Lysis of pig endothelium by IL-2 activated human natural killer cells is inhibited by swine and human major histocompatibility complex (MHC) class I gene products.

We have previously described a form of xenograft rejection, mediated by natural killer (NK) cells, occurring in pig-to-primate organ transplants beyond the period of antibody-mediated hyperacute rejection. In this study, two distinct NK activation pathways were identified as mechanisms of pig aortic endotheliual cell (PAEC) lysis by human NK cells. Using an antibody-dependent cellular cytotoxicity (ADCC) assay, a progressive increase in human NK lysis of PAEC was observed following incubation with human IgG at increasing serum titer. In the absence of IgG, a second mechanism of PAEC lysis by human NK cells was observed following activation with IL-2. IL-2 activation of human NK cells increased lysis of PAEC by over 3-fold compared with ADCC. These results indicate that IL-2 activation of human NK cells induces significantly higher levels of lytic activity than does conventional ADCC involving IgG and FcRIII. We next investigated the role of MHC class I molecules in the regulation of NK lysis following IL-2 activation. PAEC expression of SLA class I molecules was increased by up to 75% by treatment with human TNFa. Following treatment with TNFa at 1 u/ml, IL-2 activated human NK lysis of PAEC was inhibited at every effector:target (E:T) ratio tested. Maximal effect occurred at an E:T ratio of 10:1, with TNFa inhibiting specific lysis by 59% (p < 0.01). Incubation with an anti-SLA class I Mab, but not IgG isotype control, abrogated the protective effects of TNFa on NK lysis of PAEC, suggesting direct inhibitory effects of SLA class I molecules on human NK function. To investigate whether human MHC class I molecules might have similar effects on human NK lysis of PAEC, further experiments were performed using a soluble peptide derived from the alpha-helical region of HLA-B7. Incubation with the HLA-B7 derived peptide significantly reduced the IL-2 activated NK lytic activity against PAEC in a dose-dependent fashion. Maximal effect occurred at a concentration of 10 mg/ml, where an 8-fold reduction in IL-2 augmented NK lysis was observed (p < 0.01). These results suggest that IL-2 activated human NK lysis of porcine xenografts may be inhibited by strategies which increase PAEC expression of SLA class I molecules, introduce HLA class I genes into PAEC, or use soluble HLA class I peptides.

Itescu S ，Artrip JH ，Kwiatkowski PA ，Wang SF ，Minanov OP ，Morgenthau AS ，Michler RE ... -  《Annals of Transplantation》

Induction of swine major histocompatibility complex class I molecules on porcine endothelium by tumor necrosis factor-alpha reduces lysis by human natural killer cells.

Natural killer (NK) cells have been implicated in a process of delayed xenograft rejection occurring in pig-to-primate organ transplants. As tumor necrosis factor-a (TNF-a) induces expression of both adhesion receptors and major histocompatibility complex class I molecules on porcine endothelium, we investigated the effects of TNF-alpha on human NK cell adherence to and cytotoxicity of porcine aortic endothelial cell (PAEC) monolayers. Adherence of human NK cells was measured after PAEC treatment with increasing concentrations of TNF-alpha. Monoclonal antibodies (mAbs) against adhesion molecules on NK cells and PAEC were used in inhibition studies. Resting or TNF-alpha-treated PAEC were used as targets for NK lysis. Increasing titers of anti-swine leukocyte antigen (SLA) class I antibodies or pooled human immune globulin (IVIg) were used to reverse the effects of TNF-alpha on NK lysis. NK cell adhesion to TNF-a-treated PAEC increased in a dose-dependent manner by a maximum of 44%, and was inhibited by mAbs against CD49d, CD11a, CD11b, CD18, and CD2, as well as porcine vascular cell adhesion molecules. In contrast, TNF-alpha treatment of PAEC reduced human NK lysis in a dose-dependent manner. Preincubation of TNF-a-treated PAEC with increasing concentrations of anti-SLA class I mAb increased NK lysis in a titer-dependent manner, and reversed the protective effect on human NK lysis by 77%. Treatment with IVIg, containing antibodies against an a-helical region of HLA class I molecules, had a similar effect. These results imply that SLA class I molecules can bind to inhibitory receptors on human NK cells, and that these interactions can be augmented by increasing the level of SLA class I molecule expression on porcine endothelium. Strategies that can increase porcine endothelial cell expression of either swine or human major histocompatibility complex class I molecules may reduce human NK activity against porcine xenografts.

Kwiatkowski P ，Artrip JH ，John R ，Edwards NM ，Wang SF ，Michler RE ，Itescu S ... -  《TRANSPLANTATION》

Human NK cells demonstrate increased adherence and lysis of porcine endothelium following activation with IL-2.

