The Role of Membrane Bound Complement Regulatory Proteins in Tumor Development and Cancer Immunotherapy.

It has long been understood that the control and surveillance of tumors within the body involves an intricate dance between the adaptive and innate immune systems. At the center of the interplay between the adaptive and innate immune response sits the complement system-an evolutionarily ancient response that aids in the destruction of microorganisms and damaged cells, including cancer cells. Membrane-bound complement regulatory proteins (mCRPs), such as CD46, CD55, and CD59, are expressed throughout the body in order to prevent over-activation of the complement system. These mCRPs act as a double-edged sword however, as they can also over-regulate the complement system to the extent that it is no longer effective at eliminating cancerous cells. Recent studies are now indicating that mCRPs may function as a biomarker of a malignant transformation in numerous cancer types, and further, are being shown to interfere with anti-tumor treatments. This highlights the critical roles that therapeutic blockade of mCRPs can play in cancer treatment. Furthermore, with the complement system having the ability to both directly and indirectly control adaptive T-cell responses, the use of a combinatorial approach of complement-related therapy along with other T-cell activating therapies becomes a logical approach to treatment. This review will highlight the biomarker-related role that mCRP expression may have in the classification of tumor phenotype and predicted response to different anti-cancer treatments in the context of an emerging understanding that complement activation within the Tumor Microenvironment (TME) is actually harmful for tumor control. We will discuss what is known about complement activation and mCRPs relating to cancer and immunotherapy, and will examine the potential for combinatorial approaches of anti-mCRP therapy with other anti-tumor therapies, especially checkpoint inhibitors such as anti PD-1 and PD-L1 monoclonal antibodies (mAbs). Overall, mCRPs play an essential role in the immune response to tumors, and understanding their role in the immune response, particularly in modulating currently used cancer therapeutics may lead to better clinical outcomes in patients with diverse cancer types.

The role of complement inhibitors beyond controlling inflammation.

The complement system is an arm of innate immunity that aids in the removal of pathogens and dying cells. Due to its harmful, pro-inflammatory potential, complement is controlled by several soluble and membrane-bound inhibitors. This family of complement regulators has been recently extended by the discovery of several new members, and it is becoming apparent that these proteins harbour additional functions. In this review, the current state of knowledge of the physiological functions of four complement regulators will be described: cartilage oligomeric matrix protein (COMP), CUB and sushi multiple domains 1 (CSMD1), sushi domain-containing protein 4 (SUSD4) and CD59. Complement activation is involved in both the development of and defence against cancer. COMP expression is pro-oncogenic, whereas CSMD1 and SUSD4 act as tumour suppressors. These effects may be related in part to the complex influence of complement on cancer but also depend on unrelated functions such as the protection of cells from endoplasmic reticulum stress conveyed by intracellular COMP. CD59 is the main inhibitor of the membrane attack complex, and its deficiency leads to complement attack on erythrocytes and severe haemolytic anaemia, which is now amenable to treatment with an inhibitor of C5 cleavage. Unexpectedly, the intracellular pool of CD59 is crucial for insulin secretion from pancreatic ß-cells. This finding is one of several relating to the intracellular functions of complement proteins, which until recently were only considered to be present in the extracellular space. Understanding the alternative functions of complement inhibitors may unravel unexpected links between complement and other physiological systems, but is also important for better design of therapeutic complement inhibition.

[Paroxysmal nocturnal hemoglobinuria: An unknown cause of thrombosis?]. / L'hémoglobinurie paroxystique nocturne : une cause méconnue de thrombose ?

