Cell Activation Assays Research Articles

Targeting TCMR-associated cytokine genes for drug screening identifies PPARγ agonists as novel immunomodulatory agents in transplantation.

T cell-mediated rejection (TCMR) remains a significant challenge in organ transplantation. This study aimed to define a TCMR-associated cytokine gene set and identify drugs to prevent TCMR through drug repurposing. Gene expression profiles from kidney, heart, and lung transplant biopsies were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between TCMR and non-TCMR groups were identified, and their intersection with cytokine-related genes yielded an 11-gene TCMR-associated cytokine gene set (TCMR-Cs). To evaluate the effectiveness of this gene set, a diagnostic predictive model was constructed using Lasso regression and multivariate logistic regression, with validation in independent datasets. Connectivity Map (CMap) analysis was employed to screen drugs targeting TCMR-Cs. Experimental validation of the identified drug was performed in vitro using T cell activation and Th1 differentiation assays, and in vivo in a mouse skin transplant model with survival analysis. The TCMR-Cs exhibited outstanding predictive performance for TCMR, achieving an AUC of 0.99 in the training cohorts and maintaining strong performance in the test cohorts. CMap analysis identified peroxisome proliferator-activated receptor gamma (PPARγ) agonists as potential therapeutic candidates. Experimental validation showed that the PPARγ agonist rosiglitazone significantly suppressed T cell activation and reduced Th1 differentiation in vitro without cytotoxic effects. The combination of rosiglitazone and rapamycin significantly prolonged graft survival. This study defined a novel TCMR-associated cytokine gene set that effectively predicts TCMR and identified PPARγ agonists, which prevent TCMR and improve graft survival when combined with rapamycin.

CD69+CD103+CD8+ tissue-resident memory T cells possess stronger anti-tumor activity and predict better prognosis in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers worldwide. Despite advancements in therapeutic methodologies, it still causes a high rate of patient mortality. CD8+ tissue-resident memory T (TRM) cells are strategically positioned to mediate effective anti-tumor responses. However, the characteristic surface molecules and functions of CD8+ TRM cells exhibit significant heterogeneity.MethodsThe roles and anti-tumor biological functions of different CD8+ TRM subsets in CRC were determined by clinical CRC samples, bioinformatics analysis, and in vitro experiments including co-culture experiments and transwell migration assays. The signaling pathways that synergistically regulate the differentiation of CD8+ TRM cells were identified by in vitro CD8+ T cell activation and inhibition assays, and the functioning transcription factors were predicted using the UCSC and JASPAR databases.ResultsWe found that different CD8+ TRM subsets existed in CRC tumor tissues, which were identified as CD69−CD103−CD8+ TRM, CD69+CD103−CD8+ TRM (SP CD8+ TRM), and CD69+CD103+CD8+ TRM (DP CD8+ TRM) subsets. Compared with SP CD8+ TRM cells, increased infiltration of DP CD8+ TRM cells predicted better prognosis and played a protective role mainly in tumor invasion and lymph node metastasis of CRC. DP CD8+ TRM cells expressed higher levels of effector molecules and exerted stronger anti-tumor effects in a FAS/FASL pathway-dependent manner. Additionally, DP CD8+ TRM cells secreted higher levels of CXCL13 and recruited B cells into tumor tissues through the CXCL13/CXCR5 signaling axis to form tertiary lymphoid structures, participating in anti-tumor immune responses. Notch and TGF-β signaling pathways synergistically regulate the differentiation of DP CD8+ TRM cells.ConclusionsWe clarified the roles and mechanisms of different CD8+ TRM subsets in CRC and identified that DP CD8+ TRM cells exert stronger anti-tumor effects and predict better prognosis, which provides ideas for developing new clinically available therapeutic targets.

