Tomotherapy and Hematopoietic Stem Cells for Tolerance to Kidney Transplants ABSTRACT The overarching goal of this project is to develop a tolerance induction protocol for MHC disparate kidney transplants in rhesus macaques and to elucidate the underlying mechanisms of the induction and maintenance of mixed chimerism and tolerance in this model. The primary hypothesis is that tolerance to MHC mismatched kidney transplants can be safely and effectively achieved by establishing a mixed chimeric state using a newly established post‐transplant non‐myeloablative, helical tomotherapy‐based total lymphoid irradiation (TLI)‐ based conditioning regimen followed by donor bone marrow‐CD34+ hematopoietic cell (HSC) infusions. In addition, we will elucidate mechanisms of host immunoregulatory characteristics that are associated with successful HSC engraftment and maintenance of the chimeric state. We propose to test our hypotheses by means of 2 specific aims: 1.) Combined Bone Marrow Hematopoietic Cell/Kidney Transplants to determine the proportion of rhesus macaque recipients of disparate unrelated donor kidney transplants that achieve chimerism (without GVHD) and can be withdrawn from all immunosuppressive drugs for greater than 2 years while maintaining normal allograft function and without rejection. We will measure the state of mixed chimerism in recipients as a function of time post‐transplant and according to various peripheral blood and bone marrow cell types using DNA (STR) analysis, and with flow cytometry using rhesus antibodies specific for MHC class I Mamu alleles of the donor. 2) Immune Monitoring, Immunopathology and Immunocompetence. We will: a.) determine early recipient immunoregulatory changes of host myeloid cells induced by the TLI‐based conditioning regimen that correlate with the success of bone marrow engraftment, b.) determine if the mixed chimeric state induces changes of host dendritic cell acquisition of donor MHC class I antigen and PD‐LI expression at serial time points during and after withdrawing immunosuppression, c.) characterize the development of renal allograft immune and non‐immune injury by analysis of serial renal allograft biopsies, and d.) determine the degree of recipient immune‐competency after immunosuppressive drug withdrawal by testing the recall T cell responses to cytomegalovirus antigens and tetanus toxoid. Knowledge gained through this rhesus tolerance induction protocol, including the underlying immunological mechanisms, will have direct relevance to a variety of deceased donor transplants. Furthermore, it will set a new course of healthcare innovation and delivery that will greatly benefit transplant patients, and other patient populations.