CTLA4-Ig treatment induces M1–M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients

Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05). Conclusions CTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

Expression Dynamics of the O-Glycosylated Proteins Recognized by Amaranthus leucocarpus Lectin in T Lymphocytes and Its Relationship With Moesin as an Alternative Mechanism of Cell Activation

T lymphocyte activation begins with antigen/MHC recognition by the TCR/CD3 complex followed by a costimulatory signal provided by CD28. The search for novel costimulatory molecules has been extensive due to their potential use as immunotherapeutic targets. Although some molecules have been identified, they are unable to provide sustainable signaling to allow for proper T cell activation and proliferation. It has been shown that the Amaranthus leucocarpus lectin (ALL) can be used as an in vitro costimulator of CD4+ lymphocytes in the presence of anti-CD3 mAb; this lectin specifically recognizes O-glycans of the Galβ1-3GalNAc-O-Ser/Thr type, including a 70-kDa moesin-like protein that has been suggested as the costimulatory molecule. However, the identity of this molecule has not been confirmed and such costimulation has not been analyzed in CD8+ lymphocytes. We show herein that the expression kinetics of the glycoproteins recognized by ALL (gpALL) is different in CD4+ and CD8+ T cells, unlike moesin expression. Results from IP experiments demonstrate that the previously described 70-kDa moesin-like protein is an O-glycosylated form of moesin (O-moesin) and that in vitro stimulation with anti-CD3 and anti-moesin mAb induces expression of the activation molecules CD69 and CD25, proliferation and IL-2 production as efficiently as cells costimulated with ALL or anti-CD28. Overall, our results demonstrate that O-moesin is expressed in CD4+ and CD8+ T lymphocytes and that moesin provides a new costimulatory activation signal in both T cell subsets.

AC099850.3 Promotes Cell Proliferation and is One of Five Costimulatory Molecule-Related LncRNAs That Predict Overall Survival of Liver Hepatocellular Carcinoma

Background: Extensive research has revealed that costimulatory molecules play central roles in mounting anti-tumor immune responses and long non‐coding RNA (lncRNA) is an important regulatory factor in the development of various cancers. However, their roles in liver hepatocellular carcinoma (HCC) remain unexplored. In this study, we aimed to explore costimulatory molecule-related lncRNAs in HCC and construct a prognostic signature to predict prognosis and improve clinical outcomes with HCC patients.Methods: The data we used for bioinformatics analysis were downloaded from The Cancer Genome Atlas database. Costimulatory molecules were obtained from the known literature. The R software, SPSS and GraphPad Prism were used for mapping and statistical analysis.Results: A five costimulatory molecule-related lncRNAs based risk model was initially constructed through lasso and Cox regression analysis. Moreover, multivariate regression suggested that the risk score was a significant prognostic risk factor in HCC. Samples in high- and low-risk groups exhibited significantly different in gene set enrichment analysis and immune infiltration analysis. Importantly, we found that the AC099850.3 were significantly related to cell proliferation in HCC according to the colony formation and CCK8 assays.Conclusion: In summary, we first identified and validated a novel costimulatory molecule-related survival model and we found that AC099850.3 is closely associated with clinical stage and could remarkably facilitate cell proliferation in HCC, making it potential to be a novel therapeutic target.

CD226 knockout alleviates high-fat diet induced obesity by suppressing proinflammatory macrophage phenotype

Obesity is associated with chronic low-grade inflammation, contributing to an increasing prevalence of chronic metabolic diseases, such as insulin resistance, non-alcoholic fatty liver disease (NALFD), and steatohepatitis. Macrophages are the predominant immune cells in adipose tissues. Adipose tissue macrophages (ATMs) would switch to pro-inflammatory M1 state during obesity, causing local and systemic inflammation. However, the regulatory mechanism of ATMs has not yet been well described within this process. Using a high-fat diet (HFD)–induced mouse obesity model, we found that the costimulatory molecule CD226 was highly expressed on ATMs and knockout (KO) of CD226 alleviated obesity caused by HFD. Loss of CD226 reduced the accumulation of ATMs and hindered macrophage M1 polarization, with lower serum proinflammatory cytokine levels. Furthermore, deficiency of CD226 on ATMs decreased the phosphorylation levels of VAV1, AKT, and FOXO1 and thereby upregulated PPAR-γ. Further administration of PPAR-γ inhibitor restored M1 phenotype in CD226KO ATMs. In summary, loss of CD226 alleviates the HFD-induced obesity and systemic inflammation through inhibition of the accumulation and M1 polarization of ATMs in which PPAR-γ-dependent signaling pathway is involved, suggesting that CD226 may be identified as a potential molecular target for the clinical treatment of obesity.

Identification and Validation a Costimulatory Molecule Gene Signature to Predict the Prognosis and Immunotherapy Response for Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Costimulatory molecules have been proven to be the foundation of immunotherapy. However, the potential roles of costimulatory molecule genes (CMGs) in HCC remain unclear. Our study is aimed to develop a costimulatory molecule-related gene signature that could evaluate the prognosis of HCC patients.Methods: Based on The Cancer Gene Atlas (TCGA) database, univariate Cox regression analysis was applied in CMGs to identify prognosis-related CMGs. Consensus clustering analysis was performed to stratify HCC patients into different subtypes and compared them in OS. Subsequently, the LASSO Cox regression analysis was performed to construct the CMGs-related prognostic signature and Kaplan–Meier survival curves as well as ROC curve were used to validate the predictive capability. Then we explored the correlations of the risk signature with tumor-infiltrating immune cells, tumor mutation burden (TMB) and response to immunotherapy. The expression levels of prognosis-related CMGs were validated in HCC using qRT-PCR method.Results: All HCC patients were classified into two clusters based on 11 CMGs with prognosis values and cluster 2 correlated with a poorer prognosis. Next, a prognostic signature of six CMGs was constructed, which was an independent risk factor for HCC patients. Patients with low-risk score were associated with better prognosis. The correlation analysis showed that the risk signature could predict the infiltration of immune cells and immune status of the immune microenvironment in HCC. The qRT-PCR indicated six CMGs with significantly differential expression in HCC tissues and normal tissues.Conclusion: In conclusion, our CMGs-related risk signature could be used as a prediction tool in survival assessment and immunotherapy for HCC patients.

