O406 Aims: NK cells are a prominent component of the cellular infiltrate in delayed xenograft rejection, and are the main cell type responsible for human anti-pig cytotoxicity in vitro. It thus seems likely that strategies to inhibit NK cell-mediated cytotoxicity will need to be developed in order to achieve successful clinical transplantation between species. The overall activation state of an NK cell reflects the balance between activating and inhibitory signals received by that cell. Whereas signalling through most NK cell activating receptors can be overcome by inhibitory inputs, activation mediated by NKG2D is resistant to inhibition. It is not known whether porcine cells express ligands able to bind human NKG2D, but if such ligands are present, a specific strategy to interfere with NK cell activation by these molecules will be important to facilitate acceptance of porcine to human xenografts. The aims of this study were to determine whether ligands able to bind human NKG2D are expressed by various porcine cell lines, or in pig kidneys following transplantation. Methods: Chinese Hamster Ovary P (CHOP) cells were transiently transfected with constructs encoding human NKG2D and FLAG-tagged human DAP-10, alone or in combination, or with the empty vector pCDNA3 as a negative control. Cell surface expression was determined by antibody staining and flow cytometry at 48 h post-transfection. Binding of CFSE-labelled HeLa cells (known to express MICA, a natural ligand for human NKG2D) and of two porcine cell lines (PK-1; renal epithelial cells, and PIEC: endothelial cells) to confluent monolayers of CHOP transfectants was assessed concurrently. Normal pig kidney and explanted tissue from a rejecting porcine to baboon renal xenograft were stained using a recombinant soluble form of human NKG2D (NKG2D-Ig). Results: High levels of surface expression of the transfected molecules were obtained. In this transient system, DAP-10 was able to be expressed in the absence of NKG2D, although there was a further increase in surface expression in cells transfected with both constructs. All three cell types bound to the NKG2D-DAP-10 double-transfected CHOP cells in significantly greater numbers than to the empty-vector controls (for HeLa, 248±41 cf 59±26, P<0.001; for PK-1, 46±4 cf 8±4, P<0.01; and for PIEC, 30±14 cf 2.3±1.5) There was no increase in binding to cells expressing DAP-10 alone, but PK-1 cells bound to CHOP monolayers transfected with NKG2D alone (30±11 cf 8±4, P<0.01). Binding was blocked by pre-incubation of the monolayers with the anti-NKG2D monoclonal antibody 3D12, indicating that binding was a specific interaction between human NKG2D and ligands on the porcine cells. Renal tubules from a rejecting porcine kidney explanted on d5 post-transplantation were stained by NKG2D-Ig, whereas this reagent did not stain normal pig kidney. Conclusions: This study provides the first evidence that some porcine cell types express ligands able to bind to human NKG2D. NKG2D ligands were not detected in normal pig kidney, but were induced in a rejecting porcine renal graft. It will now be important to identify and characterise these molecules, in order to understand their role in the human anti-pig xenoresponse.