Expression Of CD86 Research Articles

Siraitia grosvenorii Extract Protects Lipopolysaccharide-Induced Intestinal Inflammation in Mice via Promoting M2 Macrophage Polarization.

Siraitia grosvenorii has anti-inflammatory, antioxidant, and immune-regulating effects, while macrophages play an important role in reducing inflammation. However, it is still unclear whether Siraitia grosvenorii extract (SGE) is effective in reducing inflammation by regulating macrophages. This study investigated the regulatory effect of SGE on macrophage polarization in a lipopolysaccharide (LPS)-induced intestinal inflammation model after establishing the model in vitro and in vivo. The results from the in vivo model showed that, compared with the LPS group, SGE significantly improved ileal morphology, restored the ileal mucosal barrier, and reduced intestinal and systemic inflammation by increasing CD206 and reducing iNOS proteins. In the in vitro model, compared with the LPS group, SGE significantly reduced the expression of iNOS protein and cytokines (TNF-α, IL-1β, and IFN-γ) while significantly increasing the protein expression of CD206 in RAW264.7 cells. In conclusion, SGE can alleviate intestinal inflammation, protect the mucus barrier, and block the systemic immunosuppressive response by increasing M2 macrophages.

Linderanine C regulates macrophage polarization by inhibiting the MAPK signaling pathway against ulcerative colitis

Ulcerative colitis (UC) is a chronic non-specific inflammatory disease involving the mucosa and submucosa of the rectum and colon. Lindera aggregate (Sims) Kosterm is a traditional Chinese herb used for thousands of years in the treatment of gastrointestinal diseases. Previously, we have demonstrated that the extracts of Lindera aggregate have good anti-UC effects, but their pharmacodynamic active components have not been fully clarified. Therefore, we explored the therapeutic effect of Linderanine C (LDC), a characteristic component of Lindera aggregata, on UC and its mechanism in this study. Firstly, we found that LDC could significantly reduce the disease activity index of UC and improve shortened colon and pathological changes in vivo. Colon tissue transcriptomics suggested that the anti-UC effect of LDC might be related to its anti-inflammatory activity. Cellular experiments revealed that LDC could inhibit the expression of the M1 cell marker CD86 in RAW264.7 cells, reduce the production of inflammatory mediators such as IL-6 and TNF-α, and have good anti-inflammatory activity in vitro. Cellular transcriptomics reveal the potential involvement of the MAPK signaling pathway in the anti-inflammatory effect of LDC. The co-culture assay confirmed that LDC could significantly reduce inflammation-mediated intestinal epithelial cell injury. In conclusion, LDC was able to inhibit macrophage M1 polarization and reduce inflammatory mediator production by inhibiting the MAPK signaling pathway, effectively improving UC.

Establishment of an in vitro evaluation method for immunomodulatory functions of yeast strains.

Saccharomyces cerevisiae, a widely studied yeast known for its industrial applications, is increasingly recognized for its potential in immunomodulation. This study aimed to systematically analyze and compare the immune-modulating properties of various S. cerevisiae strains under controlled experimental conditions. Three essential signals crucial for immune response activation were evaluated to elucidate the immunological responses elicited by these strains, i.e., dendritic cells (DC) cytokine secretion profiles, maturation status, and T cell polarization. Analysis of DC cytokine secretion profiles and maturation status revealed that all tested yeast strains induced DC activation, characterized by significant IL-6 secretion and modest IL-10 induction, as well as upregulation of MHC II molecules. Additionally, strain-specific effects were observed, particularly, strain AJM109 and Y1383 uniquely enhanced CD86 and PD-L1 expression, respectively, suggesting differential impacts on DC co-stimulatory signaling. Furthermore, strain Y1383 showed a unique capacity to support Treg-mediated immune suppression, demonstrating its potential in immune tolerance induction. These findings underscore the complexity of S. cerevisiae-based immune modulation and emphasize the importance of standardized evaluation methods to distinguish their specific immunological effects.

Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFβ1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFβ1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFβ1 mRNA expression and protein translation. Transfection of TGFβAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFβ1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNβ, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFβAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2’-5’-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFβAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFβ expression. These findings also suggest TGFβ as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.

Endoplasmic Reticulum Stress Promotes Telomerase Reverse Transcriptase Expression Contributes to Development of Allergic Rhinitis.

The Th2 cell polarization is a crucial factor in the pathogenesis of allergic diseases. The underlying mechanism requires further investigation. Telomerase has an immune-regulating ability. The aim of this study is to elucidate the association between telomerase and Th2 cell polarization in patients with allergic rhinitis (AR). CD4+ T cells were isolated from blood samples collected from AR patients and healthy control subjects. RNA sequencing was employed to analyze RNA samples extracted from CD4+ T cells. An AR mouse model was established using the ovalbumin-alum protocol. High telomerase gene activity and high endoplasmic reticulum (ER) stress status were observed in CD4+ T-cells in patients with AR. Positive correlation between the telomerase reverse transcriptase (TERT) gene expression in CD4+ T cells and AR response in patients with AR. TERT facilitated the degradation of Foxp3 proteins in CD4+ T cells, resulting in the polarization of Th2 cells. Sensitization with the ovalbumin-alum protocol enhanced the Tert expression in CD4+ T cells by exacerbating ER stress. Conditional inhibition of the Tert or eukaryotic translation initiation factor 2-α (Eif2a) expression in CD4+ T cells effectively attenuated experimental AR in mice. Elevated amounts of telomerase in CD4+ T cells were found in CD4+ T cells of subjects with AR. Telomerase promoted Th2 cell polarization by inducing Foxp3 protein degradation and promotes GATA3 activation. Inhibition of TERT or eIF2a alleviated experimental AR.

Abstract We075: Stimulator of Interferon Genes (STING) Regulates T Cell Fate in Cardiometabolic Heart Failure with Preserved Ejection Fraction (HFpEF)

Introduction: Combined obesity and hypertension alter the endoplasmic reticulum (ER) stress response by downregulating X-box binding protein 1 ( Xbp1 ) in T cells, which heightens T cell inflammatory potential and persistence in Heart Failure with Preserved Ejection Fraction (HFpEF), a prevalent syndrome with no cure. The ER protein Stimulator of Interferon Genes (STING), in concert with Xbp1 , coordinates T cell inflammation and death in other contexts, yet its role in the T cell inflammatory response in cardiometabolic HFpEF is unknown. Hypothesis: STING activation and dysregulated ER stress responses drive T cell inflammation in HFpEF by balancing T cell activation and persistence in response to combined comorbidities. Methods: C67BL/6J (wild type, WT) mice were fed high-fat diet/L-NAME (H/L) or standard chow (STD) for 5 weeks, and STING activation was measured in splenic CD4 + T cells by Western blotting. Splenic CD4 + T cells from WT or STING-deficient mice (STING -/- ) were activated with αCD3/αCD28 for 3 days in vitro , then treated with complete media (CM) or CM with 0.2 mM palmitate (abundant in dietary fat)/0.2 mM L-NAME (PALNA) for up to 48 hours. T cell gene expression, activation, and death were assessed using qPCR, flow cytometry, Western blotting, and Lionheart live-cell microscopy. Results: Mice fed H/L had significantly increased splenic T cell expression of phospho-STING, alongside decreased T cell Xbp1 expression and increased cardiac inflammation, compared to STD-fed mice. Ex vivo incubation of CD4 + T cells with PALNA significantly decreased Xbp1 gene expression and increased apoptosis, indicated by cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expression, compared to cells in CM. However, the remaining live PALNA-treated cells showed higher expression of the activation protein CD69, and this was sustained over time, compared to cells in CM. Strikingly, live cell imaging revealed that STING -/- T cells had improved survival and decreased CD69 expression in response to PALNA compared to WT PALNA-treated cells. Conclusions: STING contributes to T cell inflammation in experimental cardiometabolic HFpEF by synergizing with dysregulated ER stress responses to promote the emergence of hyperactivated T cells.

