Xenogeneic human NK cytotoxicity against porcine endothelial cells is perforin/granzyme B dependent and not inhibited by Bcl-2 overexpression Introduction In view of the acute shortage of human organs in clinical transplantation, xenogeneic organ transplantation has become a major focus of transplantation research and miniature swine are considered as an attractive candidate organ donor. Still, there are many immunological barriers to overcome in pig-to-human xenotransplantation, one of them being the cellular immune response mediated by natural killer (NK) cells [1,2]. NK cells appear to play a minor role in solid organ allotransplantation [3]. In contrast, there is now evidence that NK cells participate in cellular responses to xenografts in vivo, both in small animal as well as in pig-to-primate models [4,5], and in ex vivo perfusion models using human blood [6]. Xenogeneic lysis of porcine target cells by human NK cells in vitro has been demonstrated by several groups and reviewed [7]. This NK cell- mediated xenogeneic cytotoxicity is at least parti- ally related to the lack of recognition of porcine major histocompatibility complex (MHC) class I molecules by human inhibitory NK receptors [8]. Consequently, the expression of human leukocyte antigen (HLA) molecules on porcine endothelial cells (pEC) partially inhibited xenogeneic NK cytotoxicity [9–11]. On the other hand, the expres- sion of activating molecules on pEC may also contribute to the observed strong human natural killer (hNK) cell-mediated lysis of pEC [12]. Natural killer cells employ several different pathways of cytotoxicity [13], but little is known on the respective role of these pathways in xeno- geneic NK cytotoxicity. Upon target cell contact, NK cells release the contents of their lytic granules that are used for the storage of a variety of cytotoxic proteins such as perforin and serine Matter-Reissmann UB, Forte P, Schneider MKJ, Filgueira L, Groscurth P, Seebach JD. Xenogeneic human NK cytotoxicity against porcine endothelial cells is perforin/granzyme B dependent and not inhibited by Bcl-2 overexpression. Xenotransplantation 2002; 9: 325–337. Ó Blackwell Munksgaard, 2002 Abstract: Because of organ shortages in clinical allotransplantation, the potential of pig-to-human xenotransplantation is currently being explored showing a possible critical role for natural killer (NK) cells in the immune response against xenografts. Therefore, we analyzed the cytotoxic pathways utilized by human natural killer cells (hNK) against porcine endothelial cells (pEC). Transmission electron microscopy of pEC cocultured with hNK cells showed both apoptotic and necrotic cell death, whereas soluble factors such as Fas ligand or TNFa did not induce apoptosis in pEC. NK lysis of pEC was abrogated by concanamycin A and ammonium chloride, reagents inhibiting the perforin/granzyme B (grB) pathway, but only partially blocked by caspase inhibition with z-VAD-fmk. Overexpression of bcl-2 protected pEC against apoptosis induced by staurosporine or actinomycin D, but failed to prevent hNK cell-mediated lysis. In conclusion, pEC are lysed in vitro by hNK cells via the perforin/grB pathway and are not protected from NK lysis by overexpression of bcl-2. Ulrike B. Matter-Reissmann, 1 Pietro Forte, 1 Mårten K. J. Schneider, 1 Luis Filgueira, 2 Peter Groscurth 2 and Jörg D. Seebach 1 1 Laboratory for Transplantation Immunology, Department of Internal Medicine, University Hospital Zürich, and 2 Institute of Anatomy, University of Zürich, Zürich, Switzerland Key words: apoptosis – bcl-2 – caspase – cytotoxicity – human NK cells – necrosis – porcine endothelial cells – xenotransplantation Address reprint requests to Dr Jörg D. Seebach, Laboratory for Transplantation Immunology, Depart- ment of Internal Medicine, University Hospital Zürich, Rämistrasse 100, C HOER 31, CH-8091 Zürich, Switzerland (E-mail: klinseeb@usz.unizh.ch) Received 12 July 2001; accepted 18 October 2001 Xenotransplantation 2002: 9: 325–337 Printed in UK. All rights reserved Copyright Ó Blackwell Munksgaard 2002 XENOTRANSPLANTATION ISSN 0908-665X 325