Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice.

Severe aplastic anemia (SAA) is a rare, fatal disease characterized by severe cytopenias and loss of hematopoietic stem cells (HSCs). Immune-mediated destruction and inflammation are known drivers of SAA, however, the underlying mechanisms driving persistent inflammation are unknown. Current treatments for SAA rely on immunosuppressive therapies or HSC transplantation, however, these treatments are not always effective. Using an established mouse model of SAA, we observed a significant increase in apoptotic cells within the bone marrow (BM) and impaired efferocytosis in SAA mice, relative to radiation controls. Single-cell transcriptomic analysis revealed heterogeneity among BM monocytes and unique populations emerged during SAA characterized by increased inflammatory signatures and significantly increased expression of Sirpa and Cd47. CD47, a "don't eat me" signal, was increased on both live and apoptotic BM cells, concurrent with markedly increased expression of signal regulatory protein alpha (SIRPα) on monocytes. Functionally, SIRPα blockade improved cell clearance and reduced accumulation of CD47-positive apoptotic cells. Lipidomic analysis revealed a reduction in the precursors of specialized pro-resolving lipid mediators (SPMs) and increased prostaglandins in the BM during SAA, indicative of impaired inflammation resolution. Specifically, 18-HEPE, a precursor of E-series resolvins, was significantly reduced in SAA-induced mice relative to radiation controls. Treatment of SAA mice with Resolvin E1 (RvE1) improved efferocytic function, BM cellularity, platelet output, and survival. Our data suggest that impaired efferocytosis and inflammation resolution contributes to SAA progression and demonstrate that SPMs, such as RvE1, offer new and/or complementary treatments for SAA that do not rely on immune suppression.

Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

Enhancing anticancer activity of macrophages through rational drug combinations.

Targeting tumor-associated macrophages (TAMs) is an emerging approach being tested in multiple clinical trials. TAMs, depending on their differentiation state, can exhibit pro- or antitumorigenic functions. For example, the M2-like phenotype represents a protumoral state that can stimulate tumor growth, angiogenesis, metastasis, therapy resistance, and immune evasion by expressing immune checkpoint proteins. In this issue of the JCI, Vaccaro and colleagues utilized an innovative drug screen approach to demonstrate that targeting driver oncogenic signaling pathways concurrently with anti-CD47 sensitizes tumor cells, causing them to undergo macrophage-induced phagocytosis. The combination treatment altered expression of molecules on the tumor cells that typically limit phagocytosis. It also reprogrammed macrophages to an M1-like antitumor state. Moreover, the approach was generalizable to tumor cells with different oncogenic pathways, opening the door to precision oncology-based rationale combination therapies that have the potential to improve outcomes for patients with oncogene-driven lung cancers and likely other cancer types.

CD47 blockade reduces ischemia/reperfusion injury in murine heart transplantation and improves donor heart preservation.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is an important cause of early dysfunction and exacerbation of immune rejection in transplanted hearts. The integrin-related protein CD47 exacerbates myocardial ischemia-reperfusion injury by inhibiting the nitric oxide signaling pathway through interaction with thrombospondin-1 (TSP-1). In addition, the preservation quality of the donor hearts is a key determinant of transplant success. Preservation duration beyond four hours is associated with primary graft dysfunction. We hypothesized that blocking the CD47-TSP-1 system would attenuate ischemia-reperfusion injury in the transplanted heart and, thus, improve the preservation of donor hearts. METHODS: We utilized a syngeneic mouse heart transplant model to assess the effect of CD47 monoclonal antibody (CD47mAb) to treat MIRI. Donor hearts were perfused with CD47mAb or an isotype-matched control immunoglobulin (IgG2a) and were implanted into the abdominal cavity of the recipients after being stored in histidine-tryptophan-ketoglutarate (HTK) solution at 4 °C for 4 h or 8 h. RESULTS: At both the 4-h and 8-h preservation time points, mice in the experimental group perfused with CD47mAb exhibited prolonged survival in the transplanted heart, reduced inflammatory response and oxidative stress, significantly decreased inflammatory cell infiltration, and fewer apoptosis-related biomarkers. CONCLUSION: The application of CD47mAb for the blocking of CD47 attenuates MIRI as well as improves the preservation and prognosis of the transplanted heart in a murine heart transplant model.

