Expression Of SIRPα Research Articles

Targeting HDAC6 improves anti-CD47 immunotherapy

BackgroundCancer 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.

Microglia Promote Inhibitory Synapse Phagocytosis in the Spinal Cord Dorsal Horn and Modulate Pain-Like Behaviors in a Murine Cancer-Induced Bone Pain Model.

The microglial activation has been implicated in cancer-induced bone pain. Recent studies have revealed that microglia mediate synaptic pruning in the central nervous system, where the cluster of differentiation 47-signal regulatory protein α (CD47-SIRPα) axis creates a "don't eat me" signal and elicits an antiphagocytic effect to protect synapses against elimination. To date, the synaptic phagocytosis in microglia has never been investigated in the murine cancer-induced bone pain model. The present experiments sought to explore whether microglia phagocytize synapses in mice with bone cancer pain as well as the possible mechanisms. Male C3H/HeN mice were used to induce bone cancer pain. Minocycline and S-ketamine were injected into D14. The number of spontaneous flinches (NSF) and paw withdrawal mechanical thresholds (PWMT) were measured on D0, D4, D7, D10, D14, D21, and D28. Hematoxylin and eosin staining presented bone lesions. Western blotting examined the Gephyrin, CD47, and SIRPα expression. Flow cytometry evaluated the proportion of SIRPα + cells in the spine. Immunofluorescence and 3-dimensional reconstruction showed the Gephyrin puncta inside microglial lysosomes. Mice embedded with tumor cells induced persistent spontaneous pain and mechanical hyperalgesia. Hematoxylin and eosin staining revealed bone destruction and tumor infiltration in marrow cavities. Microglia underwent a responsive and proliferative burst (t = -16.831, P < .001). Western blotting manifested lowered Gephyrin expression in the tumor group (D4, D7, D10, D14, D21, and D28: P < .001). Immunofluorescence and 3-dimensional reconstruction showed larger volumes of Gephyrin puncta inside microglial lysosomes (t = -23.273, P < .001; t = -27.997, P < .001). Treatment with minocycline or S-ketamine exhibited pain relief and antiphagocytic effects (t = -6.191, P < .001, t = -7.083, P < .001; t = -20.767, P < .001, t = -17.080, P < .001; t = 11.789, P < .001, t = 16.777, P < .001; t = 8.868, P < .001, t = 21.319, P < .001). Last but not least, the levels of CD47 and SIRPα proteins were downregulated (D10: P = .004, D14, D21, and D28: P < .001; D10, D14, D21, and D28: P < .001). Flow cytometry and immunofluorescence substantiated reduced microglial SIRPα (t = 11.311, P < .001; t = 12.189, P < .001). Microglia-mediated GABAergic synapse pruning in the spinal cord dorsal horn in bone cancer pain mice, which might be associated with the declined CD47-SIRPα signal. Our research uncovered an innovative mechanism that highlighted microglia-mediated synaptic phagocytosis in a murine cancer-induced bone pain model.

A new role for erythropoietin in the homeostasis of red blood cells

The regulation of red blood cell (RBC) homeostasis is widely assumed to rely on the control of cell production by erythropoietin (EPO) and the destruction of cells at a fixed, species-specific age. In this work, we show that such a regulatory mechanism would be a poor homeostatic solution to satisfy the changing needs of the body. Effective homeostatic control would require RBC lifespan to be variable and tightly regulated. We suggest that EPO may control RBC lifespan by determining CD47 expression in newly formed RBCs and SIRP-α expression in sinusoidal macrophages. EPO could also regulate the initiation and intensity of anti-RBC autoimmune responses that curtail RBC lifespan in some circumstances. These mechanisms would continuously modulate the rate of RBC destruction depending on oxygen availability. The control of RBC lifespan by EPO and autoimmunity emerges as a key mechanism in the homeostasis of RBCs.

Anti-tumor effect of PTEN and its effect on inhibition of the bone tumor through the CD47-SIRPα signaling pathway.

