[Show abstract][Hide abstract]ABSTRACT: Clinical trials have pointed out the promising role of co-stimulation blocker Belatacept for improvement of graft function and avoidance of undesired side-effects associated with calcineurin-inhibitors (CNI). However, due to the worldwide limited availability of appropriate patients, almost no data exist to assess the effects of sustained application of this immunomodulator on the recipient's immune system. The aim of this study was to reveal specific alterations in the composition of immunologic subpopulations potentially involved in development of tolerance or chronic graft rejection following long-term Belatacept therapy. For this, peripheral lymphocyte subsets of kidney recipients treated with Belatacept (n=5; average 7.8years) were determined by flow-cytometry and compared with cells from matched patients on CNI (n=9) and healthy controls (n=10). T cells capable of producing IL-17 and serum levels of soluble CD30 were quantified. Patients on CNI showed a higher frequency of CD4(+) CD161(+) Th(17) -precursors and IL-17-producing CD4(+) T cells than Belatacept patients and controls. Significantly higher serum levels of soluble CD30 were observed in CNI patients, indicating a possible involvement of the CD30/CD30L-system in Th(17) -differentiation. No differences were found concerning CD4(+) CD25(+) CD127(low) FoxP3(+) regulatory T cells. In conclusion, patients on therapy with Belatacept did not show a comparable Th(17) -profile to that seen in individuals with chronic intake of CNI. The distinct effects of Belatacept on Th(17) -immunity might prove beneficial for the long-term outcome following kidney transplantation.
Full-text available · Article · Feb 2012 · Transplant International
[Show abstract][Hide abstract]ABSTRACT: Efficient and precise techniques for the genetic modification of pigs facilitate the generation of tailored donor animals for xenotransplantation. Numerous transgenic pig lines exist with the focus on inhibition of the complement system and of humoral immune responses. In addition, immune cell-based responses need to be controlled to prevent pig-to-primate xenograft rejection. Expression of human (hu) TNF-related apoptosis-inducing ligand (TRAIL) on porcine cells has the potential to ameliorate human T cell responses.
We generated transgenic pigs expressing human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (huTRAIL) under the control of either the mouse H2K(b) promoter or a CMV enhancer/chicken β-actin (CAG) promoter, the latter one (CAG-huTRAIL) on a GGTA1 knockout/huCD46 transgenic background. The biological activity of huTRAIL was demonstrated by its apoptosis-inducing effect on Jurkat lymphoma cells. To clarify whether huTRAIL affects also primary immune cells and whether its effects depend on the presence of co-stimulatory molecules, we exposed human peripheral blood mononuclear cells (PBMC) or isolated T cells to huTRAIL-expressing porcine fibroblasts or dendritic cells in vitro.
H2Kb-huTRAIL transgenic pigs express huTRAIL mainly in the spleen and secondary lymphoid tissues. The CAG-huTRAIL construct facilitated huTRAIL expression in multiple organs, the level being at least one order of magnitude higher than in H2Kb-huTRAIL transgenic pigs. Incubation with huTRAIL-expressing H2Kb-huTRAIL transgenic porcine dendritic cells decreased human T cell proliferation significantly without any signs of apoptosis. In spite of the high transgene expression level, CAG-huTRAIL transgenic fibroblasts did not affect proliferation of human PBMC, independent of their activation state.
These results suggest huTRAIL expression on porcine dendritic cells as a possible strategy to attenuate T cell responses against pig-to-primate xenografts.
[Show abstract][Hide abstract]ABSTRACT: The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-κB ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.
Full-text available · Article · Dec 2011 · The FASEB Journal
[Show abstract][Hide abstract]ABSTRACT: Specificity of the rabbit antiserum raised against the predicted extracellular domain of pUL11. Immunoblot with the rabbit antiserum raised against the fusion protein consisting of the predicted extracellular domain of pUL11 and the human IgG Fc domain (c.f. Figure S2). The rabbit serum was pre-absorbed to rAdV GFP transduced cells to reduce non-specific interactions. Lysates of A549 cells transduced with rAdV UL11 (UL11), rAdV GFP (GFP), or left uninfected (U) were used to prepare immunoblots and proteins were detected using pUL11 anti-serum or for comparison using an antibody specific for the V5 epitope. Bands corresponding to the major 50 kDa form (arrow) and to high molecular weight forms of pUL11 (bracket) are indicated.
[Show abstract][Hide abstract]ABSTRACT: Generation of the pUL11 and pUL6 Fc fusion proteins. (A) Cartoons of the domains of the Fc fusion proteins. (B, C) The UL11Fc and UL6Fc proteins harvested from the supernatants of transduced or transfected 293T cells and purified using protein A sepharose were separated by SDS-PAGE and detected by Coomassie blue staining or by immunoblotting using HRP conjugated anti-human IgG. (D) The UL11Fc or the control Fc protein were treated with PNGase F and detected after immunoblotting as in (C).
[Show abstract][Hide abstract]ABSTRACT: Blocking effects of CD45 antibodies on the interaction of pUL11 with Jurkat cells. Jurkat cells were left untreated (top panel) or incubated with 10 µg of the indicated CD45 antibodies for 30 min, prior to incubation with UL11Fc (black lines) or the Fc control protein (grey lines).
