Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer.

BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Reduced decay-accelerating factor expression promotes complement-mediated cystogenesis in murine ADPKD.

Patients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1-/- C3-/- mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1-/- C3-/- mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1-/- C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.

Complement decay-accelerating factor inhibits inflammation-induced myopia development.

Myopia is regarded as a worldwide epidemic ocular disease, has been proved related to inflammation. CD55, also known as decay-accelerating factor (DAF) can modulate the activation of complement through inhibiting the formation of complement 3 convertase and its dysregulation is involved in various inflammatory diseases. To investigate the association between CD55 and myopia, and to test whether CD55 can inhibit myopia development by suppressing inflammation in the eye, we use three different animal models including monocular form-deprivation myopia, myopia induced by TNF-α administration and allergic conjunctivitis animal model to reveal the CD55 in myopia development. The tears of thirty-eight participants with different spherical equivalents were collected and CD55 in the tears were also analyzed. Complement 3 and complement 5 levels increased while CD55 levels decreased in allergic conjunctivitis and myopic eyes. After anti-inflammatory drugs administration, CD55 expression was increased in monocular form-deprivation myopia model. We also found inflammatory cytokines TGF-ß, IL-6, TNF-α, and IL-1ß may enhance complement 3 and complement 5 activation while CD55 level was suppressed contrary. Moreover, lower CD55 levels were found in the tears of patients with myopia with decreased diopter values. Finally, CD55-Fc administration on the eyelids can inhibit the elongation of axial length and change of refractive error. CD55-Fc application also suppress myopia development subsequent to complement 3 and complement 5 reduction and can lower myopia-specific (MMP-2 and TGF-ß) cytokine expression in TNF-α induced myopia animal model. This suggests that CD55 can inhibit myopia development by suppression of complement activation and eventual down-regulation of inflammation.

Pozelimab, a human monoclonal immunoglobulin for the treatment of CHAPLE disease.

The complement is a crucial factor of the innate immune system. However, its activation can lead to various diseases, so it needs to be controlled. In mammals, surface-bound complement regulatory proteins safeguard cells from uncontrolled complement-mediated lysis. One of the human complement regulators is CD55, also known as the decay-accelerating factor (DAF), a single-chain, type I cell surface protein anchored to glycosylphosphatidylinositol (GPI). The genetic loss of the complement regulatory protein CD55 leads to a fatal illness known as CHAPLE disease. The complement and innate immunity become hyperactive in this disease, causing angiopathic thrombosis and protein-losing enteropathy. Patients with CHAPLE disease experience abdominal pain, nausea, vomiting, diarrhea, loss of appetite, weight loss, impaired growth, and swelling. This genetic condition has no known cure, and managing its symptoms can be challenging. Pozelimab, a human monoclonal immunoglobulin IgG4 antibody, is a drug that targets the terminal complement protein C5. The drug has a high affinity for both wild-type and variant human C5. Pozelimab has received designations such as fast track, orphan drug, and rare pediatric disease, making it a significant medical breakthrough. It is currently the only available treatment for this disease. In this review, we have summarized the preclinical and clinical data on pozelimab.

Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways.

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Critical Role of CD55 in Controlling Wound Healing.

How reparative processes are coordinated following injury is incompletely understood. In recent studies, we showed that autocrine C3a and C5a receptor (C3ar1 and C5ar1) G protein-coupled receptor signaling plays an obligate role in vascular endothelial growth factor receptor 2 growth signaling in vascular endothelial cells. We documented the same interconnection for platelet-derived growth factor receptor growth signaling in smooth muscle cells, epidermal growth factor receptor growth signaling in epidermal cells, and fibroblast growth factor receptor signaling in fibroblasts, indicative of a generalized cell growth regulatory mechanism. In this study, we examined one physiological consequence of this signaling circuit. We found that disabling CD55 (also known as decay accelerating factor), which lifts restraint on autocrine C3ar1/C5ar1 signaling, concomitantly augments the growth of each cell type. The mechanism is heightened C3ar1/C5ar1 signaling resulting from the loss of CD55's restraint jointly potentiating growth factor production by each cell type. Examination of the effect of lifted CD55 restraint in four types of injury (burn, corneal denudation, ear lobe puncture, and reengraftment of autologous skin) showed that disabled CD55 function robustly accelerated healing in all cases, whereas disabled C3ar1/C5ar1 signaling universally retarded it. In wild-type mice with burns or injured corneas, applying a mouse anti-mouse CD55 blocking Ab (against CD55's active site) to wounds accelerated the healing rate by 40-70%. To our knowledge, these results provide new insights into mechanisms that underlie wound repair and open up a new tool for accelerating healing.

