Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

University of Colorado, Denver

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.

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

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.

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

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.

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

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.

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

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.

Four decades of vascularized heterotopic cardiac transplantation in the mouse.

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.

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

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.

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

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.

Article	Year
A two-step model of acute CD4 T-cell mediated cardiac allograft rejection. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 15187123	2004
Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells. Transplantation\| PubMed ID: 17565318	2007
Acute cardiac allograft rejection by directly cytotoxic CD4 T cells: parallel requirements for Fas and perforin. Transplantation\| PubMed ID: 20061916	2010
CD4 T cells mediate cardiac xenograft rejection via host MHC Class II. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation\| PubMed ID: 22789136	2012
Four decades of vascularized heterotopic cardiac transplantation in the mouse. Journal of investigative surgery : the official journal of the Academy of Surgical Research\| PubMed ID: 23514056	2013
Inhibition of autophagy increases apoptosis during re-warming after cold storage in renal tubular epithelial cells. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 25270002	2015
Ectopic expression of Fas Ligand on cardiomyocytes renders cardiac allografts resistant to CD4(+) T-cell mediated rejection. Cellular immunology\| PubMed ID: 25497973	2015
Revised Arterial Anastomosis for Improving Murine Kidney Transplant Outcomes. Journal of investigative surgery : the official journal of the Academy of Surgical Research\| PubMed ID: 26087146	2015

Robert Plenter

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