The DNA-based Lassa vaccine INO-4500 confers durable protective efficacy in cynomolgus macaques against lethal Lassa fever

BackgroundWe have previously developed a DNA-based vaccine, INO-4500, encoding the Lassa lineage IV glycoprotein precursor. INO-4500, when delivered with electroporation, elicited humoral and cellular responses, and conferred 100% protection in cynomolgus non-human primates. Here, we expanded the characterization of INO-4500 assessing immunogenicity and protective efficacy of lower doses and single immunization, and the durability of immune responses.MethodsThe study was divided into three arms evaluating INO-4500 vaccination: Arm 1 – Dosing regimen; Arm 2 – Single immunization; and Arm 3—Durability of immune responses and protective efficacy. Humoral and T cell responses were assessed by IgG binding ELISA, IFNγ ELISpot and flow cytometry-based T cell activation assays. NHPs were challenged with a lethal dose of Lassa lineage IV 8 weeks (Arms 1 and 2) or one year (Arm 3) after immunization. NHPs were assigned clinical scores and monitored for survival. Viremia, virus neutralization and release of soluble mediators were assessed post-challenge, as well as disease pathology following NHPs death or euthanasia.ResultsINO-4500 induces dose-dependent immune responses and protective efficacy. Animals receiving two doses of 2 mg of INO-4500 show complete short- and long-term LASV protection. NHPs receiving 1 mg of INO-4500 are protected from LASV challenge one year after vaccination but are only partially protected 8 weeks post-vaccination. LASV-specific memory T cells are present in vaccinated NHPs one year after vaccination. INO-4500 vaccination prevents NHPs from developing severe disease.ConclusionsThese studies demonstrate that INO-4500 can provide short- and long-term protection in NHPs from lethal LASV challenge.

IMMU-03. EXPLORING CANCER-SPECIFIC T CELLS AND ANTIGENS IN GLIOBLASTOMA BASED ON SINGLE-CELL SEQUENCING OF CD8+ TILS

Abstract BACKGROUND The effectiveness of cancer immunotherapy against glioblastoma (GBM) remains limited. This study aims to identify cancer-specific antigens to develop antigen-based cancer immunotherapies for GBM. We investigated candidate tumor antigen-specific T cells in GBM by single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq), and we explored candidate antigens through genetic analysis of tumor tissues. METHODS Flow cytometry analysis was conducted on fresh tumor digest samples to evaluate tumor-infiltrating lymphocytes (TILs) of GBM. Single-cell RNA and TCR sequencing were performed on CD8+ T cells in TILs. Both data generated by Cell Ranger software were loaded into Seurat at R studio. The dimensional reduction for clustering was performed with uniform manifold approximation and projection (UMAP). Additionally, we performed bulk RNA sequencing (RNA-seq) and whole exome sequencing (WES) on tumor tissues. RESULTS Two out of 20 patients (10%) exhibited abundant TILs in GBM. From these two patients, approximately 20,000 CD8+ T cells in total were analyzed in single-cell analysis. Clustering based on UMAP revealed a predominance of clusters expressing exhaustion markers, with high TCR clonality centered on exhausted T cell (TEX) clusters, like our previous date of lung cancer TILs recognizing tumor antigens. Moreover, within the TEX clusters, the expression of CXCL13, often reported as an antigen-specific marker in recent years, was significantly higher in our data compared to two publicly available datasets. Subsequently, based on RNA-seq and WES, 61 candidate neoantigens were estimated using machine-learning prediction algorithms from NEC Corporation. Furthermore, 5 overexpressed cancer/testis antigens, and 3 bacterial species-derived microbial peptides using Kraken2 were selected as antigen candidates. CONCLUSIONS We identified prospective tumor antigen-specific T cell subsets with high CXCL13 expression from two GBM patients. We plan to identify cancer-specific T cells and antigens from these candidates by T cell activation assay.

Evaluation of STK17B as a cancer immunotherapy target utilizing highly potent and selective small molecule inhibitors.

