Expression Of CD86 Research Articles

IMMU-38. LEVERAGING INNATE IMMUNE SENSORS TO GENERATE DURABLE ANTI-GLIOMA ADAPTIVE IMMUNE RESPONSES

Abstract Glioblastoma harbors a tumor microenvironment (TME) that is mostly devoid of T cell effector infiltration but is dominated by immune suppressive microglia, macrophages, and myeloid-derived suppresser cells. The innate immune cells in the TME have impaired phagocytosis and antigen presentation and are inadequate for triggering the subsequent immune activating signals needed for anti-tumor effector responses. Phagocytic activity can also be blocked by the upregulation of CD47 on the tumor cells. In the immune checkpoint resistant Qki-/- Pten-/- P53-/- (QPP) glioma model, intratumoral treatment with the synthetic cyclic di-nucleotide STING agonist IACS-8803 (8803) induces global immunological reprogramming and long-term survival resistant to tumor rechallenge. More specifically, in the infiltrating myeloid stroma 8803 increases costimulatory protein CD86 while reducing the expression of suppressive markers CD163 and CD206. Conventional type 1 dendritic cells are increased in both the TME (p<0.05) and the draining cervical lymph node (p<0.01). Intratumoral infiltration of both CD8 T (p<0.05) and NK (p<0.001) cells increases. However, only T cells, but not NK cells, are necessary for 8803 therapeutic activity when assessed with in vivo NK cell depletion studies and treatment in the RAG-/- background. STING activation increases microglial phagocytic capacity, but also induces a compensatory increase in SIRPα expression (p<0.001) – the ligand for CD47. 8803 synergizes with anti-CD47 blockade to enhance microglia phagocytosis of QPP glioblastoma cells in vitro. Finally, the combination of 8803 and anti-CD47 synergizes to extend both median survival and the fraction of long-term survivors in orthotopic QPP-bearing mice (QPP8v; vehicle control MS=39d, anti-CD47 MS=40d, 8803 MS=67d 20% long-term survival, 8803 + anti-CD47 MS=107.5d 40% long-term survival). These data demonstrate that 8803 induces proinflammatory conversion of the glioblastoma TME, generates T cell-dependent survival benefits in QPP-bearing mice, and synergizes with anti-CD47 blockade to enhance microglia phagocytosis of glioblastoma cells and extend survival in QPP-bearing mice.

Single-cell landscape identified SERPINB9 as a key player contributing to stemness and metastasis in non-seminomas

AbstractEmbryonal carcinoma (EC), characterized by a high degree of stemness similar to that of embryonic stem cells, is the most malignant subtype within non-seminomatous testicular germ cell tumors (TGCTs). However, the mechanisms underlying its malignancy remain unknown. In this study, we employed single-cell RNA sequencing to analyze four non-seminoma samples. Our differential expression analysis revealed high expression of SERPINB9 in metastatic EC cells. We conducted in vitro experiments to further investigate SERPINB9’s role in the progression of EC. Functionally, the knockdown of SERPINB9 in NCCIT and NTERA-2 leads to a diminished migratory capability and decreased cis-platin resistance, as demonstrated by Transwell migration assay and drug sensitivity assay. Moreover, embryoid bodies showed reduced size and lower OCT4 expression, alongside heightened expression of differentiation markers AFP, ACTA2, and CD57 in shSERPINB9 cells. In vivo, the role of SERPINB9 in maintaining cancer stemness was validated by the limiting dilution assay. Mechanistically, Bulk RNA-seq further showed downregulation of ERK1/2 signaling and WNT signaling pathways with concomitant upregulation of differentiation pathways subsequent to SERPINB9 knockdown. Additionally, the analysis indicated increased levels of cytokines linked to tertiary lymphoid structures (TLS), such as IL6, IL11, IL15, CCL2, CCL5, and CXCL13 in shSERPINB9 cells, which were further validated by ELISA. Our research indicates that SERPINB9 plays a key role in driving tumor progression by enhancing tumor stemness and suppressing TLS. This study stands as the first to elucidate the molecular signature of non-seminomas at a single-cell level, presenting a wealth of promising targets with substantial potential for informing the development of future therapeutic interventions.

