Activated Jurkat Cells Research Articles

Discovery of Daclatasvir as a potential PD-L1 inhibitor from drug repurposing

This study employed a drug repositioning strategy to discover novel PD-L1 small molecule inhibitors. 3D-QSAR pharmacophore models were establishedand subsequently validated through various means to select a robust model, Hypo-1, suitable for virtual screening. Hypo1 was used toscreen a library of 7,475 compounds from the Drugbank database, leading to the identification of 283 molecules following molecular docking with PD-L1.19 compounds underwent HTRF assays, with 15 showing varying degrees of inhibition of the PD-1/PD-L1 interaction. Compounds2202,2204,2207, and2208were further confirmed to bind to PD-L1 using SPR experiments. Among them, compound2204(Daclatasvir, KD = 11.4 μM) showeda higher affinity for human PD-L1 than the control compound BMS-1. In the HepG2/Jurkat cell co-culture model, Daclatasvir effectively activated Jurkat cells to kill HepG2 cells. In the mouse H22 hepatocellular tumor model, Daclatasvir significantly inhibited tumor growth (TGI = 53.4 % at a dose of 100 mg/kg). Its anti-tumor effect was more pronounced when combined with Lenvatinib (TGI = 85.1 %). Flow cytometry analysis of splenocytes and tumor cells indicated that Daclatasvir activated the immune system in both models. In summary, Daclatasvir was identified as a novel PD-L1small molecule inhibitor.

The Influence of N-Acetyl-selenomethionine on Two RONS-Generating Cancer Cell Lines Compared to N-Acetyl-methionine.

Jurkat and MTC-SK cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of NASeLM and NALM solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of Jurkat and MTC-SK cells. Both substances, NASeLM and NALM, were similarly able to inhibit cell growth and mitochondrial activity of Jurkat cells in a concentration-dependent and time-dependent manner up to 70%. Only the determination of caspase activity showed that only NASeLM was able to increase this to almost 40% compared to the control as well as the same lack of NALM. However, the experiments on MTC-SK cells showed a clear difference in favor of NASeLM compared to NALM. While NASeLM was able to reduce cell growth to up to 55%, the same amount of NALM was only at around 15%, which turned out to be highly significant (p < 0.001). The same could also be measured for the reduction in MTC-SK mitochondrial activity. Time dependence could also be recognized: the longer both substances, NASeLM and NALM, were incubated, the higher the effect on cell growth and mitochondrial activity, in favour of NASeLM. Only NASeLM was able to increase caspase-3 activity in MTC-SK cells: at 250 µg/mL NASeLM, caspase-3 activity increased significantly to 28% after 24 and 48 h compared to the control (14%) or the same NALM concentration (14%). After 72 h, this could still increase to 37%. A further increase in the NASeLM concentration did not result in higher caspase-3 activity. NASeLM could clearly increase caspase-3 activity in both cell types, Jurkat or MTC-SK cells, and thus induce cell death. NALM and NASeLM showed a reduction in cell growth and mitochondrial activity in both cell lines: While NALM and NASeLM showed almost identical measurements on Jurkat cells, NASeLM was much more effective on MTC-SK than the non-selenium-containing carrier, indicating that it has additional anti-chemoprotective effects.

Abstract 4021: The development of CAR T cell therapeutics targeting tumor specific CD146