Validation of reliable reference genes for qPCR of CD4+ Tcells exposed to compressive strain.

For accurate interpretation of quantitative real-time PCR (qPCR) data, stable reference genes are essential for normalization of target genes. To date, there is no information on reliable housekeeping genes in CD4+ Tcells in athree-dimensional (3D) matrix under pressure stimulation. This in vitro study describes for the first time amethod for pressure stimulation of CD4+ Tcells in a3D matrix in the context of orthodontic tooth movement (OTM) and identifies aset of reliable reference genes. CD4+ Tcells were isolated from murine spleen and activated with anti-CD3/-CD28 Dynabeads (Thermo Fisher, Langenselbold, Germany) on standard cell culture plates or in 3D scaffolds with or without compressive strain. Expression stability of nine potential reference genes was examined using four mathematical algorithms. Gene expression of Il2 was normalized to all potential reference genes to highlight the importance of correct normalization. Cell proliferation and the expression of the surface markers CD25 and CD69 were also determined. The 3D matrix did not inhibit proliferation after immunological activation of Tcells and embedded the cells sufficiently to expose them to pressure load. Expression of ubiquitin C (Ubc) and hypoxanthine phosphoribosyltransferase (Hprt) was the most stable under all conditions tested. Acombination of these two genes was suitable for normalization of qPCR data. Normalization of Il2 gene expression showed highly variable results depending on the reference gene used. Pressure reduced cell proliferation and the number of CD69-positive Tcells. This study provides abasis for performing valid and reliable qPCR experiments with CD4+ Tcells cultured in 3D scaffolds and exposed to compressive forces simulating OTM.

Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p>0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.

Curcumin loaded hydrogel with double ROS-scavenging effect regulates microglia polarization to promote poststroke rehabilitation

Cyclodextrins are used to include curcumin to form complex, which is subsequently loaded into a reactive oxygen species (ROS) responsive hydrogel (Cur gel). This gel exhibits a dual ROS scavenging effect. The gel can neutralize extracellular ROS to lead to a ROS-sensitive curcumin release. The released curcumin complex can eliminate intracellular ROS. Furthermore, the Cur gel effectively downregulates the expression of CD16 and IL-1β while upregulating CD206 and TGF-β in oxygen and glucose-deprived (OGD) BV2 cells. Additionally, it restores the expression of synaptophysin and PSD95 in OGD N2a cells. Upon injection into the stroke cavity, the Cur gel reduces CD16 expression and increases CD206 expression in the peri-infarct area of stroke mice, indicating an in vivo anti-inflammatory polarization of microglia. Colocalization studies using PSD95 and VGlut-1 stains, along with Golgi staining, reveal enhanced neuroplasticity. As a result, stroke mice treated with the Cur gel exhibit the most significant motor function recovery. Mechanistic investigations demonstrate that the released curcumin complex scavenges ROS and suppresses the activation of the ROS-NF-κB signaling pathway by inhibiting the translocation of p47-phox and p67-phox to lead to anti-inflammatory microglia polarization. Consequently, the Cur gel exhibits promising potential for promoting post-stroke rehabilitation in clinics.

