William H. Kitchens

NK Cells Can Trigger Allograft Vasculopathy: The Role of Hybrid Resistance in Solid Organ Allografts

Progressive arterial stenosis (cardiac allograft vasculopathy (CAV)) is a leading cause of long-term failure of organ transplants. CAV remains intractable, in part because its mechanisms are insufficiently understood. A central proposition is that MHC-driven alloimmune processes play a necessary role in CAV, as shown by the absolute requirement for histoincompatibility between donor and recipient for its production. Two immunological pathways have been implicated involving reactivity to donor MHC Ags by either T or B cells. In this study, we use a novel system of semiallogeneic cardiac transplants between parental donors and F1 hybrid recipients to provide evidence that NK cells, members of the innate immune system, also contribute to the generation of CAV in mice. This finding marks the first demonstration that the hybrid resistance phenomenon occurs in solid organ allografts. Extension of these experiments to recipients deficient in T cells demonstrates that this third pathway of CAV, the NK cell-triggered pathway, involves the recruitment of T cells not responsive to donor alloantigens. Finally, transplants performed with donors or recipients deficient in IFN-γ revealed that recipient-derived IFN-γ is necessary for CAV formation in parental to F1 transplants, suggesting a possible effector mechanism by which NK cells can promote CAV. Together, these results define a previously unknown pathway toward CAV and assign a novel role to NK cells in organ allograft rejection.

Macrophage Depletion Suppresses Cardiac Allograft Vasculopathy in Mice

Cardiac allograft vasculopathy (CAV) is a major source of late posttransplant mortality. Although numerous cell types are implicated in the pathogenesis of CAV, it is unclear which cells actually induce the vascular damage that results in intimal proliferation. Because macrophages are abundant in CAV lesions and are capable of producing growth factors implicated in neointimal proliferation, they are leading end‐effector candidates. Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL/6 hearts were transplanted into (C57BL/6 × BALB/c)F1 recipients, which then received anti‐macrophage therapy with intraperitoneal carrageenan or i.v. gadolinium. Intraperitoneal carrageenan treatment depleted macrophages by 30–80% with minimal effects upon T, B or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. Carrageenan treatment led to a 70% reduction in the development of CAV, as compared to mock‐treated controls (p = 0.01), which correlated with the degree of macrophage depletion. Inhibition of macrophage phagocytosis alone with gadolinium failed to prevent CAV. Macrophages may represent the end‐effector cells in a final common pathway towards CAV independent of T‐cell or B‐cell alloreactivity and exert their injurious effects through mechanisms related to cytokine/growth factor production rather than phagocytosis.

Integrin antagonists prevent costimulatory blockade-resistant transplant rejection by CD8+ memory T cells

The success of belatacept in late‐stage clinical trials inaugurates the arrival of a new class of immunosuppressants based on costimulatory blockade, an immunosuppression strategy that disrupts essential signals required for alloreactive T‐cell activation. Despite having improved renal function, kidney transplant recipients treated with belatacept experienced increased rates of acute rejection. This finding has renewed focus on costimulatory blockade‐resistant rejection and specifically the role of alloreactive memory T cells in mediating this resistance. To study the mechanisms of costimulatory blockade‐resistant rejection and enhance the clinical efficacy of costimulatory blockade, we developed an experimental transplant system that models a donor‐specific memory CD8+ T‐cell response. After confirming that graft‐specific memory T cells mediate costimulatory blockade‐resistant rejection, we characterized the role of integrins in this rejection. The resistance of memory T cells to costimulatory blockade was abrogated when costimulatory blockade was coupled with either anti‐VLA‐4 or anti‐LFA‐1. Mechanistic studies revealed that in the presence of costimulatory blockade, anti‐VLA‐4 impaired T‐cell trafficking to the graft but not memory T‐cell recall effector function, whereas anti‐LFA‐1 attenuated both trafficking and memory recall effector function. As antagonists against these integrins are clinically approved, these findings may have significant translational potential for future clinical transplant trials.

Limiting the amount and duration of antigen exposure during priming increases memory T cell requirement for costimulation during recall.

