Expression Of CD86 Research Articles

Cholesterol promotes IFNG mRNA expression in CD4+ effector/memory cells by SGK1 activation.

IFNγ-secreting T cells are central for the maintenance of immune surveillance within the central nervous system (CNS). It was previously reported in healthy donors that the T-cell environment in the CNS induces distinct signatures related to cytotoxic capacity, CNS trafficking, tissue adaptation, and lipid homeostasis. These findings suggested that the CNS milieu consisting predominantly of lipids mediated the metabolic conditions leading to IFNγ-secreting brain CD4 T cells. Here, we demonstrate that the supplementation of CD4+CD45RO+CXCR3+ cells with cholesterol modulates their function and increases IFNG expression. The heightened IFNG expression was mediated by the activation of the serum/glucocorticoid-regulated kinase (SGK1). Inhibition of SGK1 by a specific enzymatic inhibitor significantly reduces the expression of IFNG Our results confirm the crucial role of lipids in maintaining T-cell homeostasis and demonstrate a putative role of environmental factors to induce effector responses in CD4+ effector/memory cells. These findings offer potential avenues for further research targeting lipid pathways to modulate inflammatory conditions.

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.

Autoreactive B cells remain active despite clinical disease control in rheumatoid arthritis

BackgroundAutoimmune diseases (AIDs) are frequently hallmarked by the presence of autoreactive B cell responses which are involved in disease pathogenesis. However, the dynamics of such responses and their relation to clinical disease activity in humans is poorly understood. Rheumatoid arthritis (RA), a prototypic chronic AID, is hallmarked by B cell responses directed against citrullinated proteins. ObjectiveTo determine the relation between the activity of the anti-citrullinated protein antibody (ACPA) B cell response and clinical disease activity in ACPA+ patients with RA. MethodsExpression of B cell activation markers by ACPA+, tetanus toxoid (TT)+ and ACPA− memory B cells (MBCs) from peripheral blood of ACPA+ RA patients receiving different treatments was analyzed by flow cytometry. Results were correlated to clinical disease activity. ResultsCompared to TT+ and ACPA− MBCs, ACPA+ MBCs displayed a highly activated phenotype as evidenced by increased expression of Ki-67, CD86, CD80, CD19 and CD20 and reduced expression of CD32. The activated phenotype of ACPA+ MBCs did not associate with clinical disease activity in a cross-sectional analysis of RA patients treated with various therapeutic agents. Also, in a longitudinal analysis of patients treated with Janus kinase (JAK) inhibitors, ACPA+ MBCs retained their activated phenotype despite effective control of inflammation and clinical disease. ConclusionACPA+ MBCs remain active despite clinical disease control in patients with RA across a range of interventions. This persistent activity indicates the absence of immunological remission and might explain why ACPA+ patients rarely reach sustained drug-free remission and frequently flare upon drug tapering.

Licochalcone A loaded multifunctional chitosan hyaluronic acid hydrogel with antibacterial and inflammatory regulating effects to promote wound healing

The wound healing process is characterized by persistent infection and long-term inflammation. The licochalcone A (LicA) has the potential for skin wound healing and needs a good drug-loading platform to apply its antibacterial and anti-inflammatory effects. In this study, the LicA@chitosan (CS) -hyaluronic acid (HA) hydrogel with antibacterial and anti-inflammatory was developed for wound healing in mice. The SEM displayed that the hydrogel had an obvious porous structure and was very suitable to be used as a delivery carrier for LicA. The FTIR results suggested that the LicA can be effectively loaded in the CS-HA hydrogel. Variable strain scanning, frequency scanning and temperature scanning indicated that the LicA@CS-HA hydrogel can maintain the gel state. The LicA@CS-HA hydrogel had good biological safety, can inhibit the activity of Escherichia coli and Staphylococcus aureus, and can release LicA stably. The LicA@CS-HA hydrogel also has good adhesion and hemostatic properties. Finally, the LicA@CS-HA hydrogel significantly accelerated wound healing in mice skin injury model, and reduced inflammation and orderly collagen deposition were observed by HE and Masson staining. The immunohistochemistry indicated that the LicA@CS-HA hydrogel induced the positive expression of CD31, VEGF, and HIF-1α promoted neovascularization. The LicA@CS-HA hydrogel also down-regulated the expression of M1 macrophage markers CD86, IL-6, and TNF-α, and increased the expression of M2 macrophage markers CD206, IL-4, and IL-10 proteins. The molecular docking demonstrated that the target proteins had better binding activity to LicA. Collectively, the LicA@CS-HA hydrogel has broad application prospects in promoting wound healing.

