A Standardized Warm Ischemia Time for the Induction of Injury in Murine Kidney Transplants.

One of the cornerstone research models used in our laboratories is the induction of ischemic injury through cold ischemia followed by warm ischemia to donor kidneys to mimic the clinical realities of transplantation. The experimental design of the present study included bilateral nephrectomies on the day of syngeneic kidney transplant, with serum creatinine measured 24 hours postoperatively to measure acute function. Cold ischemia time in these experiments was always 30 minutes, and warm ischemia time was not standardized but always recorded. It became apparent that some transplanted kidneys that should have displayed injury were producing close to normal serum creatinine levels on postoperative day 1. In reviewing our data, we found a potential correlation between warm ischemia time and serum creatinine, in particular a significant proportion of low serum creatinine results (0.48 ± 0.26 mg/dL vs 1.99 ± 1.11 mg/dL; P < .05) was associated with warm ischemia times that were significantly shorter than our historical average (29.2 ± 2.7 min vs 35.7 ± 2.2 min; P < .05). The kidneys with lower serum creatinine also displayed lower apoptosis and brush border injury scores and fewer tubular casts. Therefore, we concluded that establishing a minimum warm ischemia time was just as important as standardized cold ischemia time to ensure consistent injury in this model.

Anti-LFA-1 induces CD8 T-cell dependent allograft tolerance and augments suppressor phenotype CD8 cells.

The induction of tolerance to transplanted organs is a major objective in transplantation immunology research. Lymphocyte function-associated antigen-1 (LFA-1) interactions have been identified as a key component of the T-cell activation process that may be interrupted to lead to allograft tolerance. In mice, αLFA-1 mAb is a potent monotherapy that leads to the induction of donor-specific transferable tolerance. By interrogating important adaptive and innate immunity pathways, we demonstrate that the induction of tolerance relies on CD8+T-cells. We further demonstrate that αLFA-1 induced tolerance is associated with CD8+CD28-T-cells with a suppressor phenotype, and that while CD8 cells are present, the effector T-cell response is abrogated. A recent publication has shown that CD8+CD28- cells are not diminished by cyclosporine or rapamycin, therefore CD8+CD28- cells represent a clinically relevant population. To our knowledge, this is the first time that a mechanism for αLFA-1 induced tolerance has been described.

Revised Arterial Anastomosis for Improving Murine Kidney Transplant Outcomes.

AIM: One of the most challenging research microsurgical techniques is the mouse kidney transplant however, very few laboratories have made use of this important model due to its difficulty. One of the main obstacles to utilizing this procedure is the high incidence of post-operative arterial thrombosis. We believe this is caused by the path in which blood is required to flow from the recipient abdominal aorta, via the donor recipient aorta and on into the renal artery creating a tortuous route and areas of turbulence, which are prone to thrombus formation and failure of the graft. METHODS: We describe revised methods of donor artery recovery, whereby the traditional transection of the donor aorta is replaced with a heel and toe cuff, which is created by dividing the donor abdominal aorta obliquely across the face of the renal arterial ostium, which then provides for an arterial end-to-side anastomosis of a scale similar to that used for the heterotopic heart model. This technique produces an anastomosis that facilitates free blood flow from the recipient abdominal aorta at less than 90° thereby reducing the likelihood of thrombus formation. RESULTS: Utilizing this new technique the incidence of arterial thrombosis has decreased from 35% to 0% (n = 20 and 24, respectively) with no change in ischemia times. CONCLUSION: We describe a revised method of performing the arterial anastomosis during mouse kidney transplantation, which facilitates improved fluid dynamics by straightening the flow path for blood to the graft resulting in significantly reduced thrombus formation, excellent graft function, histology, and post-transplant survival.

Inhibition of autophagy increases apoptosis during re-warming after cold storage in renal tubular epithelial cells.