Abstract Tu020: Contrasting effect of Ox-PAPC and PC-KLH in atherosclerotic plaques or peripheral blood T cell, macropahges and dendritic cell activation

Background: Atherosclerosis is a chronic inflammation characterized by accumulation of immune cells, oxidized low density lipoprotein (OxLDL) and dead cells in the lesions, both of the latter have the hapten phosphorylcholine (PC) as an important antigen. PC is also exposed on microorganisms and other compounds and needs a carrier to be antigenic. Antibodies against PC (anti-PC) ameliorates deleterious effects of both OxLDL and dead cells. Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (Ox-PAPC) is a major compound in the lipid moiety of OxLDL exposing PC. Methods: We studied effect of Ox-PAPC and PC with keyhole limpet cyanid (KLH) on T cells, dendritic cells (DC) and macrophages (MQ). We examined the influence of Ox-PAPC and PC conjugated to KLH (PC-KLH) on T cells, DC, and MQ from peripheral blood or atherosclerotic plaques. We investigated phospholipid and glutathione (GSH) levels in the blood plasma of healthy controls (n=30) and patients (n=30). Results: Ox-PAPC, in contrast to PAPC and PC-KLH, independently of DC, induced proinflammatory T cell activation, which was inhibited by mito tempo, inhibitor of reactive oxygen species (ROS). Ox-PAPC triggered inflammatory activities while reducing metabolic activities in T cells. PC-KLH, on the other hand, reduced CD86 and CD11C, proinflammatory cytokines, and induced anti-inflammatory IL-10 in DC. Ox-PAPC induced ROS, GSH, and proinflammatory MQ activation, inhibited by Mito tempo or GSH inhibitor. Additionally, Ox-PAPC, in the presence of PCSK9, elevated CD86 expression and lipid peroxidation. Patients exhibited higher GSH levels comapre to control groups, associated with phospholipid levels. Conclusions: The study reveals the differential effects of Ox-PAPC on various immune cell types, showcasing T cell activation independent of DC and heightened macrophage activation in the presence of PCSK9. Ox-PAPC-induced redox imbalance regulates T cell activation, apoptosis, and MQ activation, potentially rendering plaques more vulnerable and contributing to lesion formation and thus plaque rupture. In contrast, PC-KLH had anti-inflammatory effects, inducing anti-inflammatory cytokines from T cells, DC and MQ. PC-effects thus vary from proinflammatory to anti-inflammatory depending on presentation. Inhibition of OxPAPC effects by agents such as anti-PC could thus be anti-atherogenic, while on the other hand PC presented on a protein as KLH could be anti-atherogenic and anti-inflammatory itself.

Dual role of circulating and mucosal Vδ1 T cells in the control of and contribution to persistent HIV-1 infection.

Curative strategies for human immunodeficiency virus (HIV-1) infection are hindered by incomplete characterization of the latent reservoir and limited enhancement of anti-HIV immune responses. In this study, we identified a novel dual role for peripheral and tissue-resident Vδ1 T cells within the gastrointestinal mucosa of virally suppressed people with HIV. Phenotypic analyses identified an increased frequency of highly differentiated, cytotoxic effector Vδ1 T cells that exerted potent inhibition of HIV-1 replication in vitro coinciding with direct increases in cytolytic function. Conversely, we detected an enrichment of HIV-1 DNA in tissue-resident CD4+Vδ1 T cells in situ. Despite low CD4 expression, we found circulating Vδ1 T cells also contained HIV-1 DNA which was replication-competent. We show that TCR-mediated activation of peripheral Vδ1 T cells induced de novo upregulation of CD4 providing a plausible mechanism for increased permissibility to infection. These findings highlight juxtaposing roles for Vδ1 T cells in HIV-1 persistence including significant contribution to tissue reservoirs.

Clinical Significance of TNFSF14/LIGHT and CD160 in Gastric Cancer and Peptic Ulcer Dyspepsia.

