Loss Of CD59 Research Articles

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

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.

The complement inhibitor CD59 is required for GABAergic synaptic transmission in the dentate gyrus

Complement-dependent microglia pruning of excitatory synapses has been widely reported in physiological and pathological conditions, with few reports concerning pruning of inhibitory synapses or direct regulation of synaptic transmission by complement components. Here, we report that loss of CD59, an important endogenous inhibitor of the complement system, leads to compromised spatial memory performance. Furthermore, CD59 deficiency impairs GABAergic synaptic transmission in the hippocampal dentate gyrus (DG). This depends on regulation of GABA release triggered by Ca2+ influx through voltage-gated calcium channels (VGCCs) rather than inhibitory synaptic pruning by microglia. Notably, CD59 colocalizes with inhibitory pre-synaptic terminals and regulates SNARE complex assembly. Together, these results demonstrate that the complement regulator CD59 plays an important role in normal hippocampal function.

CD47 expression is critical for CAR T-cell survival in vivo

BackgroundCD47 is an attractive immunotherapeutic target because it is highly expressed on multiple solid tumors. However, CD47 is also expressed on T cells. Limited studies have evaluated CD47-chimeric antigen receptor...

Targeting the CD47-SIRPα Axis: Present Therapies and the Future for Cutaneous T-cell Lymphoma.

The loss of CD47 on aging cells serves as a signal to macrophages to eliminate the target. Therefore, CD47 is a "do-not-eat-me" sign preventing macrophagal phagocytosis via interaction with its ligand SIRPα. Malignant lymphocytes of mycosis fungoides and Sézary syndrome express CD47 highly, thus, being ideal candidates for targeted anti-CD47 therapies. The classes of current anti-CD47-SIRPα therapeutic molecules present in a large variety and include monoclonal antibodies against CD47 and SIRPα, bioengineered SIRPα proteins, miRNAs, and bispecific antibodies. We provided a detailed analysis of all available investigational drugs in a contest of cutaneous T-cell lymphoma. A combination of blockade of the CD47-SIRPα axis and secondary targets in the tumor microenvironment (TME) may improve the clinical efficacy of current immunotherapeutic approaches. We evaluated the possible combination and outlined the most promising one.

Abstract 13606: Deletion of CD47 Specifically in Lymphatic Endothelium Augments Arterial Lymphangiogenesis and Attenuates Atherosclerosis

Thrombospondin-1 (TSP1), an extracellular glycoprotein mediates differential physio- or pathological effects via interacting with cell surface receptors including CD36 and CD47. TSP1 is a well-known angiogenesis inhibitor, however, its role in regulating lymphangiogenesis is not clear. We recently reported that global CD47-deficient mice are protected from atherosclerosis, however, myeloid cell-specific CD47 loss augments lesion formation suggesting the importance of cell-specific CD47 blockade in atherosclerosis. Our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we herein investigated the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerotic lesion formation. Our immunoblotting data demonstrated elevated TSP1 expression in human and mouse atherosclerotic aortic tissue compared to non-atherosclerotic control tissue. TSP1 at pathological concentrations inhibited VEGF-C-stimulated in vitro lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF-C-induced Akt activation in LECs, leading to decreased eNOS phosphorylation and attenuated nitric oxide production. Further, TSP1-treated CD47-silenced LECs proliferate faster and have higher Akt and eNOS activation than TSP1-treated control cells. AAV8-PCSK9-injected LEC-specific CD47 knockout ( Cd47 f/f Lyve-1 Cre +/- ) mice had reduced atherosclerosis in both aorta and aortic root compared with control ( Cd47 f/f ) mice. Additional immunostaining experiments performed on aortic root cross-sections indicated improved lymphatic vessel density in Cd47 f/f Lyve-1 Cre +/- mice. These results demonstrate that TSP1-mediated LEC CD47 activation inhibits lymphangiogenesis and blockade of LEC CD47 signaling suppresses atherosclerosis. Altogether, these findings identify LEC CD47 as a potential therapeutic target in atherosclerosis.