Prolonged cold storage and re-warming (CS/REW) of kidneys are risk factors for delayed graft function (DGF). Studies in renal tubular epithelial cells (RTECs) have determined apoptosis and autophagy in models of either cold storage (CS) or re-warming alone. The effect of both cold storage and re-warming on apoptosis and autophagy, in RTECS is not known and is relevant to DGF as the kidney is subjected to both CS and re-warming. We hypothesized that CS/REW of RTECs would induce autophagy that protects against apoptosis. In CS/REW, there was increased autophagic flux of RTECs. Autophagy inhibition using an Atg5 siRNA resulted in increased cleaved caspase-3 and increased apoptotic cells (on both morphology and annexin V staining) during CS/REW. The effect of autophagy inhibition on necrosis in RTECs is unknown. There were increased necrosis and caspase-1, a mediator of necrosis, during CS/REW, and the Atg5 siRNA had no effect on necrosis and caspase-1. In a kidney transplant model, there was an increase in LC3 II, a marker of autophagy, in kidneys transplanted after cold storage. In summary, autophagic flux is increased during CS/REW. Autophagy inhibition resulted in increased cleaved caspase-3 and increased apoptosis during CS/REW without an effect on necrosis or caspase-1. In conclusion, autophagy inhibition in RTECs after CS/REW induces apoptotic cell death and may be deleterious as a therapy to decrease DGF.

Ectopic expression of Fas Ligand on cardiomyocytes renders cardiac allografts resistant to CD4(+) T-cell mediated rejection.

Fas Ligand limits inflammatory injury and permits allograft survival by inducing apoptosis of Fas-bearing lymphocytes. Previous studies have shown that the CD4(+) T-cell is both sufficient and required for murine cardiac allograft rejection. Here, utilizing a transgenic mouse that over-expresses Fas Ligand specifically on cardiomyocytes as heart donors, we sought to determine if Fas Ligand on graft parenchymal cells could resist CD4(+) T-cell mediated rejection. When transplanted into fully immunocompetent BALB/c recipients Fas Ligand transgenic hearts were acutely rejected. However, when transplanted into CD4(+) T-cell reconstituted BALB/c-rag(-/-) recipients, Fas Ligand hearts demonstrated long-term survival. These results indicate that Fas Ligand over-expression on cardiomyocytes can indeed resist CD4(+) T-cell mediated cardiac rejection and suggests contact dependence between Fas Ligand expressing graft parenchymal cells and the effector CD4(+) T-cells.

Murine heterotopic heart transplant technique.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers. Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

Four decades of vascularized heterotopic cardiac transplantation in the mouse.

Since the first clinical heart transplant in 1967, there has been a heightened need to understand immune and inflammatory responses to "foreign" tissues. Research efforts in those early days were based on species that would now be considered "large" and were typically out-bred individuals. While this closely mirrors the clinical scenario, where genetic mismatches of donors and recipients can only be minimized in the selection process, these were not ideal models for studying the complexities and nuances of the immune system. Even when the rat was considered the standard model those early endeavors were limited by a small number of rat strains. The mouse model has provided us with an overwhelming array of strains, knockouts, knockins and transgenics that allow us to investigate the many layers of the innate and adaptive immune systems leading to a much greater understanding of immune responses. Fully vascularized heterotopic cardiac transplantation in the mouse has now been with us for four decades; the original papers describing this technique being published by Corry in 1973. In the subsequent 40 years, this technique has been used by many laboratories, including our own, and has become a powerful tool for the investigation of transplant immunity and ischemia reperfusion injury. Given the modern availability of mouse strains and mouse-related reagents, our current understanding of transplant immunity undoubtedly would not exist without such a technique.

CD4 T cells mediate cardiac xenograft rejection via host MHC Class II.