Discovery of a novel small molecule as CD47/SIRPα and PD-1/PD-L1 dual inhibitor for cancer immunotherapy.

BACKGROUND: Targeting the tumor microenvironment (TME) has emerged as a promising strategy in cancer treatment, particularly through the utilization of immune checkpoint blockade (ICB) agents such as PD-1/PD-L1 inhibitors. Despite partial success, the presence of tumor-associated macrophages (TAMs) contributes to an immunosuppressive TME that fosters tumor progression, and diminishes the therapeutic efficacy of ICB. Blockade of the CD47/SIRPα pathway has proven to be an effective intervention, that restores macrophage phagocytosis and yields substantial antitumor effects, especially when combined with PD-1/PD-L1 blockade. Therefore, the identification of small molecules capable of simultaneously blocking CD47/SIRPα and PD-1/PD-L1 interactions has remained imperative. METHODS: SMC18, a small molecule with the capacity of targeting both SIRPα and PD-L1 was obtained using MST. The efficiency of SMC18 in interrupting CD47/SIRPα and PD-1/PD-L1 interactions was tested by the blocking assay. The function of SMC18 in enhancing the activity of macrophages and T cells was tested using phagocytosis assay and co-culture assay. The antitumor effects and mechanisms of SMC18 were investigated in the MC38-bearing mouse model. RESULTS: SMC18, a small molecule that dual-targets both SIRPα and PD-L1 protein, was identified. SMC18 effectively blocked CD47/SIRPα interaction, thereby restoring macrophage phagocytosis, and disrupted PD-1/PD-L1 interactions, thus activating Jurkat cells, as evidenced by increased secretion of IL-2. SMC18 demonstrated substantial inhibition of MC38 tumor growths through promoting the infiltration of CD8+ T and M1-type macrophages into tumor sites, while also priming the function of CD8+ T cells and macrophages. Moreover, SMC18 in combination with radiotherapy (RT) further improved the therapeutic efficacy. CONCLUSION: Our findings suggested that the small molecule compound SMC18, which dual-targets the CD47/SIRPα and PD-1/PD-L1 pathways, could be a candidate for promoting macrophage- and T-cell-mediated phagocytosis and immune responses in cancer immunotherapy.

A comprehensive analysis of CD47 expression in various histological subtypes of soft tissue sarcoma: exploring novel opportunities for macrophage-directed treatments.

PURPOSE: The CD47 molecule, often referred to as the "do not eat me" signal, is frequently overexpressed in tumor cells. This signaling pathway limits phagocytosis by macrophages. Our objective was to determine CD47 abundance in various soft tissue sarcomas (STS) to investigate whether it could serve as a potential evasion mechanism for tumor cells. Additionally, we aimed to assess the prognostic value of CD47 expression by examining its association with different clinicopathological factors. This study aimed to elucidate the significance of CD47 in the context of emerging anti-tumor targeting approaches. METHODS: In this retrospective study, formalin-fixed paraffine-embedded (FFPE) tumor tissues of 55 treatment-naïve patients were evaluated by immunohistochemistry for the abundance of CD47 molecule on tumor cells. The categorization of CD47 positivity was as follows: 0 (no staining of tumor cells), 1 + (less than 1/3 of tumor area positive), 2 + (between 1/3 and 2/3 of tumor area positive), and 3 + (more than 2/3 of tumor area positive for CD47). Next, we compared CD47 abundance between different tumor grades (G1-3). We used Kaplan-Meier survival curves with log-rank test to analyze the differences in survival between patients with different CD47 expression. Moreover, we performed Cox proportional hazards regression model to evaluate the clinical significance of CD47. RESULTS: CD47 is widely prevalent across distinct STS subtypes. More than 80% of high grade undifferentiated pleiomorphic sarcoma (UPS), 70% of myxofibrosarcoma (MFS) and more than 60% of liposarcoma (LPS) samples displayed a pattern of moderate-to-diffuse positivity. This phenomenon remains consistent regardless of the tumor grade. However, there was a tendency for higher CD47 expression levels in the G3 group compared to the combined G1 + G2 groups when all LPS, MFS, and UPS were analyzed together. No significant associations were observed between CD47 abundance, death, and metastatic status. Additionally, high CD47 expression was associated with a statistically significant increase in progression-free survival in the studied cohort of patients. CONCLUSION: This study highlights the potential of the CD47 molecule as a promising immunotherapeutic target in STS, particularly given its elevated expression levels in diverse sarcoma types. Our data showed a notable trend linking CD47 expression to tumor grade, while also suggesting an interesting correlation between enhanced abundance of CD47 expression and a reduced hazard risk of disease progression. Although these findings shed light on different roles of CD47 in STS, further research is crucial to assess its potential in clinical settings.

