Associate ProfessorAlexandra Sharland

Project Opportunities

Title: Interactions between porcine ligands and the human lymphocyte activating receptor NKG2D

Summary of opportunity:

Role of NKG2D in the human cellular immune response against porcine xenotransplants.

Opportunity synopsis:

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.

----------Current Research Projects----------

Studies of the endogenous peptide repertoire in allorecognition and transplant tolerance induction

(Eric Son)

Factors determining the fate and function of alloreactive T cells (Moumita Paul-Heng)

Preclinical Studies of MHC gene transfer for transplant tolerance induction (Mario Leong)