PATH-35. INTEGRATED GENOMIC ANALYSES OF IMMUNODEFICIENCY-ASSOCIATED EPSTEIN-BARR VIRUS- (EBV) POSITIVE PRIMARY CNS LYMPHOMAS

Abstract Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. Deconvolution of bulk RNA sequencing data was performed using CIBERSORTx to characterize the tumor microenvironment. EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV negative PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. In addition to novel insights into the pathobiology of EBV+ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

Urinary sediment mRNA as a potent biomarker of IgA nephropathy

BackgroundThe quantification of mRNA expression in urinary sediments is a reliable biomarker for various diseases. However, few studies have investigated the clinical relevance of urinary mRNA levels in IgA nephropathy (IgAN). Thus, we investigated the expression of urinary mRNAs and their clinical significance in IgAN.MethodsOverall, 200 patients with biopsy-proven IgAN, 48 disease controls, and 76 healthy controls were enrolled. We identified the differential expression of mRNAs in renal tissue between patients with IgAN and normal subjects using the Gene Expression Omnibus dataset and selected candidate mRNAs. mRNA expression in the urinary sediment was measured using quantitative real-time polymerase chain reaction. Associations between urinary mRNA levels and clinicopathological parameters were analyzed and the predictive value of mRNAs for disease progression was evaluated.ResultsThe urinary expression of CCL2, CD14, DNMT1, FKBP5, Nephrin, and IL-6 was significantly upregulated in patients with IgAN compared with healthy controls. C3, FLOT1, and Podocin levels were significantly correlated with renal function, where C3, FLOT1, and TfR levels were significantly correlated with urinary protein excretion. During follow-up, 26 (13.0%) patients with IgAN experienced disease progression, defined as a greater than 50% reduction in the estimated glomerular filtration rate or progression to end-stage renal disease. Urinary mRNA levels of FLOT1 (HR 3.706, 95% CI 1.373–10.005, P = 0.010) were independently associated with an increased risk of disease progression.ConclusionsOur results suggest that urinary sediment mRNAs are a useful biomarker in IgAN patients. Further studies with larger sample sizes and longer follow-up durations are required.

CD137 Protein Expression Pattern Determines the Functional Role of Galectin-9 in Colorectal Cancer.

The rapid advancement of single-cell sequencing technology has generated extensive data, providing critical resources for colorectal cancer (CRC) research. This study conducts a detailed analysis of CRC single-cell sequencing data to develop a novel clinical prognostic tool and explore potential therapeutic targets for the LGALS9 gene. Using the Scissor algorithm, we created a CRC prognostic scoring system (SDRS) based on 13 key genes, with particular focus on LGALS9 and its protein, Galectin-9, in mice CRC model with altered CD137 expression. Our findings demonstrate that the SDRS accurately reflects clinical and pathological features of CRC patients, acting as an independent predictor of outcomes. LGALS9 expression is generally reduced in CRC tissues and is associated with poorer prognosis. We also observed a strong positive correlation between LGALS9 and CD137 expression, with CD137 showing significant variability in CRC tissues. In mouse models with CD137 overexpression, Galectin-9 treatment led to notable antitumor effects and increased infiltration of activated T cells. In contrast, in CD137-deficient models, Galectin-9 promoted tumor growth with limited T cell presence. These results suggest that the role of LGALS9 in CRC may depend on CD137 expression, highlighting the potential of LGALS9 as a therapeutic target. CD137 levels may serve as a key indicator for predicting the effectiveness of this treatment strategy.