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder of hematopoietic stem cells. Somatic mutation in the phosphatidylinositol glycan class A (PIG-A), X-linked gene, is responsible for a deficiency in glycosphosphatidylinositol-anchored proteins (GPI-AP). The lack of one of the GPI-AP complement regulatory proteins (CD55, CD59) leads to hemolysis. The disease is diagnosed with hemolytic anemia, marrow failure and thrombosis. Thromboembolic complication occurs in 30% of patient after 10 years of follow-up and is the first event in one out of 10 patients. The two most common sites are hepatic and cerebral veins. These locations are correlated with high risk of death. Currently, these data are balanced with the use of a monoclonal antibody (Eculizumab), which has significantly improved the prognosis with a survival similar to general population after 36 months of follow-up. Anticoagulant treatment is recommended after a thromboembolic event but has no place in primary prophylaxis.

The complement receptors CD46, CD55 and CD59 are regulated by the tumour microenvironment of head and neck cancer to facilitate escape of complement attack.

BACKGROUND: Membrane-bound complement restriction proteins (mCRPs) CD46, CD55 and CD59 enable tumour cells to evade complement dependent cytotoxicity and antibody-dependent killing mechanisms. But less is known about the role of these mCRPs in head and neck cancer. METHODS: In this study we determined the expression of the mCRPs on head and neck squamous cell carcinoma (HNSCC) cell lines, on tumour tissue and TDLNs (tumour-draining lymph nodes) as well as on lymphocytes from HNSCC patients. The influence of the HNSCC microenvironment on the mCRP regulation was analysed using Flow Cytometry, Western blotting and small interfering RNAs (siRNA) transfection studies. RESULTS: We examined the effects of the HNSCC tumour milieu on the expression levels of CD46, CD55 and CD59. We investigated the susceptibility of HNSCC cells to CDC (complement-dependent cytotoxicity) while silencing the mCRPs. Our results demonstrate a huge influence of the HNSCC tumour microenvironment on the regulation of mCRP expression and show a reciprocal regulation between the different mCRPs themselves. CONCLUSIONS: In summary, our data indicate that HNSCC has evolved different strategies to evade complement attacks and that the tumour microenvironment leads to the enhancement of complement resistance of the surrounding tissue.

Comparative study on signal transduction in endothelial cells after anti-a/b and human leukocyte antigen antibody reaction: implication of accommodation.

BACKGROUND: Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. METHODS: Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. RESULTS: Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. CONCLUSION: Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

The protective role of CD59 and pathogenic role of complement in hepatic ischemia and reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is a major factor influencing graft outcome in liver transplantation, but its mechanism is not well defined. Although complement, including the membrane attack complex (MAC), a terminal product of complement activation, is thought to be involved in the multiple reactions subsequent to the ischemia-reperfusion (IR) process, the role of MAC in the pathogenesis of hepatic IRI requires further investigation. We used a warm ischemia-reperfusion injury model in mice and a syngeneic orthotopic liver transplantation model in rats to define the role of complement, including MAC, in hepatic IR. CD59-deficient mice had more severe liver dysfunction, evidenced by increased aspartate aminotransferase levels and increased injury of liver parenchymal and nonparenchymal cells than did CD59-sufficient mice during warm hepatic IR. Furthermore, complement depletion in CD59-deficient mice by pretreatment with cobra venom factor (CVF) or the genetic introduction of C3 deficiency partially protected against development of the severe liver dysfunction that occurred in CD59-deficient mice. Severity of liver dysfunction correlated with MAC deposition, apoptotic cells, and increased inflammatory mediators such as tumor necrosis factor α. Moreover, depletion of complement with CVF in orthotopic liver transplantation recipient rats attenuated IRI of the donor livers. Taken together, these results highlight the protective role of CD59 and pathogenic role of complement, including MAC, in the pathogenesis of hepatic IRI.

[Eculizumab in paroxysmal nocturnal hemoglobinuria]. / Anticorps monoclonal anti-C5 (éculizumab) dans l'hémoglobinurie paroxystique nocturne.

Paroxysmal nocturnal hemoglobinuria is a rare acquired clonal of the hematopoietic stem cell due to acquired mutation of the PIG-A gene. This results in the lack of two GPI-anchored membrane proteins involved in the inhibition of complement attack, thus explaining red cells hemolysis. The development of an anti-C5 monoclonal antibody (eculizumab) had profoundly modified the treatment of the the hemolytic form of the disease.