Bicalutamide Reveals Immunomodulatory Effects in Prostate Cancer by Regulating Immunogenic Dendritic Cell Maturation

Prostate cancer poses a significant global health challenge, ranking as the second most prevalent and fifth most lethal malignancy among males. The intricate interplay between androgen signaling and the immune microenvironment underscores the complexity of prostate cancer progression. Notably, androgen receptor (AR) signaling has been shown to affect immune response mediated by tumor antigen-presenting dendritic cells (DCs). Therefore, this study aimed to explore the potential of Bicalutamide, a nonsteroidal anti-androgen, in modulating DCs-mediated immune responses. Peripheral blood mononuclear cells (PBMCs) were isolated, and monocytes were extracted, followed by their differentiation into immature dendritic cells (iDCs) using GM-CSF and IL-4. Harvested tumor cell lysates from human prostate cancer cells were then utilized to induce the transformation of iDCs into mature dendritic cells (mDCs). Then, mDCs were treated with non-toxic concentration of Bicalutamide determined by annexin V/PI assay. The morphological characteristics of mDCs were investigated using an inverted light microscope. Flow cytometry was used to determine the cell surface expression of molecular markers of DC maturation, and qRT-PCR was employed to evaluate expression levels of proinflammatory genes involved in DC maturation. The obtained results indicated that Bicalutamide treatment of monocyte-derived mDCs induces an immunogenic and matured phenotype, marked by increased expression of CD86 and HLA-DR. Besides, qRT-PCR results evidenced that Bicalutamide decreased the expression of anti-inflammatory genes, including Interleukin-10 (IL-10) and TGF-beta, as an indication of immunogenic DCs. These findings suggest that beyond its established anti-androgen role, Bicalutamide may exert anti-tumor effects through the modulation of DCs-mediated immune responses. This novel immunomodulatory feature holds promise for the development of novel therapies, including combination therapies, in prostate cancer treatment.

Genome-Wide Identification of Cell Type-Specific Susceptibility Genes for SLE Through the Analysis of RNA Modification-Associated SNPs

ABSTRACT Introduction This study aimed to elucidate the functional genes associated with systemic lupus erythematosus (SLE) in various cell types through the utilization of RNAm-SNPs. Methods Utilizing large-scale genetic data, we identified associations between RNAm-SNPs and SLE. The association between RNAm-SNPs and bulk and single-cell mRNA expression (eQTL) and protein levels (pQTL) were examined. Mendelian randomization and differential expression analyses were conducted to explore the links between gene expression, protein levels, and SLE. Results We identified 41 RNAm-SNPs that were significantly associated with SLE. The GWAS signals exhibited notable enrichment in m6A-SNPs and m7G-SNPs. These RNAm-SNPs showed both eQTL and pQTL effects. In our single-cell analysis, 16 RNAm-SNPs exhibited associations with gene expression levels across 13 distinct cell types, including HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DRB1 and IRF7. We identified 58 noteworthy associations between the expression levels of 20 genes and SLE across 12 distinct immune cell types. Notably, HLA-DQB1, HLA-DRB1 and IRF7 exhibited abnormalities in CD8+ T cells, IRF7 displayed abnormal expression in CD4+ T cells, while HLA-DRB1 and IRF7 were also distinctly perturbed in natural killer cells. Discussion This study advances our understanding of the genetic basis of SLE by highlighting the significance of RNAm-SNPs and immune cell gene expression in SLE.