Dynamic encounters with red blood cells trigger splenic marginal zone B cell retention and function.

Spleen marginal zone (MZ) B cells are important for antibody responses against blood-borne antigens. The signals they use to detect exposure to blood are not well defined. Here, using intravital two-photon microscopy in mice, we observe transient contacts between MZ B cells and red blood cells that are in flow. We show that MZ B cells use adhesion G-protein-coupled receptor ADGRE5 (CD97) for retention in the spleen. CD97 function in MZ B cells depends on its ability to undergo autoproteolytic cleavage and signaling via Gα13 and ARHGEF1. Red blood cell expression of the CD97 ligand CD55 is required for MZ B cell homeostasis. Applying a pulling force on CD97-transfected cells using an optical C-trap and CD55+ beads leads to accumulation of active RhoA and membrane retraction. Finally, we show that CD97 deficiency leads to a reduced T cell-independent IgM response. Thus, our studies provide evidence that MZ B cells use mechanosensing to position in a manner that enhances antibody responses against blood-borne antigens.

Effective clearance of rituximab-resistant tumor cells by breaking the mirror-symmetry of immunoglobulin G and simultaneous binding to CD55 and CD20.

Complement-dependent cytotoxicity (CDC), which eliminates aberrant target cells through the assembly and complex formation of serum complement molecules, is one of the major effector functions of anticancer therapeutic antibodies. In this study, we discovered that breaking the symmetry of natural immunoglobulin G (IgG) antibodies significantly increased the CDC activity of anti-CD20 antibodies. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in a rituximab-resistant cell line generated in-house, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance. Based on these findings, we developed an asymmetric bispecific antibody (SBU-CD55 × CD20) that simultaneously targets both CD55 and CD20 to effectively eliminate rituximab-resistant cancer cells. In various cancer cell lines, including rituximab-resistant lymphoma cells, the SBU-CD55 × CD20 antibody showed significantly higher CDC activity than either anti-CD20 IgG antibody alone or a combination of anti-CD20 IgG antibody and anti-CD55 IgG antibody. Furthermore, the asymmetric bispecific antibody (SBU-CD55 × CD20) exhibited significantly higher CDC activity against rituximab-resistant cancer cells compared to other bispecific antibodies with symmetric features. These results demonstrate that enhancing CDC with an asymmetric CD55-binding bispecific antibody could be a new strategy for developing therapeutics to treat patients with relapsed or refractory cancers.

Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells.

OBJECTIVES: This study aimed to evaluate the expression pattern of complement regulatory proteins (CRPs) CD46, CD59, and CD55 in HPV-positive (HPV+) & negative (HPV-) cervical cancer cell lines in search of a reliable differential biomarker. STUDY DESIGN: We analysed the expression of CRPs in HPV 16-positive SiHa cell line, HPV 18-positive HeLa cell line, and HPV-negative cell line C33a using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. RESULTS: We observed a differential expression profile of CRPs in HPV+ and HPV- cervical cancer cell lines. The mRNA level of CD59 & CD55 showed a higher expression pattern in HPV+ cells when compared to HPV- cancer cells. However, flow cytometry-based experiments revealed that CD46 was preferentially expressed more in HPV 16-positive SiHa cells followed by HPV 18-positive HeLa cells when compared to HPV- C33a cells. Interestingly, confocal microscopy revealed a high level of CD59 expression in Hela cells and SiHa cells but low expression in HPV- C33a cells. In addition, HPV 18-positive HeLa cells expressed more CD55, which was lower in SiHa cells and very weak in C33a cells. CONCLUSION: The study demonstrates the differential expression of CRPs in both HPV+ and HPV- cervical cancer cells for the first time, and their potential to serve as an early diagnostic marker for cervical carcinogenesis.