For clinical xenotransplantation of pig organs to be successful, the significant barriers of host cellular immunity against the pig will need to be overcome. Natural killer (NK) cells appear to play an important role in rejection of discordant xenografts, and IL-2 activated human NK cells are cytotoxic to human endothelium. We addressed the role of IL-2 activated human and baboon NK cells in the primate immune response to pig xenoantigens in vivo and in vitro. Human natural killer (NK) cells were isolated from pooled human peripheral mononuclear cells (PBMC) using density gradient centrifugation method. Phenotype of isolated cells was confirmed by flow cytometry. Purified NK cells were than tasted for spontaneous and IL-2 augmented adhesion and lysis of cultured pig aortic endothelial cells (PAEC) monolayers in a short-term 51 Cr-release assay. The effects of human macrophage derived cytokines TNF alpha and IL-1 on adhesion of IL-2 activated NK cells were studied. Human NK cells demonstrate prominent adherent and lytic activity against pig endothelium, which can be significantly augmented by IL-2. Macrophage derived cytokines can further augment the adherent properties of these NK cells. These result suggest that human NK cells are likely to play a significant role in rejection of pig-to-human xenografts.

Kwiatkowski B ，Artrip JH ，Michler RE ，Itescu S ... -  《Annals of Transplantation》

Circulating human mononuclear cells exhibit augmented lysis of pig endothelium after activation with interleukin 2.

In immunohistochemical studies investigating the cellular infiltrates in pig xenografts undergoing delayed rejection by newborn and adult primate recipients, we observed extensive infiltration with primate macrophages and natural killer (NK) cells. To extend these studies in vitro, we investigated the functional properties of human NK cell precursors with respect to their potential interactions with pig aortic endothelial cells (PAEC). Using a short-term 51Cr release assay, human peripheral blood mononuclear cells (PBMC) demonstrated spontaneous and interleukin (IL) 2 augmented lytic activity against PAEC which increased with increasing effector to target cell ratio. Treatment of human PBMC with anti-CD2 significantly reduced this NK lytic activity by IL-2-activated PBMC. Finally, we investigated the effects of PAEC treatment with certain macrophage-derived human cytokines on adhesion of IL-2-activated human PBMC. Treatment of PAEC with IL-1 and tumor necrosis factor-alpha, in a dose-dependent manner, increased adherence of IL-2-activated human PBMC. These results demonstrate that humans contain circulating NK cells capable of lysing PAEC after activation with IL-2, that the mechanism involves interactions between CD2 and its ligand on porcine endothelium, and that these interactions may be influenced by macrophage-derived cytokines produced at the site of xenograft rejection.

Itescu S ，Kwiatkowski P ，Wang SF ，Blood T ，Minanov OP ，Rose S ，Michler RE ... -  《TRANSPLANTATION》

Effect of redox modulation on xenogeneic target cells: the combination of nitric oxide and thiol deprivation protects porcine endothelial cells from lysis by IL-2-activated human NK cells.

:Evidence suggests that NK cells contribute to the pathogenesis of delayed rejection of vascularized xenografts, and NK cells have been suggested to participate in hyperacute xenograft rejection. Endothelial cells have been shown to be the primary target of the recipient's immune responses that mediate both hyperacute and delayed xenograft rejection. Under conditions of oxidative stress induced by thiol deprivation, but not under normal conditions, pretreatment of porcine aortic endothelial cells (PAECs) with the NO donor, S-nitroso-N-acetyl-penicillamine, dramatically inhibited killing of PAEC target cells by IL-2-activated human NK cells. This same combined treatment reduced both surface expression and mRNA levels of E-selectin. Moreover, anti-E-selectin mAb, but not Ab to VCAM-1, protected PAEC from lysis by human IL-2-activated NK cells in a dose-dependent manner. These findings suggest that expression of porcine E-selectin is important for the cytotoxicity of PAEC mediated by activated human NK cells and may be involved in the redox-mediated modulation of that cytotoxicity. It is known that NF-kappa B activation is required for transcription of E-selectin, and the current data show that the suppression of E-selectin expression by S-nitroso-N-acetyl-penicillamine pretreatment and thiol deprivation was associated with reduced NF-kappa B DNA-binding activity in PAEC. These data suggest that the regulation of porcine E-selectin may be important for modulating delayed xenograft rejection and that manipulation of cellular redox systems may provide a means to protect xenogeneic endothelial cells from NK cell-mediated cytotoxicity.

Tsuyuki S ，Horvath-Arcidiacono JA ，Bloom ET 《JOURNAL OF IMMUNOLOGY》