Biofabricated zinc oxide nanoparticles mitigate acrylamide-induced immune toxicity and modulate immune-related genes and microRNA in rats.

This study evaluated the potential efficacy of eco-friendly biofabricated zinc oxide nanoparticles (GS-ZnONP) (10mg/kg b.wt) to reduce the impacts of long-term oral acrylamide (ALD) exposure (20mg/kg b.wt) on the blood cells, immune components, splenic oxidative status, and expression of CD20, CD3, CD4, CD8, TNF-α, caspase-3, microRNA-181a-5p, and microRNA-125-5p in rats in a 60-day experiment. The study findings revealed that GS-ZnONP significantly corrected the ALD-induced hematological alterations. Additionally, the ALD-induced increase in the serum C3, splenic ROS, CD4, CD8, and MDA and histological alterations were significantly repressed in the ALD + GS-ZnONP-treated rats. Instead, the depleted splenic antioxidants and Zn contents were markedly reestablished in the ALD + GS-ZnONP-treated group. Additionally, a significant upregulation of expression of splenic CD3, CD4, CD8,CD20, TNF-α, and caspase-3, but downregulation of microRNA-181a-5p and microRNA-125-5p was detected in the ALD-exposed group. Yet, the former deviations in the gene expressions were corrected in the ALD + GS-ZnONP-treated rats. Furthermore, GS-ZnONP treatment significantly minimized the increased caspase-3 and TNF-α immunoexpression in the splenic tissues of ALD-exposed rats. Conclusively, the study findings proved the efficacy of GS-ZnONP in rescuing ALD-induced disturbances in blood cell populations, immune function, splenic antioxidant status, and immune-related gene expression.

Palmitoylation of TIM-3 promotes immune exhaustion and restrains antitumor immunity.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is an immune checkpoint that has critical roles in immune exhaustion. However, little is known about the mechanisms that regulate TIM-3 surface expression and turnover. Here, we report that human TIM-3 is palmitoylated by the palmitoyltransferase DHHC9 at residue cysteine 296 (Cys296). Palmitoylation stabilized TIM-3 by preventing binding to E3 ubiquitin ligase HRD1, thereby suppressing its polyubiquitination and degradation. DHHC9 knockdown attenuated chimeric antigen receptor T (CAR-T) cell exhaustion, and a peptidic inhibitor of TIM-3 palmitoylation accelerated TIM-3 degradation and enhanced antitumor immunity mediated by CAR-T cells and natural killer (NK) cells. In hepatocellular carcinoma, DHHC9 expression correlated with TIM-3 expression in CD8+ T cells and NK cells, and high DHHC9 expression was associated with shorter survival in patients with high TIM-3. These findings demonstrate that palmitoylation of TIM-3 catalyzed by DHHC9 promotes its stability, resulting in immune exhaustion and impaired antitumor immunity.

Cirsilineol improves anesthesia/surgery-induced postoperative cognitive dysfunction through attenuating oxidative stress and modulating microglia M1/M2 polarization