Expression of a mutant CD47 protects against phagocytosis without inducing cell death or inhibiting angiogenesis.

CD47 is a ligand of SIRPα, an inhibitory receptor expressed by macrophages, dendritic cells, and natural killer (NK) cells, and, therefore, transgenic overexpression of CD47 is considered an effective approach to inhibiting transplant rejection. However, the detrimental effect of CD47 signaling is overlooked when exploring this approach. Here, we construct a mutant CD47 by replacing the transmembrane and intracellular domains with a membrane anchor (CD47-IgV). In both human and mouse cells, CD47-IgV is efficiently expressed on the cell surface and protects against phagocytosis in vitro and in vivo but does not induce cell death or inhibit angiogenesis. Furthermore, hematopoietic stem cells expressing transgenic CD47-IgV show no detectable alterations in engraftment or differentiation. This study provides a potentially effective means of achieving transgenic CD47 expression that may help to produce gene-edited pigs for xenotransplantation and hypoimmunogenic pluripotent stem cells for regenerative medicine.

Opportunities and challenges for anti-CD47 antibodies in hematological malignancies.

CD47 is a cell-surface ligand that is overexpressed in various malignancies and that binds to SIRPα on macrophages to promote tumor cell evasion of phagocytosis. Blocking the CD47-SIRPα axis can increase the phagocytosis of macrophages to exert antitumor effects. CD47-based immunotherapy is a current research focus. The combination of anti-CD47 antibodies with other drugs has shown encouraging response rates in patients with hematological tumors, but side effects also occur. Bispecific antibodies and SIRPα/Fc fusion proteins appear to balance the efficacy and safety of treatment. We review the latest clinical research advances and discuss the opportunities and challenges associated with CD47-based immunotherapy for hematological malignancies.

Structural-functional diversity of CD47 proteoforms.

The ubiquitously expressed transmembrane glycoprotein CD47 participates in various important physiological cell functions, including phagocytosis, apoptosis, proliferation, adhesion, and migration, through interactions with its ligands, including the inhibitory receptor signal regulatory protein α (SIRPα), secreted glycoprotein thrombospondin-1 (TSP-1), and integrins. Elevated expression of CD47 is observed in a wide range of cancer cells as a mechanism for evading the immune system, blocking the interaction between the CD47 and SIRPα is the most advanced and promising therapeutic approach currently investigated in multiple clinical trials. The widely held view that a single type of CD47 protein acts through membrane interactions has been challenged by the discovery of a large cohort of CD47 proteins with cell-, tissue-, and temporal-specific expression and functional profiles. These profiles have been derived from a single gene through alternative splicing and post-translational modifications, such as glycosylation, pyroglutamate modification, glycosaminoglycan modification, and proteolytic cleavage and, to some extent, via specific CD47 clustering in aging and tumor cells and the regulation of its subcellular localization by a pre-translational modification, alternative cleavage and polyadenylation (APA). This review explores the origins and molecular properties of CD47 proteoforms and their roles under physiological and pathological conditions, mentioning the new methods to improve the response to the therapeutic inhibition of CD47-SIRPα immune checkpoints, contributing to the understanding of CD47 proteoform diversity and identification of novel clinical targets and immune-related therapeutic candidates.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed an unbiased, high-throughput screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we found that therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found that the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized antitumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations - including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of ß2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Enhancing CAR Macrophage Efferocytosis Via Surface Engineered Lipid Nanoparticles Targeting LXR Signaling.