BACKGROUND: Previous studies have shown that acute CD4 T-cell-mediated cardiac allograft rejection requires donor major histocompatibility complex (MHC) Class II expression and can be independent of "indirect" antigen presentation. However, other studies suggested that indirect antigen presentation to CD4 T cells may play a primary role in cellular xenograft immunity. Thus, the relative roles of direct/indirect CD4 T cell reactivity against cardiac xenografts are unclear. In this study we set out to determine the role for indirect CD4 T cell reactivity in cardiac xenograft rejection. METHODS: Rat hearts were transplanted heterotopically into wild-type and immunodeficient mice. Recipients were untreated, treated with depleting antibodies, or reconstituted with wild-type cells. RESULTS: Antibody depletion confirmed that rat heart xenograft rejection in C57Bl/6 mice was CD4 T-cell-dependent. Also, heart xenografts survived long term in B6 MHC Class II (C2D)-deficient mice. Graft acceptance in C2D mice was not secondary to CD4 T cell deficiency alone, because transferred B6 CD4 T cells failed to trigger rejection in C2D hosts. Furthermore, purified CD4 T cells were sufficient for acute rejection of rat heart xenografts in immune-deficient B6rag1(-/-) recipients. Importantly, CD4 T cells did not reject rat hearts in C2Drag1(-/-) hosts, in contrast to results using cardiac allografts. "Direct" xenoreactive CD4 T cells were not sufficient to mediate rejection despite vigorous reactivity to rat stimulator cells in vitro. CONCLUSIONS: Taken together, our results show that CD4 T cells are both necessary and sufficient for acute cardiac xenograft rejection and that host MHC Class II is critical in this process.

Acute cardiac allograft rejection by directly cytotoxic CD4 T cells: parallel requirements for Fas and perforin.

BACKGROUND: CD4 T cells can suffice as effector cells to mediate primary acute cardiac allograft rejection. Although CD4 T cells can readily kill appropriate target cells in vitro, the corresponding role of such cytolytic activity for mediating allograft rejection in vivo is unknown. Therefore, we determined whether the cytolytic effector molecules perforin (PFP) and/or FasL (CD95L) were necessary for CD4 T cell-mediated rejection in vivo. METHODS: Wild-type C3H(H-2) or Fas (CD95)-deficient C3Hlpr (H-2) hearts were transplanted into immune-deficient C57B6rag (H-2) mice. Then, recipients were reconstituted with naïve purified CD4 T cells from wild-type, PFP-deficient, or FasL (gld)-deficient T-cell donors. RESULTS: In vitro, alloreactive CD4 T cells were competent to lyse donor major histocompatibility complex class II+ target cells, largely by a Fas-dependent mechanism. In vivo, the individual disruption of donor Fas expression (lpr) or CD4 T-cell-derived PFP had no significant impact on acute rejection. However, FasL-deficient (gld) CD4 T cells demonstrated delayed allograft rejection. Importantly, the simultaneous removal of both donor Fas expression and CD4 T-cell PFP completely abrogated acute rejection, despite the persistence of CD4 T cells within the graft. CONCLUSIONS: Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell PFP expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo.

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells.

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from naïve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.

A two-step model of acute CD4 T-cell mediated cardiac allograft rejection.

CD4 T cells are both necessary and sufficient to mediate acute cardiac allograft rejection in mice. This process requires "direct" engagement of donor MHC class II molecules. That is, acute rejection by CD4+ T cells requires target MHC class II expression by the donor and not by the host. However, it is unclear whether CD4+ T cell rejection requires MHC class II expression on donor hemopoietic cells, nonhemopoietic cells, or both. To address this issue, bone marrow transplantation in mice was used to generate chimeric heart donors in which MHC class II was expressed either on somatic or on hemopoietic cells. We report that direct recognition of hemopoietic and nonhemopoietic cells are individually rate limiting for CD4+ T cell-mediated rejection in vivo. Importantly, active immunization with MHC class II(+) APCs triggered acute rejection of hearts expressing MHC class II only on the somatic compartment. Thus, donor somatic cells, including endothelial cells, are not sufficient to initiate acute rejection; but they are necessary as targets of direct alloreactive CD4 T cells. Taken together, results support a two-stage model in which donor passenger leukocytes are required to activate the CD4 response while direct interaction with the somatic compartment is necessary for the efferent phase of acute graft rejection.