This study aimed to explore the correlation between the expression of phosphatase and tensin homolog (PTEN), a tumor suppressor gene, and CD47-SIRPα signaling pathway, and clarify the underlying mechanisms of the bone tumor inhibition effect of PTEN. In this study, August x Copenhagen Irish (ACI) male rats were used, and 100μl of UMR-106 cell suspension (1´106 cells) was injected subcutaneously to induce the bone tumor model. The gene expression of PTEN, CD47 and SIRPα of both groups (control and bone tumor model) were analyzed by RT-PCR. For in vitro experiments, pEGFP-N1-PTEN plasmid was used to transfect the murine bone tumor UMR-106 and the human bone tumor KRIB cells. Also, we detected the invasiveness of the UMR-106 cells and KRIB cells after transfection. In this study, we observed that the gene expression of PTEN and SIRPα were significantly decreased in the ACI rats with bone tumors in comparison to the control group, however, the expression level of CD47 has been significantly increased. The tumor cells transfected with the pEGFP-N1-PTEN plasmid showed significantly higher levels of PTEN expression, however, the expression level of the CD47 gene has been decreased. Also, the invasion ability of tumor cells has been down-regulated. Also, we observed a negative correlation between the gene expression of the tumor suppressor gene PTEN and the CD47 and SIRPα genes. In summary, based on the anti-tumor effect of PTEN and its effect on inhibition of the bone tumor, it could be hypothesized that this phenomenon might be related to the phosphorylation of the CD47 and SIRPα gene.

TMIC-68. SIRPΑ BLOCKADE AS AN IMMUNOTHERAPEUTIC STRATEGY TO TREAT BREAST CANCER BRAIN METASTASIS

Abstract Triple-negative breast cancer (TNBC) has a high incidence of brain metastasis, with no standard therapeutic strategy for treatment. Therefore, new targets are urgently needed to overcome disease mortality. The CD47/SIRPα signaling pathway is implicated in tumor progression due to bypassing innate and adaptive immune surveillance. Most strategies targeting this pathway focus on targeting the receptor CD47; however, direct targeting SIRPα as a potential approach to mitigate metastatic burden remains understudied. Single-cell-RNA-sequencing indicates that SIRPα expression on basal epithelial cells, and TCGA data shows SIRPα expression is associated with a significant reduction in survival in basal-like breast cancer. Our immune staining against SIRPα in breast cancer patient biopsies shows a 3.5-fold increase in expression in metastatic lesions compared to the primary tumor (n=19; p ≤ 0.01). Staining of 4T1 parental and brain-trophic 4T1-Br3 cells showed an 84% increase in SIRPα in the metastatic cells (n=3; p ≤ 0.05). Furthermore, Real-time cell impedance analysis revealed that SIRPα blockade inhibits TNBC 4T1br3 cell migration (n=4; p≤0.01), suggesting a link between SIRPα expression and metastatic potential. Furthermore, in vivo SIRPα blockade reduced brain metastatic burden by approximately 90%, (n=4-7; p ≤ 0.05). Digital spatial profiling of tissues revealed that SIRPα blockade was associated with reductions in immune checkpoints, including CTLA-4, PD-1, PD-L1, and LAG3, and immunosuppressive T-cell/monocyte populations. Furthermore, our spatial analysis indicated that brain lesions of WT mice have increases in the extracellular matrix protein fibronectin in the immune enriched regions, potentially serving as a barrier for immune cell infiltration contributing to invasion, metastasis, and immune evasion, was reduced by 70% in SIRPα treated brain lesions (n-3-6; p ≤ 0.05). Thus, these data suggest that SIRPα blockade may influence the tumor microenvironment to limit brain metastatic breast cancer growth and potentially enhance patient survival to metastatic brain disease.

Clinicopathological analysis of CD47 and signal regulatory protein alpha expression in myeloid sarcoma patients: CD47 expression is a favourable prognostic factor