MDS-482 Impact Of Magrolimab in Combination With Azacitidine on Red Blood Cells (RBCs) in Patients With Higher-Risk Myelodysplastic Syndromes (HR MDS)

Magrolimab is an antibody blocking CD47, a "don't eat me" signal expressed on cancer cells, to escape immune surveillance and macrophage-mediated clearance. Preclinical studies found that CD47 is critical to RBC homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns that potential on-target anemia could result from the use of anti-CD47 agents. Several clinical trials demonstrated that magrolimab can be safely administered as monotherapy, with an initial lower "priming" dose yielding transient anemia with compensatory reticulocytosis and no anemia observed at higher maintenance doses. However, the underlying mechanism has not been fully defined. To describe manageable anemia in magrolimab-treated patients and further investigate the underlying mechanisms in preclinical models. Prospective analysis from a ph1 trial of magrolimab+azacitidine (NCT03248479). Complete blood counts (CBCs), peripheral blood, and bone marrow (BM) were collected from patients at prespecified time points. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for CD47 expression on RBCs and white blood cells (WBCs). Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice. 57 patients with HR MDS. Magrolimab IV 1 mg/kg (priming) then 30 mg/kg QW, then Q2W (maintenance). Azacitidine 75 mg/m2 days 1-7 (each 28-day cycle). Treatment with magrolimab+azacitidine resulted in tolerable anemia that correlated with rapid, near-complete loss of CD47 in RBCs but not WBCs. The initial 1-mg/kg priming dose was sufficient for CD47 loss, which persisted with subsequent 30-mg/kg maintenance doses. Both findings are consistent with prior clinical observations of magrolimab monotherapy in patients with solid tumors and magrolimab+rituximab in patients with lymphoma. Our preclinical studies with mouse models revealed that CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 cross-linking between RBCs and non-RBCs. These results support the idea that on-target magrolimab-mediated anemia is mitigated by a near-complete loss of RBC CD47. Patients with HR MDS treated with magrolimab+azacitidine had tolerable anemia with priming and maintenance doses.

Loss of CD47 alters CD8+ T cell activation in vitro and immunodynamics in mice

ABSTRACT CD47 has established roles in the immune system for regulating macrophage phagocytosis and lymphocyte activation, with growing evidence of its cell-intrinsic regulatory roles in natural killer and CD8+ T cells. CD47 limits antigen-dependent cytotoxic activities of human and murine CD8+ T cells, but its role in T cell activation kinetics remains unclear. Using in vitro and in vivo models, we show here that CD47 differentially regulates CD8+ T cell responses to short- versus long-term activation. Although CD47 was not required for T cell development in mice and early activation in vitro, short-term stimuli elevated pathogen-reactive gene expression and enhanced proliferation and the effector phenotypes of Cd47-deficient relative to Cd47-sufficient CD8+ T cells. In contrast, persistent TCR stimulation limited the effector phenotypes of Cd47 −/− CD8+ T cells and enhanced their apoptosis signature. CD8+ T cell expansion and activation in vivo induced by acute lymphocytic choriomeningitis virus (LCMV) infection did not differ in the absence of CD47. However, the frequency and effector phenotypes of Cd47−/− CD8+ T cells were constrained in chronic LCMV-infected as well as in mice bearing B16 melanoma tumors. Therefore, CD47 regulates CD8+ T cell activation, proliferation, and fitness in a context-dependent manner.

Single vesicle analysis of CD47 association with integrins and tetraspanins on extracellular vesicles released by T lymphoblast and prostate carcinoma cells.

CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47‐deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell‐derived EVs, β1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell‐derived EVs lacked detectable α4 integrin, specific association of CD81 with β1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced β1 and α4 levels on EVs produced by these cells while elevating CD9+, CD63+, and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non‐interacting tetraspanins via mechanisms that remain to be determined.

Impact of magrolimab treatment in combination with azacitidine on red blood cells in patients with higher-risk myelodysplastic syndrome (HR-MDS).