714 Selective expansion of antigen-specific CD8 T cells with engineered antigen presenting exosome

BackgroundExosomes are vesicular granules of about 100 nm and are secreted by many types of cells. Exosomes contain various proteins, lipids, and RNAs that are transported to target cells which induce functional and physiological changes. Exosomes are promising nano-vesicles for clinical application, owing to their high biocompatibility, low immunogenicity, and high drug delivery efficacy. Recent studies have demonstrated that exosomes from tumor cells or antigen presenting cells (APCs) regulate immune responses. Tumor derived exosomes express PD-L1 on their surface and suppress tumor immunity systemically. On the other hand, mature dendritic cells derived exosomes exert immune activation, and tumor immunotherapy using DCs exosome has been developed. However, few studies have been found to exert a significant effect on cancer treatment, may be because of low expression of costimulatory molecules and lack of cytokines on DCs derived exosomes.MethodsIt has been demonstrated that GFP can be conveyed into exosomes by conjugating GFP with tetraspanins, exosome-specific surface proteins. First, we generated a tetraspanin fusion protein with a single-chain MHCI trimer (scMHCI). IL-2 is inserted on the second extracellular loop of CD81, allowing robust and functional expression of IL-2 on the exosome. We collected exosomes from HEK293 cells culture, which stably express scMHCI-CD81-IL2 and CD80-MFGE8, and used as Antigen-presenting exosome(AP-Exo).ResultsAP-Exo expresses high expression of MHCI-peptide complex, costimulatory molecule, and cytokine, activating cognate CD8 T cells as dendritic cells do. AP-Exo selectively delivered co-stimulation and IL-2 to antigen-specific CD8 T cells, resulting in a massive expansion of antigen-specific CD8 T cells without severe adverse effects in mice. AP-Exo can expand endogenous tumor-specific CD8 T cells and induce the potent anti-tumor effect.ConclusionsOur strategy for building engineered exosomes that work like APCs might develop novel methods for cancer immunotherapy.Ethics ApprovalAll mice were housed in a specific pathogen-free facility, and all animal experiments were performed according to a protocol approved by Kanazawa University, Kanazawa, Japan.

862 Dectin-2, a novel target for tumor macrophage reprogramming in cancer immunotherapy

BackgroundTumor-associated macrophages (TAMs) are an abundant immune cell population in most cancers that support tumor progression through their immunosuppressive effects. We discovered that TAMs express the pattern recognition receptor Dectin-2 (Clec4n/CLEC6A), an activating C-type lectin receptor (CLR) that binds to high-mannose glycans on fungi and other microbes and induces protective immune responses against infectious disease. Dectin-2 is selectively expressed by myeloid cells, and upon ligation mediates enhanced phagocytosis, antigen processing and presentation, and proinflammatory cytokine production. Given these properties, we evaluated the therapeutic potential of targeting Dectin-2 using naturally derived ligands. We also generated human Dectin-2-targeted agonistic antibodies capable of robustly activating immunosuppressive ”M2” or TAM-like macrophages.MethodsDectin-2 expression was assessed by flow cytometry, immunohistochemistry, and using public databases. Mouse and human monocytes were differentiated into macrophages using recombinant cytokines or tumor-conditioned media, and stimulation was measured following overnight incubation with Dectin-2 ligands or antibodies. Mouse tumor cell lines were implanted into syngeneic hosts and mice were treated with mannan derived from S. cerevisiae via IT or IV administration.ResultsDectin-2 gene expression is minimal in normal human tissues but elevated across many tumor types, including breast, colon, lung, and kidney cancers. Dectin-2 is strongly expressed by macrophages differentiated in vitro and on primary TAMs. The fungal Dectin-2 ligand mannan stimulated proinflammatory cytokine production (e.g. TNFalpha) and costimulatory molecule expression (e.g. CD86) by macrophages in a Dectin-2-dependent manner. Treatment of tumor-bearing mice with mannan mediated tumor regression in multiple syngeneic tumor models, with high rates of tumor clearance in the MB49 bladder cancer model. These effects were Dectin-2 dependent, as efficacy was not observed when a Dectin-2-blocking antibody was co-administered or in knockout mice lacking Dectin-2 signaling components. Furthermore, depletion of either macrophages or T cells impaired efficacy, suggesting that Dectin-2-stimulated TAMs augment anti-tumor T cell responses. Based on these data, we developed novel Dectin-2 targeted agonist antibodies capable of activating human ”M2” or TAM-like macrophages in vitro to produce an array of proinflammatory cytokines and chemokines akin to tumor-destructive ”M1” macrophages.ConclusionsThe data presented demonstrate the therapeutic potential of targeting Dectin-2 using natural ligands or agonistic antibodies as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy.Ethics ApprovalAll animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.