T cell responses to allogeneic major histocompatibility (MHC) antigens present a formidable barrier to organ transplantation, necessitating long-term immunosuppression to minimize rejection. Chronic rejection and drug-induced morbidities are major limitations that could be overcome by allograft tolerance induction. Tolerance was first intentionally induced in humans via combined kidney and bone marrow transplantation (CKBMT), but the mechanisms of tolerance in these patients are incompletely understood. We now establish an assay to identify donor-reactive T cells and test the role of deletion in tolerance after CKBMT. Using high-throughput sequencing of the TCRB chain CDR3 region, we define a fingerprint of the donor-reactive T cell repertoire prior to transplantation and track those clones post-transplant. We observed post-transplant reductions in donor-reactive T cell clones in three tolerant CKBMT patients; such reductions were not observed in a fourth, non-tolerant, CKBMT patient or in two conventional kidney transplant recipients on standard immunosuppressive regimens. T cell repertoire turnover due to lymphocyte-depleting conditioning only partially accounted for the observed reductions in tolerant patients; in fact, conventional transplant recipients showed expansion of circulating donor-reactive clones, despite extensive repertoire turnover. Moreover, loss of donor-reactive T cell clones more closely associated with tolerance induction than in vitro functional assays. Our analysis supports clonal deletion as a mechanism of allograft tolerance in CKBMT patients. The results validate the significance of donor-reactive T cell clones identified pre-transplant by our method, supporting further exploration as a potential biomarker of transplant outcomes.

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5–11.4 years, while three required reinstitution of IS after 5–8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction.

A paradigm in transplantation states that graft-infiltrating T cells are largely non-alloreactive “bystander” cells. However, the origin and specificity of allograft T cells over time has not been investigated in detail in animals or humans. Here, we use polychromatic flow cytometry and high throughput TCR sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intra-graft host-vs-graft (HvG) and graft-vs-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted long-term in the graft. Early expansion of GvH clones in the graft correlated with rapid replacement of donor APCs by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ alpha beta T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady state tissue-resident phenotype, but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in non-rejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.

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Blood chimerism has been reported sporadically among visceral transplant recipients, mostly in association with graft-vs-host disease (GVHD). We hypothesized that a higher degree of mixed chimerism would be observed in multivisceral (MVTx) than in isolated intestinal (iITx) and isolated liver transplant (iLTx) recipients, regardless of GVHD. We performed a longitudinal prospective study investigating multilineage blood chimerism with flow cytometry in 5 iITx and 4 MVTx recipients up to one year post-transplant. Although only one iITx patient experienced GVHD, T-cell mixed chimerism was detected in 8 out of 9 iITx/MVTx recipients. Chimerism was significantly lower in the four subjects who displayed early moderate to severe rejection. Pre-formed high titer donor-specific antibodies, bound in vivo to the circulating donor cells, were associated with an accelerated decline in chimerism. Blood chimerism was also studied in 10 iLTx controls. Among non-sensitized patients, MVTx recipients exhibited greater T and B-cell chimerism than either iITx and iLTx recipients. Myeloid lineage chimerism was present exclusively among iLTx and MVTx (6/13) recipients, suggesting that its presence required the hepatic allograft. Our study demonstrates, for the first time, frequent T cell chimerism without GVHD following visceral transplantation and a possible relationship with reduced rejection rate in MVTx recipients.