Abstract Chimeric antigen receptor (CAR) T cells represent a promising class of "living drugs" for cancer treatment. These primary T cells are genetically engineered to express a synthetic antigen receptor, incorporating various domains to enhance their effector functions. While CAR T cells have shown success against liquid cancers, their application to solid cancers is hindered by significant "on-target/off-tumor" toxicities due to the absence of specific target proteins. In this study, we aim to enhance the tumor specificity of CAR T cells by targeting a tumor-specific version of CD146. CD146 is a glycoprotein widely expressed in several solid tumors, particularly melanoma and ovarian cancer. However, it is also expressed on benign cells, such as endothelial cells, pericytes, and smooth muscle cells. Nevertheless, a CD146 isoform exists that is enriched in cancer cells. We assessed CAR activity using flow cytometry to measure surface CD69 expression (an early activation marker) on CAR-expressing Jurkat cells co-cultured with various CD146-positive and CD146-negative human melanoma and ovarian cancer cell lines. The killing ability of primary CAR-T cells was evaluated by measuring the relative luciferase activity of target cells, and their effector functions were further analyzed by ELISA for the secretion of IFN-g and TNF-a. Through multiple rounds of protein engineering, we have developed a CAR construct capable of reprogramming T cells to respond to a tumour-enriched CD146 isoform. Our CAR demonstrated antigen-dependent activation of Jurkat cells when co-cultured with CD146-positive human melanoma and ovarian cancer cell lines, comparable to a CAR targeting pan-CD146. Moreover, primary CAR T cells exhibited proportional killing activity and other effector functions. We have successfully engineered a CAR with specificity for a tumour-enriched isoform of CD146. Our future work will involve rigorously evaluating the tumor selectivity of our CAR, as well as assessing the ability of primary CAR T cells expressing this CAR to control tumor growth in vivo. Citation Format: Erez Uzuner, Holly Mole, Chun W. Wong, Macarena L. Fernandez Carro, Rotem Leshem, Kieran Sefton, Helen Tovey, Andrew Picken, Marcel Blot-Chabaud, Adam F. Hurlstone. The development of CAR T cell therapeutics targeting tumor specific CD146 [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 4021.

Alpha-tocopherylquinone-mediated activation of the Aryl Hydrocarbon Receptor regulates the production of inflammation-inducing cytokines and ameliorates intestinal inflammation

This study investigated the role of Alpha-tocopherylquinone (TQ) in regulating the intestinal immune system and the underlying mechanisms. In the experimental dextran sodium sulfate and T cell-mediated colitis models, TQ significantly reduced the mRNA levels of interleukin (IL)-6, IL-1β, IL-17A, IL-23, and tumor necrosis factor (TNF)-α and the abundance of proinflammatory macrophages, T helper (Th)17 cells, and ILC3s in the colons of wild-type mice. TQ also prevented lipopolysaccharide (LPS)-induced activation of NFκB and signal transducer and activator of transcription (Stat)-3 pathways in the human macrophage U937 cells. Pharmacological inhibition or CRISPR-Cas-9-mediated knockout of Aryl hydrocarbon Receptor (AhR) prevented the anti-inflammatory effects of TQ in the LPS-treated U937 cells. Furthermore, TQ reduced the mRNA levels of the LPS-induced pro-inflammatory cytokines in the WT but not Ahr-/- mice splenocytes. TQ also reduced IL-6R protein levels and IL-6-induced Stat-3 activation in Jurkat cells and in vitro differentiation of Th17 cells from wild-type but not Ahr-/- mice naive T cells. Additionally, TQ prevented the pro-inflammatory effects of LPS on macrophages and stimulation of T cells in human PBMCs and significantly reduced the abundance of tumor necrosis factor-α, IL-1β, and IL-6hi inflammatory macrophages and Th17 cells in surgically resected Crohn's disease (CD) tissue. Our study shows that TQ is a naturally occurring, non-toxic, and effective immune modulator that activates AhR and suppresses the Stat-3-NFκB signaling.

A luminescence-based method to assess antigen presentation and antigen-specific T cell responses for in vitro screening of immunomodulatory checkpoints and therapeutics.