Background Cirsilineol is a trimethoxy and dihydroxy flavonoid isolated from plant species such as Artemisia vestita and has a variety of pharmacological properties. This study analyzed whether cirsilineol could prevent postoperative cognitive dysfunction (POCD). Methods A POCD mouse model induced by anesthesia/surgery induction and a cell model established with hydrogen peroxide (H2O2)-induced microglia BV-2 were employed to explore the efficacy of cirsilineol on POCD. The cognition function of the mice were assessed by carrying out behavioral tests (Morris water maze test and Y-maze test). We assessed the activation and polarization status of microglia using immunofluorescence analysis and detected the expression levels of CD86 and CD206 using the quantitative PCR (qPCR). Subsequently, cell viability was determined by CCK-8 assay and apoptosis was assessed using Calcein-AM/PI staining. Meanwhile, superoxide dismutase (SOD) and malondialdehyde (MDA) levels in plasma and cell culture medium were detected using chemiluminescence. Finally, the phosphorylation levels of JAK/STAT signaling pathway-related proteins were analyzed by Western blot. Results Cirsilineol reduced the escape latency and times of crossing island and increased spontaneous alternation (SA) rate, restoring the cognitive dysfunctions of POCD-modeled mice. Meanwhile, POCD elevated CD86 expression and malondialdehyde content and lowered the level of SOD; however, cirsilineol promoted CD206 expression and generation of SOD and inhibited malondialdehyde production. In H2O2-induced microglia BV-2, cirsilineol treatment increased SOD content and suppressed the generation of reactive oxygen species (ROS) and malondialdehyde, modulating microglia M1/M2 polarization and JAK/STAT pathway. Conclusion Cirsilineol prevented against POCD by attenuating oxidative stress and modulating microglia M1/M2 polarization, providing novel insights for the management of POCD.

CD40 agonist on patient-derived xenograft mice for the treatment of B-cell acute lymphoblastic leukemia.

Cluster of differentiation 40 (CD40) is expressed on B-cell acute lymphoblastic leukemia (B-ALL) cases. However, the effect of CD40 activation on B-ALL cells has never been tested in vivo. The aim of our preclinical study was to investigate the therapeutic potential of a CD40 agonist in the treatment of B-ALL using patient-derived xenograft (PDX) mouse models. Intravenous administration of the CD40 agonist significantly impeded B-ALL cell proliferation and growth in vivo, accompanied by rapid activation of the extracellular signal-regulated kinase (ERK) pathway, leading to the induction of apoptosis and disruption of cell cycle progression. Co-treatment with a specific inhibitor of ERK further demonstrated that CD40 stimulation induced the pro-apoptosis of B-ALL cells in an ERK-dependent manner. Proteomic analysis revealed alterations in key signaling pathways associated with B-ALL expansion and maintenance. Moreover, the CD40 agonist markedly reduced the frequency of leukemia-initiating cells and leukemia development in PDX mice. Our study showed that the CD40 agonist can be associated with chemotherapeutic agents such as vincristine and dexamethasone, and this combination showed improved effectiveness. Additionally, the CD40 agonist was more effective on pre-B-ALL (EGIL B-III) that expressed CD40, than on common B-ALL (EGIL B-II) that lacked CD40 expression. These findings suggest that CD40 agonists are promising immunotherapeutic candidates for pediatric B-ALL, warranting further clinical investigations to improve patient outcomes in CD40-expressing B-ALL.

Platelet activation through CD62P and the formation of platelet-monocyte complexes are associated with the exacerbation of mucosal inflammation in patients with ulcerative colitis.

Ulcerative colitis (UC) is a refractory, chronic inflammatory bowel disease of unknown etiology. Although platelets are activated in UC, their relevance in pathophysiology remains unclear. We analyzed the correlation of platelet activation and platelet-monocyte complexes (PMCs) with severity of mucosal inflammation using the Mayo endoscopic subscore (MES). Platelet activation marker, CD62P was upregulated in patients with UC compared with that in healthy controls (P < 0.05). CD62P expression was significantly higher in patients with MES3 (severe inflammation) than in those with MES ≤ 2 (endoscopic remission to moderate inflammation) (P < 0.001). The concentration of sCD62P in patients with MES0 (endoscopic remission) was significantly higher than in those with MES ≥ 1 (P < 0.01). The expression of CD40L, CD63, PAC-1, annexin V, and CD36, and the concentrations of sCD40L, PF4, and RANTES did not correlate with MES. The proportion of PMCs in patients with MES3 was higher than in those with MES ≤ 2 (P < 0.05). CD16 expression on monocytes with platelets was significantly higher than in monocytes without platelets (P < 0.001). Patients with complete remission after treatment showed significant reduction in PMCs 3 months after treatment (P < 0.05) but had no change in CD62P and sCD62P. Our data suggest that platelet activation via the CD62P-PMC axis is involved in UC pathophysiology.