Simultaneous Stimulation of Peripheral Blood Mononuclear Cells with CpG ODN2006 and α-IgM Antibodies Leads to Strong Immune Responses in Monocytes Independent of B Cell Activation

CpG ODN2006 is widely used both in vitro and in vivo to achieve B cell activation and has been previously applied in clinical trials as an adjuvant and anti-cancer agent. Recent studies have demonstrated the benefit of combining CpG ODN2006 with α-IgM antibodies to obtain optimal B cell activation in vitro. In this study, we expanded the knowledge of how both agents affect other types of peripheral blood mononuclear cells (PBMCs), thereby highlighting beneficial and potentially unfavorable properties of the combination of CpG ODN2006 and α-IgM when applied beyond isolated B cells. We elucidated the effects of both compounds on mixed PBMCs, as well as on B cell- and monocyte-depleted PBMCs, allowing us to distinguish between direct effects and indirect influences mediated by other interacting immune cells. Flow cytometry was used to measure the expression of surface markers and intracellular cytokines, while ELISA and multiplex assays were performed to determine cytokine secretion. Our results revealed that stimulation of mixed PBMCs with CpG ODN2006 and α-IgM strongly increased cytokine secretion, primarily originating from α-IgM-stimulated monocytes. Monocyte activation was confirmed by increased CD86 and HLA-DR expression and occurred independently of B cells. The high level of monocyte-derived cytokines after α-IgM exposure did not affect B cell activation. However, it represents a rather unfavorable property for clinical applications. In conclusion, α-IgM is a potent inducer of cytokine production in monocytes. Based on our findings we hypothesize that significant side effects on monocytes can occur when using α-IgM to enhance CpG ODN2006’s efficacy on B cells, particularly in clinical settings.

ELF1 serves as a potential biomarker for the disease activity and renal involvement in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs, yet its underlying mechanisms remain unclear, and precise biomarkers are lacking. In this study, we employed Mendelian randomization and HEIDI tools to comprehensively analyze large-scale Genome-Wide Association Study (GWAS) and expression Quantitative Trait Loci (eQTL) data, leading to the identification of seven novel potential functional genes associated with SLE, including BLK, ELF1, STIM1, B3GALT6, APOLD1, INPP5B, and FHL3. Subsequent investigations revealed a significant downregulation of ELF1 gene expression in CD4+ T cells of SLE patients compared to healthy controls. Moreover, within various SLE subgroups, such as those with decreased serum complement C3 levels, positive urinary protein, new-onset skin rashes, and SLE Disease Activity Index (SLEDAI) scores ≥ 5, ELF1 expression displayed a consistent decreasing trend. Notably, ROC curve analysis highlighted the diagnostic potential of ELF1 expression in SLE (AUC = 0.9493), as well as its value in assessing disease activity (AUC = 0.6852) and renal involvement (AUC = 0.7363). In conclusion, this study underscores the potential of ELF1 as a SLE biomarker for diagnosis and evaluation, offering insights into the underlying mechanisms of SLE and paving the way for future therapeutic research.

Prediction of Treatment Response Based on Nutritional Status and Tumor Immunity in Oropharyngeal Cancer Patients Treated With Chemoradiotherapy.

Radiotherapy (RT) for advanced oropharyngeal cancer (OPC) is effective, especially when combined with chemotherapy (CRT). However, its success can vary depending on factors, such as tumor stage, HPV infection (p16 status), and the patient's nutritional and immune status. This study examined the controlling nutritional status (CONUT) score and tumor immunity as predictive factors for treatment outcomes in OPC, aiming to develop a personalized risk score. A retrospective analysis was conducted on 84 patients with OPC treated with definitive RT or CRT, and survival outcomes were compared based on various factors, including BMI, CONUT score, CD8 expression, and HLA class II expression. We observed better overall survival (OS) rates in CD8-positive patients and those with higher HLA class II expression. The univariate analysis identified stage, p16 status, BMI, CONUT score, and CD8 expression as significantly associated with OS. In multivariate analysis, stage, BMI, and CONUT score remained significant predictors of OS. A risk scoring system was developed based on stage, p16 status, BMI, CONUT score, and CD8 expression. Patients were categorized into low-risk and high-risk groups, with significantly better survival in the low-risk group. A combined risk score incorporating clinical, nutritional, and immune factors can improve the prediction of treatment outcomes for OPC patients. This risk stratification may enable personalized treatment plans and improve ΟS rates.

