The killer cell lectin-like receptor B1 (KLRB1) 503T>C polymorphism (rs1135816) and acute rejection after liver transplantation.

The killer cell lectin-like receptor B1 (KLRB1) gene encodes for CD161 expressed by different subsets of leukocytes involved in the development of acute liver transplant rejection. The single nucleotide polymorphism (SNP) 503T>C (rs1135816) in the KLRB1 gene represents a missense mutation modifying functional properties of CD161. The aim of our study is to determine whether the SNP 503T>C is associated with acute liver transplant rejection. We genotyped the SNP for 163 liver recipients without acute rejection, 125 recipients with a single acute rejection, and 53 recipients with multiple acute rejections. The genotype frequencies within the groups did not show any significant difference. Our data suggest that the SNP 503T>C has no impact on the susceptibility of acute liver transplant rejection.

Overexpression of CD45RA isoforms in carriers of the C77G mutation leads to hyporeactivity of CD4+CD25highFoxp3+ regulatory T cells.

Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Xenotransplantation of porcine islet cells as a potential option for the treatment of type 1 diabetes in the future.

Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction [immediate blood-mediated inflammatory reaction (IBMIR)], we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.

Altered CD45 isoform expression in C77G carriers influences cytokine responsiveness and adhesion properties of T cells.

The C77G polymorphism in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of C77G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, hepatitis C and human immunodeficiency virus (HIV)-1. C77G individuals overexpress CD45RA isoforms on activated/memory T cells. We have shown previously that aberrant expression of CD45RA isoforms enhances the intensity of T cell receptor (TCR) signalling. Here we report that the C77G polymorphism also influences the responsiveness of T cells to cytokines and alters their adhesion properties. When stimulated by interleukin (IL)-2, C77G T cells proliferated more strongly than wild-type controls and showed accelerated phosphorylation of Janus kinase (Jak1). Furthermore, C77G T cells exhibited a higher tendency to form homotypic aggregates in culture which could be enhanced significantly by antibody-mediated triggering of the variant CD45RA molecules. These data indicate that the changes in CD45 isoform combination resulting from C77G may not only affect TCR signalling but also cytokine-driven T cell responses and cellular adhesion. Altered immune responsiveness may enhance susceptibility of C77G carriers for certain diseases.

Unusual case presentations associated with the CD45 C77G polymorphism.

CD45, the leucocyte common antigen, is a haematopoietic cell specific tyrosine phosphatase. Human polymorphic CD45 variants are associated with autoimmune and infectious diseases and alter the phenotype and function of lymphocytes, establishing CD45 as an important regulator of immune function. Here we report four patients with diverse diseases with unusual clinical features. All four have the C77G polymorphism of CD45 exon 4, which alters the splicing and CD45RA/CD45R0 phenotype of lymphocytes. We suggest that C77G may be a contributing factor in these unusual cases.

Enhanced frequency of a PTPRC (CD45) exon A mutation (77C-->G) in systemic sclerosis.

A point mutation in exon A (C to G transversion at position 77) of human PTPRC (CD45) has recently been associated with the development of multiple sclerosis (MS) for at least a subgroup of patients. In the present report, we studied the frequency of the 77C-->G transversion in two other autoimmune diseases namely systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). The mutation was found with significantly enhanced frequency in patients suffering from SSc suggesting that PTPRC could play a role as susceptibility gene not only in MS but also in other autoimmune diseases. Further understanding of the mode of interaction of mutant PTPRC with other susceptibility genes may uncover mechanisms common in various autoimmune disorders.

Successful outcome of acute graft-versus-host disease in a liver allograft recipient by withdrawal of immunosuppression.

BACKGROUND: Graft-versus-host disease (GVHD) after liver transplantation is uncommon, and the outcome is almost always fatal. Since 1987, about 30 cases have been described, and patient survival is mostly exceptional. METHODS: A 29-year-old man underwent retransplantation due to chronic cholestatic syndrome, 5 years after his first liver transplantation. Indication for the first liver transplantation was acute liver failure caused by exsiccosis. After the second transplantation, the patient had an initially uneventful course, developing thrombocytopenia at day 21 followed by skin rash and septic complications. Diagnosis of acute GVHD was made by using serological techniques for HLA-A and HLA-DRB and subsequently by fluorogenic sequence-specific primed polymerase chain reaction. In addition, donor lymphocytes were marked by immunohistochemical methods via biopsies of the skin. Immunosuppressive therapy was withdrawn to allow the patient's own immune system to eliminate donor cells. RESULTS: By withdrawing the immunosuppressive therapy, clinical and morphological signs of GVHD vanished. The patient is doing well without recurrence 13 months after transplantation. CONCLUSION: Withdrawal of immunosuppressive therapy is a promising approach in the treatment of acute GVHD to allow the patient's immune system to reconstitute itself, reject offending lymphocytes, and avoid lethal septic complications.

