Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-beta hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3(+)CD4(+)CD25(high) CD127(low)Foxp3(+)) 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 proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor 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 posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.