Donor-reactive memory T cells (Tmem) can play an important role in mediating graft rejection after transplantation. Transplant recipients acquire donor-reactive Tmem not only through prior sensitization with alloantigens but also through previous exposure to environmental pathogens that are cross-reactive with allogeneic peptide–MHC complexes. Current dogma suggests that most, if not all, Tmem responses are independent of the requirement for CD28 and/or CD154/CD40-mediated costimulation to mount a recall response. However, heterogeneity among Tmem is increasingly being appreciated, and one important factor known to impact the function and phenotype of Ag-specific T cell responses is the amount/duration of Ag exposure. Importantly, the impact of Ag exposure on development of costimulation independence is currently unknown. In this study, we interrogated the effect of decreased Ag amount/duration during priming on the ability of donor-reactive Tmem to mediate costimulation blockade-resistant rejection during a recall response after transplantation in a murine model. Recipients possessing donor-reactive Tmem responses that were generated under conditions of reduced Ag exposure exhibited similar frequencies of Ag-specific T cells at day 30 postinfection, but, strikingly, failed to mediate costimulation blockade-resistant rejection after challenge with an OVA-expressing skin graft. Thus, these data demonstrate the amount/duration of Ag exposure is a critical factor in determining Tmem’s relative requirement for costimulation during the recall response after transplantation.

Combined costimulatory and leukocyte functional antigen-1 blockade prevents transplant rejection mediated by heterologous immune memory alloresponses.

Background Recent evidence suggests that alloreactive memory T cells are generated by the process of heterologous immunity, whereby memory T cells arising in response to pathogen infection crossreact with donor antigens. Because of their diminished requirements for costimulation during recall, these pathogen-elicited allocrossreactive memory T cells are of particular clinical importance, especially given the emergence of costimulatory blockade as a transplant immunosuppression strategy. Methods We used an established model of heterologous immunity involving sequential infection of a naïve C57BL/6 recipient with lymphocytic choriomeningitis virus and vaccinia virus, followed by combined skin and bone marrow transplant from a BALB/c donor. Results We demonstrate that coupling the integrin antagonist anti-leukocyte functional antigen (LFA)-1 with costimulatory blockade could surmount the barrier posed by heterologous immunity in a fully allogeneic murine transplant system. The combined costimulatory and integrin blockade regimen suppressed proliferation of alloreactive memory T cells and attenuated their cytokine effector responses. This combined blockade regimen also promoted the retention of FoxP3+ Tregs in draining lymph nodes. Finally, we show that in an in vitro mixed lymphocyte reaction system using human T cells, the combination of belatacept and anti-LFA-1 was able to suppress cytokine production by alloreactive memory T cells that was resistant to belatacept alone. Conclusions As an antagonist against human LFA-1 exists and has been used clinically to treat psoriasis, these findings have significant translational potential for future clinical transplant trials.

Domino liver transplantation: indications, techniques, and outcomes

The long-term shortage of livers available for transplantation has spurred the development of many strategies to bolster the donor organ supply. One particularly innovative strategy is domino liver transplantation in which a select group of liver transplant recipients can donate their explanted native livers for use as liver grafts in other patients. Several hereditary metabolic diseases (such as familial amyloid polyneuropathy, maple syrup urine disease, and familial hypercholesterolemia) are caused by aberrant or deficient protein production in the liver, and these conditions can be cured with an orthotopic liver transplant. Although their native livers eventually caused severe systemic disease in these patients, these livers are otherwise structurally and functionally normal, and they have been used successfully in domino liver transplants for the past 15 years. This article will review the indications for donating or receiving a domino liver transplant, the surgical techniques necessary to perform these transplants, as well as the recently revealed long-term outcomes and risks of domino transplantation.

Integrin antagonists for transplant immunosuppression: panacea or peril?