Down-regulation of human alloimmune responses by genetically engineered expression of CD95 ligand on stimulatory and target cells.

Transgenic expression of apoptosis-inducing molecules could be a strategy to protect cells and tissues from destruction by apoptosis-susceptible effector T cells. Some evidence for the potency of this approach has been obtained in mouse and rat transplantation models. However, limited data are available on the capacity of apoptosis-inducing molecules to modulate human alloimmune responses. In this study we analyzed the in vitro consequences of an interaction of human T cells with allogeneic 293 cells and 293 transfectants stably expressing high levels of the apoptosis-inducing CD95 ligand (CD95L). Both, CD95L(-) and CD95L(+) 293 cells were able to activate allogeneic T cells as demonstrated by comparable CD25 expression at day 2 of culture. The analysis of viable T cells at day 7, however, revealed anti-293 cytotoxic activity only in cultures that had been stimulated with CD95L(-) 293 cells. Alloactivated effector T cells lysed CD95L(-) and CD95L(+) 293 targets with similar efficiency when tested in a 4-h 51Cr-release assay. Prolongation of the effector phase to 20 h resulted in a further increase in the destruction of CD95L(-) target cells, whereas lysis of CD95L(+) targets remained low. These data suggest that genetically engineered expression of CD95L on cells or tissues could be an approach to control human T cell reactivity towards allografts. During the induction of an alloimmune response depletion of cytotoxic precursor cells may be obtained by overexpressing CD95L on stimulatory cells; CD95L expression on graft tissue might limit T cell-mediated destruction of the transplant during the effector phase of the response.

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

A point mutation in PTPRC is associated with the development of multiple sclerosis.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is widely accepted that a dysregulated immune response against brain resident antigens is central to its yet unknown pathogenesis. Although there is evidence that the development of MS has a genetic component, specific genetic factors are largely unknown. Here we investigated the role of a point mutation in the gene (PTPRC) encoding protein-tyrosine phosphatase, receptor-type C (also known as CD45) in the heterozygous state in the development of MS. The nucleotide transition in exon 4 of the gene locus interferes with mRNA splicing and results in altered expression of CD45 isoforms on immune cells. In three of four independent case-control studies, we demonstrated an association of the mutation with MS. We found the PTPRC mutation to be linked to and associated with the disease in three MS nuclear families. In one additional family, we found the same variant CD45 phenotype, with an as-yet-unknown origin, among the members affected with MS. Our findings suggest an association of the mutation in PTPRC with the development of MS in some families.

A CD28-associated signaling pathway leading to cytokine gene transcription and T cell proliferation without TCR engagement.

Stimulation of resting human T cells with the CD28-specific mAb BW 828 induces proliferation and cytokine synthesis without further requirement for TCR coengagement. This observation prompted us to postulate that signal 2 (costimulatory signal) alone without signal 1 (TCR signal) can activate T cells. To test whether this putative function of CD28 is mediated via a particular signaling pathway, we compared early signaling events initiated in resting T cells by the stimulatory mAb BW 828 with signals triggered by the nonstimulating CD28 mAb 9.3. Stimulation of T cells with BW 828 induced an increase in intracellular Ca2+, but did not lead to detectable activation of the protein kinases p56(lck) and c-Raf-1. This pathway resulted in the induction of the transcription factors NF-kappa B, NF-AT, and proteins binding to the CD28 response element of the IL-2 promoter. On the other hand, stimulation of T cells with mAb 9.3 increased the level of intracellular Ca2+ and triggered the activation of p56(lck) and c-Raf-1, but was unable to induce the binding of transcription factors to the IL-2 promoter. In contrast to the differential signaling of BW 828 and 9.3 in resting T cells, the two mAbs exhibited a similar pattern of early signaling events in activated T cells and Jurkat cells (p56(lck) activation, association of phosphatidylinositol 3-kinase with CD28), indicating that the signaling capacity of CD28 changes with activation. These data support the view that stimulation through CD28 can induce some effector functions in T cells and suggest that this capacity is associated with a particular pattern of early signaling events.