The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy. To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo. In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model. In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

Construction and anti-pancreatic cancer activity of selenium nanoparticles stabilized by Prunella vulgaris polysaccharide

Selenium nanoparticles (SeNPs), as a potential cancer therapeutic agent, have attracted extensive attention due to their high anticancer activity and low toxicity. Polysaccharides could be the modifiers and stabilizers to improve the stability and dispersibility of SeNPs in aqueous solution. This study aimed to investigate the physicochemical characterization, stability, and anti-pancreatic cancer cell activities of SeNPs stabilized by a heteroxylan PVP3-1 extracted from the clusters of Prunella vulgaris Linn. Our results showed that PVP3-1 with Mw of 154 kDa was composed of →4)-β-D-Xylp(1→, →2, 4)-β-D-Xylp(1→, t-α-L-Araf(1→ and 4-MeO-α-D-GlcpA(1→. Red, zero-valent, and uniform spherical SeNPs with an average diameter of about 60 nm and high stability in aqueous solution were constructed successfully by polysaccharide PVP3-1. Anti-pancreatic cancer cell activity assays showed that PVP3-1-SeNPs could inhibit the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, PVP3-1-SeNPs induced apoptosis and autophagy of pancreatic cancer cells through inhibiting mTOR signaling pathway. In conclusion, these results indicated that PVP3-1-SeNPs could be potential anti-tumor nanoparticles for treating pancreatic cancer.

Development and characterization of dendritic cell internalization and activation assays contributing to the immunogenicity risk evaluation of biotherapeutics.

Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

Preparation and biological characteristics of extracellular matrix vesicle mimetics

Objective: To investigate the characteristics of extracellular matrix vesicle mimetics prepared by mechanical extrusion and their effects on the cell viability and osteogenic differentiation potential of human periodontal ligament stem cells (PDLSC). Methods: PDLSC derived extracellular matrix vesicles were prepared by collagenase digestion, while the cell derived vesicle mimetics were simulated by mechanical extrusion. The obtained extracellular matrix vesicles and parental cell derived vesicle mimetics were divided into 4 groups: matrix vesicles derived from PDLSC cultured in basic medium for 7 days (PDLSC matrix vesicles, MVs), vesicle mimetics derived from PDLSC cultured in basic medium for 7 days (PDLSC vesicle mimetics, CVMs), matrix vesicles derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC matrix vesicles, O-MVs) and vesicle mimetics derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC vesicle mimetics, O-CVMs). Vesicles morphologies and sizes were observed by transmission electron microscopy and nanoparticle tracking analysis. Vesicles uptake was detected by immunofluorescence. With PDLSC as the control group, the effects of vesicles on the viability of PDLSC were detected by cell activity assay (cell counting kit-8), and the effects of vesicles on the osteogenic differentiation potential of PDLSC were detected by alizarin red staining and Western blotting. Results: Vesicles in MVs, O-MVs, CVMs and O-CVMs were all observed with a round structure (size 50-250 nm), and could be taken up by PDLSC without affecting the cell viability. Under osteogenic inducing conditions, PDLSC incubated with O-MVs or O-CVMs could produce more mineralized nodules than those in the control group (PDLSC). MVs, O-MVs, CVMs and O-CVMs could promote the expression of osteogenic-related proteins in PDLSC. PDLSC in group O-CVMs showed significant higher expressions of osteogenic-related proteins, including alkaline phosphatase (ALP) (1.571±0.348), osteopontin (OPN) (1.827±0.627) and osteocalcin (OCN) (1.798±0.537) compared to MVs (ALP: 1.156±0.170, OPN: 1.260±0.293, OCN: 1.286±0.302) (P<0.05). Compared to CMVs-incubated PDLSC, O-CVMs-incubated PDLSC expressed more Runt-related transcription factor 2 (1.632±0.455 vs 1.176±0.128) and OPN (1.827±0.627 vs 1.428±0.427) (P<0.05). Moreover, there was no significant difference in the expression levels of osteoblast-related proteins in PDLSC cultured with MVs, O-MVs and CVMs (P>0.05). Conclusions: The vesicle mimetics prepared by mechanical extrusion method are similar in shape and size to the extracellular matrix vesicles. MVs, O-MVs, CVMs and O-CVMs do not affect the cell viability of PDLSC, and can promote the osteogenic differentiation potential of PDLSC to a certain extent.