[Show abstract][Hide abstract]ABSTRACT: A knockout (KO) of the porcine α1,3-galactosyltransferase (GGTA1) gene is crucial for controlling the hyperacute rejection after pig-to-human xenotransplantation. Porcine kidney and cardiac xenografts from Gal-KO pigs showed prolonged survival after transplantation into baboons. Unfortunately, knockouts produced by conventional targeting (homologous recombination) are rare events and normally do not lead to biallelic KO. Zinc-finger nucleases (ZFN) have been shown to be much more efficient by inducing mutations via specific cleavage followed by nonhomologous end joining (NHEJ). Zinc-finger nucleases do not require antibiotic selection. Here, we used designed ZFN to specifically target exon 9 of the GGTA1 gene encoding the catalytic domain of the Gal-transferase. Recently, we generated female pigs with a GGTA1-KO using ZFN (Hauschild et al. 2011 PNAS 108, 12013-12017). Here, we investigated whether cells of a male cell line are susceptible to ZFN-mediated genome editing in a comparable manner. Male porcine fetal fibroblasts (3×10(6)) were co-transfected with a ZFN-plasmid pair (7.5μg each) by electroporation at 250V and 400μF. One week after transfection, a Cel-I assay revealed a NHEJ rate of 5.7% of all alleles in the cell population. After magnetic bead selection, Gal-expression was analysed by fluorescence-activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. Ninety-five percent of the cells were free of Gal epitopes, indicating a biallelic KO. These Gal-negative cells served as donor cells in somatic cell nuclear transfer (SCNT). In total, 507 transgenic embryos were transferred into 6 recipient sows. By obtaining live animals by SCNT after transfer of male ZFN-GGTA1-KO embryos, we will have produced female and male ZFN-KO pigs, which can be used for further breeding experiments to circumvent the extensive and relative inefficient recloning method. These results show that ZFN work independent of the sex of the cells and that a biallelic Gal-KO can be produced in male cells by using the ZFN technology. This technology could benefit both agriculture and biomedicine and establishes the pig as a model for human diseases.
Article · Dec 2011 · Reproduction Fertility and Development
[Show abstract][Hide abstract]ABSTRACT: Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV.
Full-text available · Article · Dec 2011 · PLoS Pathogens
[Show abstract][Hide abstract]ABSTRACT: The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay.
Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC.
Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically.
These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation.
Full-text available · Article · Nov 2011 · Xenotransplantation
[Show abstract][Hide abstract]ABSTRACT: Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.
Full-text available · Article · Jul 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: Genetic modification of pigs (e.g. transgenic expression of human complement regulatory molecules or inactivation of α1,3galactosyltransferase) enabled the development of promising strategies to overcome hyperacute rejection after pig-to-primate xenotransplantation. However, cellular rejection still remains a hurdle for successful xenograft survival. Cellular rejection of porcine cells in xenotransplantation models is mediated by macrophages, T cells and NK cells. Activation of human monocytes by pig cells is partly due to the incapacity of porcine ligands to bind the inhibitory receptor SIRPα (signal regulatory protein α). Thus, one approach to impair the ability of human macrophages to phagocyte porcine cells is the overexpression of the human ligand for SIRPα in porcine cells. To inhibit human NK cell reactivity after xenotranslantation transgenic expression of HLA-E in pigs has been shown to be a promising concept. Cells from these pigs were partially protected from lysis by human NK cells. Our group focuses on manipulation of human anti-pig T cell responses by negative costimulatory signals. Thus, we asked whether overexpression of PD-L1 on porcine cells can (i) downregulate human anti-pig cellular responses in vitro, and (ii) inhibit rat anti-pig cellular immune responses in vivo. Pig cells overexpressing PD-L1 triggered reduced proliferation and low amounts of IL-2, IFNγ, TNF-alpha, IL-4, and IL-5 in human CD4+ T cells compared to control pig cells. The concentration of IL-10, however, was increased. In long-term cultures of human CD4+ T cells and PD-L1 transfectants a high frequency of CD4+ CD25high FoxP3+ cells showed up which had the capacity to suppress the activation of conventional CD4+ T cells. Cytotoxic CD8+ T cells and NK cells lysed pig control cells very efficiently. In contrast, PD-L1 transfected pig cells were partially protected from lysis by human effector cells. Overexpression of PD-L1 on porcine cells was not sufficient to prevent rejection after transplantation under the rat kidney capsule. However, in rats that had been grafted with PD-L1 expressing cells we observed reduced cellular infiltrates in the kidneys and lower antibody responses compared to rats grafted with control cells. Together these observations support the assumption that PD-1/PD-Ligand pathways are interesting targets to prevent cellular immune responses after xenotransplantation. PD-L overexpression might not only impede the initiation of an anti-pig T cell response by suppressing CD4+ T cells but may also protect pig cells from destruction by cytotoxic effectors.Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe “Xenotransplantation”, FOR 535).
[Show abstract][Hide abstract]ABSTRACT: Cellular rejection is a relevant hurdle for successful pig-to-primate xenotransplantation. We have shown previously that the induction of a human anti-pig T cell response (in vitro activation of CD4(+) T cells) can be suppressed by the overexpression of human negative costimulatory ligands (e.g. programmed death receptor ligand, PD-L1) on pig antigen presenting cells. Here, we asked whether PD-L1 mediated enhancement of negative signaling might also be efficient during the effector phase of human anti-pig cellular immune responses. The porcine B-cell line L23 was transfected with human PD-L1, and clones were selected stably expressing PD-L1 with low, medium, or high density. Mock-transfected L23 cells were effectively lysed by human cytotoxic effector cells (IL-2 activated CD8(+) T cells and CD56(+) cells). The lytic potential of the effectors decreased with increasing levels of PD-L1 and was reduced by about 50% in L23-PD-L(high) targets. A proportion of activated CD8(+) effector cells underwent apoptosis when exposed to PD-L1 expressing L23 cells. These data suggest that the overexpression of PD-L1 on target cells may (a) trigger negative signals in effector cells that prevent the release of cytolytic molecules and/or (b) induce apoptosis in the attacking effector cells thereby protecting targets from destruction.