New insights into the immunomodulatory potential of sialic acid on monocyte-derived dendritic cells

Sialic acids at the cell surface of dendritic cells (DCs) play an important immunomodulatory role, and their manipulation enhances DC maturation, leading to heightened T cell activation. Particularly, at the molecular level, the increased stability of surface MHC-I molecules in monocyte-derived DCs (MoDCs) underpins an improved DC: T cell interaction. In this study, we focused on the impact of sialic acid remodelling by treatment with Clostridium perfringens sialidase on MoDCs' phenotypic and functional characteristics. Our investigation juxtaposes this novel approach with the conventional cytokine-based maturation regimen commonly employed in clinical settings.Notably, C. perfringens sialidase remarkably increased MHC-I levels compared to other sialidases having different specificities, supporting the idea that higher MHC-I is due to the cleavage of specific sialoglycans on cell surface proteins. Sialidase treatment induced rapid elevated surface expression of MHC-I, MHC-II and CD40 within an hour, a response not fully replicated by 48 h cytokine cocktail treatment. These increases were also observable 48 h post sialidase treatment. While CD86 and PD-L1 showed significant increases after 48 h of cytokine maturation, 48 h post sialidase treatment showed a higher increase in CD86 and shorter increase in PD-L1. CCR-7 expression was significantly increased 48 h after sialidase treatment but not significantly affected by cytokine maturation. Both treatments promoted higher secretion of the IL-12 cytokine. However, the cytokine cocktail induced a more pronounced IL-12 production. SNA lectin staining analysis demonstrated that the sialic acid profile is significantly altered by sialidase treatment, but not by the cytokine cocktail, which causes only slight sialic acid upregulation. Notably, the lipid-presenting molecules CD1a, CD1b and CD1c remained unaffected by sialidase treatment in MoDCs, a finding also further supported by experiments performed on C1R cells. Inhibition of endogenous sialidases Neu1 and Neu3 during MoDC differentiation did not affect surface MHC-I expression and cytokine secretion. Yet, sialidase activity in MoDCs was minimal, suggesting that sialidase inhibition does not significantly alter MHC-I-related functions. Our study highlights the unique maturation profile induced by sialic acid manipulation in MoDCs. These findings provide insights into the potential of sialic acid manipulation as a rapid immunomodulatory strategy, offering promising avenues for targeted interventions in inflammatory contexts.

Kinetics of viral hemorrhagic septicemia virus (VHSV) and immune response in olive flounder (Paralichthys olivaceus) at altered temperature

Viral hemorrhagic septicemia virus (VHSV) is a pathogen that causes hemorrhagic septicemia in olive flounder at water temperatures below 15 °C, leading to symptoms such as abdominal swelling due to ascites and muscle hemorrhaging, and in severe cases, mortality. In this study, we investigated the proliferation of NCCs, the transcriptional analysis of CD4 and CD8, and the expression of pro-inflammatory cytokines (IL-1β, IL6, TNFα) and the cytokines (IL12, IL15, IFN-1β, IFNγ) involved in cytotoxic cell activation in the kidney of olive flounder during VHSV infection at suboptimal temperature (17 °C) and following a shift to optimal temperature (10 °C). Following viral infection, the population of NCCs and CD8 gene expression steadily increased. Meanwhile, IL12, IL15, and IFN-1β levels exhibited an early surge, reaching their peak within 1–2 days post-challenge at 17 °C. Notably, in olive flounder that were re-challenged with VHSV at 10 °C after an initial challenge at 17 °C, there was a rapid and robust expansion of NCCs and increased CD4 and CD8 gene expression, demonstrating the mounting of immune memory by NCCs and an efficient adaptive immune response from CD4+ and CD8+ T cells against VHSV infection.