Characterization and biological evaluation of 3D printed composite ink consisting of collagen, hyaluronic acid and calcium phosphate for bone regeneration

In large bone defects the self-healing capacity is insufficient, and the current standard treatment, autologous bone grafting, has severe disadvantages such as limited availability and donor site morbidity. Alternatively, clinically available bone graft substitutes lack spatial control over scaffold architecture to anatomically match complicated bone defects. Therefore, the aim in this study was to develop a 3D printable composite biomaterial-ink to promote healing of large bone defects. The composite biomaterial-ink consisted of an organic biopolymer matrix with tyramine modified hyaluronic acid (THA) and collagen type I (Col) mixed with osteoinductive calcium phosphate particles (CaP). The biopolymer was combined with 0, 10, 20 and 30 % of either 45–63 µm or 45–106 µm CaP. µCT imaging showed a homogeneous distribution of CaP in the THA-Col hydrogel and all composites were 3D printable. In vitro cell activity assays revealed no indirect cytotoxicity using L929 cells and high cell cytocompatibility using human mesenchymal stromal cells (hMSCs). Additionally, all composites supported in vitro osteogenic differentiation of hMSCs. This study highlights the development of a 3D printable composite biomaterial-ink using CaP and THA-Col hydrogel that holds significant potential to be used as patient-specific bone graft substitute for the regeneration of large bone defects. Statement of significanceThis paper introduces a 3D printable composite biomaterial-ink made of osteoinductive calcium phosphate particles combined with matrix biopolymers collagen and hyaluronic acid, which was chemically modified to introduce shear thinning and shape fixation properties for 3D printing. The chemical modification only involves a small percentage of functional groups, preserving hyaluronan's biological properties. We demonstrated printability, the homogeneous distribution of the mineral phase, cytocompatibility and that the composites support osteogenesis of primary human mesenchymal stromal cells from multiple donors. The printability of the composite biomaterial-ink allows the creation of patient-specific implants with controlled geometry on porosity. This study contributes towards engineering personalized implants for replacing autologous bone grafting in all clinical situations where the bone self-healing capacity is insufficient.

Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6 mg/kg VB6), VB6-deprived (0.5 mg/kg VB6) or VB6-enhanced (12 mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5 mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5 mg/kg VB6 groups, and in the 12.5 mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

Inhibitory Effects of Fermented Sprouted Oat Extracts on Oxidative Stress and Melanin Overproduction.

Hyperpigmentation occurs due to irregular secretion of melanin pigment in the skin. This can affect quality of life depending on its severity, so prevention and management are essential. Oats (Avena sativa L.), a grain consumed worldwide, are known to offer improved health benefits upon germination and fermentation. This study is aimed to investigate the protective effects of lactobacilli-fermented sprouted oat extracts on oxidative stress and melanin overproduction in vitro. The anti-melanogenic effect was investigated using melanin content and tyrosinase activity assays in B16F10 cells, as well as a mushroom tyrosinase-based enzyme inhibition assay. The results showed that L. casei-fermented oat extracts were the most effective for reducing melanin formation by reducing the mRNA expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein 2. Furthermore, L. casei fermentation was effective in improving the total phenolic, flavonoid, and avenanthramide A contents of sprouted oat extracts. The results also demonstrated the antioxidant effects of L. casei-fermented sprouted oat extracts in promoting DPPH radical-scavenging activity, superoxide dismutase-like activity, and reduction in reactive oxygen species levels. Overall, the findings indicate that fermented sprouted oat extracts are promising candidates for antioxidant and anti-hyperpigmentation treatments.