Previous studies have reported the role of the Herpes Virus Entry Mediator (HVEM) in various cancer including gastric cancer. However, the expression level and clinical significance of CD160 and Tumor Necrosis Factor Ligand Superfamily Member 14 (TNFSF14) pathways in gastric cancer and gastric dyspepsia patients have remained unexplored. The study involved the collection of gastric tissue biopsies from 42 patients with non-ulcerative dyspepsia (NUD) as the control group, 43 gastric cancer (GC) patients, and 48 patients with peptic-ulcerative dyspepsia (PUD). All the patients were endoscopically examined at Imam Khomeini Hospital in Sari, Mazandaran, Iran. The expression levels of TNFSF14 and CD160 mRNA were assessed using quantitative real-time PCR (qPCR) with the SYBR Green method. Statistical analysis was performed to investigate the potential association between the clinical and experimental data. Among the 133 gastric endoscopic biopsies examined, LIGHT exhibited a significant overexpression in GC patients (p-value < 0.01). Moreover, the expression of TNFSF14 was higher in GC patients with stages I and II (p-value<0.05). Furthermore, GC patients with TNM stages III+IV were accompanied by high expression levels of LIGHT (p-value < 0.01) as well as CD160 (p-value<0.05). The expression of CD160 was also higher in younger adults with PUD (p-value<0.05). Whereas TNFSF14 exhibited higher expression in older adults with GC (p-value<0.05). Furthermore, this research provided insights into the potential biological pathways and significant gene enrichment of TNFSF14 and CD160, suggesting the potential role of CD160 and TNFSF14 in the regulation of immune system in GC and PUD. These findings suggest the possible role of LIGHT and CD160 expression in gastric cancer patients in immune dysregulation toward gastric cancer. Targeted immunotherapy that harnessing co-stimulatory molecules like LIGHT and CD160 could be a promising approach in the treatment of GC as well as potential GC tumor markers.

Yttrium-90 Induces an Effector Memory Response with Neoantigen Clonotype Expansion: Implications for Immunotherapy.

90Y can safely treat HCC; however, it causes transient lymphopenia. In this article, 90Y stimulates a peripheral effector memory response independent of initial treatment response. TCR sequencing revealed that polyclonal profiles in patients without an OR to treatment were associated with rapid progression rates 6 months after 90Y.

Research on the mechanism of exosomes from different sources influencing the progression of lung cancer.

As a key regulator of intercellular communication, exosomes are essential for tumor cells. In our study, we will explore the mechanisms of exosomes from different sources on lung cancer. We isolated CD8+T cells and cancer-associated fibroblasts (CAFs) from venous blood and tumor tissues of lung cancer patients, and isolated exosomes. MiR-2682 was high expression in CD8+T-derived exosomes, and lncRNA-FOXD3-AS1 was upregulated in CAF-derived exosomes. Online bioinformatics database analysis showed that RNA Binding Motif Protein 39 (RBM39) was identified as the target of miR-2682, and eukaryotic translation initiation factors 3B (EIF3B) was identified as the RNA binding protein of FOXD3-AS1. CD8+T-derived exosomes inhibited the growth of A549 cells and promoted apoptosis, while miR-2682 inhibits reversed these effects of CD8+T-derived exosomes. CAF-derived exosomes promoted the growth of A549 cells and inhibited apoptosis, while FOXD3-AS1 siRNA reversed the effect of CAF-derived exosomes. Mechanism studies have found that miR-2682 inhibits the growth of lung cancer cells by inhibiting the expression of RBM39. FOXD3-AS1 promoted the growth of lung cancer cells by binding to EIF3B. In vivo experiments showed that CD8+T cell-derived exosome miR-2682 inhibited lung cancer tumor formation, while CAF-derived exosome FOXD3-AS1 promoted lung cancer tumor formation. This study provides mechanistic insights into the role of miR-2682 and FOXD3-AS1 in lung cancer progression and provides new strategies for lung cancer treatment.

SIT1 identifies circulating hypoactive T cells with elevated cytotoxic molecule secretion in systemic lupus erythematosus patients.