Polycythemia as an Uncommon Finding in 2 Children, One With Systemic Capillary Leak Syndrome and the Other With Protein-losing Enteropathy Caused by CD55 Deficiency.

We report 2 children with distinct causes of polycythemia, 1 from systemic capillary leak syndrome (SCLS) and the other from protein-losing enteropathy (PLE) caused by CD55 deficiency. There is only a single case series about polycythemia in children with SCLS, but none on polycythemia in children with PLE. We present a 10-year-old girl with hypoalbuminemia, polycythemia, and edema who died as a result of an SCLS attack and a 1-year-old girl with PLE who was successfully treated with eculizumab. Our experience suggests that hematologists should be alert for SCLS and PLE in children with relative polycythemia.

Knockdown of membrane-bound complement regulatory proteins suppresses colon cancer growth in mice through inducing tumor cell apoptosis.

CD46, CD55 and CD59 are membrane-bound complement regulatory proteins (mCRPs) and highly expressed in many tumor tissues. Our analysis by RNA sequencing and qRT-PCR revealed that the expression of mCRPs was significantly elevated in cancer tissues of 15 patients with colon cancer. To further investigate the role of mCRPs in the development of colon cancer, we suppressed the expression of mCRPs by CD46-shRNA, CD55-shRNA and CD59-shRNA in colon cancer cell lines, SW620 and HT-29 cells. The results indicated that CD46-shRNA, CD55-shRNA and CD59-shRNA effectively reduced the expression of mCRPs, accompanied with the increased LDH release and the percentage of Annexin V + 7-AAD- early phase of apoptotic cells. The similar cytotoxic effects were also observed in the cells treated with CD46 neutralizing antibody (aCD46), associated with the increased C5b-9 deposition, cleaved caspase-3 and Bax expression in the treated cells. The cytotoxic effects by mCRPs knock-down were potentiated in the cells co-treated with doxorubicin (Dox). In addition, STAT3, STAT6, and p38 MAPK inhibitors, including C188-9, AS1517499 and SB203580 effectively reduced the expression of CD46 in the treated colon cells, associated with increased cell apoptosis and LDH release. Further study with mouse model revealed that mCRPs knockdown by mCRPs-shRNA significantly reduced colon cancer growth, associated with increased expression of Bax, cleaved caspase-3 and C5b-9 deposition, but reduced expression of Bcl-2, IL-6 and IL-1beta in tumor tissues of nude mice transplanted with SW620 cells. Thereby, mCRPs expression in human colon cancer cells were upregulated by STAT3/STAT6/p38 MAPK signaling and mCRPs knockdown reduced colon cancer growth in mice through inducing tumor cell apoptosis.

Relationship between the expression of complement inhibitory proteins and therapeutic efficacy of antibodies in breast cancer.

Complement regulatory proteins (mCRPs) CD55, CD46 and CD59 have been proposed as key elements in therapeutic resistance against cancer. mCRP-expressing tumor cells, in addition to hindering trastuzumab, pertuzumab and sacituzumab-govitecan therapeutic activity in breast cancer, can regulate biological processes that promote tumor progression. This review describes the structure of mCRPs and analyzes their expression using transcriptomic databases from breast cancer patients, in addition to collecting information on mCRPs interactions and signaling in tumor cells. Given that mCRPs are relevant targets, several strategies that have been explored for their inhibition and regulation in order to increase therapeutic efficacy and prevent cancer resistance and progression are described.