Investigations into the strength of antigen-specific responses in vitro is becoming increasingly relevant for decision making in early-phase research of novel immunotherapeutic approaches, including adoptive cell but also immune checkpoint inhibitor (ICI)-based therapies. In the latter, antigen-specific rapid and high throughput tools to investigate MHC/antigen-specific T cell receptor (TCR) activation haven't been implemented yet. Here, we present a simple and rapid luminescence-based approach using the human papillomavirus 16 (HPV16) E711-20 peptide as model antigen and E7-TCR transgenic Jurkat.NFAT-luciferase reporter cells. Upon E7 peptide pulsing of HLA-A2+ cell lines and macrophages, an effector to target ratio dependent increase in luminescence compared to non-pulsed cells was observed after co-incubation with E7-TCR expressing Jurkat, but not with parental cells. Analogous experiments with cells expressing full-length HPV16 identified that E7-specific activation of Jurkat cells enabled detection of endogenous antigen processing and MHC-I presentation. As proof of concept, overexpression of established checkpoints/inhibitory molecules (e.g., PD-L1 or HLA-G) significantly reduced the E7-specific TCR-induced luminescence, an effect that could be restored after treatment with corresponding targeting antagonistic antibodies. Altogether, the luminescence-based method described here represents an alternative approach for the rapid evaluation of MHC-dependent antigen-specific T cell responses in vitro. It can be used as a rapid tool to evaluate the impact of the immunosuppressive tumor microenvironment or novel ICI in triggering effective T cell responses, as well as speeding up the development of novel therapeutics within the immune-oncology field.

DDB1 regulates the activation-induced apoptosis of T cells via downregulating the expression of histone methyltransferase SETD7.

The damaged DNA-binding protein 1 (DDB1) enhances the survival and maintenance of multipotent cells through promoting the Cullin 4 E3 ligase complex-dependent ubiquitination and subsequent degradation of downstream substrates. Naive T cells could be activated and differentiated into effector and memory T cells by exogenous stimulatory molecules, which are essential in immune response and inflammation. However, possible regulation and molecular mechanisms of DDB1 in T-cell activation-induced apoptosis were largely unknown. Here, in this study, we uncovered that DDB1 could downregulate the expression of histone methyltransferase SETD7 through decreasing its mRNA level and then regulated activation-induced apoptosis of T-cell line Jurkat cells. Furthermore, RNA-sequencing assay on activated Jurkat cells confirmed that the SETD7 attenuated the activation of Jurkat cells. Our study revealed the non-enzymatic functions of DDB1 on the activation-induced apoptosis of T cells.

Inhibitory Peptide of Soluble Guanylyl Cyclase/Trx1 Interface Blunts the Dual Redox Signaling Functions of the Complex.

Soluble guanylyl cyclase (GC1) and oxido-reductase thioredoxin (Trx1) form a complex that mediates two NO signaling pathways as a function of the redox state of cells. Under physiological conditions, reduced Trx1 (rTrx1) supports the canonical NO-GC1-cGMP pathway by protecting GC1 activity from thiol oxidation. Under oxidative stress, the NO-cGMP pathway is disrupted by the S-nitrosation of GC1 (addition of a NO group to a cysteine). In turn, SNO-GC1 initiates transnitrosation cascades, using oxidized thioredoxin (oTrx1) as a nitrosothiol relay. We designed an inhibitory peptide that blocked the interaction between GC1 and Trx1. This inhibition resulted in the loss of a) the rTrx1 enhancing effect of GC1 cGMP-forming activity in vitro and in cells and its ability to reduce the multimeric oxidized GC1 and b) GC1's ability to fully reduce oTrx1, thus identifying GC1 novel reductase activity. Moreover, an inhibitory peptide blocked the transfer of S-nitrosothiols from SNO-GC1 to oTrx1. In Jurkat T cells, oTrx1 transnitrosates procaspase-3, thereby inhibiting caspase-3 activity. Using the inhibitory peptide, we demonstrated that S-nitrosation of caspase-3 is the result of a transnitrosation cascade initiated by SNO-GC1 and mediated by oTrx1. Consequently, the peptide significantly increased caspase-3 activity in Jurkat cells, providing a promising therapy for some cancers.