Dual C–Cl isotope fractionation offers potential to assess biodegradation of 1,2-dichloropropane and 1,2,3-trichloropropane by Dehalogenimonas cultures

1,2-dichloropropane (1,2-DCP) and 1,2,3-trichloropropane (1,2,3-TCP) are hazardous chemicals frequently detected in groundwater near agricultural zones due to their historical use in chlorinated fumigant formulations. In this study, we show that the organohalide-respiring bacterium Dehalogenimonas alkenigignens strain BRE15 M can grow during the dihaloelimination of 1,2-DCP and 1,2,3-TCP to propene and allyl chloride, respectively. Our work also provides the first application of dual isotope approach to investigate the anaerobic reductive dechlorination of 1,2-DCP and 1,2,3-TCP. Stable carbon and chlorine isotope fractionation values for 1,2-DCP (ƐC = −13.6 ± 1.4 ‰ and ƐCl = −27.4 ± 5.2 ‰) and 1,2,3-TCP (ƐC = −3.8 ± 0.6 ‰ and ƐCl = −0.8 ± 0.5 ‰) were obtained resulting in distinct dual isotope slopes (Λ12DCP = 0.5 ± 0.1, Λ123TCP = 4 ± 2). However direct comparison of ΛC-Cl among different substrates is not possible and investigation of the C and Cl apparent kinetic isotope effects lead to the hypothesis that concerted dichloroelimination mechanism is more likely for both compounds. In fact, whole cell activity assays using cells suspensions of the Dehalogenimonas-containing culture grown with 1,2-DCP and methyl viologen as electron donor suggest that the same set of reductive dehalogenases was involved in the transformation of 1,2-DCP and 1,2,3-TCP. This study opens the door to the application of isotope techniques for evaluating biodegradation of 1,2-DCP and 1,2,3-TCP, which often co-occur in groundwaters near agricultural fields.

Network pharmacology prediction and experiment validation of anti liver cancer activity of curcumae rhizoma and hedyotis diffusa willd

Objective: This study aims to elucidate anti liver cancer components and potential mechanisms of Curcumae Rhizoma and Hedyotis diffusa Willd (CR-HDW). Methods: Effective components and targets of CR-HDW were identified from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Liver cancer-related genes were collected from GeneCards, Gene-Disease Association (DisGeNET), and National Center for Biotechnology Information (NCBI). Protein-protein interaction networks, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were conducted to analyze the identified genes. Molecular docking was used to simulate binding of the active components and their target proteins. Cell activity assay, Western blot, and Senescence-associated β-galactosidase (SA-β-gal) experiments were conducted to validate core targets identified from molecular docking. Results: Ten active compounds of CR-HDW were identified including quercetin, 3-epioleanic acid and hederagenin. The primary core proteins comprised Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Protein Kinase B（AKT1）, etc. The pathways for Phosphoinositide 3-kinase (PI3K)/ AKT, cellular senescence, Fork head boxO (FOXO) were revealed as important for anti-cancer activity of CR-HDW. Molecular docking demonstrated strong binding between liver cancer target proteins and major active components of CR-HDW. In vitro experiments confirmed that hederagenin and 3-epioleolic acid inhibited HuH-7 cell growth, reduced expression of PI3K, AKT, and mechanistic target of rapamycin (mTOR) proteins. Hederagenin also induced HuH-7 senescence. Conclusion: In summary, our results suggest that the CR-HDW component (Hederagenin, 3-epoxy-olanolic acid) can inhibit the proliferation of HuH-7 cells by decreasing PI3K, AKT, and mTOR. Hederagenin also induced HuH-7 senescence.

A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

BackgroundGlioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM.MethodsWe conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells.ResultsWe established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy.ConclusionsOur study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

The role of FPR2-mediated ferroptosis in formyl peptide-induced acute lung injury against endothelial barrier damage and protective effect of the mitochondria-derived peptide MOTS-c