Se ha propuesto a las proteínas reguladoras de complemento (mCRP) CD55, CD46 y CD59 como piezas clave en la resistencia terapéutica contra el cáncer. Las células tumorales que expresan las mCRP, además de obstaculizar la actividad terapéutica de trastuzumab, pertuzumab y sacituzumab-govitecan en cáncer de mama, pueden regular procesos biológicos que promueven la progresión tumoral. Esta revisión describe la estructura de las mCRP y analiza su expresión a partir de bases de datos transcriptómicos de pacientes con cáncer de mama; también recopila información de interacciones y señalización de las mCRP en células tumorales. Dado que estas mCRP son dianas relevantes, se describen diversas estrategias para su inhibición y regulación para incrementar la eficacia terapéutica y evitar la resistencia y progresión del cáncer.

Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.

The active form of Helicobacter pylori vacuolating cytotoxin induces decay-accelerating factor CD55 in association with intestinal metaplasia in the human gastric mucosa.

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1-type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Human FcRn Is a Two-in-One Attachment-Uncoating Receptor for Echovirus 18.

Virus-receptor interactions determine viral host range and tissue tropism. CD55 and human neonatal Fc receptor (FcRn) were found to be the binding and uncoating receptors for some of the echovirus-related enterovirus species B serotypes in our previous study. Echovirus 18 (E18), as a member of enterovirus species B, is a significant causative agent of aseptic meningitis and viral encephalitis in children. However, it does not use CD55 as a critical host factor. We conducted CRISPR/Cas9 knockout screening to determine the receptors and entry mechanisms and identified FcRn working as a dual-function receptor for E18. Knockout of FCGRT and B2M, which encode the two subunits of FcRn, prevented infection by E18 and other echoviruses in the same physiological cluster. We then elucidated the underlying molecular mechanism of receptor recognition by E18 using cryogenic electron microscopy. The binding of the FCGRT subunit to the canyon region rotates the residues around the pocket, triggering the release of the pocket factor as observed for other enterovirus species B members. IMPORTANCE E18 is a member of enterovirus species B. As one of the most common enterovirus serotypes in nonpolio enterovirus detection, it easily infects children and causes various clinical symptoms. Aseptic meningitis and viral encephalitis are the most commonly reported syndromes associated with E18. No effective antiviral drugs or approved vaccines are available. Previous studies showed that CD55 and FcRn were the binding and uncoating receptors for some echoviruses. However, we found that CD55 is not the critical host factor for E18. Thus, we want to determine the receptors and elucidate the entry mechanism of E18. Our findings reveal that FcRn is a two-in-one attachment-uncoating receptor for E18.

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model.

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

CD55 is upregulated by cAMP/PKA/AKT and modulates human decidualization via Src and ERK pathway and decidualization-related genes.

Decidualization is an essential process for embryo implantation and maintenance of pregnancy, and abnormal decidualization contributed to several pregnancy disorders like a miscarriage. The objective of this study was to explore the regulation and function of CD55 in human decidualization. By immunohistochemical staining, it was found that CD55 expression was higher in first-trimester decidua than in the endometrium. In both primary endometrial stromal cells and immortalized cell line T-hESCs, CD55 was upregulated by induction of in vitro decidualization with medroxyprogesterone acetate (MPA) and 8-Br-cAMP. During decidualization in vitro, CD55 was stimulated by 8-Br-cAMP in a time- and concentration-dependent manner, which was reversed by a PKA inhibitor H89 and partially by an AKT activator SC79. Knocking down CD55 expression diminished the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1), accompanied by inhibition of Src, aberrant activation of ERK and decreased expression of several decidualization-related genes, including FOXO1, EGFR, and STAT3. Furthermore, the decidua of unexplained miscarriage women and the endometrium of unexplained infertile women both exhibited decreased CD55 expression. Collectively, these findings revealed that 8-Br-cAMP promotes CD55 expression via PKA activation and AKT dephosphorylation, and decreased CD55 impairs decidualization by inactivation of Src, aberrant activation of ERK pathway, and compromised expression of decidualization-related genes, indicating that CD55 deficiency may contribute to the pathogenesis of spontaneous miscarriage and infertility.

CD97 promotes spleen dendritic cell homeostasis through the mechanosensing of red blood cells.

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα13 and adhesion G protein-coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.