We recently reported long-term organ allograft survival without ongoing immunosuppression in 4 of 5 patients receiving combined kidney and bone marrow transplantation from haploidentical donors following non-myeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4+CD25+CD127−FOXP3+ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in 2 of 4 patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥18 months) studies on all 4 patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney and bone marrow transplantation (CKBMT) that led to transient chimerism under a previously-published non-myeloablative conditioning regimen (Immune Tolerance Network study ITN036). Polychromatic flow cytometry (FCM) and high throughput sequencing of TCRβ hypervariable regions of DNA from peripheral blood T regulatory cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of regulatory T cells (CD3+CD4+CD25highCD127lowFoxp3+) in blood that resulted from peripheral proliferation (Ki67+), possibly new thymic emigration (CD31+) and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large fraction of the T cell clones detected in post-transplant biopsy specimens by TCR sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early post-transplant period may contribute to tolerance following CKBMT. Furthermore, most conventional T cell clones detected in immunologically quiescent post-transplant biopsies appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Background We have previously reported operational tolerance in patients receiving HLA-mismatched combined kidney and bone marrow transplantation (CKBMT). We now report on transient multilineage hematopoietic chimerism and lymphocyte recovery in five patients receiving a modified CKBMT protocol, and evidence for early donor-specific unresponsiveness in one of these patients. Methods Five patients with end-stage renal disease received CKBMT from HLA-mismatched, haploidentical living related donors following modified non-myeloablative conditioning. Polychromatic flow cytometry (FCM) was used to assess multilineage chimerism where evaluable and lymphocyte recovery post-transplant. Limiting dilution analysis was used to assess helper-T-lymphocyte reactivity to donor antigens. Results Transient multilineage mixed chimerism was observed in all patients but chimerism became undetectable by 2 weeks post-CKBMT. A marked decrease in T and B lymphocyte counts immediately following transplant was followed by gradual recovery. Initially recovering T cells were depleted of CD45RA+/CD45RO− “naïve-like” cells, which have shown strong recovery in two patients and CD4/CD8 ratios increased immediately following transplant but then declined markedly. NK cells were enriched in the peripheral blood of all patients following transplant. For Subject 2, a pre-transplant limiting dilution assay revealed T helper cells recognizing both donor and third-party PBMCs. However, the anti-donor response was completely undetectable by Day 24, while third-party reactivity persisted. Conclusion These results characterize the transient multilineage mixed hematopoietic chimerism and recovery of lymphocyte subsets in patients receiving a modified CKBMT protocol. The observations are relevant to the mechanisms of donor-specific tolerance in this patient group.