The research of this proposal outlines a new strategy for improving transplantation tolerance in combinations of mismatch donor/recipient combinations such as parent/child, non-identical sibling combinations, and most combinations of unrelated donors. It has been shown in the past that a transfer of adult stem cells (hematopoietic stem cell) from a prospective organ donor in some cases have been able to establish a new parallel immune system (chimerism), which have made the transplant patient tolerant to the new organ graft. In connection with research on the autoimmune disease type 1 diabetes in humans, we have over the past 15 years developed a humanized mouse model that exclusively expresses human transplantation antigens (HLA-DR and DQ genes). The immune function of these mice have over these years been optimized with several new human transgenes to better reflect the function of the original immune and autoimmune responses that these HLA genes control in humans. Thus, we have transgenic mice that represent diabetes susceptible human HLA genes and other mice with HLA genes with known protective effects against development of type 1 diabetes. The proposal promotes the idea to manipulate and engineer the adult stem cell graft before transfer with a particular transient/short-lived defective virus infection, which boosts the capacity of these stem cells to survive in the recipient organism to further preserve donor specific tolerance in the future and allow for long-term survival of the "second graft" for example a kidney graft or new beta islets in diabetes. The humanized HLA class II transgenic mice will be used to evaluate the tolarizing function of the manipulated/engineered stem cell graft to protect and prolong graft survival after established immune chimerism followed by a new transplantation challenge of the mice with a donor specific fetal heart grafts or donor specific beta-islets transplantation of the mice. This re-education of the immune system may then allow us to transfer new donor beta cells from mice that carry transplantation antigens that are protective to the development diabetes into mice, where the insulin producing beta cells of the pancreas have been completely eliminated by means of high dose streptozotocin treatment. Since we are dealing with human transplantation antigens, we hope that our findings will inform the use of similar approaches to allow for transplantation of human stem and beta cells into patients with diabetes.
Statement of Benefit to California:
This proposal is beneficial to the State of California because it will establish an unique method to establish chimerism with human MHC class II molecules in the mouse. At the same time, it will investigate the possibility that improving antigen processing and presentation (APP) in the context of forced expression of tissue-restricted antigens (TRAs) by Aire and lack of co-stimulation, we will create a situation analogous to central tolerance in the thymus with cells in the periphery. A precedent for this notion exists already with extra-thymic Aire-expressing cells (ETACs) in the spleen. With subsequent tranplantation of allogeneic beta cells into diabetic mice, we will have established a paradigm for how to treat human T1D in the future. All systems that will be investigated in the mouse can be adapted directly to humans and we have all human transcription factors already. Thus, our humanized mice will inform directly how to approach chimerism and organ transplantation in humans, using stem cells and/or more mature organs.
This proposal is aimed at improving transplantation tolerance through the establishment of mixed chimerism, an approach previously demonstrated to produce tolerance to engrafted organs. Mixed lymphoid chimerism has been difficult to achieve reliably in humans. The applicant plans to establish lymphoid chimerism in a humanized mouse model and demonstrate induction of tolerance to a donor-specific organ graft. The investigator hypothesizes that such tolerance can be achieved by engineering a hematopoietic stem cell (HSC) graft to promote its survival and ability to regulate tolerance. In Aim 1, the investigator proposes to transfer donor HSCs into MHC mismatched, humanized mice that are either immunodeficient or immunocompetent and to establish lymphoid chimerism that promotes the survival of a second donor-specific cardiac graft. In the second aim, the applicant plans to promote long-term chimerism in the immunocompetent animals and boost tolerance by engineering the donor HSCs through viral transduction to express molecules involved in regulation of central tolerance, prevention of autoimmunity, and induction of T-regulatory cell (Treg) responses. Engineered HSCs will be transferred to recipient mice to establish functional chimerism, and tolerance to subsequent cardiac and beta islet grafts will be tested in functionally chimeric animals. Reviewers agreed that use of the molecules selected by the applicant represent a novel approach to generating peripheral tolerance through mixed chimerism and, if successful, could generate a new method for the induction of chimerism and transplantation tolerance. However, the reviewers thought that the potential impact was seriously limited and found it difficult to imagine how this complex combined approach could be translated into an effective therapy for overcoming immune rejection of stem cells in humans. Reviewers appreciated the novel and innovative hypothesis that the proposed molecules would promote establishment of mixed chimerism; however, they felt the scientific rationale was not appropriately justified. It was unclear to reviewers how transient transduction of the proposed molecules would increase the tolerogenicity of the transduced HSCs. The proposed molecules are normally expressed in the immature thymus, and, other than citing work indicating these molecules can be expressed in the spleen, the applicant did not discuss how these molecules might behave in mature cells in the periphery to produce tolerance. Further, some of the proposed molecules are nonspecific in action, and this issue was not addressed nor were alternatives considered. The reviewers appreciated the use of the humanized mouse model and thought this aspect of the proposal could provide useful information. However, they were concerned that Aim 1 is primarily a validation of the humanized mouse model and does not demonstrate the feasibility of inducing tolerance using the proposed molecules. A more logical approach would be to demonstrate proof of concept in an allogeneic mouse model before going to the considerable time, effort, and expense of confirming that rejection occurs in the humanized mouse model. Reviewers noted several additional concerns with the experimental design. For example, the proposal inadequately addressed feasibility issues such as transduction efficiency and concerns regarding long-term transgene expression that are associated with the use of the lentiviral system. Further, rejection of the cardiac graft proposed in Aims 1 and 2 is often B-cell mediated and the applicant did not propose experiments to address this issue. Due to these serious flaws in the experimental approach, reviewers did not believe the aims were achievable. Reviewers considered the Principle Investigator (PI) and the team well equipped to carry out the proposed studies. The PI has a long track record in cellular immunology, and the proposed collaborations are long standing and productive. The research environment is excellent. In summary, while reviewers thought this was an interesting proposal from a competent PI, the approach lacked a convincing scientific rationale. The experimental design was considered overly complex, inadequately developed, and unlikely to provide novel insights into the induction of tolerance in humans.