The removal of dying cells, or efferocytosis, is an indispensable part of resolving inflammation. However, the inflammatory microenvironment of the atherosclerotic plaque frequently affects the biology of both apoptotic cells and resident phagocytes, rendering efferocytosis dysfunctional. To overcome this problem, a chimeric antigen receptor (CAR) macrophage that can target and engulf phagocytosis-resistant apoptotic cells expressing CD47 is developed. In both normal and inflammatory circumstances, CAR macrophages exhibit activity equivalent to antibody blockage. The surface of CAR macrophages is modified with reactive oxygen species (ROS)-responsive therapeutic nanoparticles targeting the liver X receptor pathway to improve their cell effector activities. The combination of CAR and nanoparticle engineering activated lipid efflux pumps enhances cell debris clearance and reduces inflammation. It is further suggested that the undifferentiated CAR-Ms can transmigrate within a mico-fabricated vessel system. It is also shown that our CAR macrophage can act as a chimeric switch receptor (CSR) to withstand the immunosuppressive inflammatory environment. The developed platform has the potential to contribute to the advancement of next-generation cardiovascular disease therapies and further studies include in vivo experiments.

Restoration of miR-299-3p promotes macrophage phagocytosis and suppresses malignant phenotypes in breast cancer carcinogenesis via dual-targeting CD47 and ABCE1.

Immunoevasion has been a severe obstacle for the clinical treatment of breast cancer (BC). CD47, known as an anti-phagocytic molecule, plays a key role in governing the evasion of tumor cells from immune surveillance by interacting with signal-regulated protein α (SIRPα) on macrophages. Here, we report for the first time that miR-299-3p is a direct regulator of CD47 with tumor suppressive effects both in vitro and in vivo. miRNA expression profiles and overall survival of BC cohorts from the Cancer Genome Atlas, METABRIC, or GSE19783 datasets showed that miR-299-3p is downregulated in BC tissues and that BC patients with low levels of miR-299-3p have poorer prognoses. Using dual-luciferase reporter, qRT-PCR, Western blot, and phagocytosis assays, we proved that restoration of miR-299-3p can suppress CD47 expression by directly targeting the predicted seed sequence "CCCACAU" in its 3'-UTR, leading to phagocytosis of BC cells by macrophages, whereas miR-299-3p inhibition or deletion reversed this effect. Additionally, Gene Ontology (GO) analysis and a variety of confirmatory experiments revealed that miR-299-3p was inversely correlated with cell proliferation, migration, and the cell cycle process. Mechanistically, miR-299-3p can also directly target ABCE1, an essential ribosome recycling factor, alleviating these malignant phenotypes of BC cells. In vivo BC xenografts based on nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice further proved that restoration of miR-299-3p resulted in a significant suppression of tumorigenesis and a promotion of macrophage activation and infiltration. Overall, our study suggested that miR-299-3p is a potent inhibitor of CD47 and ABCE1 to exhibit bifunctional BC-suppressing effects through immune activation conjugated with malignant behavior inhibition in breast carcinogenesis and thus can potentially serve as a novel therapeutic target for BC.

Tolerating CD47.

Cluster of differentiation 47 (CD47) occupies the outer membrane of human cells, where it binds to soluble and cell surface receptors on the same and other cells, sculpting their topography and resulting in a pleiotropic receptor-multiligand interaction network. It is a focus of drug development to temper and accentuate CD47-driven immune cell liaisons, although consideration of on-target CD47 effects remain neglected. And yet, a late clinical trial of a CD47-blocking antibody was discontinued, existent trials were restrained, and development of CD47-targeting agents halted by some pharmaceutical companies. At this point, if CD47 can be exploited for clinical advantage remains to be determined. Herein an airing is made of the seemingly conflicting actions of CD47 that reflect its position as a junction connecting receptors and signalling pathways that impact numerous human cell types. Prospects of CD47 boosting and blocking are considered along with potential therapeutic implications for autoimmune diseases and cancer.

Targeting HDAC6 improves anti-CD47 immunotherapy.

BACKGROUND: Cancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages. METHODS: We tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models. RESULTS: We observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment. CONCLUSIONS: Our results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies.

Reprogramming Exosomes to Escape from Immune Surveillance for Mitochondrial Protection in Hepatic Ischemia-Reperfusion Injury.

