Pietro Forte

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.

HLA-G Inhibits Rolling Adhesion of Activated Human NK Cells on Porcine Endothelial Cells

Human NK cells adhere to and lyse porcine endothelial cells (pEC) and therefore may contribute to the cell-mediated rejection of vascularized pig-to-human xenografts. Since MHC class I molecules inhibit the cytotoxic activity of NK cells, the expression of HLA genes in pEC has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity. HLA-G, a minimally polymorphic HLA class I molecule that can inhibit a wide range of NK cells, is an especially attractive candidate for this purpose. In this study we tested whether the expression of HLA-G on pEC inhibits the molecular mechanisms that lead to adhesion of human NK cells to pEC and subsequent xenogeneic NK cytotoxicity. To this end two immortalized pEC lines (2A2 and PED) were stably transfected with HLA-G1. Rolling adhesion of activated human NK cells to pEC monolayers and xenogeneic cytotoxicity against pEC mediated by polyclonal human NK lines as well as NK clones were inhibited by the expression of HLA-G. The adhesion was partially reversed by masking HLA-G on pEC with anti-HLA mAbs or by masking the HLA-G-specific inhibitory receptor ILT-2 on NK cells with the mAb HP-F1. The inhibition of NK cytotoxicity by HLA-G was only partially mediated by ILT-2, indicating a role for other unknown NK receptors. In conclusion, transgenic expression of HLA-G may be useful to prevent human NK cell responses to porcine xenografts, but is probably not sufficient on its own. Moreover, the blocking of rolling adhesion by HLA-G provides evidence for a novel biological function of HLA molecules.

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.

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.

Adhesive Interactions between Human NK Cells and Porcine Endothelial Cells

Human natural killer (NK) cells are able to adhere to xenogeneic porcine endothelial cells (EC) and evidence from in vitro studies as well as animal models suggests a potential role for NK cells in the cellular recognition and damage of porcine xenogeneic tissues. One possible explanation for the observed NK cell‐mediated xenogeneic cytotoxicity against porcine EC is the molecular incompatibility between porcine major histocompatibility complex (MHC) class I molecules and MHC‐specific inhibitory receptors on human NK cells. In this review we attempt to summarize the current knowledge concerning adhesive interactions between human NK cells and porcine EC under special considerations of the cross‐species receptor–ligand interactions. Methodological differences in assessing adhesion between various studies are reviewed and comparisons to the syngeneic/allogeneic adhesion mechanisms are made. Finally, the therapeutic potential of blocking antibodies and transgenic HLA expression in preventing NK‐cell adhesion and xenogeneic cytotoxicity is discussed.

Porcine aortic endothelial cells transfected with HLA-G are partially protected from xenogeneic human NK cytotoxicity.

In this study we tested whether the expression of HLA-G protects porcine endothelial cells (PEC) from the lysis mediated by human natural killer (NK) cells. Because HLA-E is not present in PEC, this model provides an ideal tool to study the direct role of HLA-G in NK inhibition. Immortalized porcine aortic endothelial cells (PED) were stably transfected with a vector coding for the HLA-G1 protein and surface expression was demonstrated by flow cytometry analysis. Although the adhesion of human NK cells to PED was not compromised by HLA-G, the expression of HLA-G partially protected PED from the lysis mediated by polyclonal NK lines derived from different donors. A decrease of the surface expression of HLA-G on PED corresponded to a loss of the capacity of PED to inhibit NK cytotoxicity, indicating that the surface density of HLA-G molecules must exceed a certain threshold to protect target cells. In summary, these data show that HLA-G, independent from the presence of HLA-E, can only partially and inefficiently protect PED from human NK cell-mediated cytotoxicity. Because ILT-2/LIR-1 expression did not correlate with HLA-G mediated inhibition, we hypothesize that other yet unidentified receptors expressed by peripheral blood NK cells are involved in the recognition of HLA-G.

Xenogeneic human NK cytotoxicity against porcine endothelial cells is perforin/granzyme B dependent and not inhibited by Bcl-2 overexpression.

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 TNFα 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.

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.

Human CD26 expression in transgenic mice affects murine T-cell populations and modifies their subset distribution

CD26 is a type II transmembrane glycoprotein with dipeptidyl peptidase (DPPIV) activity, constitutively expressed in different cell types and contributing to T-cell activation by acting as costimulatory molecule. Although data suggest an important role for CD26 within the immune system, the physiologic function of this molecule is still unknown. To investigate the role of CD26 in vivo we have produced transgenic mice expressing the human molecule in T cells. Human CD26 (huCD26) is constitutively expressed in all thymocytes and peripheral T lymphocytes of these transgenic mice and is endowed with an enhanced DPPIV activity. CD26 transgene expression induces major phenotypic changes to T-cell populations within the thymus and in peripheral blood. After the onset of sexual maturity, huCD26 expression induces an age-related overreduction of thymus cellularity accompanied by a relative impairment of thymocyte proliferation following lectin stimulation. Also the peripheral blood T-cell pool is reduced in huCD26 transgenic mice and this is accompanied by an increase of the apoptotic rate of CD4+ and CD8+ subpopulations. Taken together these data suggest that CD26 interferes with transduction pathway(s) needed for the maturation of T cells and plays an important role in T lymphocyte homeostasis in peripheral blood.