strains to stimulation with porcine xenoantigen. Furthermore, this project provides a functional monitoring program to assess the biological activity of transgenes in pigs and the immunogenicity of different porcine MHC haplotypes (project group B). Data from this project will help define MHC and NKC haplotypes associated with differential risk of cellular xenograft rejection and might also unravel novel targets for immunomodulation after clinical xenotransplantation.

The endothelium of the donor is the first cell lining exposed to the recipient’s immune system and thus of particular importance in the initiation of transplant rejection. Project A2 will modify the endothelium, applying innovative strategies to prevent rejection and achieve adaptation. Adeno-associated virus (AAV) vectors, which have been successfully used by this group for transduction of pig myocardium, will be coated with nanoparticles aiming at highly efficient, homogenous transduction of target cell populations. Nano-AAVs will be equipped with novel transgenes as adjuvant immunosuppression therapy complementing transgenic techniques (project group B). As a second approach to achieve adaptation, vascular progenitor cells or mesenchymal stem cells syngeneic to the recipient endothelium will be inserted into the graft vasculature. The recruitment will utilise CXCR4 expression of progenitor / stem cell populations, which will be attracted by an artificial homing molecule, S1FG, and may initiate replacement of the graft endothelium by host endothelial cells. Furthermore, the question will be addressed as to whether stepwise elimination of donor endothelial cells can be obtained by inducible suicide gene activation. This project will also provide the consortium with viral vectors and assays for endothelial adaptation.

Hyperacute rejection mediated by anti-Gal antibodies and complement does not play an important role after pig-to-primate islet transplantation. Survival of pig islets is limited by the immediate blood-mediated inflammatory reaction (IBMIR), which is in part mediated by the infiltration of neutrophils and monocytes/macrophages. Understanding the molecular mechanisms of initial leukocyte infiltration in the early phases of pig islet transplantation is the central question addressed by project A3. A thorough analysis of the interactions of human leukocytes with porcine islets will be performed, and the mechanisms of leukocyte recruitment and macrophage polarisation in the early immune responses to porcine islet xenografts will be characterised. In vitro experiments will be complemented by in vivo studies using a novel mouse model (Rag2nullIL2rgammacnull Kitwv/wv) reconstituted with a human immune system, established by Dr. Waskow as recipients for porcine islets. Results from this project (e.g. identification of targets to prevent early immune responses) will be translated into non-human primate models (project group C) and/or will influence selection of new transgenes for genetic engineering of pig (project group B).

Regulatory T cells (Treg) are known to play an important role in the balance between induction and maintenance of tolerance and could thus be used as therapeutic reagents to control aberrant or unwanted immune responses in the case of autoimmunity or transplantation. Project A4 is a “bridging project” combining the Treg expertise present at LMU and MHH. They will explore strategies to reduce T cell-mediated xenogeneic responses in recipients by means of recipient-derived Treg. The central goal is to induce tissue-specific xenogeneic tolerance. Xenospecific Treg will be generated by transfecting T cells with FOXP3 after in vitro sensitisation to xenoantigen (mixed lymphocyte reactions). Islet antigen-specific Treg will be generated by creating a chimeric antigen receptor (CAR) using the beta cell specific single chain antibody Sca-1, or a xenospecific single chain antibody. Sca-1 recognises a beta cell specific target antigen in vivo. Furthermore, natural Treg populations (nTreg) from humans and non-human primates will be prepared and expanded ex vivo. The capacity of different Treg populations to diminish xenogeneic T cell responses will be assessed in vitro. The in vivo potential of the Treg concept will be studied in humanised mouse models of porcine islet transplantation, pig-to-baboon heart transplantation and pig-to-primate islet transplantation (project group C).