This study aims to elucidate the expression and functionality of SIT1 in circulating CD8/CD4 + T cells in humans and to delineate its significance in systemic lupus erythematosus (SLE) patients. We employed multiparametric flow cytometry to investigate the expression of SIT1 in circulating CD8/CD4 + T cells and their respective subsets, comparing healthy controls (HCs) with SLE patients. Furthermore, we assessed the levels of granzyme B, perforin, IL-17, and IFN-γ in SIT1-related CD8/CD4 + T cells from both HCs and SLE patients, both before and after PMA stimulation. Clinically, we conducted receiver operating characteristic curve analysis and correlation analysis to evaluate the clinical relevance of SIT1-related CD8/CD4 + T cells in SLE patients. SIT1 exhibited higher expression in CD4 + T cells, with SIT1 - T cells demonstrating elevated levels of granzyme B, perforin, and IFN-γ compared to SIT1 + T cells. PMA-stimulated T cells exhibited reduced SIT1 expression compared to unstimulated T cells. SLE patients displayed increased SIT1 + proportions in CD8 + T cells and decreased SIT1 + CD4 + T cell numbers. Additionally, SIT1 + cells in SLE patients exhibited significantly higher levels of granzyme B and perforin compared to HCs. SIT1 + cells demonstrated significant associations with clinical indicators in SLE patients, with indicators related to SIT1 proving valuable in the diagnosis of SLE patients. SIT1 is inversely correlated with T cell activation. In SLE patients, SIT1 expression is altered in T cells concomitant with an augmented secretion of cytotoxic molecules. This upregulation may contribute to the pathogenesis of SLE and enhance its diagnostic potential.

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Generation of Devil Facial Tumour Cells Co-Expressing MHC With CD80, CD86 or 41BBL to Enhance Tumour Immunogenicity.

The major histocompatibility complex (MHC) molecules play an integral role in the adaptive immune response to transmissible cancers through tumour antigen presentation and recognition of allogeneic MHC molecules. The transmissible devil facial tumours 1 and 2 (DFT1 and DFT2) modulate MHC-I antigen presentation to evade host immune responses and facilitate transmission of tumours cells to new Tasmanian devil (Sarcophilus harrisii) hosts. To enhance T-cell-driven tumour immunogenicity for vaccination and immunotherapy, DFT1 and DFT2 cells were co-transfected with (i) NLRC5 for MHC-I expression or CIITA for MHC-I and MHC-II expression, and (ii) a co-stimulatory molecule, either CD80, CD86 or 41BBL. The co-transfected DFT cells presented enhanced expression of MHC-I and/or MHC-II. As few devil-specific monoclonal antibodies exist, we used recombinant CTLA4 and 41BB fused to a fluorescent protein to confirm expression of cell surface CD80, CD86 and 41BBL. The capacity for these cells to induce T-cell responses including PD1 and IFNG expression was evaluated in in vitro co-culture assays with captive devil peripheral blood mononuclear cells (PBMCs). Although PBMC viability had increased, there was no evidence of enhanced T-cell activation. This system can be used to identify additional factors required to promote activation of naïve devil T-cells in vitro.

4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury.

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success with mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns. We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in animals treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

