Benjamin G. Lilienfeld

HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity

Background. Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, &bgr;2-microglobulin (&bgr;2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules. Methods. To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human &bgr;2-microglobulin (hu&bgr;2m) into zygotes. Results. Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and hu&bgr;2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and hu&bgr;2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/hu&bgr;2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/hu&bgr;2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-&ggr; by co-cultured human NK cells. Conclusions. This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation.

Human NK Cytotoxicity against Porcine Cells Is Triggered by NKp44 and NKG2D

Pig-to-human xenotransplantation has been proposed as a means to alleviate the shortage of human organs for transplantation, but cellular rejection remains a hurdle for successful xenograft survival. NK cells have been implicated in xenograft rejection and are tightly regulated by activating and inhibitory receptors recognizing ligands on potential target cells. The aim of the present study was to analyze the role of activating NK receptors including NKp30, NKp44, NKp46, and NKG2D in human xenogeneic NK cytotoxicity against porcine endothelial cells (pEC). 51Cr release and Ab blocking assays were performed using freshly isolated, IL-2-activated polyclonal NK cell populations as well as a panel of NK clones. Freshly isolated NK cells are NKp44 negative and lysed pEC exclusively in an NKG2D-dependent fashion. In contrast, the lysis of pEC mediated by activated human NK cells depended on both NKp44 and NKG2D, since a complete protection of pEC was achieved only by simultaneous blocking of these activating NK receptors. Using a panel of NK clones, a highly significant correlation between anti-pig NK cytotoxicity and NKp44 expression levels was revealed. Other triggering receptors such as NKp30 and NKp46 were not involved in xenogeneic NK cytotoxicity. Finally, Ab-dependent cell-mediated cytotoxicity of pEC mediated by human NK cells in the presence of xenoreactive Ab was not affected by blocking of activating NK receptors. In conclusion, strategies aimed to inhibit interactions between NKp44 and NKG2D on human NK cells and so far unknown ligands on pEC may prevent direct NK responses against xenografts but not xenogeneic Ab-dependent cell-mediated cytotoxicity.

Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity

Abstract:  Background:  The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)‐specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK‐mediated cytotoxicity afforded by the stable expression of HLA‐E single chain trimers (SCT) composed of a canonical HLA‐E binding peptide antigen, VMAPRTLIL, the mature human β2‐microglobulin, and the mature HLA‐E heavy chain, and (ii) to test whether HLA‐E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells.

Porcine UL16-Binding Protein 1 Expressed on the Surface of Endothelial Cells Triggers Human NK Cytotoxicity through NKG2D

Cellular rejection mechanisms, including NK cells, remain a hurdle for successful pig-to-human xenotransplantation. Human anti-pig NK cytotoxicity depends on the activating receptor NKG2D. Porcine UL16-binding protein 1 (pULBP1) and porcine MHC class I chain-related protein 2 (pMIC2) are homologues of the human NKG2D ligands ULBP 1–4 and MICA and B, respectively. Although transcribed in porcine endothelial cells (pEC), it is not known whether pULBP1 and pMIC2 act as functional ligands for human NKG2D. In this study, surface protein expression of pULBP1 was demonstrated by flow cytometry using a novel pULBP1-specific polyclonal Ab and by cellular ELISA using NKG2D-Fc fusion protein. Reciprocally, pULBP1-Fc bound to primary human NK cells, whereas pMIC2-Fc did not. Transient and stable down-regulation of pULBP1 mRNA in pEC using short-interfering RNA oligonucleotide duplexes and short hairpin RNA, respectively, resulted in a partial inhibition of xenogeneic NK cytotoxicity through NKG2D in 51Cr release assays. In contrast, down-regulation of pMIC2 mRNA did not inhibit NK cytotoxicity. Human NK cytotoxicity against pEC mediated by freshly isolated or IL-2-activated NK cells through NKG2D was completely blocked using anti-pULBP1 polyclonal Ab. In conclusion, this study suggests that pULBP1 is the predominant, if not only, functional porcine ligand for human NKG2D. Thus, the elimination of pULBP1 on porcine tissues represents an attractive target to protect porcine xenografts from human NK cytotoxicity.

Endothelial cells derived from pigs lacking Gal alpha(1,3)Gal: no reduction of human leukocyte adhesion and natural killer cell cytotoxicity.

Background. The expression of galactose-&agr;(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal−/− pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal−/− and Gal+/+ pigs, and (2) the susceptibility of such Gal−/− porcine endothelial cells to human natural killer (NK) cell cytotoxicity. Methods. Primary porcine aortic endothelial cells (PAEC) were isolated from Gal−/− (PAEC-Gal−/−) and Gal+/+ (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays. Results. Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal−/− cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal−/− and PAEC-Gal+/+ was detected. Tumor-necrosis factor-&agr; stimulation of PAEC-Gal−/− and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal expression on PAEC-Gal−/− cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2–activated NK cells. Conclusions. Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.

Porcine cells express more than one functional ligand for the human lymphocyte activating receptor NKG2D

Abstract:  Background:  Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically‐modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti‐pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8+ and γδ T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co‐stimulation of T cells.

Characterization of porcine UL16-binding protein 1 endothelial cell surface expression.

Abstract:  Background:  Natural killer (NK) cells participate in the immune response against solid organ allo‐ and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL‐16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell‐surface expression on primary porcine aortic endothelial cells (PAEC).