BackgroundAcute lung injury (ALI) has garnered significant attention in the field of respiratory and critical care due to its high mortality and morbidity, and limited treatment options. The role of the endothelial barrier in the development of ALI is crucial. Several bacterial pathogenic factors, including the bacteria-derived formyl peptide (fMLP), have been implicated in damaging the endothelial barrier and initiating ALI. However, the mechanism by which fMLP causes ALI remains unclear. In this study, we aim to explore the mechanisms of ALI caused by fMLP and evaluate the protective effects of MOTS-c, a mitochondrial-derived peptide. MethodsWe established a rat model of ALI and a human pulmonary microvascular endothelial cell (HPMVEC) model of ALI by treatment with fMLP. In vivo experiments involved lung histopathology assays, assessments of inflammatory and oxidative stress factors, and measurements of ferroptosis-related proteins and barrier proteins to evaluate the severity of fMLP-induced ALI and the type of tissue damage in rats. In vitro experiments included evaluations of fMLP-induced damage on HPMVEC using cell activity assays, assessments of inflammatory and oxidative stress factors, measurements of ferroptosis-related proteins, endothelial barrier function assays, and examination of the key role of FPR2 in fMLP-induced ALI. We also assessed the protective effect of MOTS-c and investigated its mechanism on the fMLP-induced ALI in vivo and in vitro. ResultsResults from both in vitro and in vivo experiments demonstrate that fMLP promotes the expression of inflammatory and oxidative stress factors, activates ferroptosis and disrupts the vascular endothelial barrier, ultimately contributing to the development and progression of ALI. Mechanistically, ferroptosis mediated by FPR2 plays a key role in fMLP-induced injury, and the Nrf2 and MAPK pathways are involved in this process. Knockdown of FPR2 and inhibition of ferroptosis can attenuate ALI induced by fMLP. Moreover, MOTS-c could protect the vascular endothelial barrier function by inhibiting ferroptosis and suppressing the expression of inflammatory and oxidative stress factors through Nrf2 and MAPK pathways, thereby alleviating fMLP-induced ALI. ConclusionOverall, fMLP disrupts the vascular endothelial barrier through FPR2-mediated ferroptosis, leading to the development and progression of ALI. MOTS-c demonstrates potential as a protective treatment against ALI by alleviating the damage induced by fMLP.

Abstract 2921: Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models

Abstract Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis induced by radiotherapy (RT) and chemotherapy. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved locoregional control at 18 months, 3-year progression-free survival and nearly doubled 5-year overall survival vs. placebo + CRT (53% vs. 28%). Two phase 3 trials, in which patients receive 6 cycles of xevinapant or placebo monotherapy (3 cycles in combination with standard-of-care (C)RT followed by 3 cycles of monotherapy), are ongoing. Based on the roles of IAPs in apoptosis and tumor immunity, continual dosing of xevinapant post RT may deliver additional therapeutic benefit through modulation of multiple tumor microenvironment (TME) compartments. Methods: The impact on antitumor efficacy of xevinapant dosing duration was evaluated in A1419 (head and neck), LLC (lung) and MC38 (colorectal) syngeneic tumor-bearing mice treated with vehicle control, RT alone (3.6Gy, QD, 5 days), or RT + xevinapant (100 mg/kg, QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation in MC38 tumors, RNAseq and FACS-based immune profiling was performed, followed by functional validation with ex vivo ELISpot assays, in vitro immunogenic cell death assays, T cell activation assays, and macrophage polarization and viability assays. To evaluate the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation and CAF-modulated immune phenotype, activation assay and cytokine profiling was performed. Results: In the tumor models tested, RT in combination with extended xevinapant dosing markedly improved efficacy and prolonged survival compared to treatment arms of RT with shorter xevinapant dosing durations, RT alone, and vehicle control. RNAseq and FACS analyses suggested a TME with enhanced antitumor immunity, while ELISpot and in vitro T cell assays confirmed that extended xevinapant dosing promoted antigen-specific T cell response and T cell activation. Furthermore, macrophage polarization and viability assays suggested that xevinapant reduced the viability of M2 macrophages which may have contributed to the increased M1:M2 macrophage ratio and the improved antitumor immune response observed in vivo. In addition, via the suppression of RT-mediated CAF activation, xevinapant may promote M1 polarization of macrophages and recruitment of T cells by upregulating GM-CSF and CXCL10 in CAFs. Conclusions: Extended dosing of xevinapant post concurrent RT improved antitumor efficacy and prolonged survival of tumor-bearing mice. Mechanistic investigation suggested that such benefit may be, in part, modulated by xevinapant enhanced antitumor immunity. Citation Format: Tsz-Lun Yeung, Feng Jiang, Hui Huang, Huakui Yu, Li-Ya Chiu, Mathilde Bonnemaison, Bo Marelli, Roberta Ferretti, Ralph Lindemann, Christina Esdar. Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2921.