Myeloid sarcoma is a rare extramedullary haematopoietic malignancy. Interaction between CD47 and signal regulatory protein α (SIRPα) inhibits phagocytosis. CD47-positive tumours confer poor prognoses in various malignant tumours, including acute myeloid leukaemia. This study aimed to investigate the clinicopathological effects of CD47 and SIRPα expression in myeloid sarcoma. Immunohistochemistry (IHC) of CD47 and SIRPα was performed in 84 biopsy samples obtained from patients with myeloid sarcoma, some of which were CD47-positive. Patients were categorised into the following two groups based on IHC of SIRPα: those with SIRPα-positive neoplastic cells (nSIRPα) and, SIRPα expression on non-neoplastic stromal cells in tumour microenvironment (miSIRPα). In addition, patients with CD47 positivity had higher lymphocytic infiltration into the tumour microenvironment. Overall, these patients had significantly higher overall survival, however, no significant difference was observed in progression-free survival. No significant prognostic differences were observed between the nSIRPα and miSIRPα groups. This is the first study to demonstrate an association between CD47 expression and improved prognosis in myeloid sarcoma. Nonetheless, it will be necessary to conduct additional research on gene expression and genomic abnormalities to elucidate the corresponding pathogenesis of myeloid sarcoma.

SIRP-alpha-IL-6 axis induces immunosuppressive macrophages in non-small-cell lung cancer

Signal regulatory protein-alpha (SIRPα) and IL-6 participate in the induction of tumor immune suppressive environment and facilitate tumor growth. In this study, we found that SIRPα was significantly elevated in macrophages of non-small cell lung cancer (NSCLC) tissues, which was positively correlated to the expression of CD163, PD-1, IL-6, and lung cancer progression. SIRPα in peripheral blood mononuclear cells (PBMCs) of NSCLC patients was also associated with CD163, PD-1, and plasma IL-6. Blockade of SIRPα signaling in SIRPα ± and SIRPα−/− mice attenuated lung cancer growth and reduced IL-6 expression in LLC cells-transplanted murine lung cancer model. Co-targeting SIRPα and IL-6 additively suppressed the expression of IL-6 and activation of STAT3, accompanied with a reduced population of pro-tumorigenic CD206+ M2 subtype of macrophages, PD-1+ tumor-associated macrophages (TAMs), and PD-1+CD8+ T cells in tumor tissues of anti-IL-6 antibody (aIL-6)-treated mice deficient in SIRPα. Further in vitro studies showed that blockade of SIRPα signaling by anti-SIRPα effectively improved phagocytosis of human PBMCs. IL-6 treatment improved polarization of M2 subtypes and the expression of PD-1 in bone marrow-derived macrophages (BMDMs); whereas both aIL-6 and STAT3 inhibitor C188-9 suppressed the expression of PD-1 and SIRPα in BMDMs. M2 cell-biased polarization was also reduced in aIL-6 or C188-9 treated BMDMs. Thereby, SIRPα and IL-6 form a positive feedback loop and regulate each other through STAT3 signaling in macrophages. The increased SIRPα/IL-6 axis may promote immune suppressive environment and lung cancer growth, which may be a potential target for clinical treatment.

Airway macrophages display decreased expression of receptors mediating and regulating scavenging in early cystic fibrosis lung disease.

Cystic fibrosis (CF) airway disease is characterized by chronic inflammation, featuring neutrophil influx to the lumen. Airway macrophages (AMs) can promote both inflammation and resolution, and are thus critical to maintaining and restoring homeostasis. CF AM functions, specifically scavenging activity and resolution of inflammation, have been shown to be impaired, yet underlying processes remain unknown. We hypothesized that impaired CF AM function results from an altered expression of receptors that mediate or regulate scavenging, and set out to investigate changes in expression of these markers during the early stages of CF lung disease. Bronchoalveolar lavage fluid (BALF) was collected from 50 children with CF aged 1, 3 or 5 years. BALF cells were analyzed using flow cytometry. Expression levels of surface markers on AMs were expressed as median fluorescence intensities (MFI) or percentage of AMs positive for these markers. The effect of age and neutrophilic inflammation, among other variables, on marker expression was assessed with a multivariate linear regression model. AM expression of scavenger receptor CD163 decreased with age (p = 0.016) and was negatively correlated with BALF %neutrophils (r = -0.34, p = 0.016). AM expression of immune checkpoint molecule SIRPα also decreased with age (p = 0.0006), but did not correlate with BALF %neutrophils. Percentage of AMs expressing lipid scavenger CD36 was low overall (mean 20.1% ± 16.5) and did not correlate with other factors. Conversely, expression of immune checkpoint PD-1 was observed on the majority of AMs (mean PD-1pos 72.9% ± 11.8), but it, too, was not affected by age or BALF %neutrophils. Compared to matched blood monocytes, AMs had a higher expression of CD16, CD91, and PD-1, and a lower expression of CD163, SIRPα and CD36. In BALF of preschool children with CF, higher age and/or increased neutrophilic inflammation coincided with decreased expression of scavenger receptors on AMs. Expression of scavenging receptors and regulators showed a distinctly different pattern in AMs compared to blood monocytes. These findings suggest AM capacity to counter inflammation and promote homeostasis reduces during initiation of CF airway disease and highlight new avenues of investigation into impaired CF AM function.

