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A phase 2 protocol was developed to attempt to induce donor-specific tolerance to renal allografts in related and unrelated donor/recipient combinations (IND 13947 phase 2). Subjects were conditioned with fludarabine (days -5, -4, -3), cyclophosphamide (50 mg/ky days -3 and +3), and 200 cGy total body irradiation. The kidney transplant was performed day 0. G-CSF mobilized peripheral blood stem cells processed to remove GVHD-producing cells and retain graft facilitating cells (FC) was administered on day +1. The conditioning was well tolerated and the subjects were managed as outpatients after post-operative day 2. This has resulted in high levels of durable chimerism and immunosuppression-free graft survival without GVHD or engraftment syndrome in mismatched related and unrelated recipients of living donor FC/hematopoietic stem cell/kidney allografts. Nine subjects are completely off immunosuppression from 2 months to 3 years. A number of others are in the process of tapering. In the present study, we have prospectively analyzed recovery of immune function, persistence of vaccination memory, and response to vaccination in subjects who exhibit high levels of chimerism. Chimerism testing was performed using molecular short tandem report (sensitivity ±5%). Three of four subjects who had been vaccinated to hepatitis B prior to transplantation and whose donors had not been vaccinated retained their immunity following transplantation. All subjects tested exhibited memory for varicella and the majority did as well for measles (9/11), mumps (8/11), and rubella (5/10). A blood group disparity was present in 9 chimeric donor/recipient pairs. One chimeric subject converted to donor blood type, 3 exhibited mixed donor/host RBC chimerism, and 5 retained their own blood type. Six chimeric subjects have been immunized with pneumococcal vaccine after transplantation. All generated an immune response to vaccination, confirming immunocompetence to generate an antibody response to antigen. Notably, recovery of CD8+ and CD4+ central memory, naïve, and effector memory T cells occurred within one year post-transplantation to levels that were not significantly different from pre-transplantation. In addition, CD31+/CD45RA+ CD8+ and CD4+ T cells representative of recent thymic emigrants were present by 3 months, demonstrating de novo thymic production of T cells after transplantation. Four patients were randomly selected for study of Tcell repertoire (TCR) generation after transplantation. Two had achieved durable full donor chimerism and the other two did not have durable chimerism. Peripheral blood samples freshly obtained from donors and recipients and T cell subsets were isolated using MACs microbead system. DNA was extracted and sequenced by ImmunoSeq (Adaptive Biotech, Seattle, WA) to evaluate TCR clonal repertoires in recipients. Although clonal diversity in TCR repertoire was reduced in post-Tx recipients (0.9 ± 0.05 pre-Tx vs. 0.79 ± 0.09 post-Tx), the repertoire was diverse enough to suggest recovery of immune competence. Interestingly, at least 97% of the unique sequences observed in post-Tx recipient were not present in either donor or recipient pre-Tx. Within the pool of shared sequences, full chimerism correlated with a shift towards homology with the donor, while loss of chimerism correlated with recipient pre-Tx. In addition, the chimeric patients also exhibited reduced diversity of TCR sequences and increased clonality. Top 20 “high frequency” clones are most stably expressed. CD8+ cells had the highest number of “high frequency” clones. Notably, the pattern of “low frequency” clones was highest in the CD127-CD4+CD25+ regulatory T cell subset, indicating an extensive and rapidly changing TCR repertoire. Taken together, these data suggest that immunologic recovery is robust in these nonmyeloablatively conditioned tolerant chimeric subjects. Disclosures: Bozulic: Regenerex, LLC: Employment. Badder:Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.