Fibroblast growth factor 10 alleviates LPS-induced acute lung injury by promoting recruited macrophage M2 polarization.

Acute lung injury (ALI) is characterized by damage to the alveoli and an overabundance of inflammation. Representing a serious inflammatory condition, ALI lacks a precise treatment approach. Despite the recognized benefit impacts of Fibroblast growth factor-10 (FGF10) on ALI, the underlying mechanisms remain unelucidated. To study the role of FGF10 in ALI, C57BL/6J mice were intratracheally injected with 5mg/kg Lipopolysaccharide (LPS) with FGF10 (5mg/kg) or an equal volume of PBS. Inflammatory factors were quantified in bronchoalveolar lavage fluid (BALF) and plasma using ELISA. RNA sequencing of F4/80+Ly6G- macrophages in BALF explored changes in macrophage phenotype and potential mechanisms. Macrophage polarization in BALF was assessed using qRT-PCR, flow cytometry, and Western blot analysis. In vitro, a Transwell co-culture of mouse lung epithelial cells (MLE12) and bone marrow macrophages (BMDM) validated the role of FGF10 in modulating LPS-induced macrophage phenotypic changes. FGF10 ameliorated LPS-induced ALI by diminishing pro-inflammatory factors (IL-1β, TNF-α, and IL-6) and the neutrophil accumulation in BALF. FGF10 also increased the levels of anti-inflammatory factor IL-10. The FGF10 intervention group exhibited enhanced gene expression of macrophage arginine biosynthesis marker (ARG1), and expression of M2-type marker CD206 in monocytes and macrophages. In addition, phosphorylated STAT3 expression increased in isolated monocyte-derived macrophages. Experiments in vitro confirmed that FGF10 could elevate macrophage M2 marker ARG1 expression through the JAK2/STAT3 pathway. FGF10 ameliorates acute LPS-induced lung injury by modulating the polarization of monocyte-derived macrophages recruited in the alveolar space to the M2 type.

Lactobacillus johnsonii JERA01 upregulates the production of Th1 cytokines and modulates dendritic cells-mediated immune response.

Lactic acid bacteria are known to have various effects on the immune system. The type and extent of the effect differ, depending on the type of lactic acid bacteria. This study aimed to investigate the effects of Lactobacillus johnsonii bacterin on mouse-derived immune cells. Treating splenocytes with L. johnsonii bacterin slightly increased the metabolic activity. Additionally, the expression of the activation marker CD25 and production of the Th1-type inflammatory cytokine interferon (IFN)- gamma increased. We confirmed that the increase in IFN-gamma production due to L. johnsonii stimulation was mainly due to T and B cells among splenocytes. Treating dendritic cells (DCs) with L. johnsonii bacterin at concentrations of 106 and 107 cfu/ ml significantly increased tumor necrosis factor-alpha, a pro-inflammatory cytokine, and interleukin-12, a cell-mediated immunity cytokine. Additionally, the expression of surface markers increased. Allogeneic mixed lymphocyte reactions showed that L. johnsonii reduced the antigen-presenting ability of DCs. In cocultures of DCs and splenocytes, L. johnsonii decreased cellular metabolic activity and increased cell death. L. johnsonii upregulated the expression of programmed death ligand 1 on DCs. The findings of this study indicate that L. johnsonii bacterin has immunomodulatory and immunostimulatory effects. While L. johnsonii increased the expression of cytokines and surface markers of immune cells, it modulated DC-mediated immune response. Further studies are needed to determine the effects of L. johnsonii bacterin on DCs and related immune cells.