Serum exosomal m6A demethylase FTO promotes gefitinib resistance in non-small cell lung cancer by up-regulating FLRT3, PTGIS and SIRPα expression

This study investigates the molecular mechanism of FTO m6A demethylase in non-small cell lung cancer (NSCLC) and gefitinib resistance using GEO and TCGA databases. Differentially expressed genes (DEGs) were screened from RNA-seq data sets of serum exosomes of gefitinib-resistant NSCLC patients in the GEO database and the NSCLC data set in the GEPIA2 database. From this analysis, FTO m6A demethylase was found to be significantly upregulated in the serum exosomes of gefitinib-resistant NSCLC patients. To identify downstream genes affected by FTO m6A demethylase, weighted correlation network analysis and differential expression analysis were performed, resulting in the identification of three key downstream genes (FLRT3, PTGIS, and SIRPA). Using these genes, the authors constructed a prognostic risk assessment model. Patients with high-risk scores exhibited a significantly worse prognosis. The model could predict the prognosis of NSCLC with high accuracy measured by AUC values of 0.588, 0.608, and 0.603 at 1, 3, and 5 years respectively. Furthermore, m6A sites were found in FLRT3, PTGIS, and SIRPA genes, and FTO was significantly positively correlated with the expression of these downstream genes. Overall, FTO m6A demethylase promotes gefitinib resistance in NSCLC patients by upregulating downstream FLRT3, PTGIS, and SIRPA expression, with these three downstream genes serving as strong prognostic indicators.

Development of a quantitative systems pharmacology (QSP) model to support dose optimization of DS-1103a (anti SIRPa antibody) in combination with trastuzumab deruxtecan in patients with cancer.

e14509 Background: DS-1103a, an anti-human signal regulatory protein α (SIRPα) monoclonal antibody (mAb), is designed to block the SIRPα-CD47 axis “Don’t Eat Me” signal and trigger phagocytosis of tumor cells when combined with an Fc-active antitumor mAb. DS-1103a is being developed as a combination therapy with trastuzumab deruxtecan (T-DXd; DS-8201a) as the antibody-dependent cellular phagocytosis (ADCP) enabling antibody. We developed a QSP model to support the first in human (FIH) intravenous dosing (IV) based on predicted intratumoral receptor occupancy (RO) within immunological synapses (the intercellular contact zone between macrophages and cancer cells) and corresponding ADCP activity. Methods: The QSP model mechanistically integrated DS-1103a plasma pharmacokinetics (PK), tumor distribution and binding of receptors to both antibodies and their ligands to simulate RO in the tumor microenvironment (TME). A proximal pharmacodynamics pathway model was first developed to estimate the intratumoral RO required to induce desired levels of ADCP for DS-1103a+T-DXd combination therapy. A human model was then developed to integrate DS-1103a PK with derived intratumormal RO in patients considering key human pathophysiological parameters, including SIRPα expression, tumor lesion permeability, SIRPα+ cell fractions in TME, tumor capillary radius, SIRPα internalization, CD47 expression and tumor lesion size. Virtual patient simulations were conducted in various cancer types to facilitate FIH dose selection. Results: The proximal pharmacodynamics pathway model quantitatively recapitulated the dose dependent in vitro ADCP activities for DS-1103a+T-DXd combination therapy. The intratumoral RO (within synapses) required to induce desired levels of ADCP activity were also derived. The human model predicted similar dose dependent intratumoral RO in various cancer types. Virtual patient simulations indicated that a flat dose of 100 mg Q3W IV DS-1103a could achieve steady-state intratumoral RO sufficient to reach half of the maximal ADCP activity and would be a suitable FIH starting dose. The predicted steady-state intratumoral RO after a flat dose of ≥1000 mg Q3W IV DS-1103a is well above the predicted intratumoral RO threshold to achieve maximum ADCP activity. Conclusions: The QSP model mechanistically characterized DS-1103a mechanism of action and linked PK to intratumoral RO and corresponding ADCP activity in both in vitro and in vivo setting. The model was used to derive the FIH starting dose and potentially clinically effective doses for DS-1103a in combination with T-DXd. This QSP model will be integrated into a large-scale disease platform to support dose expansion and further dose optimization as new clinical data emerge.