7054 Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal expressed on cancer cells to escape immune surveillance and macrophage-mediated clearance. Prior preclinical studies have shown that CD47 is critical to RBC homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns for potential on-target anemia from anti-CD47 agents via multiple mechanisms. Notwithstanding, several clinical trials have demonstrated that magrolimab can be safely administered as a monotherapy with initial lower “priming” dose yielding transient anemia with compensatory reticulocytosis, with anemia not observed at subsequent higher maintenance doses. However, the mechanism underlying this observed protection has not been fully defined. Here we describe manageable anemia in patients (pts) with HR-MDS treated with magrolimab in combination with azacitidine (AZA) (NCT03248479) and further investigate these underlying mechanisms in preclinical models. Methods: In a multicenter prospective study, CBCs, peripheral blood, and bone marrow (BM) were collected at prespecified timepoints from HR-MDS pts (n = 57) treated with magrolimab in combination with AZA. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for expression of CD47 on RBCs and WBCs. Magrolimab was initially dosed with a priming dose (1mg/kg) followed by an initial weekly maintenance dosing (30mg/kg) before transitioning to every 2 weeks maintenance dosing. AZA 75mg/m2 was administered on days 1-7 of the 28-day cycle. Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice. Results: Combination treatment of magrolimab with AZA resulted in a tolerable anemia that correlated with rapid, near complete loss of CD47 from RBCs, but not WBCs. The initial 1mg/kg priming dose was sufficient for this CD47 loss, which persisted under subsequent 30mg/kg maintenance doses. Both findings are consistent with prior clinical observations in solid tumor pts with magrolimab monotherapy and lymphoma pts in combination with rituximab. Our preclinical studies with mouse models revealed that the CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 crosslinking between RBCs and non-RBCs. Conclusions: Overall, these results support that on-target magrolimab mediated anemia is mitigated by a near complete loss of RBC CD47. HR-MDS patients treated with magrolimab in combination with AZA exhibit a tolerable anemia through priming and maintenance doses. Clinical trial information: NCT03248479.

The bovine dialysable leukocyte extract IMMUNEPOTENT CRP induces immunogenic cell death in breast cancer cells leading to long-term antitumour memory

BackgroundCancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells.MethodsImmunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice.ResultsICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes.ConclusionsICRP induces ICD in BC cells, leading to long-term antitumour memory.

CD47-SIRPα Axis as a Biomarker and Therapeutic Target in Cancer: Current Perspectives and Future Challenges in Nonsmall Cell Lung Cancer.

CD47 is a cell surface protein in the immunoglobulin superfamily which is normally expressed at low levels in every healthy cell. It´s main physiologic function is to act as an inhibitor of phagocytosis; this occurs throughout interaction with SIRPa expressed on macrophages. Interaction between CD47 and SIRPa leads to activation of tyrosine phosphatases that inhibit myosin accumulation at the submembrane assembly site of the phagocytic synapse, resulting in phagocytosis blockade. In this way CD47 acts as a “don´t eat me signal” for healthy self-cells; accordingly, loss of CD47 leads to phagocytosis of aged or damaged cells. Taking advantage of this anti-phagocytic signal provided by CD47, many types of tumors overexpress this protein, thereby avoiding phagocytosis by macrophages and aiding in the survival of cancer cells. The aim of this review is to describe the physiologic the pathophysiologic role of CD47; summarize the available high-quality information about this molecule as a potential biomarker and/or therapeutic target in cancer; finally, we present an in-depth analysis of the available information about CD47 in association with nonsmall cell lung cancer, EGFR mutations, and tumor microenvironment.

Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo.

Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.

Molecular Mechanisms of Stem Cell Reprogramming by CD47 Antagonists in Primary Human Cells