[Inhibition of mutant CD59 protein on proliferation of ovarian cancer A2780 cells].

BACKGROUND AND OBJECTIVE: Overexpression of CD59 is found in a variety of solid tumor cells, which is related to the uncontrolled cell growth of tumors and malignant transformation. This study was to investigate the anti-complement capacity of mutant CD59 (at the W40 site), and its inhibitory effect on ovarian cancer cell line A2780 in combination with lip polysaccharide (LPS). METHODS: The pIRES-WTCD59 plasmid and the pIRES-M1CD59 plasmid were transfected into A2780 cells. Stable expression clones were selected by G418. CD59 gene and protein were successfully expressed in A2780 cells. The anti-complement capacity of wide-type CD59 and mutant CD59, and the effect of combination treatment with mutant CD59 protein and LPS on A2780 cells were both detected by MTT assay. RESULTS: Stable expressions of wild type and mutant CD59 were established in A2780 cells. Mutant CD59 lost its inhibition of human complements compared with wide-type CD59 (P<0.05), but this was not significantly different compared with the control group. After incubation with 5 mg/L LPS for half an hour, the inhibition rates were (26.9+/-2.95)%, (36.3+/-4.87)%, (29.6+/-3.16)% in A2780 cells transfected with wide-type CD59, mutant CD59, and without transfection respectively (P<0.05). CONCLUSIONS: The W40 site is important to the activity of human CD59 protein. Inhibition of the W40 site may be a potential method to increase the complement capacity and promote the anti-proliferation activity of LPS.

Deficiency of the complement regulator CD59a exacerbates Wallerian degeneration.

The complement system is implicated in Wallerian degeneration (WD). We have previously shown that the membrane attack complex (MAC), the terminal activation product of the complement cascade, mediates rapid axonal degradation and myelin clearance during WD after peripheral nerve injury. In this study we analyzed the contribution of CD59a, a cell membrane negative regulator of the MAC, to WD. Following injury, the level of MAC deposition was higher in the CD59a deficient mice than wildtypes whereas the residual axonal content was lower in CD59a deficient mice than wildtypes, strongly implicating MAC as a determinant of axonal damage during WD. The number of endoneurial macrophages was significantly higher in CD59a deficient mice compared to wildtypes at 1 day post-injury. These findings are relevant to the understanding of the mechanisms of axon loss in injury and disease.

CD59 efficiently protects human NT2-N neurons against complement-mediated damage.

The complement regulatory protein CD59 controls cell survival by the inhibition of C5b-9 formation on the cell membrane. Loss of CD59 increases the susceptibility of cells to complement-mediated damage and lysis. Deposition of IgM can induce complement activation with subsequent cell death. We have previously demonstrated the presence of CD59 on human NT2-N neurons. In this study, we investigated the functional role of CD59 for NT2-N cell survival after IgM-mediated complement activation. Complement activation was induced on NT2-N neurons with human serum following incubation with the IgM monoclonal antibody A2B5 reacting with a neuronal cell membrane epitope. Deposition of C1q and C5b-9 was detected on the cell membrane and sC5b-9 in the culture supernatant. Specific inhibition of complement was obtained by the C3 inhibitor compstatin, and by anti-C5/C5a MoAb. CD59 was blocked by the MoAb BRIC 229. Membrane damage of propidium iodide-stained NT2-N cells was confirmed by immunofluorescence microscopy and degeneration of neuronal processes was shown with crystal violet staining. A2B5, but not the irrelevant control IgM antibody, induced complement activation on NT2-N neurons after incubation with a human serum, as detected by the deposition of C1q. A marked membrane deposition of C5b-9 on NT2-N neurons with accompanying cell death and axonal degeneration was found after the blocking of CD59 with MoAb BRIC 229 but not with an isotype-matched control antibody. Compstatin and anti-C5 monoclonal antibodies which blocked C5 activation efficiently inhibited complement activation. In conclusion, CD59 is essential for protecting human NT2-N neurons against complement-mediated damage, which is known to occur in a number of clinical conditions including stroke.