Background: Therapeutic interventions such as synthetic drugs and microRNA (miR) modulators have created opportunities for mitigating hepatic ischemia/reperfusion injury (HIRI) by alleviating mitochondrial dysfunction. However, delivering multi-therapeutic ingredients with low toxicity to hepatocytes still lags behind its development. Methods: In this study, we endowed exosomes with delivery function to concentrate on hepatocytes for multidimensionally halting mitochondria dysfunction during HIRI. Concretely, exosomes were reprogrammed with a transmembrane protein CD47, which acted as a "camouflage cloak" to mimic the "don't eat me" mechanism to escape from immune surveillance. Besides, HuR was engineered bridging to the membrane by fusing with CD47 and located in the cytoplasm for miR loading. Results: This strategy successfully delivered dual payloads to hepatocytes and efficiently protected mitochondria by inhibiting the opening of mitochondrial permeability transition pore (mPTP) and upregulating mitochondrial transcription factor A (TFAM), respectively. Conclusions: The reprogramming of exosomes with CD47 and HuR for targeted delivery of CsA and miR inhibitors represents a promising therapeutic strategy for addressing HIRI. This approach shows potential for safe and effective clinical applications in the treatment of HIRI.

Sirpα on tumor-associated myeloid cells restrains antitumor immunity in colorectal cancer independent of its interaction with CD47.

Immunosuppressive myeloid cells hinder immunotherapeutic efficacy in tumors, but the precise mechanisms remain undefined. Here, by performing single-cell RNA sequencing in colorectal cancer tissues, we found tumor-associated macrophages and granulocytic myeloid-derived suppressor cells increased most compared to their counterparts in normal tissue and displayed the highest immune-inhibitory signatures among all immunocytes. These cells exhibited significantly increased expression of immunoreceptor tyrosine-based inhibitory motif-bearing receptors, including SIRPA. Notably, Sirpa-/- mice were more resistant to tumor progression than wild-type mice. Moreover, Sirpα deficiency reprogramed the tumor microenvironment through expansion of TAM_Ccl8hi and gMDSC_H2-Q10hi subsets showing strong antitumor activity. Sirpa-/- macrophages presented strong phagocytosis and antigen presentation to enhance T cell activation and proliferation. Furthermore, Sirpa-/- macrophages facilitated T cell recruitment via Syk/Btk-dependent Ccl8 secretion. Therefore, Sirpα deficiency enhances innate and adaptive immune activation independent of expression of CD47 and Sirpα blockade could be a promising strategy to improve cancer immunotherapy efficacy.

CD133, CD47, and PD-L1 Expression in Ovarian High-grade Serous Carcinoma and Its Association with Metastatic Disease: A Cross-sectional Study.

INTRODUCTION: Ovarian cancer is a primary cause of cancer-related death in women. At the time of diagnosis, the majority of ovarian malignancies had metastasized. It is believed that cancer stem cells (CSCs) and immune evasion play a crucial role in the metastatic process. The objective of this study was to describe the expression profiles of cluster of differentiation (CD)133, CD47, and programmed death ligand 1 (PD-L1) in high-grade serous ovarian cancer (HGSC) as commonly utilized markers for CSCs and immune evasion. MATERIAL AND METHODS: Using an immunohistochemical procedure, 51 HGSC tissue samples were stained with anti-CD133, anti-CD47, and anti-PDL1 antibodies. The samples contained 31 HGSC with metastases and 20 HGSC absent metastases. The expression of CD133, CD47, and PD-L1 was compared between groups. RESULTS: Strong expression of CD133 and CD47 was seen in 52% and 66% of tissue samples, respectively. Twenty of the thirty-one patients with metastases had a significant level of CD133 expression, with a p-value of 0.039. CD47 expression was increased in 26 of 31 samples with metastatic disease. A 62.7 percent of samples were negative for PD-L1 expression, significantly inversely correlated with HGSC metastatic disease (p=0.023). Although there was no significant association between CD133, CD47, or PD-L1 expression and age, Tumor Infiltrating Lymphocytes demonstrated a significantly varied relationship. CONCLUSION: Our findings suggested that expression of CD133, CD47, and PD-L1 may have dynamically increased as the primary lesion progressed to the metastatic lesion, implying that these proteins may be involved in the progression of high-grade serous ovarian cancer from the primary to the metastatic stage.

Pretreatment with Daudi cells eliminates anti-CD47 monoclonal antibody interference in immunohematology testing.