Nicotine promotes M2 macrophage polarization through α5-nAChR/SOX2/CSF-1 axis in lung adenocarcinoma

α5-nicotinic acetylcholine receptor (α5-nAChR) plays a vital part in lung adenocarcinoma (LUAD). However, it is not comprehensively understood that how the α5-nAChR affects LUAD. Through diverse bioinformatics analyses and immunohistochemistry, the expressions of α5-nAChR and SOX2 as well as their relations were dissected. α5-nAChR regulated the differentiation of monocytes into M2 macrophages by targeting the STAT3/SOX2/CSF-1 signaling in the coculture system by western blotting and ChIP. α5-nAChR-mediated macrophage-mediated LUAD cell migration via SOX2/CSF-1 signaling in the cocultured medium. Correlations of α5-nAChR, SOX2 and M2 phenotype tumor-associated macrophages (TAMs) were validated in mouse LUAD models and clinical samples. α5-nAChR expression was connected to SOX2 expression, smoking and bad prognosis of LUAD among clinical samples. Nicotine-induced SOX2 expression was mediated by α5-nAChR via STAT3. Additionally, SOX2-mediated macrophage colony-stimulating factor (CSF-1) expression contributed to LUAD progression in vitro. Furthermore, α5-nAChR expression was strongly linked to pSTAT3, SOX2 and M2 macrophage marker CD206 expression and negatively correlated with M1 macrophage marker CD86 expression in vivo. It is indicated that M2 macrophages are mediated by the new α5-nAChR /SOX2/CSF-1 axis in nicotine-related LUAD, which is a potential therapeutic strategy for cancer.

Qingrexiaoji Recipe Regulates the Differentiation of M2 TAM via miR-29 in GC.

Gastric cancer, one of the most familiar adenocarcinomas of the gastrointestinal tract, ranks third in the world in cancer-related deaths. Traditional Chinese medicine can suppress the growth of tumors, and the underlying mechanism may be associated with the tumor microenvironment. Here, we investigated the anti-cancer effects of the Qingrexiaoji recipe on gastric cancer and the underlying molecular mechanism. An in vivo nude mouse model was established, and the expression of CD206, CD80, and M2 phenotype-related proteins (Arg-1, Fizz1) was obtained by flow cytometry and western blotting. The expressions of the M2 phenotype-related cytokines were examined by ELISA. Qingrexiaoji recipe inhibited gastric tumor growth and downregulated the expression of CD206, IFN-γ, IL-13, IL-4, and TNF-α in vivo. Qingrexiaoji recipe deceased M2 phenotypic polarization by upregulating microRNA (miR)-29a-3p level. Luciferase activity assays showed that HDAC4 is a potential target of miR-29a-3p. In cells co-transfected with HDAC4 siRNA and miR-29a-3p inhibitor and treated with IL-4 and Qingrexiaoji recipe, the miR-29a-3p inhibitorinduced increase of M2 phenotypic polarization was reversed. In summary, these results suggested that the Qingrexiaoji recipe regulated M2 macrophage polarization by regulating miR-29a-3p/HDAC4, providing a different and innovative treatment for gastric cancer.

A polysaccharide from Periplaneta americana promotes macrophage M2 polarization, exhibiting anti-inflammatory and wound-healing activities

A miscellaneous polysaccharide, PAP55–3-1, with a molecular weight of 23.03 kDa, was isolated from Periplaneta americana through extraction with dilute alkali solution, ethanol precipitation, and column chromatography purification. Structural analysis shows that PAP55–3-1 is mainly composed of five monosaccharides: galactosamine hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose. Its main glycosidic bonds are: Manp-(1→, Galp-(1→, →3)-Galp-(1→, →3,6)-Manp-(1→, →2,6)-Manp-(1→, →6)-Manp-(1→, →4)-Galp-(1→, →6-Glcp-(1→, →6)-Galp-(1→, →2)-Manp-(1 →, →3,4)-Glcp-(1→, →3,6)-Galp-(1→. In vitro experiments demonstrated that PAP55–3-1 can effectively inhibit reactive oxygen species (ROS) and O2− production following H2O2−induction. After H2O2−induction, HIF-1α (hypoxia-inducible factor) was translocated in mitochondria PAP55–3-1 increased localization of HIF-1α was located on mitochondria to maintain the stability of mitochondrial function stability, thereby effectively inhibiting H2O2-induced mitochondrial oxidative damage. Additionally, PAP55–3-1 inhibited the M1 polarization of macrophages stimulated by H2O2 and promoted the phenotype polarization of macrophages from M1 to M2, displaying anti-inflammatory and pro-repair properties. In vivo experimental results indicated that PAP55–3-1 promoted wound healing in mice. Immunohistochemical experiments revealed a reduction in CD68 expression and increase in CD206 expression in both positive and the high-dose polysaccharide group control group. This further demonstrated that PAP55–3-1 promotes the phenotype polarization of macrophages from M1 to M2, exerting anti-inflammatory and wound-healing activities.

