Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4 + T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4 + T cells, and a high ratio of forkhead box P3 to α-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Mitochondrial 2-enoyl-CoA hydratase (mECH) and 3,2-trans-enoyl-CoA isomerase (mECI), two enzymes which catalyze totally different reactions in fatty acid beta-oxidation, belong to the low-similarity hydratase/isomerase enzyme superfamily. Their substrates and reaction mechanisms are similar [Müller-Newen, G. & Stoffel, W. (1993) Biochemistry 32, 11,405-11,412]. Glu164 of mECH is the only amino acid with a protic side chain that is conserved in these monofunctional and polyfunctional enzymes with 2-enoyl-CoA hydratase and 3,2-trans-enoyl-CoA isomerase activities. We tested our hypothesis that Glu164 of mECH is the putative active-site amino acid responsible for the base-catalyzed alpha-deprotonation in the hydratase/dehydrase and isomerase reaction. We functionally expressed rat liver mECH wild-type and [E164Q] mutant enzymes in Escherichia coli. Characterization of the purified wild-type and mutant enzymes revealed that the replacement of Glu164 by Gln lowers the kcat value more than 100,000-fold, whereas the Km value is only moderately affected. We have demonstrated in a previous study that Glu165 is indispensable for the 3,2-trans-enoyl-CoA isomerase activity. Taking these results together, we conclude that the conserved glutamic acid is the essential basic group in the active sites of 2-enoyl-CoA hydratase (Glu164) and 3,2-trans-enoyl-CoA isomerase (Glu165), and that these enzymes are not only evolutionarily but also functionally and mechanistically related.