YIV-906 enhances nuclear factor of activated T-cells (NFAT) activity of T cells and promotes immune checkpoint blockade antibody action and CAR T-cell activity.

YIV-906 is a systems biology botanical cancer drug, inspired by a traditional Chinese herbal formulation. Results from eight Phase I/II to II clinical studies demonstrated the potential of YIV-906 to prolong survival and improve the quality of life of cancer patients. As an immunomodulator in the tumor microenvironment, YIV-906 can turn cold tumors hot and potentiate anti-tumor activity for different classes of anticancer agents; and as a cytoprotector in the GI, YIV-906 can reduce non-hematological side effects and speed up damaged tissue recovery. YIV-906 enhanced anti-PD1 action against hepatoma in mice by stimulating both innate and adaptive immunity. In a Jurkat cell-staphylococcal superantigen E (SEE)-Raji cell culture model, YIV-906 promoted T cell activation with upregulation of CD69 by enhancing NFAT activity, with or without PD1-PD-L1 interaction. YIV-906 could trigger the phosphorylation of TCR downstream signaling cascades without the involvement of TCR. YIV-906 could inhibit SHP1 and SHP2 activities, which dephosphorylates TCR downstream proteins due to the PD1-PD-L1 interaction. Therefore, YIV-906 could enhance anti-PD1 action to rescue the depressed NFAT activity of Jurkat cells due to the PD1-PD-L1 interaction. In addition, YIV-906 enhanced the NFAT activity and killing capability of Jurkat cells expressing chimeric antigen receptor (CAR-CD19-CD3z) toward CD19 expressing cells, such as Raji cells, with or without PD1-PD-L1 overexpression. Ingredient herb S (Scutellaria baicalensis Georgi) of YIV-906 and some S compounds were found to play key roles in these activities. In conclusion, YIV-906 modulates adaptive immunity by activating T effector cells mainly through its action on SHP1/2. YIV-906 could also facilitate immune checkpoint blockade therapy or CAR-T cell therapy for cancer treatment.

Site-Selective Tyrosine Phosphorylation in the Activation of the p50 Subunit of NF-κB for DNA Binding and Transcription.

The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

Penicillactonin and preaustinoid C, lactone-containing metabolites from a hot spring sediment Penicillium sp.

Phytochemical investigation of Penicillium sp. RO-11 strain, collected from the sediments of a hydrothermal spring located in the southwestern area of Saudi Arabia, afforded, along with previously isolated compounds, the undescribed polyketides penicillactonin (1), penipyranicin D (4) and isopyrenulin B (5) and the undescribed meroterpenoid preaustinoid C (7). The structures of these compounds were elucidated based on data from mass spectrometry, 1D and 2D NMR, and comparison between experimental and calculated ECD spectra. Penicillactonin and preaustinoid C bring unprecedented structural features, for which a biosynthetic rationale is proposed, further extending the chemodiversity associated to Penicillium fungi. Preaustinoid C showed significant activity against LPS-induced NO production and selective effect on IL-2 and IFN-γ gene regulation in activated Jurkat cells.

β-ADRENERGIC RECEPTOR BLOCKERS AS A REGULATOR OF T CELL VIABILITY (IN THE MODEL SYSTEM OF THE JURKAT CELLS)

Our research aimed to establish the effectiveness of various β-adrenergic receptor blockers in the regulation of T cell proliferation in the model system of the Jurkat cells.Material and Methods. The research was conducted on the human leukemic mature T cells (Jurkat cells) (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany)) activated by phytohemagglutinin (PHA). Viability and proliferative activity of intact and PHA-stimulated Jurkat cells under the influence of Nebilet, Egilok, Betalok Zok, and Propranolol were studied with the MTT test. Results. Study results show that β-adrenergic receptors blockers selectively influence the activity of mitochondrial dehydrogenases, and therefore viability in both intact and mitogen-stimulated Jurkat cells. In particular, Nebilet, Betalok Zok, and Propranolol induced a statistically significant decrease of intact Jurkat cells viability by 63%, 20%, and 32%, respectively; Egilok and Betalok Zok (25mg) didn’t statistically significantly affect the PHA- activated Jurkat cells viability. Nebilet, Betalok Zok (50 mg), and Propranolol decreased the viability of the mitogen (PHA) activated Jurkat cells (for PHA dose 20μg/ml) and didn’t affect at a PHA dose of 50μg/ml.