CD47 is a ubiquitously expressed transmembrane receptor whose interaction with signal regulatory protein alpha on macrophages initiates anti‐phagocytic signaling. Additionally, thrombospondin‐1 (THBS1) binding to CD47 induces cell‐autonomous signaling that regulates cell proliferation, differentiation, and recovery from genotoxic stress. Previous studies showed that CD47‐ or THBS1‐null primary lung endothelial cells exposed to ionizing radiation have higher viability than primary wildtype cells. The mechanisms that enhance the viability of CD47‐null primary cells include enhanced autophagy, anabolic metabolism, and increased expression of c‐Myc and other stem cell transcription factors. The latter enable null cells to spontaneously form embryoid bodies. These results suggest that loss of CD47 may generally facilitate cell reprogramming and self‐renewal. Induced pluripotent stem cells (iPS) can be induced by introducing Yamanaka stem cell factors (MYC, KLF4, SOX2, and OCT4) into primary somatic cells, but iPS cells can form teratomas and thus pose risk of cancer when used for regenerative medicine. Therefore, alternative approaches are needed to overcome these limitations. We propose that CD47 gene knock out (KO) via CRISPR or treating with CD47 antagonists from primary human cells could be a new, simpler, and more effective way to develop multipotent stem cells. To investigate this, we have studied the effects of CD47 knockout and antagonists on embryoid body formation and mRNA expression of stem cell factors using fibroblasts, adipocytes, and human umbilical vein cells (HUVEC). Reprogramming was measured by quantifying embryoid body formation, cell viability, and mRNA expression using real‐time PCR. Preliminary results with HUVEC, fibroblast, and adipocyte primary cells treated with CD47 knockout or antagonists have shown enhanced cell proliferation and expression of stem cell factors similar to murine cd47‐null lung endothelial cells. These results suggest that CD47 plays a key regulatory role in stem cell reprogramming of human primary cells.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIH/National Cancer Institute.

CD47 Regulates Red Blood Cell Alloimmunization in Mice

Background: Exposure to red blood cell (RBC) alloantigens during pregnancy or transfusion can lead to the development of alloantibodies and result in transfusion-related complications, including hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. However, the factors that regulate RBC alloimmunization remain incompletely understood. Several studies suggest that alterations in factors that regulate RBC clearance may impact RBC uptake and antigen presentation, directly influencing the likelihood of RBC alloimmunization. To test this, we directly examined the potential role of CD47, a master regulator of RBC removal previously shown to be altered during RBC senescence and cold storage. To accomplish this, we crossed transgenic mice that express the model HOD antigen (a fusion protein consisting of hen egg lysozyme fused to ovalbumin and human Duffy b) with CD47 knock out (KO) mice to generate HOD RBC donors with wild type, heterozygous or homozygous KO levels of CD47 and used these donors to define the impact of CD47 on antibody formation following RBC transfusion. Methods: HOD transgenic mice expressing the HOD antigen exclusively on RBCs were crossed with CD47-/- mice to produce HOD CD47+/- or HOD CD47-/- mice. HOD and CD47 levels were assessed by flow cytometric analysis using anti-HOD and anti-CD47 antibodies. HOD CD47+/+, HOD CD47+/- or HOD CD47-/- RBCs were transfused into C57BL6 recipients, followed by serum collection on days 14 and 28 post transfusion and evaluation of anti-HOD antibodies by flow cytometry crossmatch. To determine the CD4 T cell response to transfusion, TCR transgenics specific to ovalbumin (OTII) were labeled with CFSE, followed by adoptive transfer, transfusion of HOD CD47+/+, HOD CD47+/- or HOD CD47-/- RBCs and evaluation of T cell proliferation, activation and cytokine secretion. Cellular removal of HOD RBCs was determined by flow cytometric examination of CFSE-labeled HOD RBCs. Finally, antigen levels on HOD RBCs was determined by staining cells with anti-HEL antibodies followed by flow cytometric examination. All three groups were subjected to one-way ANOVA analysis with a p value <0.05 considered significant. Results: While HOD CD47+/+ RBCs expressed levels of CD47 comparable to WT RBCs, HOD CD47+/- RBCs exhibited significantly reduced CD47 levels (nearly half that observed on WT HOD RBCs) and HOD CD47-/-RBCs failed to express any detectable CD47 (p < 0.0001). Following transfusion, HOD CD47+/- and HOD CD47-/- RBCs produced significantly higher levels of IgG anti-HOD antibodies than HOD CD47+/+ RBCs on days 14 and 28 post-transfusion (p < 0.001). However, while HOD CD47-/- RBCs displayed increased clearance consistent with the possible enhancement of a CD4 T cell response (p < 0.0001), HOD CD47+/- RBCs failed to exhibit any different in clearance when compared to HOD CD47+/+ RBCs overtime. To examine the potential impact of differences in HOD RBC clearance on CD4 T cell activation, OTII T cell proliferation was evaluated. While HOD CD47-/- RBC transfusion resulted in significantly increased 33D1+ dendritic cell uptake, OTII proliferation, activation and cytokine secretion (p < 0.05), no difference in 33D1+ dendritic cell uptake or T cell response was observed following HOD CD47+/+ RBC or HOD CD47+/- RBC transfusion. Instead, HOD CD47+/- RBCs exhibited enhanced antigen removal, while also displaying an increased ability to activate HEL specific B cells when compared to HOD CD47+/+ or HOD CD47-/- RBC transfusion. Conclusions: These results demonstrate that alterations in CD47, which occur during normal RBC senescence and cold storage, directly influence RBC alloimmunization through different mechanisms depending on the extent of CD47 loss. While complete loss of CD47 results in enhanced RBC clearance, antigen presentation and CD4 T cell activation, reductions in CD47 to half WT levels failed to impact RBC clearance or T cell activation, but instead enhanced antigen specific B cell activation. These results demonstrate that even partial loss of CD47 is capable of significantly enhancing alloantibody formation completely independent of its role in regulating RBC clearance. In doing so, these findings provide novel insight into the role of CD47 as a key regulator of RBC alloimmunization. Disclosures Stowell: Grifols: Honoraria.

Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial β1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-β1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.

Increased lymphocyte activation and atherosclerosis in CD47-deficient mice

CD47, also known as integrin-associated protein (IAP), is a transmembrane protein with multiple biological functions including regulation of efferocytosis and leukocyte trafficking. In this study we investigated the effect of CD47-deficiency on atherosclerosis using a model of adeno-associated virus (AAV)-induced hypercholesterolemia. We observed increased plaque formation in CD47 null mice compared to wild-type controls. Loss of CD47 caused activation of dendritic cells, T cells and natural killer (NK) cells, indicating an important role for CD47 in regulating immunity. In particular, Cd47 deficiency increased the proportion of IFN-γ producing CD90+ NK cells. Treatment with depleting anti-NK1.1 monoclonal antibody (mAb), but not depleting anti-CD4/CD8 mAbs, equalized atherosclerotic burden, suggesting NK cells were involved in the enhanced disease in Cd47 deficient mice. Additional studies revealed that levels of CD90+ and IFN-γ+ NK cells were expanded in atherosclerotic aorta and that CD90+ NK cells produce more IFN-γ than CD90- NK cells. Finally, we demonstrate that anti-CD47 (MIAP410) causes splenomegaly and activation of DCs and T cells, without affecting NK cell activation. In summary, we demonstrate that loss of CD47 causes increased lymphocyte activation that results in increased atherosclerosis.

Extracellular Matrix Protein Tenascin C Increases Phagocytosis Mediated by CD47 Loss of Function in Glioblastoma.

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47-SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47-/-) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor-TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47-/- tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway-mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47-/- xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47-/- tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFα via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells. SIGNIFICANCE: These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system-based therapies for brain tumors.

CHAPLE syndrome uncovers the primary role of complement in a familial form of Waldmann's disease.

Primary intestinal lymphangiectasia (PIL) or Waldmann's disease was described in 1961 as an important cause of protein-losing enteropathy (PLE). PIL can be the sole finding in rare individuals or occur as part of a multisystemic genetic syndrome. Although genetic etiologies of many lymphatic dysplasia syndromes associated with PIL have been identified, the pathogenesis of isolated PIL (with no associated syndromic features) remains unknown. Familial cases and occurrence at birth suggest genetic etiologies in certain cases. Recently, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (the CHAPLE syndrome) has been identified as a monogenic form of PIL. Surprisingly, loss of CD55, a key regulator of complement system leads to a predominantly gut condition. Similarly to other complement disorders, namely paroxysmal nocturnal and hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), CHAPLE disease involves pathogenic cross-activation of the coagulation system, predisposing individuals to severe thrombosis. The observation that complement system is overly active in CHAPLE disease introduced a novel concept into the management of PLE; anti-complement therapy. While CD55 deficiency constitutes a treatable subgroup in the larger pool of patients with isolated PIL, the etiology remains to be identified in the remaining patients with intact CD55.