Complement-mediated Cytolysis Research Articles

Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.

The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specificorgan transplantation. In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.

Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model.

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.

Author response: Rare side effects of alemtuzumab remind us of the need for postmarketing surveillance.

We thank Drs. Whiteside and Trip for their comment on our editorial.1 The authors highlight noninfectious pneumonitis as a rare potential complication of alemtuzumab.2,3 The time course of onset in the 2 reported cases was different, with symptoms developing a few days or 1 month after infusion. Although not proven, this suggestion of immune-mediated mechanisms seem reasonable.2 This reaction may arise from antibody-dependent cell-mediated or complement-mediated cytolysis, which leads to cytokine release and then type III/IV hypersensitivity reaction and lung-resident T-cell activation. As for the side effects highlighted in our editorial,1 clinicians should recognize noninfectious pneumonitis as a potential rare complication of alemtuzumab, as it can respond well to corticosteroids.

Generation of GTKO Diannan Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT.

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assaysusing human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

Molecular mechanism of inhibitory effects of C-phycocyanin combined with all-trans-retinoic acid on the growth of HeLa cells in vitro

We studied the effects of all-trans-retinoic acid (ATRA), C-phycocyanin (C-PC), or ATRA+C-PC on the growth of cervical cells (HeLa cells), cell cycle distribution, and apoptosis. The anticancer mechanism of the drug combination was revealed. MTT assay was adopted to determine the effects of C-PC and ATRA on the growth of HeLa cells. The expression quantities of cyclin-dependent kinase (CDK) 4, cyclin D1, Bcl-2, caspase-3, and CD59 were determined by in situ hybridization, immunofluorescence, immunohistochemistry staining, Western blot, and RT-PCR. TUNEL assay was adopted to determine the cellular apoptosis levels. Both C-PC and ATRA could inhibit the growth of HeLa cells, and the combination of ATRA+C-PC functioned cooperatively to induce apoptosis in HeLa cells. The dosage of ATRA was reduced when it cooperated with C-PC to reduce the toxicity. ATRA treated with C-PC could induce more cell cycle arrests than the single drug used by decrease in cyclin D1 and CDK4 expression. The combination of the two drugs could upregulate caspase-3 and downregulate the Bcl-2 gene and induce cell apoptosis. Moreover, the combination therapy has an important immunological significance in decreased expression of the CD59 protein. Singly, C-PC or ATRA could inhibit the growth of HeLa cells, and the effects of treatment were further enhanced in the combination group. In combination with C-PC, the dosage of ATRA was effectively reduced. The C-PC + ATRA combination might take effect by inhibiting the progress of the cell cycle, inducing cell apoptosis and promoting complement-mediated cytolysis.

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.

Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum)

Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.

α1,3Galactosyltransferase knockout pigs produce the natural anti‐Gal antibody and simulate the evolutionary appearance of this antibody in primates

Anti-Gal is the most abundant natural antibody in humans and Old World primates (apes and Old World monkeys). Its ligand, the α-gal epitope (Galα1-3Galβ1-4GlcNAc-R), is abundant in nonprimate mammals, prosimians and New World monkeys whereas it is absent in humans and Old World primates as a result of inactivation of the α1,3galactosyltransferase (α1,3GT) gene in ancestral Old World primates, as recent as 20-28 million years ago. Since anti-Gal has been a "forbidden" autoantibody for >140 million years of evolution in mammals producing α-gal epitopes it was of interest to determine whether ancestral Old World primates could produce anti-Gal once α-gal epitopes were eliminated, i.e. did they carry anti-Gal encoding immunoglobulin genes, or did evolutionary selection eliminate these genes that may be detrimental in mammals synthesizing α-gal epitopes. This question was studied by evaluating anti-Gal prodution in α1,3GT knockout (GT-KO) pigs recently generated from wild-type pigs in which the α-gal epitope is a major self-antigen. Anti-Gal antibody activity in pig sera was assessed by ELISA, flow cytometry and complement mediated cytolysis and compared to that in human sera. The study demonstrates abundant production of the natural anti-Gal antibody in GT-KO pigs at titers even higher than in humans. The fine specificity of GT-KO pig anti-Gal is identical to that of human anti-Gal. Pigs and probably other mammals producing α-gal epitopes carry immunoglobulin genes encoding anti-Gal as an autoantibody. Once the α-gal epitope is eliminated in GT-KO pigs, they produce anti-Gal. These findings strongly suggest that similar to GT-KO pigs, inactivation of the α1,3GT gene in ancestral Old World primates enabled the immediate production of anti-Gal, possibly as a protective antibody against detrimental microbial agents carrying α-gal epitopes.

Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Molecular characterization and expression analysis of complement component C9 gene in the whitespotted bambooshark, Chiloscyllium plagiosum

Complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions. Activation of the terminal pathway of the complement system leads to the assembly of terminal complement complexes (C5b-9), which induces the characteristic complement-mediated cytolysis. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) is identified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum by RACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42% and 43% identity with human and Xenopus C9 respectively. Through sequence alignment and comparative analysis, the CpC9 protein was found well conserved, with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogenetic analysis places it in a clade with C9 orthologs in higher vertebrate and as a sister taxa to the Xenopus. Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much less or even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response, though it remains to be confirmed by further study.