Analysis of different protein expression levels in peripheral blood circulating tumor cells from patients with diffuse large B-cell lymphoma and their predictive efficiency for recurrence

Objective: To analyze the expression levels of differentiation cluster 47 (CD47), signal regulatory protein α (SIRP-α), proto-oncogene (MYC) and proliferating cell associated antigen (Ki67) proteins in peripheral blood circulating tumor cells (CTC) from patients with diffuse large B-cell lymphoma and their predictive efficiency for tumor recurrence. Methods: The data of 82 patients with diffuse large B-cell lymphoma who were confirmed by histopathology and were in remission after chemotherapy in the Hematology Department of Linyi People's Hospital from January 2018 to January 2021 were retrospectively analyzed. There were 44 males and 38 females, and aged from 50 to 75 (63.8±4.6) years. The patients were divided into recurrent group (n=36) and non-recurrent group (n=46) according to their recurrence within 1 year after remission. The fasting peripheral venous blood samples (4 ml) from patients in the morning were collected, and the CTC were isolated. The expression levels of CD47, SIRP-α, MYC and Ki67 proteins in CTC were detected by Western blotting. The correlations between CD47 expression level and SIRP-α, MYC and Ki67 expression levels were analyzed by Pearson correlation analysis. The predictive efficiency of CD47, SIRP-α, MYC and Ki67 expression levels on tumor recurrence was evaluated by receiver operating characteristic (ROC) curves, and the areas under the curve (AUC) were calculated. Results: The expression levels of CD47, SIRP-α, MYC and Ki67 in recurrent group were 2.24±0.23, 1.17±0.12, 1.98±0.20 and 2.63±0.27, while those in non-recurrent group were 2.04±0.21, 1.31±0.13, 1.53±0.16 and 2.24±0.25. The expression levels of CD47, MYC and Ki67 in the recurrent group were higher than those in the non-recurrent group, while the expression levels of SIRP-α were lower than those in the non-recurrent group (all P<0.001). In 82 patients, the expression levels of CD47, SIRP-α, MYC and Ki67 were 2.13±0.22, 1.25±0.13, 1.73±0.18 and 2.41±0.26, respectively. The expression level of CD47 was negatively correlated with the expression level of SIRP-α (r=-0.308, P=0.005), but positively correlated with the expression level of MYC and Ki67 (r=0.484 and 0.332, P=0.012 and 0.003). The sensitivity of CD47, SIRP-α, MYC and Ki67 expression levels in predicting recurrence of diffuse large B-cell lymphoma was 66.7%, 72.2%, 72.2% and 66.7%, with the specificity of 67.4%, 71.7%, 67.4% and 71.7%, and AUC (95%CI) of 0.694 (0.582-0.791), 0.693 (0582-0.790), 0.714 (0.603-0.808) and 0.709 (0.598-0.804), respectively. The sensitivity of the combined detection of the above four indicators was 83.3%, with the specificity of 78.3% and the AUC (95%CI) of 0.864 (0.771-0.930), which was higher than those of the individual detection of each indicator (all P<0.05). Conclusions: The expression level of CD47 was negatively correlated with the expression level of SIRP-α, but positively correlated with the expression level of MYC and Ki67. The expression levels of CD47, SIRP-α, MYC and Ki67 have certain predictive value for tumor recurrence in patients with diffuse large B-cell lymphoma, and the predictive efficiency of combined detection is higher than single indicator detection.

