Jörg D. Seebach

Xenogeneic human anti‐pig cytotoxicity mediated by activated natural killer cells

Abstract: Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti‐pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, NK cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3‐, CD20‐, CD33‐) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)‐stimulated lymphoblasts or bone marrow‐ or aortic‐derived endothelial cell lines in the presence of serum‐free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(α1,3)Gal‐reactive antibodies, suggesting that antibody dependent cell‐mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL‐2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK‐specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3‐ NK cells composed ca. 25% of short‐term (6 days) xenogeneic, allogeneic, or IL‐2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell‐ mediated killing, xenogeneic human anti‐porcine cytotoxicity includes an important contribution from NK cells.

Species differences in the expression of major histocompatibility complex class II antigens on coronary artery endothelium: implications for cell-mediated xenoreactivity

BACKGROUND There is controversy in the literature as to whether swine coronary endothelium expresses major histocompatibility complex (MHC) class II antigens constitutively. METHODS Because this issue has implications for cell-mediated human anti-swine xenogeneic responses, we stained tissue sections from human, pig, rat, and mouse hearts with the anti-class II monoclonal antibody ISCR3, which has a similar specificity and titer when binding to human, porcine, and rodent class II molecules. RESULTS Immunoperoxidase staining of human and porcine hearts with ISCR3 resulted in a dense reaction on the coronary endothelium of epicardial arteries, intramuscular arterioles, and capillaries. In contrast, the coronary endothelium of rat and mouse hearts did not stain with ISCR3. When freshly harvested porcine aortic endothelial cells were placed in culture, class II MHC antigen expression was lost within three to four passages. CONCLUSIONS Thus, using a single antibody with cross-species reactivities, we demonstrate that swine coronary endothelium, unlike rodent coronary arteries, expresses similar basal amounts of class II MHC antigens to human coronary vessels. The constitutive expression of class II MHC antigens on swine coronary artery endothelium may contribute to host T cell-mediated xenogeneic responses in clinical pig-to-human cardiac xenotransplantation and thus become a target for therapeutic intervention.

Immortalized bone-marrow derived pig endothelial cells.

Abstract: Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti‐pig immune responses in vitro, pig microvascular bone‐marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM‐derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNγ. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xeno‐antigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium‐phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV.15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.

Human anti‐pig T‐cell mediated cytotoxicity

Abstract: Miniature swine have a variety of advantages as potential donors for human xenotransplantation, including size, physiological similarities, and breeding characteristics. To investigate the nature of the human anti‐pig xenogeneic cellular response, we performed standard 51Cr‐release cell‐mediated lympholysis (CML) experiments. The major histocompatibility complex (MHC) allele specificity of the xenogeneic cellular response was tested on porcine target cells of three distinct homozygous MHC haplotypes (SLAaa, SLACC, SLAdd) and three intra‐MHC recombinant haplotypes (SLAjj, SLAgg, SLAkk), obtained from our herd of partially inbred miniature swine. After stimulation with irradiated porcine peripheral blood mononuclear cells (PBMC) of SLAaa haplotype, a strong nonspecific cytotoxic response of the bulk culture against xenogeneic targets of all three haplotypes was observed. However, SLA allele specificity could be demonstrated after T cell enrichment, and mapping experiments revealed predominantly SLA class I restriction of xenoreactive cytotoxic T lymphocytes (CTLs), although some class II restriction was also observed. The experiments were repeated in the presence of anti‐T cell monoclonal antibodies, anti‐CD3 (OKT3), anti‐CD2 (35.1), anti‐CD4 (OKT4), or anti‐CD 8 (OKT8). The bulk xenogeneic CML was not inhibited by any of the anti‐T cell antibodies tested. However, after T cell‐enrichment, lysis of porcine targets was significantly inhibited by anti‐CD3 or anti‐CD8 antibody and partially inhibited by anti‐CD2 antibody. In comparable assays, the human allogeneic CML was blocked by anti‐CD3 and anti‐CD8, but not by anti‐CD2 or anti‐CD4 antibodies. Finally, the cytotoxic activity of A3b3, a human CD4+ T‐cell clone, was tested. A3b3 lysed xenogeneic targets of SLAaa haplotype, but not SLACC or allogeneic targets, and was inhibited by anti‐CD4, anti‐CD2, and anti‐CD3 antibodies, but not by anti‐CD8. With the aid of intra‐MHC recombinant haplotypes, the xenogeneic CML reactivity of A3b3 was mapped to SLA class II, suggesting direct xenogeneic recognition of porcine MHC class II antigens by human T cells. Thus, the human anti‐pig cell‐mediated cytotoxic response is similar in magnitude to comparable allogeneic responses, and involves both SLA class I and class II restricted T‐cell mediated cytotoxicity, as well as additional nonspecific killing, possibly by NK cells.

Human Fas-ligand expression on porcine endothelial cells does not protect against xenogeneic natural killer cytotoxicity *

Abstract:  Several human leukocyte subsets including natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and polymorphonuclear neutrophils (PMN) participate in cellular immune responses directed against vascularized pig‐to‐human xenografts. As these leukocytes express the death receptor Fas either constitutively (PMN) or upon activation (NK, CTL), we explored in vitro whether the transgenic expression of Fas ligand (FasL) on porcine endothelial cells (EC) is a valuable strategy to protect porcine xenografts. The porcine EC line 2A2 was stably transfected with human FasL (2A2‐FasL) and interactions of 2A2‐FasL with human leukocytes were analyzed using functional assays for apoptosis, cytotoxicity, chemotaxis, adhesion under shear stress, and transmigration. FasL expressed on porcine EC induced apoptosis in human NK and T cells, but did not protect porcine EC against killing mediated by human NK cells. 2A2‐FasL released soluble FasL, which induced strong chemotaxis in human PMN. Adhesion under shear stress of PMN on 2A2‐FasL cells was increased whereas transendothelial migration was decreased. In contrast, FasL had no effect on the adhesion of NK cells but increased their transmigration through porcine EC. Although FasL expression on porcine EC is able to induce apoptosis in human effector cells, it did not provide protection against xenogeneic cytotoxicity. The observed impact of FasL on adhesion and transendothelial migration provides evidence for novel biological functions of FasL.

Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride

Ammonium-chloride-containing solutions (AC) are routinely used to lyse red blood cells during preparation of PBMC. Although exposure to AC has been described to affect the ultrastructural appearance of large granular lymphocytes and to temporarily inhibit cytolytic activity of PBMC preparations, the cellular basis of this phenomenon has not been studied. Here, the inhibitory effect of AC on human CTL and NK-mediated cytotoxicity has been analyzed in 4-h 51Cr-release assays. The results show that NK killing of K562 leukemia cells and xenogeneic endothelial cells is inhibited by AC exposure. The effect is dose-dependent and reversible, because recovery of cytotoxicity is observed within 15 h of re-culturing. AC does not reduce the viability of NK cells and the inhibitory effect is not mediated by the exhaustive release of granzymes upon AC treatment. In contrast, antigen-specific CTL killing of EBV-transformed B-lymphoblastoid cell lines and xenogeneic PHA lymphoblasts was less sensitive to AC and data are presented suggesting that FasL-induced apoptosis is not inhibited by AC. In conclusion, perforin-mediated NK killing is AC-sensitive whereas CTL killing and FasL-mediated killing appear to be AC-resistant. Therefore, AC represents a powerful tool to study different mechanisms of cell-mediated cytotoxicity and may be helpful in assessing antigen-specific CTL cytotoxicity without the influence of NK cell-mediated background killing.