Abstract With the advent of mixed chimerism as a clinical transplantation tolerance strategy, understanding the mechanisms leading to stable tolerance has become critical. We now have evidence that a key component of transplant tolerance is provided by tiny, virus-sized particles called exosomes. Exosomes derived from Treg cells were not only critical to the mechanism of donor-specific transfusion (DST) co-stimulation (CoS) blockade-induced tolerance, but they may also be the basis of tolerance effects resulting from bi-directional regulation between host and living-related kidney donor . Besides Treg-derived exosomes, the recent discovery of a major role for exosomes from donor “passenger” DC (dendritic cells) in transplant rejection led us to test the hypothesis that DC exosomes could also do the opposite, i.e. promote transplant tolerance. We found that maternal DC microchimerism, resulting from the interaction of mother and child in utero and persisting in the neonate and throughout adult life, produce exosomes that amplify the tolerogenic impact of rare allogeneic cells. This discovery could finally explain the higher probability of graft survival in a transplant between siblings that differ for HLA at the non-inherited maternal haplotype. We propose, herein, a pilot immune monitoring study of: 1) normal HLA-identical and 1 haplotype mismatch living related donor (LRD) kidney transplants, and 2) the same patient-donor types, but enrolled in a combined total lymphoid irradiation (TLI)+ kidney/hematopoetic stem cell (HSC) transplantation trial at the UW-Madison. The goal of the trial would be to determine if monitoring of Treg- and DC-derived exosomes will predict which patients are successful, and which will fail to achieve tolerance. We hypothesize that in living-related donor-recipient pairs with pre-transplant “bi-directional regulation”, host and graft-resident Tregs will produce exosomes that cross-dressed bystander T & B cells with IL35, promoting infectious tolerance. Similarly, we hypothesize that DC exosomes capable of inducing PD-L1 expression in HLA-cross- dressed (XD) DC will correlate with excellent graft outcome. We predict that both of these positive exosome effects will be amplified in the context of RTx/TLI/HSC pre-conditioning. Failure to detect IL35 on LC exosomes, and PD-L1 miRs/lncRNAs within DC exosomes in successful tolerance induction will support a “null” hypothesis. Our goal is to discover the role of Treg- and DC-derived exosomes in a clinical tolerance trial.