IGF2BP3/CTCF Axis-Dependent NT5DC2 Promotes M2 Macrophage Polarization to Enhance the Malignant Progression of Lung Squamous Cell Carcinomas.

Lung squamous cell carcinoma (LUSC) is a type of lung cancer that develops in the squamous cells. It is known to be promoted by the activation of various signaling pathways and the dysregulation of key regulatory molecules. One such molecule, 5'-nucleotidase domain containing 2 (NT5DC2), has been identified as a critical regulator in various cancers including lung cancer. However, there are no data regarding its role in LUSC. The mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), CCCTC-binding factor (CTCF), and NT5DC2 was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR), whereas their protein expression was assessed using a western blotting assay. Cell proliferation was determined using a cell counting kit-8 (CCK-8) assay. Cell apoptosis, CD11b expression, and CD206 expression were analyzed using flow cytometry. Tube formation was assessed through a tube formation assay. Glucose consumption, lactate production, and ATP levels were measured using colorimetric methods. The effect of NT5DC2 on the malignant progression of LUSC cells was analyzed using a xenograft mouse model assay. The levels of transforming growth factor-beta 1 (TGF-β1) and interleukin-10 (IL-10) were detected using enzyme-linked immunosorbent assays. The associations among IGF2BP3, CTCF and NT5DC2 were identified using dual-luciferase reporter assay, RNA immunoprecipitation assay and m6A RNA immunoprecipitation assay. The expression of NT5DC2 was found to be upregulated in LUSC tissues and cells when compared with normal lung tissues and normal human bronchial epithelial cells. Silencing of NT5DC2 inhibited LUSC cell proliferation, tube formation, glycolysis, M2 macrophage polarization, and tumor formation while inducing cell apoptosis. In addition, CTCF was found to transcriptionally activate NT5DC2 in LUSC cells. IGF2BP3 stabilized the mRNA expression of CTCF through m6A methylation. Further, overexpression of CTCF or NT5DC2 attenuated the effects of IGF2BP3 silencing in both NCI-520 and SK-MES-1 cells. The IGF2BP3/CTCF axis-dependent NT5DC2 promotes M2 macrophage polarization, thereby enhancing the malignant progression of LUSC. This study was the first to reveal the role of NT5DC2 in LUSC and the underlying mechanism. The result suggests that targeting the IGF2BP3/CTCF/NT5DC2 axis may have clinical significance in the treatment of LUSC.

ESTRIOL IN FORMATION OF MONONUCLEAR CELLS TOLEROGENIC FEATURES

Estriol (E3) is one of hormones whose synthesis is mainly associated with pregnancy. The hormone can also regulate immune cells functions. E3 influence on monocyte indoleamine-2,3-dioxygenase (IDO1) activity and Treg and NK cells’ markers expression was investigated. LPS and IFN-γ stimulated IDO1 activity was augmented by Е3 independently of the hormone doses. Both Е3 concentrations increased CD4+Foxp3+ lymphocytes percentage. This hormonal effect was mediated by protein kinase A. CD16 expression upon NK cells was reduced as a result of Е3 influence. The data indicated that Е3 is one of the factors which maintained immune tolerance during pregnancy and protected fetus preservation.

Spatial multiplex analysis of lung cancer reveals that regulatory T cells attenuate KRAS-G12C inhibitor-induced immune responses.