BACKGROUND: Anti-CD47 monoclonal antibodies have increasing clinical applications in the treatment of cancer. However, anti-CD47 monoclonal antibodies interfere with immunohematology testing in patients who require blood transfusion. As the current approaches to removing any interferences have technical problems, new methods need to be developed to resolve anti-CD47 interference in immunohematology testing. MATERIALS AND METHODS: We evaluated the Daudi cell line for the adsorption of free anti-CD47 monoclonal antibodies from patients' plasma to facilitate immunohematology testing in patients treated with anti-CD47 monoclonal antibody. CD47 expression was identified on the Daudi cells using flow cytometry and confocal microscopy. Next, we tested the ability of intact Daudi cells mixed with simulating plasma and clinical samples to achieve efficient removal of interfering anti-CD47 monoclonal antibodies. The indirect antiglobulin test was used to verify whether interference from anti-CD47 monoclonal antibodies in plasma was eliminated and whether the detection of other irregular antibodies was affected. The effect of eliminating interference was also investigated in relation to the time that the Daudi cells were stored after having been fixed with paraformaldehyde. RESULTS: CD47 expression was higher on Daudi cells than on red blood cells. Analysis of the indirect antiglobulin test results revealed that anti-CD47 monoclonal antibody-treated patients' plasma absorbed by Daudi cells for 15 min at 37°C could completely prevent the interference of anti-CD47 monoclonal antibodies in immunohematology testing while the detection of the tested antibodies, including anti-D and anti-K, was unaffected. DISCUSSION: By decreasing the incubation time, we discovered that interferences in samples with agglutination strengths below 2+ could be eliminated after incubation for 5 min. Of importance, Daudi cells can be preserved with 4% paraformaldehyde for 14 days as short-term storage reagents. This is the first study in which Daudi cells were used to effectively resolve the interference of anti-CD47 monoclonal antibodies in pretransfusion tests.

Immune proteins C1q and CD47 may contribute to aberrant microglia-mediated synapse loss in the aging monkey brain that is associated with cognitive impairment.

Cognitive impairment in learning, memory, and executive function occurs in normal aging even in the absence of Alzheimer's disease (AD). While neurons do not degenerate in humans or monkeys free of AD, there are structural changes including synapse loss and dendritic atrophy, especially in the dorsolateral prefrontal cortex (dlPFC), and these correlate with cognitive age-related impairment. Developmental studies revealed activity-dependent neuronal properties that lead to synapse remodeling by microglia. Microglia-mediated phagocytosis that may eliminate synapses is regulated by immune "eat me" and "don't eat me" signaling proteins in an activity-dependent manner, so that less active synapses are eliminated. Whether this process contributes to age-related synapse loss remains unknown. The present study used a rhesus monkey model of normal aging to investigate the balance between the "eat me" signal, complement component C1q, and the "don't eat me" signal, transmembrane glycoprotein CD47, relative to age-related synapse loss in dlPFC Area 46. Results showed an age-related elevation of C1q and reduction of CD47 at PSD95+ synapses that is associated with cognitive impairment. Additionally, reduced neuronal CD47 RNA expression was found, indicating that aged neurons were less able to produce the protective signal CD47. Interestingly, microglia do not show the hypertrophic morphology indicative of phagocytic activity. These findings suggest that in the aging brain, changes in the balance of immunologic proteins give microglia instructions favoring synapse elimination of less active synapses, but this may occur by a process other than classic phagocytosis such as trogocytosis.

Emerging functions of thrombospondin-1 in immunity.

Thrombospondin-1 is a secreted matricellular glycoprotein that modulates cell behavior by interacting with components of the extracellular matrix and with several cell surface receptors. Its presence in the extracellular matrix is induced by injuries that cause thrombospondin-1 release from platelets and conditions including hyperglycemia, ischemia, and aging that stimulate its expression by many cell types. Conversely, rapid receptor-mediated clearance of thrombospondin-1 from the extracellular space limits its sustained presence in the extracellular space and maintains sub-nanomolar physiological concentrations in blood plasma. Roles for thrombospondin-1 signaling, mediated by specific cellular receptors or by activation of latent TGFß, have been defined in T and B lymphocytes, natural killer cells, macrophages, neutrophils, and dendritic cells. In addition to regulating physiological nitric oxide signaling and responses of cells to stress, studies in mice lacking thrombospondin-1 or its receptors have revealed important roles for thrombospondin-1 in regulating immune responses in infectious and autoimmune diseases and antitumor immunity.