LINC00892 Is an lncRNA Induced by T Cell Activation and Expressed by Follicular Lymphoma-Resident T Helper Cells.

Successful immunotherapy in both solid tumors and in hematological malignancies relies on the ability of T lymphocytes to infiltrate the cancer tissue and mount an immune response against the tumor. Biomarkers able to discern the amount and the types of T lymphocytes infiltrating a given tumor therefore have high diagnostic and prognostic value. Given that lncRNAs are known to have a highly cell-type-specific expression pattern, we searched for lncRNAs specifically expressed by activated T cells and at the same time in a kind of lymphoma, follicular lymphoma, where the microenvironment is known to play a critical role in the regulation of antitumor immunity. We focused on a non-coding transcript, annotated as LINC00892, which reaches extremely high expression levels following cell activation in Jurkat cells. Interestingly LINC00892 has an expression pattern resembling that of genes involved in T cell memory. Accordingly, LINC00892 is mostly expressed by the effector memory and helper CD4+ T cell sub-types but not by naïve T cells. In situ analyses of LINC00892 expression in normal lymph nodes and in follicular lymphoma biopsies show that its expression is limited to CD4+ PD1hi T cells, with a subcellular localization within the germinal center matching that of follicular helper T cells. Our analysis therefore suggests that the previously uncharacterized lncRNA LINC00892 could be a useful biomarker for the detection of CD4+ memory T cells in both normal and tumor tissues.

Disruption of Wnt/β-catenin Pathway Elevates the Sensitivity of Gastric Cancer Cells to PD-1 Antibody.

Gastric Cancer (GC) is the fifth most common malignancy tumor and the third cause of cancer-related death around the world. Immune checkpoint inhibitors (ICIs) such as programmed cell death-1 (PD-1) antibodies play an active role in tumor therapy. A recent study reveals that Wnt/β-catenin signaling pathway is negatively correlated with T-cell infiltration in tumor microenvironment (TME), thereby influencing the therapeutic efficacy of PD-1 antibody. In this study, we aimed to uncover the relationship of Wnt/β-catenin pathway to CD8+ T cell activity as well as its effect on anti-PD-1 therapeutic efficacy in GC. We first collected clinical samples and went through an immunohistochemical analysis and found that a high β-catenin expression in GC tissues was often associated with a significant absence of CD8+ T-cell infiltration. In addition, our data further indicated that disruption of the Wnt/β-catenin pathway in GC cells inhibited their migratory and invasive ability. Meanwhile, enhanced sensitivity of GC cells to PD-1 blockade therapy was evident by decreased Jurkat cell apoptosis rate and increased GC cell apoptosis rate in a tumor and Jurkat cells co-culture system with the presence of Wnt/β-catenin pathway inhibition. Collectively, these findings indicated Wnt/β-catenin pathway may play a significant role in modulating the activity of Jurkat cells and downregulation of Wnt/β-catenin may enhance the sensitivity of GC cells to PD-1 antibody in vitro. This result further indicated that β-catenin and PD-1 targeted inhibition might become a potential and effective therapy for GC patients.