Kirsten rat sarcoma virus (KRAS)-G12C inhibition causes remodeling of the lung tumor immune microenvironment and synergistic responses to anti-PD-1 treatment, but only in T cell infiltrated tumors. To investigate mechanisms that restrain combination immunotherapy sensitivity in immune-excluded tumors, we used imaging mass cytometry to explore cellular distribution in an immune-evasive KRAS mutant lung cancer model. Cellular spatial pattern characterization revealed a community where CD4+ and CD8+ T cells and dendritic cells were gathered, suggesting localized T cell activation. KRAS-G12C inhibition led to increased PD-1 expression, proliferation, and cytotoxicity of CD8+ T cells, and CXCL9 expression by dendritic cells, indicating an effector response. However, suppressive regulatory T cells (Tregs) were also found in frequent contact with effector T cells within this community. Lung adenocarcinoma clinical samples showed similar communities. Depleting Tregs led to enhanced tumor control in combination with anti-PD-1 and KRAS-G12C inhibitor. Combining Treg depletion with KRAS inhibition shows therapeutic potential for increasing antitumoral immune responses.

LAG-3 Expression, γδ-T cell/MHC-I Interactions and Prognosis in Merkel Cell Carcinoma

Merkel Cell Carcinoma (MCC) is an aggressive cutaneous malignancy with a poor prognosis. One of the major mechanisms of immune evasion in MCC involves downregulation of MHC-I. Anti-PD-1/PD-L1 checkpoint inhibitors (CKIs) have revolutionized treatment for MCC, producing objective responses in ∼50% of patients, and are now standard of care; however, a substantial proportion of patients either fail to respond or develop resistance to CKIs. Given these recent successes, identification of other targetable immune checkpoints in the MCC tumor microenvironment (TME) is of great interest. Additionally, γ-delta (γδ) T cells may play critical roles in the response to MHC-I deficient cancers; therefore, evaluating γδ-T cells as a prognostic biomarker is warranted. We characterized the expression of PD-L1, PD-1, CD3, CD8, LAG-3, MHC-I, and γδ-T cells by IHC in a pre-immunotherapy retrospective cohort of 54 cases of MCC, and quantified expression levels and marker density via HALO. The increased density of LAG-3 and γδ-T cells correlated with other markers of an inflamed TME, with significant positive associations across all six markers (p<.002). Reflective of their putative role in the response to MHC-I suppressed cancers, cases with low HLA-I density showed a trend towards a higher ratio of γδ-T cells:CD3+ T cells (Spearman's r=-0.1582, p=0.21). Importantly, high CD3 density (HR=0.23, p=0.002), LAG-3 density (HR=0.47, p=0.037), γδ-T cell density (HR=0.26, p=0.02), and CD8 density (HR=0.27, p=0.03) showed associations with improved progression-free survival. Conditional tree analysis demonstrated that high CD8 and TCRδ expression were non-significant predictors of improved PFS and OS. Overall, LAG-3 is expressed in MCC infiltrates and is prognostic in pre-immunotherapy MCC, suggesting a potential role for LAG-3 inhibition in MCC. Additionally, CD8 and γδ-T cells may play a critical role in the response to MCC, and γδ-T cell density may represent a novel biomarker in MCC.

Activation of receptor-independent fluid-phase pinocytosis promotes foamy monocyte formation in atherosclerotic mice

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death worldwide. Clinical and experimental data demonstrated that circulating monocytes internalize plasma lipoproteins and become lipid-laden foamy cells in hypercholesterolemic subjects. This study was designed to identify the endocytic mechanisms responsible for foamy monocyte formation, perform functional and transcriptomic analysis of foamy and non-foamy monocytes relevant to ASCVD, and characterize specific monocyte subsets isolated from the circulation of normocholesterolemic controls and hypercholesterolemic patients. We hypothesized that activation of fluid-phase macropinocytosis contributes to foamy monocyte formation in vitro and in hypercholesterolemic mice in vivo. High resolution scanning electron microscopy (SEM) and quantification of FITC/TRITC-dextran internalization demonstrated macropinocytosis stimulation in human (THP-1) and wild type murine monocytes. Stimulation of macropinocytosis induced foamy monocyte formation in the presence of unmodified, native LDL (nLDL) and oxidized LDL (ox-LDL) in vitro. Genetic blockade of macropinocytosis (LysMCre+Nhe1f/f) inhibited foamy monocyte formation in hypercholesterolemic mice in vivo and attenuated monocyte adhesion to atherosclerotic aortas ex vivo. Mechanistic studies identified NADPH oxidase 2 (Nox2)-derived superoxide (O2⋅−) as an important downstream signaling molecule stimulating macropinocytosis in monocytes. qRT-PCR identified CD36 as a major SR that increases in response to lipid loading in monocytes and deletion of CD36 (Cd36-/-) inhibited foamy monocyte formation in hypercholesterolemic mice. Bulk RNA-sequencing characterized transcriptional differences between non-foamy and foamy monocytes versus macrophages. Finally, flow cytometry analysis of CD14 and CD16 expression demonstrated a significant increase in intermediate monocytes in hypercholesterolemic patients compared to normocholesterolemic controls. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets for the treatment of atherosclerosis.

