Interactions between porcine ligands and the human lymphocyte activating receptor NKG2D
Role of NKG2D in the human cellular immune response against porcine xenotransplants.
The initial results of αGT-/- pig to baboon transplantation suggest that hyperacute rejection can be overcome. However, gains in graft survival resulting from the use of αGT-/- donors have been procured in the context of very significant immunosuppression of recipients, and this remains a barrier to clinical applicability. Increasing our understanding of the function of NK cells and other components of the human anti-pig cellular immune response is now essential if progress towards ultimate clinical xenotransplantation is to continue. NKG2D is an activating immunoreceptor, present on NK cells, γδ T cells, CD8+ αβ T cells and macrophages. In NK cells, activation through NKG2D results in cytotoxicity and cytokine secretion, while in CD8+ T cells it functions as a costimulatory molecule. In macrophages, stimulation through NKG2D triggers TNFα production, and the release of nitric oxide. All these effector functions contribute to xenograft destruction. Homologues of the human NKG2D ligands MIC and ULBP are encoded in the porcine genome, and our data demonstrate that some porcine cell types express molecules able to bind to human NKG2D. Cellular stress caused by ischaemia-reperfusion injury, calcineurin inhibitors, and acute rejection may lead to the induction of NKG2D ligands within porcine xenografts. Preliminary data support this hypothesis: an Fc-fusion protein of human NKG2D stains renal tubules in a rejecting pig to baboon xenograft, but not normal pig kidney. Interactions between human NKG2D and porcine ligands are potential targets for therapeutic intervention to facilitate xenograft acceptance, and further understanding of these interactions is now imperative.
- Aim 1. Characterisation. We will characterise porcine NKG2D ligands as follows: Northern blotting and RT-PCR to determine mRNA levels in normal tissues and porcine renal transplants. Production of soluble ligands, and raising of mouse monoclonal and chicken antibodies against these. Antibodies will be used in immunostaining of porcine tissues to correlate expression of protein with mRNA. Ligand-receptor interactions will be studied in real-time by surface plasmon resonance, and by Cell-Cell binding assays.
- Aim 2. Functional studies. Porcine ligands will be expressed in target cells which are otherwise protected from lysis by human NK cells bearing the inhibitory receptor CD158b. NK cell clones positive for both CD158b and NKG2D will be used as effector cells in cytotoxicity assays, to determine whether the presence of NKG2D ligands abrogates HLA class I-mediated NK cell inhibition. We will also study the ability of the porcine ligands to cause recruitment of the p85 subunit of PI 3-kinase.
Other current research projects: Interplay between the innate immune response and rejection in a preclinical transplantation model Outline: Organs from cadaveric donors are subjected to a number of insults during the process of organ procurement. These insults not only affect the immediate function of transplanted organs, but result in an innate immune response which can initiate and amplify graft rejection. We are currently evaluating different donor management protocols in a preclinical model of brain-death and multi-organ transplantation. Our early results indicate that optimal resuscitation of brain-dead donors not only leads to improvement in the immediate function of organs, but reduces the upregulation of immunogenic and inflammatory molecules within the grafts in the six-hour period following brain-death, potentially reducing their susceptibility to subsequent rejection. In the next stage of this protocol, we will be extending these studies to examine the additive effects of harvesting, cold storage and reperfusion on transplant immune responses. Techniques involved in this project will include real-time RT-PCR, immunohistochemistry and western blotting.
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End-stage organ failure, transplantation, Transplant rejection, Xenotransplantation, cellular immunity, Lymphocyte activationCytotoxicity, NK cells, T cells, Graft rejection, Organ replacement, Cell biology, Infection & immunity
The opportunity ID for this research opportunity is: 172