Homeostatic proliferation is a normal physiological process triggered by lymphopenia to maintain a constant level of T cells. It becomes the predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire the memory phenotype, cause autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. However, it is not known whether the homeostatic proliferation that follows will have the opposite effect and accelerate rejection. We show that T cells that have undergone homeostatic proliferation acquire a memory phenotype, spontaneously skews toward the Th1 phenotype, even in the absence of antigenic stimulus. Interestingly, in contrast, the percentage of Foxp3+ regulatory T cells increased by 28-fold following homeostatic proliferation. Using a mouse life-sustaining kidney transplant model, we showed that T cells that have gone through homeostatic proliferation in lymphopenic hosts transformed chronic rejection to acute rejection of a single MHC class II-mismatched kidney allograft. T cells that have undergone homeostatic proliferation consistently cause reliable rejection even when bona fide memory T cells cannot. These functional changes are long-lasting and not restricted to the acute phase of homeostatic proliferation. Our findings have important implications for tolerance induction or graft-prolonging protocols involving leukocyte depletion such as irradiation bone marrow chimera, T cell-depleting Abs, and lymphopenia induced by infections such as CMV and HIV.

Kidney transplantation is the best form of treatment for end-stage renal failure. Despite a high success rate with graft survival of more than 90% in the 1st year, acute rejection remains the principal cause of graft loss within the 1st year of transplantation and contributes to chronic damage. Although serum creatinine is a good measurement of renal function, rejection can only be reliably diagnosed by histological analysis of biopsy samples.

The complement system plays a pivotal role in the pathogenesis of ischemia–reperfusion injury in solid organ transplantation. Mirococept is a potent membrane‐localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia‐reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5‐25 mg would be tested, starting with 10 mg in cohort 1. No significant difference between Mirococept at 10 mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80 mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3 hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80 mg (equivalent to 120 mg in human kidney) that provides a basis for further clinical development.

Complement activation occurs in at least two phases when an organ is transplanted into a naive recipient: during reperfusion with recipient blood particularly when the donor organ has undergone a significant period of ischaemia and then during acute rejection once the recipient immune system has recognised the donor tissue as non-self. Both of these reactions are most obvious in the extravascular compartment of the transplanted organ and involve local synthesis of some of the key complement components as well as loss of controls that limit the activation of the pivotal component C3. In contrast, sensitised individuals with pre-existing circulating antibodies have an immediate reaction against the transplant organ that is also complement dependent but is enacted in the intravascular space. All three types of injury (ischaemia-reperfusion, acute rejection, hyperacute rejection) have a critical effect on transplant outcome. Here we discuss therapeutic strategies that are designed to overcome the impact of these factors at the start of transplantation with the aim of improving long-term transplant outcomes. These include the concept of treating the donor organ with modified therapeutic regulators that are engineered to be retained by the donor organ after transplantation and prevent inflammatory injury during the critical early period. By targeting the donor organ with anchored therapeutic proteins, the systemic functions of complement including host defence remain intact. The control of complement activation during the first stages of transplantation, including the possibility that this will reduce the capacity of the graft for stimulating the adaptive immune system, offers an important prospect for increasing the longevity of the transplant and offsetting demand on the limited supply of donor organs. It also provides a model in which the benefits and indications for localised therapy to maximise therapeutic efficiency and minimise the systemic disturbance may be instructive in other complement-related disorders.

Allograft transplantation into sensitized recipients with antidonor antibodies results in accelerated antibody‐mediated rejection (AMR), complement activation, and graft thrombosis. We have developed a membrane‐localizing technology of wide applicability that enables therapeutic agents, including anticoagulants, to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLNs). Using a rat model of hyperacute rejection, we investigated the potential of one such inhibitor (thrombalexin‐1 [TLN‐1]) to prevent acute antibody‐mediated thrombosis in the donor organ. TLN‐1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.

Background Anticoagulants induce atherosclerosis regression in animal models but exploiting this clinically is limited by bleeding events. Here we test a novel thrombin inhibitor, PTL 060, comprising hirulog covalently linked to a synthetic myristoyl electrostatic switch to tether to cell membranes. Methods and Results ApoE−/− mice were fed chow or high‐fat diets, before transplantation of congenic aortic segments or injection of PTL 060, parental hirulog, control saline, or labeled CD 11b positive cells. Aortic transplants from transgenic mice expressing anticoagulants on endothelium did not develop atherosclerosis. A single intravenous injection of PTL 060, but not hirulog inhibited atheroma development by >50% compared with controls when assessed 4 weeks later. Mice had prolonged bleeding times for only one seventh of the time that PTL 060 was biologically active. Repeated weekly injections of PTL 060 but not hirulog caused regression of atheroma. We dissected 2 contributory mechanisms. First, the majority of CCR2+ (C‐C chemokine receptor type 2+) monocytes recruited into plaques expressed CCR7 (C‐C chemokine receptor type 7), ABCA1 (ATP‐binding cassette transporter – 1), and interleukin‐10 in PTL 060 mice, a phenotype seen in <20% of CCR2+ recruits in controls. Second, after several doses, there was a significant reduction in monocyte recruits, the majority of which were CCR2‐negative with a similar regression‐associated phenotype. Regression equivalent to that induced by intravenous PTL 060 was induced by adoptive transfer of CD 11b+ cells pre‐coated with PTL 060. Conclusions Covalent linkage of a myristoyl electrostatic switch onto hirulog in PTL 060 uncouples the pharmacodynamic effects on hemostasis and atherosclerosis, such that plaque regression, mediated predominantly via effects on monocytes, is accompanied by only transient anticoagulation.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the noncollagenous domain of the ␣ 3 chain of type IV collagen, ␣3(IV)NC1. Recent studies have identified an immunodominant peptide, pCol (24-38), from the N-terminus of rat ␣3(IV)NC1; this peptide contains the major B-and T-cell epitopes in EAG and can induce crescentic nephritis. In this study, we investigated the mechanisms of mucosal tolerance in EAG by examining the effects of the nasal administration of this peptide after the onset of disease. A dose-dependent effect was observed: a dose of 300 g had no effect, a dose of 1000 g resulted in a moderate reduction in EAG severity, and a dose of 3000 g produced a marked reduction in EAG severity accompanied by diminished antigen-specific, T-cell proliferative responses. These results demonstrate that mucosal tolerance in EAG can be induced by nasal administration of an immunodominant peptide from the N-terminus of ␣3(IV)NC1 and should be of value in designing new therapeutic strategies for patients with Goodpasture's disease and other autoimmune disorders.

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