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

Abstract

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.

Deletion of donor-reactive T cell clones after human liver transplant

“…phenomena may develop over longer periods in most patients who achieve tolerance. In contrast to the sequence proposed here for liver allograft tolerance, we have proposed a sequential Treg-dependent/deletion-dependent model to explain the tolerance observed in CKBMT recipients with transient chimerism 16,[27][28][29]. In contrast to the sequence proposed here for liver allograft tolerance, we have proposed a sequential Treg-dependent/deletion-dependent model to explain the tolerance observed in CKBMT recipients with transient chimerism 16,[27][28][29].…”

Mechanisms of Mixed Chimerism-Based Transplant Tolerance

“…The rare kidney transplant recipients who spontaneously develop a state of operational tolerance demonstrate an increased frequency of highly suppressive memory Tregs with a fully demethylated FOXP3 Treg-Demethylated Region (TSDR), a hallmark epigenetic change of stable Tregs [65]. Similarly, marked enrichment of FOXP3-expressing Tregs during the early post-transplant course has been observed following the non-myeloablative anti-CD2 mAb-based CKHCT protocol that leads to transient chimerism [31, 66]. Consistently, a recent study suggested donor-specific FOXP3+ Treg expansion in tolerant NHPs receiving a T cell depletion/costimulatory blockade-based transient chimerism CKHCT regimen [67].…”

“…Moreover, accumulation of FOXP3+ Tregs in tolerated grafts has been extensively reported in mice [62, 112], and the graft itself was found to be a major site for suppression [69]. Similarly, FOXP3+ T cells are detected in biopsies from tolerant CKHCT recipients with transient chimerism [1, 66]. …”

Alloimmune T cells in transplantation

“…TCR sequencing of post-transplant biopsies from tolerated allografts of CKBMT recipients revealed similar sequence dominance to that observed in the peripheral circulation at the same time, including a paucity of donor-reactive clones. In view of the absence of infiltrates observed histologically, these results suggest that the sequences obtained may have been from T cells in the kidney microvasculature (96).…”

“…Comparison of the pretransplant stimulated and unstim- circulating alloreactive CD4 and CD8 clones during rejection may be different, as suggested in our early study (91). Furthermore, identification of the subset to which circulating or lymphoid tissue T cells belong before transplant provides additional information about clones identified in unsorted specimens from, for example, tissue biopsies (96,98). However, because FACS is not without error, it is important to perform postsequencing data processing to eliminate ambiguous clones (those present at similar frequencies in sorted CD4 and CD8 populations) and to reassign mis-sorted clones (those that are clearly present at much greater frequency in one subset than the other).…”