Genetic link between gut microbiota, immune cells, and rheumatoid arthritis: Mechanism of action of CD28 proteins

The gut microbiota serves a crucial function in modulating the immune responses of the host, as well as in managing inflammation within the body. In this particular study, the researchers sought to delve deeper into the specific mechanisms through which CD28 interacts with the gut microbiota and influences the functionality of immune cells. The study collected intestinal microbial samples from RA patients and healthy controls, analyzed microbial composition by high-throughput sequencing, and detected CD28 expression in T cells in combination with cellular immunology methods. At the same time, the effects of CD28 deletion on intestinal microbiota changes and inflammatory responses were evaluated using animal models. The findings from this study revealed a notable distinction in the gut microbiota profiles of individuals diagnosed with rheumatoid arthritis (RA) when compared to those of healthy control subjects. Specifically, the abundance of certain microbial species was observed to have a negative correlation with the expression levels of CD28, highlighting a complex interaction between the gut microbiome and the immune regulatory mechanisms involved in RA. Furthermore, experiments conducted on mice lacking CD28 demonstrated considerable alterations in their gut microbiota composition. These Cd28-deficient mice exhibited elevated levels of inflammatory markers, indicating an interplay between CD28 and the regulation of both the microbiota and the immune response.

MiR-126 accelerates renal injury induced by UUO via inhibition PI3K/ IRS-1/ FAK signaling induced M2 polarization and endocytosis in macrophages

To investigate the role and molecular mechanism of miR-126 in unilateral ureteral occlusion (UUO). We used bioinformatics to analyse miRNAs specifically expressed in UUO. The mouse model of UUO was established using RAW264.7 cells cultured in vitro and in vivo. The mice were divided into control group, miR-126-NC (negative control) group and miR-126-KD (knockdown) group. Then the relative expression of miR-126 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the renal fibrosis was detected by Masson staining, and the protein expression of CD68, collagen I and collagen III in the kidney was detected by immunofluorescence assay. Immunohistochemistry detects α-SMA expression. Moreover, Western blotting was performed to measure the expressions of p-PI3K, CD163, CD206, CD86, iNOS, IL-1β, p-FAK, p-Rac-1, p-IRS-1 and MMP9. The relative fluorescence intensity of F-actin and p-FAK was detected by immunofluorescence assay, and the phagocytosis ability of macrophages was determined by phagocytosis assay with fluorescent microspheres. Bioinformatics analysis reveals miR-126-specific overexpression in UUO. Successful transfection of miR-126-NC and miR-126-KD was confirmed by RT-PCR. The selective reduction of miR-126 was validated by Masson, immunohistochemistry and immunofluorescence staining to decrease the area of UUO-induced renal fibrosis and to lower the expression of CD68, α-SMA, collagen I, and collagen III. The reduction of iNOS expression may also be achieved with selective knockdown of miR-126, as verified by cell tests. enhances the phagocytic ability of macrophages and the expression of p-PI3K, CD206, p-FAK, F-actin, p-Rac-1, p-IRS-1 and MMP9. MiR-126 can inhibit the PI3K signaling pathway, promote M1 macrophage polarization, and suppress the activation of FAK and Rac-1, thus accelerating the progression of UUO.