S‐acylation of STIM1 regulates store‐operated calcium entry

The initiating events happening during store‐operated calcium entry are not completely understood despite the tremendous importance of this process in immune responses. Upon endoplasmic reticulum (ER) calcium depletion, STIM1 and Orai1 form calcium release‐activated calcium (CRAC) channels to allow calcium influx from the extracellular milieu. S‐acylation is a reversible post‐translational modification of cysteine residues that affects protein structure, stability, and targeting to subdomains in the plasma membrane. Previously, we have shown that Orai1 is rapidly S‐acylated following T cell activation in Jurkat cells as well as ER calcium store depletion in HEK293 cells. Here we demonstrate that STIM1 is S‐acylated at C437 residue upon T cell activation and ER calcium store depletion. This is an interesting observation because very little is known about S‐acylation and its consequences in ER‐resident proteins. Upon store depletion, STIM1 adopts an extended conformation that permits binding to Orai1. We hypothesize that S‐acylation of STIM1 promotes and stabilizes the extended conformation of STIM1. Consistent with this hypothesis, we show that substitution of cysteine 437 in the STIM1 Orai1 activating region to a serine decreases calcium entry after store depletion. We further show that the ability of STIM1 to form puncta depends on its S‐acylation at this residue. Together with S‐acylation of Orai1, these data demonstrate that CRAC channel components are rapidly S‐acylated after ER calcium store depletion. In summary, based on our previous published literature on Orai1 S‐acylation, and current discovery of S‐acylation of STIM1, it is evident that S‐acylation recruits Orai1 to specialized membrane subdomains to form puncta with STIM1, whose extended conformation is stabilized by S‐acylation at its 437 residue. Hence, S‐acylation of Orai1 and STIM1 play a crucial role facilitate store‐operated calcium entry.

HDAC inhibitor cowanin extracted from G. fusca induces apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathways in Jurkat cells

Cowanin, a xanthone derivative extracted from the Garcinia fusca plant, has been recognized for various biological activities including, antimicrobial, anti-inflammatory, and anticancer activities. However, the mechanism to induce cancer cell death in cancer cells remains to be fully elucidated. Our previous report showed that other xanthones from these plants could act as histone deacetylase inhibitors (HDACi), so we deeply analyzed the role of cowanin, a major compound of G.fusca, and investigated through the mode of cell death both apoptosis and autophagy that have never been reported. As a result, it was demonstrated that cowanin indicated the role of HDACi as other xanthones. The molecular docking analysis showed that cowanin could interact within the catalytic pocket region of HDAC class I (HDAC2, 8) and II (HDAC4, 7) proteins and inhibit their activity. Also, the level of protein expression of HDAC2, 4, 7, and 8 was distinctly decreased, and the level of histone H3 and H4 acetylation increased in cowanin treated cells. For the mode of cell death, cowanin demonstrated both apoptosis and autophagy activation in Jurkat cells. Besides, cowanin significantly suppressed phosphorylation of PI3K, Akt, and mTOR signaling. Therefore, these findings revealed that cowanin represents a new promising candidate for development as an anticancer agent by inducing apoptosis and autophagy via PI3K/AKT/mTOR pathway and effectively inhibiting HDAC activity.

Actin Polymerization Is Required for Filopodia Formation Supporting HSV-1 Entry into Activated T Cells.

Enhanced HSV-1 production is found in activated T-lymphocytes, but the mechanism is still unknown. In this paper, the HSV-1 entry step in CD3+CD4-CD8-Jurkat T lymphocytes was investigated. Observation under electron microscopy revealed the level of filopodia formation on the surface of activated Jurkat cells was significantly higher than that of non-activated Jurkat cells especially after adding HSV-1 for 15min. A significant increase of actin protein was demonstrated in HSV-1 infected, activated Jurkat cells compared to HSV-1 infected, non-activated Jurkat cells. After the cells were treated with 2.5 and 5µg/mL cytochalasin D, an inhibitor of actin polymerization that causes depolymerization of actin's filamentous form, the actin protein was decreased significantly, resulting in an absence of filopodia formation. In summary, this is the first study revealing that HSV-1 induced filopodia formation through actin polymerization in activated T cells similar to epithelial, mucosal and neuronal cells. This phenomenon supported the virus entry resulting to increased yield of HSV-1 production.

