Cooperation between human DAF and CD59 in protecting cells from human complement-mediated lysis

The complement (C) regulatory proteins decay accelerating factor (DAF, CD55) and CD59 could protect host cells using different mechanisms from C-mediated damage at two distinct levels within the C pathway. Co-expression of DAF and CD59 would be an effective strategy to help overcome host C-induced xenograft hyperacute rejection. In this study, we made a construct of recombinant expression vector containing DAF and CD59 cDNA and the stable cell lines were obtained by G418 selection. Extraneous genes integration and co-expression were identified by PCR, RT-PCR and Western blot analysis. Human c-mediated cytolysis assays showed that NIH/3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-DAF, and pcDNA3-CD59DAF-DP were protected from Cmediated damage and that synchronously expressed human CD59 and DAF provided the most excellent protection for host cells as compared with either human CD59 or DAF expressed alone. Therefore, the construct represents an effective and efficacy strategy to overcome C-mediated damage in cells and, ultimately, in animals.     

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF,CD55) and membrane cofactor protein (MCP,CD46) on a human colonic adenocarcinoma cell line

 To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab′)₂ fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β). Cells incubated with interferon γ (IFNγ) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1β, but not by TNFα or IFNγ. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape. Received: 18 July 1995 / Accepted: 4 January 1996     

Complement-mediated lysis of cultured osteosarcoma cell lines using chimeric mouse/human TP-1 IgG1 and IgG3 antibodies

Osteosarcoma is the commonest malignant tumour of the bones. The presence of micrometastases at the time of primary diagnosis is associated with poor prognosis. Despite developments in surgery and aggressive chemotherapy, about 50% of the patients still succumb to the disease. Thus, there is a need to develop alternative treatment modalities. One such strategy is to use antibodies with improved effector functions. The two monoclonal antibodies, TP-1 and TP-3, recognize a tumour-associated antigen on human osteosarcoma cells. In the present study, we describe the cloning of the TP-1 variable genes, and the production of complete chimeric mouse/human monoclonal antibodies. Constructs containing the constant genes from human IgG1, IgG3 or a mutant IgG3 with a shortened hinge region, called m15, were expressed in the mouse myeloma cell line, NS0. The m15 mutant has been shown to be very potent in triggering complement-mediated lysis. Our goal was to investigate whether this mutant could overcome the complement protection on human osteosarcoma cells, which is generally present on all human cells. We found that the target cells expressed several membrane-bound complement inhibitors, and that masking of these inhibitors rendered the cells sensitive to lysis. The m15 mutant exhibited greater lytic activity than both IgG3 and IgG1, although it could not cause extensive killing of the target cells alone. Received: 21 January 1999 / Accepted: 3 June 1999     

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.     

Up-regulation of fibrinogen-like protein 2 in porcine endothelial cells by xenogeneic CD40 signal

Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.     

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

To harness the potent tumor-killing capacity of T cells for the treatment of CD19⁺ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19⁺ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19⁺ malignancies with an advantageous safety risk profile and anticipated dosing regimen.     

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

To harness the potent tumor-killing capacity of T cells for the treatment of CD19⁺ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19⁺ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19⁺ malignancies with an advantageous safety risk profile and anticipated dosing regimen.     

Expression of CD55 and CD59 on peripheral blood cells from systemic lupus erythematosus (SLE) patients

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p = 0.005 and p = 0.019, respectively), and CD59-granulocytes (p = 0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.     

Red blood cells promote survival and cell cycle progression of human peripheral blood T cells independently of CD58/LFA-3 and heme compounds

Red blood cells (RBC) are known to modulate T cell proliferation and function possibly through downregulation of oxidative stress. By examining parameters of activation, division, and cell death in vitro, we show evidence that the increase in survival afforded by RBC is due to the maintenance of the proliferative capacity of the activated T cells. We also show that the CD3+CD8+ T cell subset was preferentially expanded and rescued from apoptosis both in bulk peripheral blood lymphocyte cultures and with highly purified CD8+ T cells. The ability of RBC to induce survival of dividing T cells was not affected by blocking the CD58/CD2 interaction. Moreover, addition of hemoglobin, heme or protoporphyrin IX to cultures of activated T cells did not reproduce the effect of intact RBC. Considering that RBC circulate throughout the body, they could play a biological role in the modulation of T cell differentiation and survival in places of active cell division. Neither CD58 nor the heme compounds studied seem to play a direct relevant role in the modulation of T cell survival.     

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3x10(-7) M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.     

Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells

BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.     

Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid-rafts and its internalization in human endometrial cells

Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the GM1-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.     

Knockdown of CD146 reduces the migration and proliferation of human endothelial cells

Our previous study has demonstrated that CD 146 molecule is a biomarker on vascular endothelium,which is involvedin angiogenesis and tumor growth.However the mechanism behind is not clear.Here we have for the first time devel-oped a novel CD146 blockade system using CD146 siRNA to study its function on endothelial cells.Our data showedthat CD146 siRNA specifically blocked the expression of CD146 on both mRNA and protein levels,leading to thesignificant suppression of HUVEC proliferation,adhesion and migration.These results demonstrate that CD146 playsa key role in vascular endothelial cell activity and angiogenesis,and CD146 siRNA can be used as a new inhibitor foranti-angiogenesis therapy.     

