Yael Zlotnikov Klionsky

Pig Embryonic Pancreatic Tissue as a Source for Transplantation in Diabetes: Transient Treatment With Anti-LFA1, Anti-CD48, and FTY720 Enables Long-Term Graft Maintenance in Mice With Only Mild Ongoing Immunosuppression

OBJECTIVE Defining an optimal costimulatory blockade–based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice. RESEARCH DESIGN AND METHODS Considering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels. RESULTS Fetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes. CONCLUSIONS This novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.

Deletion of cognate CD8 T cells by immature dendritic cells: a novel role for perforin, granzyme A, TREM-1, and TLR7

Immature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4(+) and CD8(+) T cells after encountering cognate or noncognate imDCs. Whereas CD4(+) T cells were deleted via an MHC-independent mechanism through the NO system, CD8(+) T-cell deletion was found to occur through a unique MHC-dependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforin-expressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration.

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.

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.

Enhancement of Pig Embryonic Implants in Factor VIII KO Mice: A Novel Role for the Coagulation Cascade in Organ Size Control

Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs) or indirectly by cleaving osteopontin (OPN) on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN) were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN). Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control.

Growth enhancement by embryonic fibroblasts upon cotransplantation of noncommitted pig embryonic tissues with fully committed organs.

Background. We recently defined the optimal gestational time windows for the transplantation of several embryonic tissues. We showed that the liver and kidney obtained from E28 pig embryos can grow and differentiate normally after transplantation, whereas 1 week earlier in gestation, these tissues develop into teratoma-like structures or fibrotic mass. In this study, we investigated whether cotransplantation of E28 with E21 tissue could control its tumorogenic potential, or alternatively whether the stem cells derived from the earlier tissue contribute to the growth of the more committed one. Methods. Pig embryonic precursors from E21 and E28 gestational age were transplanted alone or together, into nonobese diabetic/severe combined immunodeficiency mice, and their growth and differentiation was evaluated by immunohistology. In situ analysis, based on sex disparity between the E21 and E28 tissues, was used to identify the tissue source. In some experiments, mouse embryonic fibroblasts (MEF) were cotransplanted with E28 liver, and their effect was evaluated. Results. E28 tissues could not abrogate the propensity of the cells within the undifferentiated tissue to form teratoma-like structures. However, E21 kidney or liver tissue markedly enhanced the growth and function of E28 kidney, liver, and heart grafts. Moreover, similar growth enhancement was observed on coimplantation of E28 liver tissue with MEF or on infusion of MEF culture medium, indicating that this enhancement is likely mediated through soluble factors secreted by the fibroblasts. Conclusion. Our results suggest a novel approach for the enhancement of growth and differentiation of transplanted embryonic tissues by the use of soluble factors secreted by embryonic fibroblasts.