Exosomes loaded with programmed death ligand-1 promote tumor growth by immunosuppression in osteosarcoma

ABSTRACT Osteosarcoma (OS) is a malignant tumor commonly observed in adolescents, who experience relapse and metastasis (30% of the total cases). Its progression is attributed to immune escape mediated by immune checkpoints. However, the intercellular connection between tumor cells and T cells remain unclear. This study was conducted to explore the effects of PD-L1-loaded exosomes on the tumor growth of OS. The exosomes were extracted from cells and tissues through ultracentrifugation. IFN-γ production was determined to evaluate the activity of Jurkat cells. The in vivo growth of OS cells was examined using a C3H xenograft model in mice, tumor volumes were monitored, and the proportion of CD3 + T cells in tumor tissues was detected. Results revealed that PD-L1 was significantly upregulated in the OS cell lines. MG63 and Saos-2 cells were the most abundant in PD-L1, so they were selected as investigation targets. PD-L1 was found to be also highly expressed in the exosomes isolated from MG63 and Saos-2 cells. The exosomes elicited significant inhibitory effects on IFN-γ secretion in Jurkat cells, which were abolished by the PD-L1 antibody or siRNAs. The in vivo growth of C3H cells was significantly facilitated by the overexpression of mPD-L1 or by the administration of mPD-L1-overloaded exosomes. The infiltration of CD3 + T cells was also decreased. The exosomes extracted from clinical PD-L1-positive OS tissues showed a promising inhibitory property against activated T cells. Therefore, PD-L1-loaded exosomes extracted from OS cells aggravated OS progression by suppressing T cell activities.

Evaluation of skin sensitization based on interleukin‑2 promoter activation in Jurkat cells

Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T-cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T-cell activation to supplement data on skin sensitization related to KE4. To evaluate T-cell activation, the Jurkat T-cell line stably expressing luciferase downstream of the pro-inflammatory cytokine interleukin-2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T-cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno-suppressiveness of chemical substances and supplements KE4 T-cell activation data, and may thus contribute to reducing the use of animal experiments.

Targeting T-Cell Receptor Signaling Pathway in Angioimmunoblastic T-Cell Lymphoma