Syndecan-2 expression increases serum-withdrawal-induced apoptosis, mediated by re-distribution of Fas into lipid rafts, in stably transfected Swiss 3T3 cells

To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycans in fibroblasts, we obtained stably transfected Swiss 3T3 clones. We examined the effects of stable syndecan-2 overexpression on programmed cell death, finding that syndecan-2 transfected cells were more sensitive to apoptosis induced by serum-withdrawal than control cells. In addition, overexpression of syndecan-2 correlates with increased membrane levels of the Fas/CD95 receptor, suggesting that the increased serum-withdrawal apoptosis observed in Swiss 3T3 cells might be Fas receptor-dependent. Differences in Fas membrane levels between both control and syndecan-2 transfected cells result from a redistribution of the Fas receptor. Our data clearly demonstrate that increased Fas levels are primarily related to lipid rafts and that this increase is a key factor in Fas/CD95-mediated apoptosis. Moreover, disruption of lipid rafts with methyl-beta-cyclodextrin or filipin significantly reduced apoptosis in response to serum withdrawal. The differences in Fas/CD95 membrane distribution could explain why syndecan-2 transfected cells have a higher susceptibility to serum-withdrawal-induced apoptosis.     

Pre-sorting endosomal transport of the GPI-anchored protein, CD59, is regulated by EHD1

EHD1 regulates the trafficking of multiple receptors from the endocytic recycling compartment (ERC) to the plasma membrane. However, the potential role of EHD1 in regulating the family of glycosylphosphatidylinositol-anchored proteins (GPI-APs) has not been determined. Here we demonstrate a novel role for EHD1 in regulating the trafficking of CD59, an endogenous GPI-AP, at early stages of trafficking through the endocytic pathway. EHD1 displays significant colocalization with newly internalized CD59. Upon EHD1 depletion, there is a rapid Rab5-independent coalescence of CD59 in the ERC region. However, expression of an active Arf6 mutant (Q67L), which traps internalized pre-sorting endosomal cargo in phosphatidylinositol(4,5)-bisphosphate enriched vacuoles, prevents this coalescence. It is of interest that sustained PKC activation leads to a similar coalescence of CD59 at the ERC, and treatment of EHD1-depleted cells with a PKC inhibitor (Go6976) blocked this rapid relocation of CD59. However, unlike sustained PKC activation, EHD1 depletion does not induce the translocation of PKCα to ERC. The results presented herein provide evidence that EHD1 is involved in the control of CD59 transport from pre-sorting endosomes to the ERC in a PKC-dependent manner. However, the mechanisms of EHD1-induced coalescence of CD59 at the ERC differ from those induced by sustained PKC activation.     

Cloning,sequencing and expression of porcine CD40 ligand in Escherichia coli and human and porcine cells

The CD40L ligand (CD40L) plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. The porcine CD40L encoding gene was isolated from porcine peripheral blood mononuclear cells (PBMC) using RT-PCR. Sequence analysis of the cloned CD40L gene showed an open reading frame of 786 base pairs encoding a 262 amino acid protein with a predicted molecular mass of 29 kD. The deduced amino acid sequence of the porcine CD40L shared 82%, 88% and 93% similarity with the CD40L protein of mouse, human and cattle. The isolated CD40L sequence was expressed as a hexahistidine fusion protein in Escherichia coli and purified by affinity chromatography. The analysis of the CD40L-expression in human 293 and porcine MAX cells by immunofluorescence showed its location on the cell surface.     

Rapid Degradation of the Complement Regulator,CD59, by a Novel Inhibitor

There is increased interest in immune-based monoclonal antibody therapies for different malignancies because of their potential specificity and limited toxicity. The activity of some therapeutic monoclonal antibodies is partially dependent on complement-dependent cytolysis (CDC), in which the immune system surveys for invading pathogens, infected cells, and malignant cells and facilitates their destruction. CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that protects cells from CDC. However, in certain tumors, CD59 expression is enhanced, posing a significant obstacle for treatment, by hindering effective monoclonal antibody-induced CDC. In this study, we used non-small lung carcinoma cells to characterize the mechanism of a novel CD59 inhibitor: the 114-amino acid recombinant form of the 4th domain of intermedilysin (rILYd4), a pore forming toxin secreted by Streptococcus intermedius. We compared the rates of internalization of CD59 in the presence of rILYd4 or anti-CD59 antibodies and determined that rILYd4 induces more rapid CD59 uptake at early time points. Most significantly, upon binding to rILYd4, CD59 is internalized and undergoes massive degradation in lysosomes within minutes. The remaining rILYd4·CD59 complexes recycle to the PM and are shed from the cell. In comparison, upon internalization of CD59 via anti-CD59 antibody binding, the antibody·CD59 complex is recycled via early and recycling endosomes, mostly avoiding degradation. Our study supports a novel role for rILYd4 in promoting internalization and rapid degradation of the complement inhibitor CD59, and highlights the potential for improving CDC-based immunotherapy.