Anti-inflammatory role of low-intensity pulsed ultrasound in inhibiting lipopolysaccharide-induced M1 polarization of RAW264.7 cells via Wnt2b/AXIN/β-catenin

Background Low-intensity pulsed ultrasound (LIPUS) is a special type of low-intensity ultrasound. In periodontal disease, LIPUS is applied as an adjuvant and non-invasive treatment. It has been reported that LIPUS significantly shifts the macrophage phenotype from M1 to M2, but the specific mechanism behind this shift is still unknown. Methods RAW264.7 cells were induced to M1/M2 polarization with lipopolysaccharide (LPS)/interleukin-4 (IL4). LIPUS was performed for 25 min two times, 24 h apart, at an intensity of 45 mW/cm2 to stimulate RAW264.7 cells. PolyA mRNA sequencing was conducted of both the LPS-induced RAW264.7 cells and the LPS-induced RAW264.7 cells with LIPUS treatment. The expression of Wnt2b in RAW264.7 cells was downregulated by siRNA. The macrophage surface markers and downstream inflammatory cytokines were detected using flow cytometry. The relative expression of proteins in the Wnt2b/AXIN/β-catenin pathway was assessed using reverse transcription real-time polymerase chain reaction (RT-qPCR) and Western blot. Results LIPUS reversed the M1 polarization of RAW264.7 cells, with decreased expression of CD80 and CD86. In addition, LIPUS enhanced the M2 polarization of RAW264.7 cells, with upregulated expression of CD163 and CD206. The polyA mRNA sequencing results indicated that the Wnt signaling pathway participated in the M1 polarization of LIPUS-treated RAW264.7. The results of the RT-qPCR showed a higher expression of Wnt2b in LIPUS-treated and M1- or M2-polarized RAW264.7 cells. Knocking down Wnt2b was shown to reverse the inhibitory effect of LIPUS on M1 polarization and increase the expression of CD80 and CD86. Wnt2b knockdown also regulated downstream AXIN, β-catenin, and inflammatory factors such as tumor necrosis factor alpha (TNFα) and interleukin-6 (IL6). Conclusions LIPUS plays an anti-inflammatory role by inhibiting LPS-induced M1 polarization of RAW264.7 cells in a Wnt2b/AXIN/β-catenin-dependent way. LIPUS may play a therapeutic role in periodontal diseases by inhibiting inflammation through the regulation of macrophage differentiation.

Magnesium-related gene ITGAL: a key immunotherapy predictor and prognostic biomarker in pan-cancer

BackgroundIntegrin subunit alpha L (ITGAL) is crucial for activating CD8+ T cells through magnesium-mediated immune synapse formation and specific cytotoxicity. ITGAL might exert an important function in the growth and transformation of cancer.MethodsOur study comprehensively analyzed ITGAL expression across various cancers, validated by Immunochemistry (IHC) in the laboratory. ITGAL showed prognostic significance in pan-cancer patients, correlated with clinical features, and associated with specific signaling pathways. We also observed a relationship between ITGAL and immune cell infiltration. In HNSCC, ITGAL demonstrated prognostic value and potential implications for immunotherapy response and novel drug targets.ResultsITGAL expression linked to tumor prognosis across 27 cancers. Elevated ITGAL correlated with good prognosis in CESC, LUAD, SARC, HNSCC, and SKCM. ITGAL involved in immune regulation pathways and showed positive correlation with immune cell infiltration. ITGAL associated with CD8+ T cell infiltration. And high ITGAL expression in CD8+ T cells and NK cells. In HNSCC, ITGAL linked to favorable prognosis and sensitivity to immunotherapy. Predicted potential drugs for HNSCC.ConclusionITGAL is remarkably associated with CD8+T cells and crucial in the tumor immune microenvironment of pan-cancer. Furthermore, our findings may provide a targeted anti-tumor strategy for ITGAL by influencing the tumor immune microenvironment.

Recruitment and polarization typing of tumor-associated macrophages is associated with tumor progression and poor prognosis in Wilms tumor patients.

