Anthony Dorling

Transplant Accommodation in Highly Sensitized Patients: A Potential Role for Bcl-xL and Alloantibody

Transplantation of renal allografts into recipients with circulating anti‐HLA antibodies results in hyperacute rejection. In some cases, however, antibodies return without causing harm; this phenomenon has been termed ‘accommodation’. We have investigated this process in human allotransplantation.

Modified Dendritic Cells Coexpressing Self and Allogeneic Major Histocompatability Complex Molecules: An Efficient Way to Induce Indirect Pathway Regulation

A feature of the tolerance that has been described in experimental models is that it can be transferred by CD4+ T cells to a naive recipient. Described is a novel approach to induce indirect pathway regulatory T cells in a rat model that exploits the natural processing and presentation of major histocompatability complex (MHC) molecules as peptide by the MHC class II molecules of the same cell. Dendritic cells (DC) coexpressing donor (AUG) and recipient (LEW) MHC molecules were rendered tolerogenic by treatment with dexamethasone. After injection into LEW animals followed by a single low dose of CTLA4-Ig, T cells were rendered unresponsive to indirectly presented AUG alloantigens, but retained direct pathway responsiveness to fully allogeneic AUG cells. The T cells from the DC-injected rats were unresponsive to (LEW x AUG)F1 stimulator cells, suggesting the presence of indirect pathway regulatory cells whose activity depended on the presence of LEW MHC molecules. Depletion of CD25+ cells from the responder population led to a marked increase in proliferation, and the T cells from the DC-injected rats inhibited the response of naive LEW T cells to (LEW x AUG)F1, but not to AUG, stimulator cells, further indicating indirect pathway-mediated regulation. Most importantly, pretreatment of LEW rats with the dexamethasone-treated DC led to the indefinite survival of AUG kidney grafts after a short course of cyclosporin to inhibit the early direct pathway response. Similarly treated AUG DC had no effect, confirming the privileged status of F1 cells in the induction of indirect pathway regulation.

Current status of xenotransplantation and prospects for clinical application

Abstract:  Xenotransplantation is one promising approach to bridge the gap between available human cells, tissues, and organs and the needs of patients with diabetes or end‐stage organ failure. Based on recent progress using genetically modified source pigs, improving results with conventional and experimental immunosuppression, and expanded understanding of residual physiologic hurdles, xenotransplantation appears likely to be evaluated in clinical trials in the near future for some select applications. This review offers a comprehensive overview of known mechanisms of xenograft injury, a contemporary assessment of preclinical progress and residual barriers, and our opinions regarding where breakthroughs are likely to occur.

Complete Inhibition of Acute Humoral Rejection Using Regulated Expression of Membrane-tethered Anticoagulants on Xenograft Endothelium

Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane‐tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non‐transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T‐cell‐mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.

HLA-G inhibits the transendothelial migration of human NK cells

The expression of the non‐classical MHC class I molecule HLA‐G is normally restricted to the placenta during pregnancy, where it is found on fetal endothelial cells and on invasive cytotrophoblast cells, specifically those at the maternal / fetal interface. Its precise physiological role has yet to be defined. HLA‐G may have nonimmune functions relating to angiogenesis and placentation, but most evidence suggests that it protects fetal cells from lysis by maternal uterine NK cells, which are found in large numbers around invading trophoblast cells. This effect is due to specific interaction with inhibitory receptors expressed on NK cells. We have examined the hypothesis that another function of HLA‐G is to inhibit NK cell migration. Using an in vitro transmigration assay system, we present data to support this hypothesis. NK cell migration across porcine endothelial cells transfected with HLA‐G1 was specifically inhibited compared to migration across HLA‐A2‐transfected monolayers. HLA‐ G1 had no influence on the migration of a control T lymphocyte line. These results support the idea that in vivo, HLA‐G may inhibit NK cell traffic across the placenta.

T cell-mediated xenograft rejection: Specific tolerance is probably required for long term xenograft survival

Abstract: T cell‐mediated mechanisms of xenograft rejection appear resistant to standard immunosuppression protocols used to prevent allograft rejection and, consequently, higher doses of immunosuppressive drugs are required to promote xenograft compared to allograft survival. Evidence from recent studies suggests that porcine xenografts may be especially immunogenic in humans because of a prominent and vigorous indirect xenoresponse and because of the ability of porcine endothelium to activate human T cells. This has led to an anxiety that systemic immunosuppressives, used as the mainstay of therapy for clinical xenotransplantation, may not allow the long‐term survival of porcine organs transplanted into human recipients. This article will review the biology of T cell xenoresponses, present the case for the development of novel graft‐specific immunosuppressive regimes in clinical xenotransplantation, and review recent experimental progress in this area.

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1–dependent leukocyte recruitment in vivo

Thrombin, acting through a family of protease-activated receptors (PARs), is known to amplify inflammatory responses, but the in vivo importance of PARs in inflammation is not fully appreciated. In a mouse heart-to-rat transplant model, where it is possible to distinguish graft (mouse) from systemic (rat) chemokines, we show that donor PAR-1 is required to generate the local monocyte chemoattractant protein (MCP)-1 needed to recruit rat natural killer cells and macrophages into the hearts. We have confirmed the importance of this mechanism in a second model of thioglycollate-induced peritonitis and also show that PAR-1 is important for the production of MCP-3 and MCP-5. Despite the presence of multiple other mediators capable of stimulating chemokine production in these models, these data provide the first evidence that thrombin and PAR activation are required in vivo to initiate inflammatory cell recruitment.

Recipient tissue factor expression is associated with consumptive coagulopathy in pig-to-primate kidney xenotransplantation.

Consumptive coagulopathy (CC) remains a challenge in pig‐to‐primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild‐type (n = 2), α1,3‐galactosyltransferase gene‐knockout (GT‐KO; n = 1) or GT‐KO and transgenic for the complement‐regulatory protein, CD46 (GT‐KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post‐Tx day 1. Subsequently, platelet‐leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig‐to‐primate kidney Tx.

Clinical xenotransplantation of solid organs

A possible solution to the chronic shortage of allografts is xenotransplantation, the use of tissue from an animal donor. Most experts believe that the pig will provide the most suitable solid organs for use in human beings. Although porcine organs are rapidly rejected by a process called hyperacute rejection (HAR), there is hope that several novel therapeutic strategies, already tested in animal models, will overcome this hurdle in patients. Successful clinical trials of these strategies, expected within the next few years, may herald the era of clinical xenotransplantation. However, there is increasing evidence that other barriers, both immune and non-immune, might exist to limit the survival of xenografts beyond the HAR phase. New strategies to overcome these barriers will be needed if long-term xenograft survival equivalent to, or better than, that of allografts is ever to be achieved.

Critical roles for thrombin in acute and chronic inflammation

Summary.  Thrombin can amplify inflammation induced by other stimuli, either through ischemia (consequent upon thrombosis), indirectly through generation of downstream mediators such as activated protein C, or directly via signals through protease activated receptors (PAR). This paper will summarize recent data from our laboratory indicating that thrombin is required to initiate CCR2‐dependent leukocyte recruitment and that it is the principal determinant of the outcome after vascular injury, via PAR‐1 activation of a distinct subset of smooth muscle cell progenitors. In both, tissue factor (TF) initiates thrombin generation and the thrombin acts locally, exemplifying that the initiation phase can generate autocrine or paracrine signalling molecules. Thrombin is an important constituent of innate immunity, able to amplify and modify responses to invading pathogens or tissue damage. With novel anti‐thrombin therapeutics and agents to target PAR, a new understanding of the importance of thrombin may allow the development of innovative anti‐inflammatory strategies.