Bettina C. Baumann

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity.

The galactose-α-1,3-galactose (αGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcγRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of αGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an αGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the αGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on αGal expression. In conclusion, removal of αGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that αGal is not a dominant target for direct human NK cytotoxicity against porcine cells.

Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs

Background. Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-&agr;(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. Methods. Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by 51chromium-release assays using human serum and freshly isolated NK cells. Results. Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. Conclusions. The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.

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.

HLA-E expression on porcine cells: protection from human NK cytotoxicity depends on peptide loading.

Human NK cells lyse porcine cells and may play an important role in the cell‐mediated rejection of pig‐to‐human xenografts. Lysis is probably a consequence of the failure of human MHC‐specific killer inhibitory receptors to recognize porcine MHC class I molecules. A majority of activated human NK cells express the HLA‐E‐specific inhibitory receptor CD94/NKG2A. The aim of this study was therefore to test the hypothesis that stable surface expression of HLA‐E on porcine cells protects against xenogeneic NK‐mediated cytotoxicity. Porcine lymphoblastoid (13 271) and endothelial (pEC) cell lines were transfected with constructs coding for HLA‐E together with the leader sequence of HLA‐B7 or ‐A2. HLA‐E was correctly expressed on 13 271 cells while pEC required peptide‐pulsing and/or IFN‐γ stimulation to express the HLA‐E complex on the cell surface. HLA‐E‐expressing porcine cells were partially protected from lysis mediated by human polyclonal NK populations and completely protected from killing by NKG2Abright NK clones. In conclusion, the capability of different porcine cell types to express HLA‐E on the cell surface can differ considerably depending decisively on the availability of peptides. These findings are important for the applicability of transgenic HLA‐E expression as an approach to protect porcine tissues from human NK cytotoxicity.

Characterization of Natural Human Anti-non-gal Antibodies and Their Effect on Activation of Porcine Gal-deficient Endothelial Cells

Background. The generation of Gal&agr;1-3Gal (Gal) transferase deficient pigs has increased the interest in non-Gal antigens potentially representing important targets for xenoreactive antibody binding leading to xenograft rejection. The present study addressed the levels and immunoglobulin isotypes of preformed human anti-non-Gal antibodies and their potential to activate porcine endothelial cells. Methods. Porcine endothelial cells lacking the Gal epitope (Gal−/−) were used to measure immunoglobulin (Ig) M and IgG subclass anti-non-Gal antibodies, using sera from 80 blood donors and pooled human AB serum. Antibodies specific for the non-Gal Hanganutziu-Deicher (HD) xenoantigen were measured by enzyme-linked immunosorbent assay. Activation of Gal−/− and Gal+/+ endothelial cells by human serum was measured, in the presence or absence of complement inhibitors, by E-selectin cell-surface expression using flow cytometry. Results. Anti-non-Gal antibody levels varied considerably among individual sera and comprised approximately 10% of total anti-porcine antibodies without sex or age differences. Among the IgG subclasses only IgG1 and IgG2 were detected. Human serum-induced E-selectin expression on Gal−/− cells was less than 20% compared with Gal+/+ cells, correlated with anti-HD IgM and IgG antibody levels (P=0.027 and 0.032, respectively), and was largely complement-independent in accordance with the lack of IgG3 anti-non-Gal antibodies. In contrast, E-selectin upregulation on Gal+/+ cells was reduced in complement blocking experiments. Conclusion. Preformed anti-non-Gal antibodies, in particular anti-HD antibodies, were present in all human sera samples, activated porcine endothelial cells, and may therefore play a role in xenograft rejection using organs from GalT−/− pigs.

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.

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.

HLA‐Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells

Abstract: Background:  Human natural killer (NK) cell‐mediated cytotoxicity represents a hurdle in pig‐to‐human xenotransplantation. It was previously reported that the expression of human major histocompatibility complex class I molecules, including HLA‐B27, ‐Cw3, ‐E, and ‐G, partially protects porcine endothelial cells (pEC) from human NK‐mediated cytotoxicity and that HLA‐G inhibits NK adhesion to pEC. Here, we tested if HLA‐Cw4 expression on pEC alone, or concurrently with HLA‐Cw3, prevents human NK adhesion and cytotoxicity against pEC via recognition of the killer‐cell immunoglobulin‐like receptors (KIR) CD158a (KIR2DL1) and CD158b (KIR2DL2/3), respectively.