Pathophysiology of acute kidney injury: roles of potential inhibitors of inflammation.

The pathogenesis of acute kidney injury (AKI) is complex and varies to some extent based on the particular cause. Inflammation contributes to this pathophysiology in a variety of contexts. Inflammation can result in reduction in local blood flow to the outer medulla with adverse consequences on tubule function and viability. Both the innate and adaptive immune responses are important contributors. With ischemia/reperfusion endothelial cells upregulate a number of adhesion molecules which have counterreceptors on leukocytes. A number of vasoactive mediators that are released with injury, such as nitric oxide, may also affect leukocyte- endothelial interactions. Tubule epithelial cells generate proinflammatory and chemotactic cytokines. We and others have found that injection of mesenchymal stem (stromal) cells is protective against renal injury as assessed by serum creatinine measured 24 h after ischemia. The mechanism of such protection may be through intrarenal paracrine effects to decrease inflammation or by systemic immune modulation. Resolvins (Rv) and protectins (PD) have been identified as two newly identified families of naturally occurring n-3 fatty acid docosahexaenoic acid metabolites. In collaboration with Serhan et al. we recently reported that, in response to bilateral ischemia/reperfusion injury, mouse kidneys produce D series resolvins (RvDs) and PD1 [J Immunol 2006;177:5902-5911]. Administration of RvDs or PD1 to mice prior to, or subsequent to, ischemia resulted in a reduction in functional and morphological kidney injury. Understanding how these anti-inflammatory processes are regulated may provide insight into how we might intervene to facilitate and enhance them so that we might prevent or mitigate the devastating consequences of AKI.

[Research progression on complement regulatory proteins CD46, CD55, and CD59 in tumor immunotherapy].

Immunologic mechanism of tumor escaping from complements' attack was unclear in the past. The efficacy of immunotherapy, especially humoral immunotherapy, to tumor is unsatisfactory. At present, with the progression in immunology, various tumors were found to highly express one or several kinds of complement regulatory proteins, such as CD46/MCP, CD55/DAF, and CD59/potectin. Complement system of the organism is inhibited because of high expression of complement regulatory proteins; therefore, the tumor can escape from the attack of complement system. Recently, the mechanism of complement regulatory proteins expressing in tumor has been studied in deep; some immunotherapies aim directly at complement regulatory proteins, including monoclonal antibody of complement regulatory proteins and cytokines, have been applied to animal experiments and clinical trails, and got some success. This review elucidated the progression on complement regulatory proteins in tumor immunotherapy.

Expression of glycosylphosphatidylinositol-anchored CD59 on target cells enhances human NK cell-mediated cytotoxicity.

NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs previously shown to lack blocking capacity for C-mediated lysis. We investigated the consequences for alternative membrane attachment modalities, namely bis-myristoylated-peptidyl (BiMP) and GPI anchoring, on CD59-negative U937 cells. Expression of GPI-anchored CD59 either via transfection or incorporation rendered U937 targets more susceptible to NK cytotoxicity, whereas incorporation of CD59 via a BiMP anchor to similar levels did not alter susceptibility to NK cytotoxicity. Localization of both BiMP- and GPI-anchored CD59 proteins was shown to be within the lipid raft microdomain. A role for the GPI anchor and independence from glycosylation status was confirmed by expression of transmembrane-anchored CD59 or unglycosylated CD59 and by testing in NK cytotoxicity assays. To investigate mechanisms, we compared the signaling capacity of the various forms of expressed and incorporated CD59 following Ab cross-linking in calcium flux assays. GPI-anchored CD59, with or without glycosylation, mediated activation events, whereas CD59 forms lacking the GPI anchor did not. The data show that the increased susceptibility of target cells expressing CD59 to NK cytotoxicity requires GPI anchor-mediating signaling events, likely mediated by interactions between GPI-anchored CD59 on targets and NK receptors.