Molecular mechanism of a novel CD59-binding peptide sp22 induced tumor cells apoptosis

Some short peptides discovered by phage display are found to be able to inhibit cancer growth and induce cancer cell apoptosis. In this study, a novel cancer-targeting short peptide which was composed of 22 amino acids (ACHWPWCHGWHSACDLPMHPMC, abbreviated as sp22) and specifically bound to human CD59 was screened from a M13 phage display library so as to counteract tumor immune escape activity. The mechanism of exogenous sp22 peptide in inducing apoptosis of MCF-7 cells was investigated. The results suggested that sp22 could lower CD59 expression level, downregulate Bcl-2 expression, activate Fas and caspase-3, and finally increase apoptotic cell numbers of MCF-7 cells. However, sp22 had no obvious influence on normal human embryonic lung cells. In addition, the effects of endogenous sp22 gene on CD59 expression and NKM cell apoptosis were explored using the recombinant plasmid sp22-PIRES. It showed that sp22 gene was efficiently expressed in transfected NKM cells. Compared with normal NKM cells, NKM cells transfected with sp22 displayed reduced mRNA and protein expression levels of CD59, increased sensitivity to complement-mediated cytolysis, decreased cell survival ratio, changes of the expression of apoptosis associated proteins, increased number of apoptotic cells and the appearance of apoptotic morphology. The results suggested that sp22 protein could bind to CD59 and inhibit the expression of CD59. The cytolytic activity of complement on tumor cells strengthened and apoptosis signal was stepwise transferred which might be a potential way to kill tumor cells.

Assessment of In Vitro Heparin Complement Regulation Capacity During Real-Time Cell Analyzer Antibody-Mediated Cytolysis Assay: Compatibility Studies for Pig-to-Baboon Xenotransplantation

ObjectiveTo assess the effect of sodium heparin concentrations on antibody- and complement-mediated cytolysis by means of a real-time cell analyzer system (RTCA) investigating the complement regulation ability of heparin to reduce or prevent hyperacute in an in vitro model of pig-to-baboon xenotransplantation. Materials and methodsFibroblasts isolated from the skin of two transgenic pigs were cultured in microelectronic 96-well plates for 9 hours. Then, we added 20 μL of normal sera from two healthy adult olive baboons (Papio anubis) or two volunteer healthy humans. Simultaneous cultures had added heparin at 3.5, 5, 7.5, 15, and 30 IU. Moreover, rabbit complement was added for the exogenous complement group (ExC) versus the other group only with the complement present in the sera as an endogenous complement group (EnC). Cellular cultures were monitored over 150 hours after challenge. With cellular index (CI) data recorded by the xCELLigence software system, we calculate area under the curve versus concentration (AUC) and minimum CI (CImin) versus concentration. ResultsAll cultures showed decreased CI after challenge with human or baboon sera. There was a high correlation for AUC (r2 > 0.90) and CImin versus concentration (r2 > 0.970) during the first 40 hours postchallenge among the EnC group, regardless of human or baboon sera. However, there was no correlation for AUC and CImin for the ExC group. There was a reduction of CImin related to increased heparin concentrations. ConclusionsThe addition of heparin did not reduce antibody- and complement-mediated cytolysis assessed in vitro by RTCA in pig-to-baboon compatibility assays.

Glycoprotein G of Herpes Simplex Virus 2 as a Novel Vaccine Antigen for Immunity to Genital and Neurological Disease

The envelope glycoproteins of herpes simplex virus 1 (HSV-1) and HSV-2, with the exception of glycoprotein G, elicit cross-reactive B- and T-cell responses. Human vaccine trials, using the cross-reactive glycoproteins B and D, have shown no protection against genital HSV-2 infection or disease. In this study, the mature form of glycoprotein G (mgG-2) of HSV-2 was used for immunization of mice, either alone or in combination with adjuvant CpG, followed by an intravaginal challenge with a lethal dose of a fully virulent HSV-2 strain. Mice immunized with mgG-2 plus CpG showed low disease scores and a significantly higher survival rate (73%) than mice immunized with mgG-2 alone (20%) or controls (0%). Accordingly, limited numbers of infectious HSV-2 particles were detected in the spinal cord of mice immunized with mgG-2 plus CpG. The observed protection was associated with a gamma interferon (IFN-γ) response by splenic CD4(+) T cells upon antigen restimulation in vitro and in vaginal washes 1 day postinfection. The majority of sera collected from mice immunized with mgG-2 plus CpG showed macrophage-mediated antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis, while no neutralization activity was observed. In conclusion, we have shown that immunization with the type-specific mgG-2 protein in combination with CpG could elicit protective immunity against an otherwise lethal vaginal HSV-2 challenge. The mgG-2 protein may therefore constitute a promising HSV-2 vaccine antigen to be considered for future human trials.