Despite a drought of innovation that has afflicted the transplant drug pipeline over the previous decade, the recent success of belatacept (a novel CD28 antagonist) in phase III clinical trials [1] may soon usher in a new era of transplant immunosuppression regimens that are designed around protein-derived “biologics.” The initial wave of these transplant biologics will likely target essential costimulatory molecules required for T cell activation, such as the CD28-B7 interaction disrupted by belatacept. However, a growing body of both experimental and clinical literature has highlighted the role that adhesion molecules such as integrins may serve as additional therapeutic targets for transplant immunosuppression. While integrin blockade potentially holds much promise for the transplant field, enthusiasm for its adoption must be tempered by a reasonable consideration of its risk profile. These integrins are heterodimeric cell surface receptors found on a variety of immune cells, including T cells, B cells, macrophages and neutrophils [2]. Integrins mediate adhesion between these immune cells and other cells in their environment, playing vital roles in both leukocyte activation and trafficking to sites of inflammation. Two prototypic integrins are LFA-1 (leukocyte function-associated antigen-1, an αLβ2 integrin) and VLA-4 (very late antigen-4, an α4β1 integrin). LFA-1 in particular has been shown to play a vital role in the formation of an immunological synapse between T cells and antigen presenting cells (APCs). Both LFA-1 and VLA-4 have also been implicated in the “arrest” of rolling lymphocytes at sites of inflammation and the subsequent transendothelial migration of T cells into this inflamed tissue [2]. These immunomodulatory properties of integrins spurred the clinical development of integrin antagonists against both LFA-1 (efalizumab) and VLA-4 (natalizumab) to treat various autoimmune diseases [3]. Specifically, efalizumab was approved by the FDA for the treatment of psoriasis and natalizumab has found use in both multiple sclerosis and Crohn’s disease patients. While the initial clinical applications of integrin blockade were focused on autoimmunity, multiple experimental and even clinical trials have emerged over the last decade supporting the use of these therapies in the clinical realm of transplantation. Monotherapy with either LFA-1 or VLA-4 antagonists proved efficacious in prolonging graft survival in a variety of murine transplant systems, including skin [4], cardiac [5] and islet [6,7] allograft models. In addition to suppressing acute rejection, integrin blockade was also found to diminish chronic rejection in a murine model of cardiac allograft vasculopathy [8]. Combined integrin blockade with both anti-VLA-4 and anti-LFA-1 demonstrated potent synergy in a murine islet transplant system, with islet grafts lasting >60 days compared to 7–9 days with integrin antagonist monotherapy [9]. To further augment the efficacy of integrin blockade, several investigators coupled it with standard costimulatory blockade drugs such as anti-CD154 or CTLA-4 Ig, achieving prolonged graft survival in a variety of murine transplant systems [10,11]. Dual integrin/costimulatory blockade was even shown to prolong survival of xenografts such as porcine islets in murine recipients [12]. This regimen of dual costimulatory and integrin blockade was also recently utilized successfully in a primate islet transplant system (using belatacept and efalizumab), demonstrating a substantial prolongation in islet graft survival [13]. All of these encouraging preclinical studies established the critical groundwork that informed later human clinical trials with these integrin antagonists for transplantation. The initial clinical trials of LFA-1 antagonists in transplantation utilized a mouse anti-human CD11a monoclonal antibody (odulimomab); small pilot studies with this monoclonal were mixed, but at least one study demonstrated that induction therapy with odulimomab was as effective as rabbit anti-thymocyte globulin in preventing acute rejection [14]. Subsequent multicenter trials utilized efalizumab, a fully humanized IgG1 anti-LFA-1 monoclonal antibody. In one early multicenter trial, patients were randomized to either high (2 mg/kg) or low (0.5 mg/kg) dose efalizumab in new renal transplant recipients who were treated with either half-dose cyclosporine/sirolimus/prednisone or routine cyclosporine/MMF/prednisone immunosuppression regimens [15]. Despite using half-dose cyclosporine and sirolimus, the cumulative rejection rates with these efalizumab-based regimens (10.4%) were comparable to historic controls with full-dose calcineurin inhibitor-based regimens. However, in the subset of patients receiving the high dose of efalizumab coupled with conventional full-dose cyclosporine/MMF/prednisone, almost 30% of patients developed post-transplant lymphoproliferative disease, a concerning development (of note, none of the patients treated with either low-dose efalizumab regimens or high-dose efalizumab with half-dose conventional agents developed PTLD in this study). More recently, efalizumab was used successfully by two different groups to promote engraftment and insulin-independence in recipients of islet transplants from single donors [16,17]. While the initial clinical experience with integrin antagonists to treat autoimmune diseases was highly encouraging, early enthusiasm for these drugs was eventually tempered by the occurrence of progressive multifocal leukoencephalopathy (PML) in several patients receiving chronic treatment with these drugs [18,19]. PML is an opportunistic viral infection resulting from reactivation of latent JC virus (a human polyomavirus) in the brain of immunosuppressed patients [18]. PML is highly lethal, with most patients succumbing to disease after it has developed. The risk of these biologics was first revealed in patients chronically treated with natalizumab, a very small handful of whom began to manifest clinical signs of PML. Importantly, the risk of PML is almost solely confined to those patients receiving long-term integrin blockade, with patients receiving at least 18 monthly infusions of natalizumab estimated to have a 1:1000 risk of developing PML [18]. The PML risk signature of natalizumab initially led to its voluntary withdrawal from the market, but vocal support from multiple sclerosis patients led to the reintroduction of natalizumab under tight controls in June 2006. As of July 2010, a total of 61 patients on natalizumab have developed PML [19]. Similar to natalizumab, efalizumab also is associated with an elevated risk of PML and JC virus reactivation. Of the estimated 46,000 patients who had been treated with efalizumab worldwide since its FDA approval in 2003, a total of four PML cases (three definite and one probable) were reported by 2009, yielding a total PML incidence of 1 in 10,000 [18]. However, of those patients treated for >3 years with efalizumab (which included every patient who developed PML), the estimated incidence rate was 1 in 400 patients [18]. On April 8, 2009, Genentech announced that they would voluntarily withdraw efalizumab from the market due to this PML risk, and efalizumab was phased out completely by June 2009. While a remote risk of developing a fatal neurodegenerative condition may be utterly unacceptable for patients with a relatively benign condition such as psoriasis, it may certainly be tolerable in the transplant arena, especially if the novel integrin blockade therapy prolongs allograft function compared to conventional immunosuppression. The argument for exploring transplant indications for integrin antagonists is further bolstered by the fact that many drugs currently used to treat transplant patients (such as mycophenolate mofetil and rituximab) actually carry similar risks for PML compared to either natalizumab or efalizumab [18,20]. Furthermore, the likely duration of therapy for integrin antagonists used in a transplant setting would likely prove protective against PML development. The epidemiology of PML in patients with autoimmune diseases treated with integrin antagonists confirms that the risk of PML increases substantially with duration of therapy: the incidence of PML is 0.01 per 1000 patients for those who received 1 to 12 infusions of natalizumab, 1.27/1,000 for those who received >12 infusions and 1.71/1,000 in those who received >24 infusions [19]. Integrin antagonists would likely be employed as short-term perioperative induction immunosuppression therapy in transplant patients, which should substantially mitigate the risks posed by integrin blockade. Thus, the clinical calculus of risk versus benefit for integrin antagonists differs dramatically when applied to organ transplantation compared to their initial indications for autoimmunity. Given the encouraging preclinical and clinical trial data, perhaps the time has come to re-evaluate the potential applications of integrin blockade (such as VLA-4 and LFA-1 antagonists) for organ transplantation. Future clinical trials, perhaps studying regimens incorporating induction therapy with integrin antagonists and maintenance therapy with belatacept, certainly seem not only justified but indeed essential to fully clarify the efficacy and safety of integrin antagonists for transplantation.