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59.

Expression of the complement-regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement-dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single-chain variable fragments (scFv) to CD55 and CD59 from a human phage-display library and from these scFv we have produced two miniantibodies (MB), MB-55 (against CD55) and MB-59 (against CD59), containing the human hinge-CH2-CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB-55 and MB-59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement-mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB-55 and MB-59 to the lymphoma cell line Karpas 422. The two MB induced a two-fold increase in the complement-dependent killing of these cells stimulated by Rituximab, a chimeric anti-CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB-55 or MB-59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB-55 and MB-59 may represent a therapeutic tool to increase the complement-dependent killing activity of Rituximab in the treatment of non-Hodgkin's lymphoma.

Human CD8+ T cell blasts are more sensitive than CD4+ T cell blasts to regulation by APO2L/TRAIL.

The mechanisms responsible for the down-modulation of the activation of separated CD4(+) or CD8(+) human T cell blasts were studied using cells obtained from healthy donors. In the presence of IL-2, human CD8(+) T cell blasts were more sensitive than CD4(+) T cell blasts to regulation by APO2 ligand/TNF-related apoptosis-inducing ligand (APO2L/TRAIL), while both T cell subsets were equally sensitive to Fas/CD95 regulation. This regulation was defined as inhibition of IL-2-dependent T cell growth in the absence of cell death induction, characterized by cell cycle arrest in G(2)/M. The physiological validity of these observations was corroborated by the demonstration of intracellular FasL and APO2L/TRAIL expression in CD4(+) and CD8(+) T cell blasts, which were secreted in their bioactive form into the supernatant upon PHA, CD3 or CD59 reactivation. Additionally, the inhibition of IL-2-dependent CD4(+) or CD8(+) T cell blast growth upon CD3 or CD59 ligation was dependent, at least partially, on FasL and/or APO2L/TRAIL. These data precisely define the role of APO2L/TRAIL in the regulation of human T cell activation.

CD59a deficiency exacerbates ischemia-reperfusion injury in mice.

The terminal complement components C5a and the membrane attack complex are involved in the pathogenesis of ischemia-reperfusion injury in many organs. CD59 is the major regulator of membrane attack complex formation. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in renal ischemia-reperfusion injury. Unilateral ischemia-reperfusion injury was induced by clamping the left renal pedicle for 30 minutes under general anesthetic. Mice were studied at 72 hours and 2 weeks after ischemia-reperfusion injury. mCd59a-/- mice developed significantly greater tubular injury (P = 0.01), tubulointerstitial apoptosis (P = 0.02), and neutrophil influx (P = 0.04) than controls at 72 hours after ischemia-reperfusion. Two weeks after ischemia-reperfusion, mCd59a-/- mice exhibited more severe tubular damage predominantly in a corticomedullary distribution than controls (P = 0.02). Quantification of interstitial leukocytes revealed significantly greater numbers of infiltrating lymphocytes (but not macrophages) in mCd59a-/- mice than controls (P = 0.04) at 2 weeks. At both time points, significantly more C9 (as a marker of membrane attack complex) deposition occurred in a peritubular distribution in mCd59a-/- mice than controls. In conclusion, these results demonstrate that the lack of CD59a, by allowing unregulated membrane attack complex deposition, exacerbates both the tubular injury and the interstitial leukocyte infiltrate after ischemia-reperfusion injury in mice.

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity.

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, during an attempt to identify novel NK receptors or co-receptors, we found that CD59 functions as a co-receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in markedly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3zeta chains associated with these NCR, but not those associated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physical interaction of this GPI-linked protein with primary triggering NK receptors.

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor.

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.