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

To analyze the protective effects against complement-mediated cytolysis of the MCP, DAF, and CD59 human complement regulatory proteins, alone and in combination, on NIH 3T3 mouse fibroblast cells. We constructed 3 double and 3 single-human complement regulatory protein plasmids (pIRES-hMCP-hDAF, pIRES-hMCP-hCD59, pIRES-hDAF-hCD59, pIRES-A-hMCP, pIRES-B-hDAF, and pIRES-B-hCD59). The plasmids were transfected into NIH 3T3 cells, and stable transfectants were obtained by treatment with 200 kg/m3 G418 for 2 weeks. Normal human serum (50%) as a source of complement was added to the culture medium of stable transfectants. The 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay was used to analyze the protective ability of different human complement regulatory protein plasmids on complement-dependent cytolysis. The viability of double-human complement regulatory protein stable transfectants was significantly higher than that of single-human complement regulatory protein stable transfectants (P < .05). Among the double-transfectants, cells expressing pIRES-hMCP-hDAF and pIRES-hMCPhCD59 survived better than cells expressing pIREShDAF- hCD59 (91.75% ± 3.30% and 84.88% ± 2.36% vs 66.19% ± 6.52%; P < .05). Among the single transfectants, cells expressing pIRES-A-hMCP or pIRES-B-hDAF survived better than cells expressing pIRES-B-hCD59 or pIRES empty vector (53.76% ± 3.84% and 56.32% ± 2.83% vs 43.28% ± 0.96% and 40.27% ± 1.11%; P < .05). These results suggest that the MCP+DAF and MCP+CD59 combinations could be more effective than DAF+CD59 in protecting the NIH 3T3 cells from injury caused by complement-dependent cytolysis, whereas MCP or DAF alone is stronger than CD59 alone in inhibiting membrane attack complex formation.

Penetration of antibody‐opsonized cells by the membrane attack complex of complement promotes Ca2+influx and induces streamers

We have reported that during complement-mediated cytolysis of B cells promoted by the CD20 mAbs rituximab or ofatumumab (OFA), long, thin structures that we call streamers (≥ 3 cell diameters) are rapidly generated and grow out from the cell surface. Streamers appear before cells are killed and contain opsonizing mAbs and membrane lipids. By exploiting the differential Ca(2+) requirements of discrete steps in the complement cascade, we determined that mAb-opsonized cells first tagged with C3b using C5-depleted serum are killed on addition of serum and EDTA, but the cells do not produce streamers. Also, cells first opsonized with OFA are lysed in serum containing Mg-EGTA by the alternative complement pathway but streamers are not produced. These findings indicate that Ca(2+) influx is necessary for streamer formation. Other mAbs that promote complement-mediated cytolysis also induce streamers on target cells. Streamer-like structures called nanotubes have been reported in several cellular systems, and are thought to promote intercellular communication/signaling. We tested whether this signaling could influence the susceptibility of neighboring cells contacted by streamers to complement attack and found that complement-mediated cytolysis of OFA-opsonized cells increases the resistance of unopsonized indicator cell populations to subsequent lysis when these cells are exposed to OFA and complement.

Human CD59 inhibitor sensitizes rituximab-resistant lymphoma cells to complement-mediated cytolysis.

Rituximab efficacy in cancer therapy depends in part on induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex, thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell non-Hodgkin's lymphoma (NHL), and upregulation of hCD59 is an important determinant of the sensitivity of NHL cells to rituximab treatment. Here, we report that the potent hCD59 inhibitor rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing rituximab-resistant lymphoma cells and primary chronic lymphocytic leukemia cells (CLL) to rituximab treatment. By defining pharmcokinetic/pharmacodynamic profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. Increasing expression levels of the complement regulators CD59 and CD55 in rituximab-resistant cells occur due to selection of preexisting clones rather than de novo induction of these proteins. Moreover, lymphoma cells overexpressing CD59 were directly responsible for the resistance to rituximab-mediated CDC therapy. Our results rationalize the use of rILYd4 as a therapeutic adjuvant for rituximab treatment of rituximab-resistant lymphoma and CLL. Furthermore, they suggest that preemptive elimination of CD59-overexpressing subpopulations along with rituximab treatment may be a useful approach to ablate or conquer rituximab resistance.

The effects of CD59 gene as a target gene on breast cancer cells

The retroviral-vector-targeted CD59 gene (pSUPER-siCD59) was constructed and transfected into breast cells (MCF-7). The results demonstrated that the retroviral vector-mediated RNAi successfully suppressed human CD59 gene. The expression of CD59 decreased at both mRNA and protein levels. Knockdown of CD59 abrogated its protective effect on complement-mediated cytolysis. Fas and caspase-3 were remarkably upregulated, which induced apoptosis and tumor growth suppression in MCF-7 cells. In addition, overexpression of CD59 promoted the proliferation of MCF-7 cells and inhibited anti-apoptotic Bcl-2 expression. In conclusion, CD59 may be a promising target in the gene therapy of breast cancer.

A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement

AbstractThe clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway–mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.