Ultrahigh-plex Spatial Phenotyping of the Glioblastoma Multiforme Immune Microenvironment

Abstract The glioblastoma multiforme (GBM) microenvironment is highly immunosuppressive, leading to poor clinical outcomes and median survival of less than 15 months. Information about the nature of immunosuppressive and immunostimulatory cell types in GBM is fragmented, and a better understanding is urgently needed. We have developed a spatial phenotyping application that permits comprehensive characterization of the human GBM immune microenvironment. The Phenocycler-Fusion is a fast spatial biology solution that affords ultrahigh-plex single-cell spatial readouts. We used this solution for deep immune phenotyping of &amp;gt;50 proteins, comprising immune cell lineages, activation states and checkpoints, as well as markers for tumor, vascular and neuronal landscapes of GBM. Via single cell spatial phenotyping, we then isolated spatial signatures within the GBM tissue immune microenvironment. We focused on large numbers of glioma-associated macrophages that were identified via the combinatorial expression of key markers CD68, CD163, STAT1, STAT6, Iba-1 and SIRPα. The macrophages and their biomarker expression profiles displayed significant inter- and intra-tumoral heterogeneity, indicative of the complex biology of GBM. In addition, we profiled other immunosuppressive elements, including Tregs and exhausted T cells. Our data provide a comprehensive account of diverse immune cell populations in GBM and they may uncover systematic differences among GBM patient samples. Our application has enormous potential to further our understanding of the GBM immune microenvironment and is ready to be deployed by other laboratories.

Characteristics, prognostic determinants of monocytes, macrophages and T cells in acute coronary syndrome: protocol for a multicenter, prospective cohort study

BackgroundAcute coronary syndrome(ACS) is the leading cause of mortality and disability worldwide. Immune response has been confirmed to play a vital role in the occurrence and development of ACS. The objective of this prospective, multicenter, observational study is to define immune response and their relationship to the occurrence and progressive of ACS.MethodsThis is a multicenter, prospective, observational longitudinal cohort study. The primary outcome is the incidence of major adverse cardiovascular events (MACE) including in-stent restenosis, severe ventricular arrhythmia, heart failure, recurrent angina pectoris, and sudden cardiac death, and stroke one year later after ACS. Demographic characteristics, clinical data, treatments, and outcomes are collected by local investigators. Furthermore, freshly processed samples will be stained and assessed by flow cytometry. The expression of S100A4, CD47, SIRPα and Tim-3 on monocytes, macrophages and T cells in ACS patients were collected. Follow-up: during hospitalization, 3, 6 and 12 months after discharge.DiscussionIt is expected that this study will reveal the possible targets to improve the prognosis or prevent from occurrence of MACE in ACS patients. Since it’s a multicenter study, the enrollment rate of participants will be accelerated and it can ensure that the collected data are more symbolic and improve the richness and credibility of the test basis.Ethics and disseminationThis study has been registered in Chinese Clinical Trial Registry Center. Ethical approval was obtained from the Affiliated Hospital of Guizhou Medical University. The dissemination will occur through the publication of articles in international peer-reviewed journals.Trial RegistrationChinese Clinical Trial Registry: ChiCTR2200066382.

Leishmania Infection-Induced Proteolytic Processing of SIRPα in Macrophages.

The shedding of cell surface receptors may bring synergistic outcomes through the loss of receptor-mediated cell signaling and competitive binding of the shed soluble receptor to its ligand. Thus, soluble receptors have both biological importance and diagnostic importance as biomarkers in immunological disorders. Signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal, is expressed by myeloid cells where its expression and function are in part regulated by proteolytic cleavage. However, reports on soluble SIRPα as a biomarker are limited. We previously reported that mice with experimental visceral leishmaniasis (VL) manifest anemia and enhanced hemophagocytosis in the spleen accompanied with decreased SIRPα expression. Here, we report increased serum levels of soluble SIRPα in mice infected with Leishmania donovani, a causative agent of VL. Increased soluble SIRPα was also detected in a culture supernatant of macrophages infected with L. donovani in vitro, suggesting the parasite infection promotes ectodomain shedding of SIRPα on macrophages. The release of soluble SIRPα was partially inhibited by an ADAM proteinase inhibitor in both LPS stimulation and L. donovani infection, suggesting a shared mechanism for cleavage of SIRPα in both cases. In addition to the ectodomain shedding of SIRPα, both LPS stimulation and L. donovani infection induced the loss of the cytoplasmic region of SIRPα. Although the effects of these proteolytic processes or changes in SIRPα still remain unclear, these proteolytic regulations on SIRPα during L. donovani infection may explain hemophagocytosis and anemia induced by infection, and serum soluble SIRPα may serve as a biomarker for hemophagocytosis and anemia in VL and the other inflammatory disorders.

