Immune tolerance induction by nonmyeloablative haploidentical HSCT combining T-cell depletion and posttransplant cyclophosphamide.

The establishment of safe approaches to attain durable donor-type chimerism and immune tolerance toward donor antigens represents a major challenge in transplantation biology. Haploidentical hematopoietic stem cell transplantation (HSCT) is currently used for cancer therapy either as a T-cell-depleted megadose HSCT following myeloablative conditioning or with T-cell-replete HSCT following nonmyeloablative conditioning (NMAC) and high-dose posttransplant cyclophosphamide (PTCY). The latter approach suffers from a significant rate of chronic graft-versus-host disease (GVHD), despite prolonged immunosuppression. The use of T-depleted grafts, although free of GVHD risk, is not effective after NMAC because of graft rejection. We now demonstrate in mice conditioned with NMAC that combining the power of high-dose PTCY with T-cell-depleted megadose HSCT can overcome this barrier. This approach was evaluated in 2 patients with multiple myeloma and 1 patient with Hodgkin lymphoma. The first myeloma patient now followed for 25 months, exhibited full donor-type chimerism in the myeloid and B-cell lineages and mixed chimerism in the T-cell compartment. The second myeloma patient failed to attain chimerism. Notably, the low toxicity of this protocol enabled a subsequent successful fully myeloablative haploidentical HSCT in this patient. The third patients was conditioned with slightly higher total body irradiation and engrafted promptly. All patients remain in remission without GVHD. Both engrafted patients were able to control cytomegalovirus reactivation. Enzyme-linked immunospot analysis revealed immune tolerance toward donor cells. Our results demonstrate a novel and safer nonmyeloablative haplo-HSCT offering a platform for immune tolerance induction as a prelude to cell therapy and organ transplantation.

Novel immunoregulatory role of perforin-positive dendritic cells.

The recently described generation of a highly defined population of dendritic cells which express perforin and granzyme A (termed "perf-DCs") and their ability to selectively delete cognate CD8+ T cell has raised the possibility that these cells play a role in the maintenance of peripheral tolerance. Using bone marrow transplantation, we generated mice selectively lacking perforin expressing dendritic cells. These mice progressively gain weight and exhibit features resembling metabolic syndrome as well as an enhanced susceptibility to autoimmunity induction. Interestingly, these pathological phenotypes were reversed upon treatment with CD4/CD8 neutralizing antibodies. Thus, it appears that this rare subpopulation of dendritic cells (perf-DCs) displays a major regulatory role in adipose tissue inflammatory processes and in autoimmunity.

Perforin-Positive Dendritic Cells Exhibit an Immuno-regulatory Role in Metabolic Syndrome and Autoimmunity.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure.

Analysis of hematopoietic stem cells (HSCs) in factor VIII knockout (FVIIIKO) mice revealed a novel regulatory role for the coagulation cascade in hematopoiesis. Thus, HSCs in FVIIIKO mice had reduced proportions of CD34(low) cells within Lin(-)Sca(+)Kit(+) progenitors, and exhibited reduced long-term repopulating capacity as well as hyper granulocyte-colony-stimulating factor (G-CSF)-induced mobilization. This disregulation of HSCs is likely caused by reduced levels of thrombin, and is associated with altered protease-activated receptor 1 (PAR1) signaling, as PAR1 KO mice also exhibited enhanced G-CSF-induced mobilization. Analysis of reciprocal bone marrow (BM) chimera (FVIIIKO BM into wild-type recipients and vice versa) and the detection of PAR1 expression on stromal elements indicates that this phenotype is likely controlled by stromal elements. Micro-computed tomography analysis of distal tibia metaphyses also revealed for the first time a major impact of the FVIII/thrombin/PAR1 axis on the dynamic bone structure, showing reduced bone:tissue volume ratio and trabecular number in FVIIIKO and PAR1KO mice. Taken together, these results show a critical and novel role for the coagulation cascade, mediated in part by thrombin-PAR1 interaction, and regulates HSC maintenance and a reciprocal interplay between HSCs and the dynamic bone structure.

Embryonic pig pancreatic tissue for the treatment of diabetes: potential role of immune suppression with "off-the-shelf" third-party regulatory T cells.

BACKGROUND: Xenogeneic embryonic pancreatic tissue can provide an attractive alternative for organ replacement therapy. However, immunological rejection represents a major obstacle. This study examines the potential of regulatory T cells (Tregs) in the prevention of E42 pancreas rejection. METHODS: To develop new approaches to combat rejection, we evaluated engraftment, growth, and development of E42 pig pancreatic tissue in mice treated with ex vivo expanded Tregs in combination with T-cell debulking and the conventional immunosuppressive drugs, rapamycin and FTY720. RESULTS: Transplantation of E42 pig pancreas into C57BL/6 mice immunosuppressed by this protocol resulted in complete rejection within less than 6 weeks. In contrast, additional treatment with a single infusion of ex vivo expanded third-party Tregs markedly delayed the onset of graft rejection to 10 weeks. The infusion of Tregs was associated with a significant reduction in CD4 and CD8 expansion in the lymph nodes and other peripheral organs at the priming stages after implantation. Freezing and thawing of the Tregs did not affect their efficacy, indicating the potential of Tregs banking. CONCLUSION: Considering the technical difficulties encountered in the generation of Tregs from patients or from specific donors, our results demonstrate the feasibility of using "off-the-shelf" fresh or frozen third-party Tregs to control rejection in organ transplantation.