Abstract 1372: BTN1A1 regulates T cell-mediated cancer immunotherapy by interacting with Galectin-9, PD-1, and TIM3

Abstract Butyrophilin 1A1 (BTN1A1) is a novel immune checkpoint protein belonging to the butyrophilin family and is expressed on immune cells such as macrophages, B cells, or activated CD8 T cells and in human tumors. Our results indicate that BTN1A1 and PD-L1 expression are mutually exclusive in various human solid tumors. Furthermore, it was observed that BTN1A1 had a significantly higher detection level than PD-L1 in cancer patient samples. We have reported that BTN1A1 plays an immunomodulatory role in T cell activation and proliferation. BTN1A1 inhibited the proliferation of T cells that are activated by anti-CD3 and anti-CD28 antibodies in vitro. Overexpression of BTN1A1 in PC3 cells also inhibits T cell-mediated killing of cancer cells. BTN1A1 is a bona fide immune checkpoint inhibitor and has anti-tumor activity in a human immune setting. BTN1A1 represents a novel cancer immunotherapy target that goes beyond the low expression levels of PD-1/PD-L1 in tumors. BNT1A1 is an immune checkpoint and cell membrane protein that, together with its binding partners, serves as a starting point for signaling pathways. The BNT1A1 complex, which has various abilities by combining with a specific partner, plays a vital role as an immune checkpoint. In this study, we identified binding partners of BTN1A that regulate immune activity in a mutually interaction relationship with BTN1A1 in various ways. We identified binding partners for the extracellular domain of human BTN1A1 using cell microarrays from Retrogenix (Whaley Bridge, UK). Through this screening approach, we found that BTN1A1 binds to galectin-1 (Gal1), galectin-9 (Gal9), and neuropilin 2 (NRP2). These three putative binding partners could specifically bind to wild-type BTN1A1 but not to this protein's unglycosylated (2NQ) form. Of these three targets, immunoprecipitation and Biacore binding assays revealed that Gal9 exhibited the greatest affinity for human BTN1A1. Since Gal9 is a known PD-1 and TIM3 binding protein, we predicted the potential formation of a BTN1A1/Gal9/PD-1 or BTN1A1/Gal9/TIM3 complex. Indeed, it reveals that immunoprecipitation and confocal microscopy assays performed using cells expressing Myc-tagged versions of these four proteins demonstrated the formation of BTN1A1/Gal9, PD-1/Gal9, TIM3/Gal9, BTN1A1/Gal9/TIM3, and BTN1A1/Gal9/PD-1 complexes. In T cell activation assay using these complexes, when present BTN1A1 alongside the complex inhibits activated T cells. As high Gal9 expression levels are correlated with poor prognosis in multiple cancers, our results highlight this BTN1A1-Gal9-PD-1 or BTN1A1-Gal9-TIM3 axis as a novel therapeutic target for immunotherapeutic drug development. Citation Format: Seung Hoon Lee, Young-Seung Kim, Chunai Wu, Andrew H Park, Stephen S. Yoo. BTN1A1 regulates T cell-mediated cancer immunotherapy by interacting with Galectin-9, PD-1, and TIM3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1372.

Abstract 2371: BGB-B167, a first-in-class 4-1BB/CEACAM5 bispecific antibody, exhibits potent in vitro and &amp;lt; for vivo antitumor activity and &amp;lt; for superior safety profile in preclinical models