[Backgrounds] Angioimmunoblastic T-cell lymphoma (AITL) belongs to nodal T-cell lymphoma with T follicular helper (TFH) phenotype. Both the G17V RHOA mutation and the loss-of-function TET2 mutation have been identified in 70% of AITLs. We previously demonstrated that G17V mutant RHOA augmented T-cell receptor (TCR) signaling though binding to VAV1, an essential component of the T-cell signaling pathway (Leukemia, in press). However, it remains elusive whether inhibition of TCR pathway serves as an effective treatment in AITL. [Methods] Activation of VAV1 and PLCgamma1 in response to TCR stimulation was examined in Jurkat cells inducibly expressing wild-type and G17V mutant RHOA under the treatment with dasatinib, a multi-kinase inhibitor. Nuclear factor of activated T cell (NFAT) activity was similarly examined in Jurkat cells transiently transfected with wild-type and G17V mutant RHOA. Mice expressing G17V RHOA under CD2 promoter (G17VRHOA mice) were crossed with MxCre Tet2f/f mice and pIpC was given to the offspring 4 weeks after birth to obtain Tet2-/- and Tet2-/-G17VRHOA mice. Whole transcriptome analysis was performed for CD4+ T cells purified from the spleen of tumor developed mice. Serum cytokines were examined by BD Cytometric Beads Assay. Cells derived from tumors developed in Tet2-/-G17VRHOA mice were transplanted into nude mice. Dasatinib was orally given at 5 mg/kg/day for 14 days. [Results] The G17V mutant RHOA enhanced phosphorylation of VAV1 and PLCgamma1, and NFAT reporter activity in Jurkat cells. The phosphorylation and the NFAT activity were efficiently blocked by dasatinib at 10 nM. Approximately 70% of Tet2-/-G17VRHOA mice died around 40 wo, while 14% of Tet2-/- and none of G17VRHOA mice died at the same age (p<0.05). All the moribund Tet2-/-G17VRHOA mice presented splenomegaly and lymphadenopathy. The lymph nodes and the spleens showed diffuse infiltration of tumor cells expressing TFH markers, i.e., CD4, PD1, and ICOS. The aberrant increase of high endothelial venules was observed in the swollen lymph nodes, mimicking human AITL. Whole transcriptome analysis followed by gene-set enrichment analysis revealed enrichment of Il-2/STAT5, Il-6/JAK-STAT3, and TNFα/NFκB signaling pathways in Tet2-/-G17VRHOA CD4+ spleen cells. The serum concentrations of Il-2, Il6, and TNFα in Tet2-/-G17VRHOA were significantly higher than control at the same age (p<0.05). The CD4+ cells in the enlarged spleen and the swollen lymph nodes in Tet2-/-G17VRHOA mice at 40 wo showed oligoclonal rearrangement of TCR genes. Nude mice transplanted with tumor-derived cells showed splenomegaly and lymphadenopathy preceded by elevation ofthe serum concentrations of Il-2, Il-6 and TNFα (before vs. after the transplantation, 4.33±0.14 vs. 4.80±0.37, 5.85±2.96 vs. 34.92±15.49, and 7.68±2.08 vs. 45.70±33.72, p<0.01, respectively). Mice given with dasatinib starting at 10 days after the transplantation showed higher overall survival compared to the vehicle control (p<0.05, Figure 1). The serum concentrations of Il-2, Il-6, and TNFα were decreased in dasatinib group (before vs. after the dasatinib treatment, 4.85±0.40 vs. 4.28±0.18, 38.23±18.74 vs. 10.01±7.38, and 48.37±34.6 vs. 18.67±12.37, p<0.05, respectively).Discussions and Conclusion: The combination of G17V mutantRHOAexpression and Tet2 deletion led to development of AITL-like tumors in mice, which were hughly effectively treated by dasatinib. This model may also be useful to examine novel treatment strategies for AITL, other than dasatinib. [Display omitted] DisclosuresChiba:Nippon Shinyaku: Honoraria, Research Funding.

Orexin A Suppresses the Expression of Exosomal PD-L1 in Colon Cancer and Promotes T Cell Activity by Inhibiting JAK2/STAT3 Signaling Pathway.

Colon cancer, ranked third in cancer related mortality, is the most common malignant cancer of digestive tract. Though immune checkpoint inhibitors show promising efficacy in colon cancer, a rather high unresponsive rate and recurrence rate requires further elucidation of the underlying regulatory mechanism of cancer-related immunity. To study the regulatory function of Orexin A in the expression of exosomal PD-L1 and T cell activity. Orthotopic colon cancer transplantation mice model were established to study the cancer growth and immune infiltration between Orexin A treated group and untreated group. In vitro studies using mouse CT-26 and human HCT-116 colon cancer cell model studied the effect of Orexin A on cellular and exosomal PD-L1 expression. Co-culturing Jurkat cells with exosomes delivered by cancer cells treated with Orexin A, PD-L1 knockdown and PBS studied different effects on T cell. Comparing Orexin A with WP1066, a JAK2/STAT3 inhibitor verified the mechanism of these changes. The growth rate of orthotopic transplanted colon cancer was slower in Orexin A treated group, with lower PD-L1 expression and higher immune infiltration. Orexin A could inhibit cellular and exosomal PD-L1 expression. The decreased expression of PD-L1 in exosomes could promote the activity of Jurkat cells secreting higher level of IFN-γ and IL-2. Orexin A showed a similar effect like WP1066 which proved JAK2/STAT3 signaling pathway was its downstream signaling pathway. Orexin A could suppress the expression of exosomal PD-L1 in colon cancer cells and promote T cells activity by inhibiting JAK2/STAT3 signaling pathway.