Engineered Immunomodulatory Nanoparticles Inhibit Root Resorption and Ankylosis

IntroductionExternal root resorption following avulsion injury is a complex process wherein differentiation of macrophages (Mϕ) to multinucleated osteoclasts is temporally regulated by resident periodontal fibroblasts (PDLF). The current study aims to assess the effect of engineered bioactive chitosan nanoparticles (CSNP), sustained released dexamethasone conjugated CSNP (CS-DEX) and CSNP functionalized with photosensitizer Rose Bengal (CSRB) for application in root resorption using an in-vitro PDLF-Mϕ direct coculture model and in-vivo delayed reimplantation model. MethodsPDLF-Mϕ direct coculture system was exposed to lipopolysaccharide (LPS), macrophage colony stimulating factor, receptor activator of nuclear factor kappa β ligand with or without CSNP/CS-DEX for 7 days. Clastic differentiation was assessed by tartrate resistant acid phosphatase (TRAP) staining on day 7. On day 2 and 7, immunofluorescence analysis was conducted to assess the expression of Mϕ polarization markers (CD80, CD206), multinucleation markers (NFATc1, STAT6) in Mϕ and matricellular protein periostin in PDLF and cytokine profiling in cell culture supernatants. Delayed replantation model with extraoral air dry/LPS exposure for 1h followed by root surface treatment with CS-DEX/CSRB was used in Wistar rats. After 21 days, rats were euthanized for histologic and immunofluorescence analysis. Statistical analysis one-way ANOVA with Tukey's multiple comparisons was used to analyze the data (P < .05). ResultsCS-DEX significantly reduced TRAP+ multinucleated cells and CSNP treatment showed no TRAP+ cells. Immunofluorescence analysis showed that CSNP/CS-DEX reduced CD80, NFATc1 and STAT6 expression and increased periostin as expressed by fluorescence intensity. CSNP/CS-DEX significantly reduced TNFα, MMP9 and increased IL10, TGFβ1. Osteoprotegerin was upregulated only by CSNP. Root surface treatment in delayed replantation model showed that CS-DEX and CSRB substantially reduced the degree of resorption and ankylosis. Further, CD80, CD206, and MMP2 expression in groups with root surface treatment with CS-DEX and CSRB was lower than airdry/LPS group and similar to healthy control and NFATc1, STAT6, and MMP9 expressions were lower than healthy control. ConclusionThe engineered nanosized immunomodulatory bioactive materials chitosan nanoparticles functionalized with photosensitizer and dexamethasone effectively reduced the clastic differentiation of Mϕ in in-vitro coculture and minimized the resorption and ankylosis in a delayed reimplantation model. These biomaterials have the potential to serve as root modification agents, promoting favorable healing outcomes in cases of dental avulsion.

AB046. Dual role of POSTN in maintaining glioblastoma stem cells and immune-suppressive microglia in glioblastoma.

Glioblastoma (GBM) is an immunosuppressive, universally lethal cancer driven by GBM stem cells (GSCs). The interplay between GSCs and the immunosuppressive microglia plays crucial roles in promoting malignant growth of GBM, however, the molecular mechanisms underlying this crosstalk are incompletely understood. We performed RNA sequencing to explore the mechanism by which periostin (POSTN) regulates GSCs and microglia. The biological function of POSTN in GBM development was confirmed in vitro and in vivo. Specifically, tumorsphere formation assay, proliferation analysis, migration assays, enzyme-linked immunosorbent assay, immunoblotting, and intracranial mouse model were performed. We identified POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/β-catenin/FOS like antigen 1 (FOSL1) pathway. In addition to its GSC intrinsic effects, POSTN is able to recruit microglia and upregulate cluster of differentiation 70 (CD70) expression through PI3K/AKT/nuclear factor-kappa B (NFκB) pathway in microglial cells, which in turn promotes the Treg development and functionality, and generates an immunosuppressive tumor microenvironment. Inhibition POSTN disrupts the GSC maintenance, inhibits recruitment of immunosuppressive microglial, reduces Treg development and function, and suppresses GBM growth, suggesting that targeting POSTN may effectively improve GBM treatment. In conclusion, our study defined POSTN as a key regulator in mediating the molecular crosstalk between GSCs and immune-suppressive Microglia in the tumor microenvironment in GBM. POSTN activates the PI3K/AKT/β-catenin/FOSL1 pathway in an autocrine manner to promote GSC self-renewal and tumor growth. At the same time, POSTN recruits microglia in a paracrine manner and upregulates the expression of CD70 in microglia through the PI3K/AKT/NFκB pathway, thereby promoting the development and function of Treg and generating an immunosuppressive tumor microenvironment. Our findings indicate that targeting the POSTN gene may be a promising approach to ablating GSCs, breaking the immunosuppressive environment and overcoming treatment resistance in GBM.