Different protein kinase C isoenzymes regulate IL-2 receptor expression or IL-2 synthesis in human lymphocytes stimulated via the TCR.

Stimulation of purified human PBL with mAbs raised against the T cell receptor resulted in an immediate and transient activation of protein kinase C-alpha (PKC-alpha) and PKC-theta, peaking at 10 min, whereas PKC-beta, -delta, and -epsilon were translocated with a delay of >90 min and remained activated for up to 2 h. To characterize specific functions of distinct PKC isoenzymes, Abs against different PKC isoenzymes were introduced by means of electropermeabilization. Neutralization of PKC-alpha and -theta resulted in the complete inhibition of IL-2R expression, whereas anti-PKC-beta, -delta, and -epsilon Abs inhibited IL-2 synthesis. Extensive control experiments have shown that neither electropermeabilization nor control Ig influenced PKC activity and cellular functions. Our data thus clearly show that specific PKC isoenzymes regulate different cellular functions in stimulated human lymphocytes.

[The CD95/CD95L system as target for modulation of allogeneic immune responses]. / Das CD95/CD95L System als Zielstruktur für die Modulation von allogenen Immunantworten.

The effects of COS7 cells which constitutively express the apoptosis-inducing human CD95 ligand (COSh95L cells) on in vitro alloimmune responses were investigated. In the presence of COSh95L cells a clear-cut inhibition of alloantigen-induced proliferation was observed. In these cultures fully activated blastoid T cells were absent suggesting that deletion of activated T cells is one mechanism of COSh95L-mediated inhibition of proliferation. Non-fully activated cells were not eliminated in MLR by COSh95L cells but achieved a hyporesponsive state as shown by their failure to proliferate in response to restimulation with alloantigen or mitogen. These data indicate that the effects of CD95L on T cells are heterogeneous. Further analysis of the actions of CD95L in T cells of different activation states and subsets will be crucial to estimate the therapeutical potential of this molecule as target for immunomodulation after allo- and xenotransplantation.

Phenotypic features of selective T cell deficiency characterized by absence of CD8+ T lymphocytes and undetectable mRNA for ZAP-70 kinase.

Selective T cell deficiency is a rare immune deficiency characterized by the absence of CD8+ T lymphocytes and depressed/absent T cell function. This syndrome has been associated with mutations in the gene for ZAP-70, a tyrosine kinase that has profound effects on signaling via the T cell receptor. In this paper we describe a patient with selective T cell deficiency and certain phenotypic features that are unique among the small number of patients described. The patient had virtually absent T cell function, hypogammaglobulinemia, and no response to vaccination. The T lymphocytes failed to respond to mitogenic stimuli, even in the presence of exogenous interleukin 2. Similar to other patients with this disorder, the T cells were capable of proliferating when stimulated by pharmacologic agents such as phorbol ester and ionomycin. While peripheral blood T cells had limited capability to increase cytosolic Ca2+ levels in response to mitogenic stimulation, thymocytes responded to a large panel of antibodies and mitogens. This report broadens the spectrum of clinical presentations associated with selective T cell deficiency and, for the first time, compares the responses of both peripheral T cells and thymocytes. The data support the concept that the defect in signal transduction resulting from the absence of ZAP-70 is primarily manifested following export of T lymphocytes from the thymus and that selection of CDS-positive T cells is dependent on the presence of ZAP-70.

CD28-mediated activation of resting human T cells without costimulation of the CD3/TCR complex.

Stimulation of resting human T cells by crosslinked CD28 monoclonal antibodies (mAb) induces some early signaling events but does not lead to IL-2 secretion and proliferation. The induction of these functions usually requires the delivery of additional signals such as that provided by costimulation of the T cell receptor (TCR). We analyzed the capacity of a panel of different CD28 mAb to induce cellular functions in purified human T cells. Two patterns of reactivity were observed. "Costimulatory" CD28 mAb like 9.3 required coengagement of the CD3/TCR complex for the induction of IL-2 gene transcription and proliferation. On the other hand, a "stimulatory" pathway could be defined by the use of the CD28 mAb BW 828, which triggered IL-2 synthesis, IL-2R expression, and proliferation without further requirement for additional stimuli. BW 828-induced proliferation was sensitive to inhibition by cyclosporin A and was mainly found in the CD4+CD45R0+ ("memory") T cell subset. These data suggest that T cell stimulation with mAb BW 828 defines a CD28-associated signaling pathway which leads to the induction of effector functions without the need for CD3/TCR coengagement. This pathway might play a role in antigen-independent activation and expansion of T cells.