Abstract 2934: BYON4228, an antagonistic SIRPα mAb with a unique and favorable preclinical profile compared to three comparator SIRPα mAbs

Abstract A growing body of data from early clinical studies supports that targeting the myeloid immune checkpoint CD47-SIRPα represents a successful strategy towards establishing effective new therapies for cancer patients. Initial studies focused on CD47 blockers and more recently also SIRPα antagonistic mAbs entered clinical studies. The latter approach is thought to be more advantageous since SIRPα expression is primarily myeloid restricted whereas CD47 is broadly expressed and -besides SIRPα- binds other ligands as well.We generated BYON4228, an antibody directed against the two existing allelic variants of human SIRPα that does not recognize the closely related T-cell expressed SIRPγ. Functional studies demonstrate that BYON4228 potentiates both macrophage- and neutrophil-mediated elimination of hematologic and solid cancer cells in vitro in the presence of a variety of tumor targeting antibodies, including rituximab, daratumumab, trastuzumab and cetuximab. In vivo, BYON4228 increases the anti-tumor activity of rituximab in a B-cell Raji xenograft model in mice and shows a favorable safety profile in cynomolgus monkeys.To position BYON4228 in the emerging field of myeloid checkpoint inhibitors, head-to-head comparison studies were performed. For these studies, previously described anti-SIRPα mAbs HEFLB, 1H9, and SIRPAB-11 were generated in house that in all probability are representative for three clinical stage SIRPα mAbs. These comparative studies focused on principal characteristics of this class of mAbs that are anticipated to affect their clinical profile, namely 1) recognition of both common allelic forms of SIRPα which determines the broadness of its clinical applicability; 2) binding to SIRPγ on T-lymphocytes which is reported to affect T-cell extravasation and activation and thereby the adaptive immune response; 3) functionality of the Fc-region of the mAb which can mediate depletion of SIRPα-positive host myeloid immune effector cells thereby compromising host defense and undermining the effect of immunotherapy. The comparative studies show that in contrast to HEFLB, BYON4228 recognizes both common allelic forms of SIRPα. Furthermore, lack of binding to T-cell expressed SIRPγ differentiates BYON4228 from 1H9 and SIRPAB-11. Finally, with its impaired Fc-region BYON4228 does not deplete SIRPα+ myeloid cells, in contrast to SIRPAB-11.Thus, BYON4228 differentiates from all three comparator SIRPα mAbs based on at least one fundamental characteristic, thereby underlining its potential to become a best-in-class drug. We therefore endorse its further development for which clinical studies are planned to start in 2023. Citation Format: Mary van Helden, Seline Zwarthoff, Roel Arends, Inge Reinieren-Beeren, Marc Parade, Lilian Driessen-Engels, Karin de Laat-Arts, Desiree Damming, Ellen Santegoeds-Lenssen, Daphne van Kuppeveld, Imke Lodewijks, Ellen Mattaar, Hugo Olsman, Hanke Matlung, Katka Franke, Marloes Stokman, Benny de Wit, Dirk Glaudemans, Danielle van Wijk, Lonnie Joosten Stoffels, Jan Schouten, Paul Boersema, Monique van der Vleuten, Jorien Sanderink, Wendy Kappers, Diels van den Dobbelsteen, Marco Timmers, Ruud Ubink, Gerard Rouwendal, Gijs Verheijden, Miranda M.C. van der Lee, Wim Dokter, Timo van den Berg. BYON4228, an antagonistic SIRPα mAb with a unique and favorable preclinical profile compared to three comparator SIRPα mAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2934.

Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy

Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy

ASO Visual Abstract: Clinical Significance of Signal Regulatory Protein Alpha (SIRPα) Expression in Hepatocellular Carcinoma.

ASO Visual Abstract: Clinical Significance of Signal Regulatory Protein Alpha (SIRPα) Expression in Hepatocellular Carcinoma.

Clinical Significance of Signal Regulatory Protein Alpha (SIRPα) Expression in Hepatocellular Carcinoma.