Tumor-associated macrophages (TAMs) play a crucial role in shaping various tumor microenvironments. However, their recruitment in Wilms tumor (WT), the predominant malignant renal tumor in children, has been inadequately explored. This retrospective cohort study involved the analysis of 148 WT samples to investigate the recruitment and polarization typing of TAMs in WT tissues. WT tissues underwent Western blotting (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and immunofluorescence (IF) to measure the expression of TAM markers CD68, CD86, and CD163. Statistically analyze the relationship between TAM recruitment levels and patient clinical characteristics, and use Kaplan-Meier curves and the log-rank test to evaluate the association between TAM levels and survival outcomes. The findings indicated a positive correlation between the recruitment levels of total macrophages (Mtotal) and M2 tumor-associated macrophages (M2 TAM) in both chemotherapy and non-chemotherapy groups with the clinical stage. Elevated recruitment of Mtotal and M2 TAM in tumor tissues was linked to a poorer prognosis. Notably, patients with persistently higher recruitment of M2 TAM following preoperative chemotherapy exhibited the worst prognosis. The recruitment and polarization typing of TAM exhibit significant differences in WT patients with various stages and prognosis outcomes, suggesting a potential avenue for future diagnosis and treatment of WT.

EXTH-85. ASSESSMENT OF ANTI-CD69 ANTIBODY THERAPY ALONE OR IN COMBINATION WITH ANTI-PD-1 IN MURINE GBM

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressive central nervous system malignancy with a dismal prognosis. There is continued interest in trying multiple immunotherapy modalities for this intractable cancer. Cluster of differentiation 69 (CD69), is an early marker of T and NK cell activation. CD69 play multiple roles in T cell-mediated immunoregulation with immune inhibitory role in antitumor immunity. Previous preclinical work in non-CNS malignancies has demonstrated that targeting CD69 can improve clinical outcome and anti-tumor immunity. We aimed to assess anti-CD69 Ab therapy alone or in combination with anti-PD-1 in murine GBM. METHODS We explored transcriptomic data from 163 GBM patients using the TCGA database. We then tested the therapeutic value of anti-CD69 immunotherapy in a preclinical model of GBM and assessed the ideal timing of treatment with anti-CD69 Ab by evaluating survival and immune tumor-microenvironment by flow cytometry. Two treatment-timings with anti-CD69 Ab were further evaluated either alone, or in combination with anti-PD1 Abs and compared to control and to anti-PD1 only treatment. Survival data was collected. RESULTS CD69 expression was significantly increased in GBM patients tissues as compared with normal brains and increased CD69 expression was associated with worse progression-free survival. Treatment-timing with single dose anti-CD69 Ab at day 1 or three doses at days 4, 8 and 12 post tumor-injection minimally improved the median survival time of animals. In addition, effective CD69 blockage and significantly elevated PD-1 expression on NK cells was found in these timings, making them good candidates to evaluate anti-CD69 Ab therapy alone or in combination with anti-PD-1 Ab. However, when assessing these treatment-timings with anti-CD69 Ab alone or in combination with anti-PD1 no survival benefit was found. CONCLUSION This is the first study assessing anti-CD69 Ab therapy alone or in combination with anti-PD-1 in murine GBM. While some effect was found on the cellular level, this didn’t translate to survival benefit and results are overall negative.