Abstract 4-1BB (CD137) is a key costimulatory immunoreceptor and a promising therapeutic target in cancer. CEACAM5 (CEA) is a well-established tumor associated antigen overexpressed in many cancers, including colorectal, gastric, lung, pancreatic cancer, liver, breast and thyroid cancers. BGB-B167 is a novel immunoglobulin G (IgG)-based bispecific antibody targeting 4-1BB and CEA and is under clinical development for the treatment of advanced or metastatic solid tumors in humans. BGB-B167 binds to its target proteins with high specificity and affinity. Potent and CEA-dependent functional activities were demonstrated using peripheral blood mononuclear cell (PBMC)-based immune cell activation and cytotoxicity assays. In humanized 4-1BB knock-in mice bearing human CEA-expressing tumors, BGB-B167 exhibited potent, dose-associated single-agent efficacy as well as synergistic antitumor activity in combination with anti-PD-1 antibody. BGB-B167 was well tolerated in 1-month repeat-dose toxicology study in cynomolgus monkeys. Here, we describe the characterization of BGB-B167 with regard to preclinical proof-of-concept and basic drug-like properties. The combined dataset provides an overview on the design, mode of action, preclinical pharmacology and safety profile of BGB-B167. Citation Format: Zhuo Li, Liang Qu, Xin Chen, Liu Xue, Jie Li, Qi Liu, Jian Sun, Hanzi Sun, Yun Chen, Yuanyuan Xie, Wanyi Wang, Lin Zhu, Penghao Wang, Xiaosui Zhou, Hongjia Hou, Jie Chen, Xinyi Liu, Yilu Zhang, Ning Liu, Xinyi Liang, Shuo Zhang, Xiaoyan Tang, Jing Song, Tong Zhang, Xiaomin Song, Xuesong Liu, Kang Li, Chichi Huang. BGB-B167, a first-in-class 4-1BB/CEACAM5 bispecific antibody, exhibits potent in vitro and &amp;lt; for vivo antitumor activity and &amp;lt; for superior safety profile in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2371.

Abstract 1343: BSI-111, a highly selective anti-CD16a monoclonal antibody with potent agonist activity for building a NK cell engager platform

Abstract Background: Natural killer cells (NK) are innate lymphocytes endowed with the ability to recognize and kill cancer cells. CD16a, a well-characterized activating receptor predominantly expressed on NK cells, is a low-affinity receptor for the IgG Fc domain and is crucial for stimulating NK cell-mediated tumor cell killing. Unlike CD16a, CD16b is mainly expressed on granulocytes as a GPI-anchored receptor and is not involved in the lysis of tumor cells although CD16a and CD16b share 97% sequence identity in their extracellular domains. Due to the high homology of extracellular domains of CD16a and CD16b, it is extremely challenging to develop CD16a-specific agonistic antibodies without binding CD16b+ granulocytes, the most abundant subset in leukocytes. Here we report the development of an anti-CD16a-specific monoclonal antibody with potent NK cell activation. Experimental procedures: Humanized mice were immunized with recombinant CD16a-ECD-Fc. The Biosion proprietary H3 (High-throughput, High-content and High-efficiency) antibody screening platform was used to identify an anti-CD16a monoclonal antibody lead candidate - BSI-111. The target binding specificity, binding activity, and affinity of BSI-111 were evaluated by protein-based ELISA, cell-based FACS and Biacore-based SPR. A cell-based agonist reporter assay and a primary NK cell activation assay were used to evaluate the bioactivity of BSI-111. Summary: BSI-111 is a fully human monoclonal antibody with the following critical properties: (1) specifically binds to CD16a without recognizing CD16b; (2) binds the two allelic variants of CD16A - 158F and 158V with similar high affinities; (3) shows cross-reactivity to cynomolgus CD16a; (4) can significantly activate NFAT signaling in a cell-based reporter assay; (5) exhibits strong activity on activating primary NK cells; and (6) has potentially longer half-life and silenced Fc-effector function through Fc-engineering. Conclusion: BSI-111 demonstrates great biophysical properties and functional characteristics, supporting the development of anti-CD16a-based NK cell engagers for potential benefit of cancer patients. Citation Format: Jinyu Liu, Wenwen Dai, Hongyan Li, Xiaodong F. Liu, Jun Li, Shukai Xia, Qun Lyu, Hugh M. Davis, Mingjiu Chen, Zeyu Peng. BSI-111, a highly selective anti-CD16a monoclonal antibody with potent agonist activity for building a NK cell engager platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1343.