Pathogen Stimulation History Impacts Donor‐Specific CD8+ T Cell Susceptibility to Costimulation/Integrin Blockade–Based Therapy

Recent studies have shown that the quantity of donor‐reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade‐based immunosuppression. Using a murine skin graft model of CD8+ memory T cell–mediated costimulation blockade resistance, we elicited donor‐reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor‐reactive memory T cell response. Intriguingly, the most immunosuppression‐sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi‐cytokine producers. These data, therefore, demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor‐reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation.

Partial abdominal evisceration and intestinal autotransplantation to resect a mesenteric carcinoid tumor

BackgroundMidgut carcinoids are neuroendocrine tumors that commonly metastasize to the intestinal mesentery, where they predispose to intestinal obstruction, ischemia and/or congestion. Because of their location, many mesenteric carcinoid tumors are deemed unresectable due to the risk of uncontrollable bleeding and prolonged intestinal ischemia.Case PresentationWe report the case of a 60-year-old male with a mesenteric carcinoid tumor obstructing his superior mesenteric vein, resulting in intestinal varices and severe recurrent GI bleeds. While his tumor was thought to be unresectable by conventional techniques, it was successfully resected using intestinal autotransplantation to safely gain access to the tumor. This case is the first described application of this technique to carcinoid tumors.ConclusionsIntestinal autotransplantation can be utilized to safely resect mesenteric carcinoid tumors from patients who were not previously thought to be surgical candidates. We review the literature concerning both carcinoid metastases to the intestinal mesentery and the use of intestinal autotransplantation to treat lesions involving the mesenteric root.

Hepatic Retransplant: What Have We Learned?

Hepatic retransplant accounts for 5% to 15% of liver transplants in most series and is associated with significantly increased hospital costs and inferior patient survival when compared with primary liver transplant. Early retransplants are usually due to primary graft nonfunction or vascular thrombosis, whereas later retransplants are most commonly necessitated by chronic rejection or recurrent primary liver disease. Hepatic retransplant remains the sole option for survival in many patients facing allograft failure after liver transplant. With improved techniques to match retransplant candidates with appropriate donor grafts, it is hoped that the outcomes of retransplant will continue to improve in future.