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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‐β hypervariable regions of DNAfrom peripheral blood regulatory T cells (Tregs) and CD4 non‐Tregs revealed marked early enrichment of Tregs (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 proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptorsequencing 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. In patients receiving HLA‐mismatched combined kidney–bone marrow transplantation to induce transplantation tolerance, polychromatic flow cytometry and high‐throughput T cell receptor sequencing reveal an early enrichment of regulatory T cells, probably arising from new thymic emigration and lymphopenia‐driven peripheral proliferation, though the T cell receptors detected are likely from circulating T cells in the allograft microvasculature rather than infiltrating T cells.

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.

The spontaneous chronic colitis in TCR alpha mutant (TCRalpha(-/-)) mice mediated by CD4(+) TCRalpha(-)beta(+) T cells is more severe in the absence of mature B cells, suggesting a suppressive role of B cells and Ig in the development of chronic colitis. To investigate the direct role of B cells in the suppression of this colitis, cell transfer studies were performed in TCRalpha(-/-) x Igmu(-/-) (alphamu(-/-)) double-knockout mice. The chronic colitis was markedly attenuated in alphamu(-/-) mice after the adoptive transfer of peripheral B cells from TCRalpha(-/-) mice into 3- to 4-week-old alphamu(-/-) mice prior to the development of colitis. Furthermore, transfer of mature B cells from TCRalpha(-/-) mice markedly decreased the number of pathogenic colonic CD4(+) TCRalpha(-)beta(+) T cells in alphamu(-/-) mice with established colitis. This B cell effect required the presence of functional co-stimulatory molecules CD40 and B7-2 (CD86) but not B7-1 (CD80). These results indicate that mature B cells play an important role in the development of chronic colitis in TCRalpha(-/-) mice by directly regulating the pathogenic T cells (CD4(+) TCRalpha(-)beta(+) T cells).

CD7+CD3- cells purified (greater than 99.99%) by FACS from the peripheral blood of healthy adults include precursors for mature T cells that have the capacity to differentiate into TCR-1+ or TCR-2+ CD3+ cells. Extrathymic differentiation was demonstrable from all eight healthy donors in the presence of a high concentration of IL-2, mitogenic levels of PHA, and irradiated blood mononuclear feeder cells, after a lag of approximately 40 d in vitro. The extrathymic T (ET) cells were predominantly TCR-1+, although TCR-2+ cells were also derived. ET TCR-1+ cells were CD4-CD8-, CD4-CD8DIM+, and CD4+CD8-, and were distinguished from natural T TCR-2+ cells by a variety of cell surface markers. The ET cells had phenotypes generally displayed by normal mature T cells, although the CD5DIM+ on ET cells was more typical of thymocytes. Acquisition of CD3 on purified CD7+CD3- cells was not due to antigenic modulation or growth of contaminants, and ET cells could be demonstrated at the clonal level. Studies in athymic mice and bone marrow recipients support the view that extrathymic maturation does occur in vivo. Whether the CD7+CD3- cell population was unexposed to the thymus, or exposed but not processed, is unknown. In any case, unusual or "forbidden" autoreactive specificities are predicted since ET cells differentiate without thymic selection of the TCR.