BIOM-09. CLINICAL EFFECT OF CD25 ON THE MTX-BASED CHEMOSENSITIVITY OF PRIMARY CENTRAL NERVUS SYSTEM LYMPHOMA

Abstract INTRODUCTION Primary central nervous system diffuse large B-cell lymphoma (PCNSL) is treated with chemotherapy, mainly with high-dose methotrexate (HD-MTX), but some cases are resistant to treatment and relapse in a short period. However, a biomarker that predicts the chemosensitivity and recurrence has not yet been identified. In this study, we used surface marker analysis of tumor tissue using flow cytometry to search for biomarkers related to chemosensitivity and early recurrence. METHODS From 2017 to 2023, 25 patients were diagnosed with PCNSL by biopsy in Kobe University Hospital, and flow cytometry of the biopsy tissues were performed. Cerebrospinal fluid (CSF) examination including sIL2R, β2MG, and IL-10 was performed in all cases before surgery, and treatment based on methotrexate was administered postoperatively. We retrospectively analyzed patient characteristics, flow cytometry data, treatment details, response rate, time to recurrence, and overall survival. RESULTS The mean age of the patients was 71 (range, 37-80) years. In univariate analysis, high expression level of CD25 (interleukin-2 receptor, IL-2R) in the tissue was associated with a lower response rate to MTX-based chemotherapy (Fisher’s exact test, p=0.0072) and had a tendency of shorter time to recurrence. However, there were no significant differences in progression-free survival and overall survival. There was no correlation between expression level of CD25 in tissue and sIL-2R in CSF. Expression level of sIL-2R in CSF was independent of CD25 expression in tissue. CONCLUSION There are some reports that CD25 can be a prognostic factor in follicular lymphoma, but no reports in PCNSL. Here, we suggested that high expression level of CD25 in PCNSL tissue was associated to chemoresistance and early recurrence. In cases of PCNSL with high CD25 expression level, additional whole-brain irradiation and transition to tirabrutinib be considered because there are possibilities of chemoresistance and early recurrence.

TMIC-27. PDPN ACTIVATION OF CLEC5A IN THE PERI-NECROTIC NICHE DRIVES TAM POLARIZATION AND TME IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common malignant glioma of adults and is characterized by a severely hypoxic and immunosuppressive tumor microenvironment (TME). CLEC5A regulates immune responses in inflammatory and infectious diseases. However, its role in GBM immune regulation remain unclear. Here, we found that CLEC5A has the strongest association with poor clinical outcomes among all immune-related genes in GBM. Tumor-associated macrophages (TAMs) account for the majority of immune cells in the GBM and CLEC5A expression in this population is highest in hypoxic, peri-necrotic zones. Both hypoxia and GBM conditioned media cause increased CLEC5A expression by THP-1 cells, and overexpression of CLEC5A enhanced TAM polarization, migration toward glioma cells, and increased secretion of cytokines that mediate immunosuppression. We show that CLEC5A is a cell surface receptor activated by podoplanin (PDPN) and induces TAM polarization through Syk-JAK-STAT3 signaling. Co-IP assay showed that CLEC5A and PDPN could be binded together using antibodies for CLEC5A, Flag-CLEC5A or PDPN when we cocultured control THP-1 cells and THP-1 cells overexpressing Flag-CLEC5A with primary GBM cells (NU02068). PDPN blocking antibody (α-PDPN) reduced the specific binding between PDPN and CLEC5A, as well as inhibited the TAM-IM polarization. Overexpression of CLEC5A significantly upregulated the expression of p-Syk, Jak2, p-Jak2, STAT3, p-STAT3, CD163, CD206 and PD-L1. Moreover, Bay 61-3606, a selective Syk inhibitor, rescued CLEC5A overexpression-mediated TAM-IM polarization and Syk-JAK-STAT3 activation in THP-1 cells. In vivo, both silencing CLEC5A in TAMs and silencing PDPN in GBM cells, as well as pharmacologically targeting Syk delayed glioma growth, prolonged survival and was associated with diminished immunosuppressive TAMs. Collectively, we found that PDPN-CLEC5A-Syk-JAK-STAT3 signaling causes TAM polarization and enhances TME immunosuppression in GBM and can be targeted to suppress growth.

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&amp;lt;0.05) and the draining cervical lymph node (p&amp;lt;0.01). Intratumoral infiltration of both CD8 T (p&amp;lt;0.05) and NK (p&amp;lt;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&amp;lt;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.