Signal regulatory protein alpha (SIRPα), expressed in the macrophage membrane, inhibits phagocytosis of tumor cells via CD47/SIRPα interaction, which acts as an immune checkpoint factor in cancers. This study aimed to clarify the clinical significance of SIRPα expression in hepatocellular carcinoma (HCC). This study analyzed SIRPα expression using RNA sequencing data of 372 HCC tissues from The Cancer Genome Atlas (TCGA) and immunohistochemical staining of our 189 HCC patient cohort. The correlation between SIRPα expression and clinicopathologic factors, patient survival, and intratumor infiltration of immune cells was investigated. Overall survival (OS) was significantly poorer with high SIRPα expression than with low expression in both TCGA and our cohort. High SIRPα expression correlated with lower recurrence-free survival (RFS) in our cohort. High SIRPα expression was associated with higher rates of microvascular invasion and lower serum albumin levels and correlated with greater intratumor infiltration of CD68-positive macrophages and myeloid-derived suppressor cells (MDSCs). Multivariate analysis showed that SIRPα expression and high infiltration of CD8-positive T cells and MDSCs were predictive factors for both RFS and OS. Patients with high SIRPα expression and infiltration of CD8-positive T cells and MDSCs had significantly lower RFS and OS rates. In spatial transcriptomics sequencing, SIRPα expression was significantly correlated with CD163 expression. High SIRPα expression in HCC indicates poor prognosis, possibly by inhibiting macrophage phagocytosis of tumor cells, promoting MDSC infiltration and inducing antitumor immunity. Treatment alternatives using SIRPα blockage should be considered in HCC as inhibiting macrophage antitumor immunity and MDSCs.

A humanized mouse model to study NK cell biology during HIV infection.

NK cells are an important subset of innate immune effectors with antiviral activity. However, NK cell development and immune responses in different tissues during acute and chronic HIV infection in vivo have been difficult to study due to the impaired development and function of NK cells in conventional humanized mouse models. In this issue of the JCI, Sangur et al. report on a transgenic MISTRG-6-15 mouse model with human IL-6 and IL-15 knocked into the previously constructed MISTRG mice. The predecessor model was deficient in Rag2 and γ chain (γc) with knock-in expression of human M-CSF, IL-3, GM-CSF, and TPO, and transgenic expression of human SIRPα. The researchers studied tissue-specific NK cell immune responses during HIV infection and clearly show that the endogenous human NK cells in the humanized mouse model suppressed HIV-1 replication in vivo. These findings provide insight into harnessing the innate immune response for clinical antiviral therapies.

Lack of SIRP-alpha reduces lung cancer growth in mice by promoting anti-tumour ability of macrophages and neutrophils.

Signal regulatory protein-alpha (SIRPα) is a transmembrane glycoprotein specifically expressed on myeloid cells. Blockade of SIRPα/CD47 interaction is effective in combinational therapy of some cancers. This study aimed to explore into the role and underlying molecular mechanisms of SIRPα in lung cancer growth. A mouse model with lung cancer in wild-type (WT) and SIRPα-knockout mouse (KO) mice was established by subcutaneous injection of Lewis murine lung cancer cells (LLC). Circulating monocytes and neutrophils were depleted in mice by intraperitoneal administration of clodronate liposomes and anti-Ly6G antibody, respectively. Phenotypes and phagocytosis of macrophages and neutrophils were analysed by flow cytometry. Transwell assay was used to analyse LLC cells migration and invasion. Lack of SIRPα inhibited LLC cells growth in KO mice, associated with reduced infiltrating PD-1+ CD8+ T cells and production of IL-6 from infiltrating macrophages and neutrophils in tumour tissues. Depletion of circulating monocytes and neutrophils reduced LLC cells growth in WT mice, which was abolished in KO mice. Studies in vitro showed that lack of SIRPα increased M1/M2 ratio, and reduced LLC cell migration and invasion via attenuated IL-6 secretion. Lack of SIRPα expression in neutrophils effectively increased the cytotoxic activity to LLC cells in vitro. Lack of SIRPα suppressed lung cancer cell growth in mice, dependent on circulating macrophages and neutrophils, in association with improved phagocytosis and reduced IL-6 expression.