T cell receptor gene-modified allogeneic T cells with siRNA for endogenous T cell receptor induce efficient tumor regression without graft-versus-host disease.

Adoptive immunotherapy using genetically engineered patient-derived lymphocytes to express tumor-reactive receptors is a promising treatment for malignancy. However, utilization of autologous T cells in this therapy limits the quality of gene-engineered T cells, thereby inhibiting the timely infusion of the cells into patients. In this study, we evaluated the anti-tumor efficacy and the potential to induce graft-versus-host disease (GVHD) in T cell receptor (TCR) gene-engineered allogeneic T cells that downregulate the endogenous TCR and HLA class I molecules with the aim of developing an "off-the-shelf" cell product with expanded application of genetically engineered T cells. We transduced human lymphocytes with a high-affinity TCR specific to the cancer/testis antigen NY-ESO-1 using a novel retrovirus vector with siRNAs specific to the endogenous TCR (siTCR vector). These T cells showed reduced expression of endogenous TCR and minimized reactivity to allogeneic cells in vitro. In non-obese diabetic/SCID/γcnull mice, TCR gene-transduced T cells induced tumor regression without development of GVHD. A lentivirus-based CRISPR/Cas9 system targeting ß-2 microglobulin in TCR gene-modified T cells silenced the HLA class I expression and prevented allogeneic CD8+ T cell stimulation without disrupting their anti-tumor capacity. This report is the first demonstration that siTCR technology is effective in preventing GVHD. Adoptive cell therapy with allogeneic T cells engineered with siTCR vector may be useful in developing an "off-the-shelf" therapy for patients with malignancy.

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement.

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.

Chemical augmentation of mitochondrial electron transport chains tunes T cell activation threshold in tumors.

BACKGROUND: Cancer immunotherapy shows insufficient efficacy for low immunogenic tumors. Furthermore, tumors often downregulate antigen and major histocompatibility complex expression to escape recognition by T cells, resulting in insufficient T cell receptor (TCR) stimulation in the tumor microenvironment. Thus, augmenting TCR-mediated recognition of tumor antigens is a useful strategy to improve the efficacy of cancer immunotherapy. METHODS: We screened 310 small molecules from our library and identified PQDN, a small molecule that activates CD8 T cells after TCR engagement, even when antigen stimulation is too weak for their activation. We used inhibitors of mitochondrial functions and Seahorse Flux Analyzer to investigate the mechanism underlying the effect of PQDN on T cells. Effect of PQDN on tumor-infiltrating CD8 T cells was examined using flow cytometry and TCR repertoire analysis. RESULTS: PQDN increased mitochondrial reciprocal capacity through enhancement of electron transport chains (ETCs) and facilitated glycolysis via mTOR/AKT signaling, resulting in augmented CD8 T cell activation, even when antigen stimulation is extremely weak. Intratumoral administration of this compound into tumor-bearing mice tunes inactivated T cell with tumor antigen recognition potent and expanded functional T cell receptor diversity of tumor-infiltrating T cells, augmenting antitumor immune responses and retarding tumor growth. Furthermore, PQDN has a synergistic potent with T cell dependent immunotherapy, such as checkpoint inhibitory therapy or adoptive cell therapy, even in a low immunogenic tumor. We also demonstrated that this compound enhances the activation of human CD8 T cells. CONCLUSIONS: These data suggest that tuning the T cell activation threshold by chemical activation of mitochondrial ETC is a new strategy for improving therapeutic efficacy through the activation of low-avidity tumor-specific T cells.

Nucleosome assembly protein 1 is a regulator of histone H1 acetylation.

Acetylation of histone H1 is generally considered to activate transcription, whereas deacetylation of H1 represses transcription. However, the precise mechanism of the acetylation is unknown. Here, using chromatography, we identified nucleosome assembly protein 1 (NAP-1) as having inhibitory activity against histone H1 acetylation by acetyltransferase p300. We found that native NAP-1 interacts with H1 in a Drosophila crude extract. We also found that it inhibits the deacetylation of histone H1 by histone deacetylase 1. The core histones in nucleosomes were acetylated in a GAL4-VP16 transcriptional activator-dependent manner in vitro. This acetylation was strongly repressed by hypoacetylated H1 but to a lesser extent by hyperacetylated H1. Consistent with these findings, a micrococcal nuclease assay indicated that hypoacetylated H1, which represses activator-dependent acetylation, was incorporated into chromatin, whereas hyperacetylated H1 was not. To determine the contribution of NAP-1 to transcriptional regulation in vivo, we compared NAP-1 knockdown (KD) with coactivator CREB-binding protein (CBP) KD using RNA sequencing in Drosophila Schneider 2 cells. Most genes were downregulated rather than upregulated by NAP-1 KD, and those downregulated genes were also downregulated by CBP KD. Our results suggest that NAP-1 plays a role in transcriptional regulation by fine-tuning the acetylation of histone H1. Graphical Abstract.

A novel RORγt inhibitor is a potential therapeutic agent for the topical treatment of psoriasis with low risk of thymic aberrations.

BACKGROUND: Retinoic acid receptor-related orphan receptor gamma t (RORγt) has critical roles in the development, maintenance and function of interleukin (IL)-17-producing cells and is a highly attractive target for the treatment of IL-17-mediated autoimmune disease, particularly psoriasis. On the other hand, RORγt is also critical for controlling apoptosis during thymopoiesis, and genetic RORγt ablation or systematic RORγt inhibition cause progressive thymic aberrations leading to T cell lymphomas. OBJECTIVE: We investigated whether topical administration of our novel RORγt inhibitor, S18-000003 has therapeutic potential for psoriasis with low risk of thymic aberrations. METHODS: We evaluated the effect of topical S18-000003 on psoriasis-like skin inflammation and influence on the thymus in a 12-O-tetradecanoylphorbol-13-acetate-induced K14.Stat3C mouse psoriasis model. RESULTS: S18-000003 markedly inhibited the development of psoriatic skin inflammation via suppression of the IL-17 pathway. In the skin, S18-000003 suppressed all subsets of IL-17-producing cells that we previously identified in this psoriasis model: Th17 cells, Tc17 cells, dermal γδ T cells, TCR- cells that probably included innate lymphoid cells, and CD4-CD8- double-negative αß T cells. Notably, neither reduction of CD4+CD8+ double-positive thymocytes nor dysregulation of cell cycling was observed in S18-000003-treated mice, even at a high dose. CONCLUSION: Our topically administered RORγt inhibitor is a potential therapeutic agent for psoriasis with low risk of thymic lymphoma.

MicroRNA-3662 expression correlates with antiviral drug resistance in adult T-cell leukemia/lymphoma cells.

Interferon regulatory factor (IRF) 4 and the proto-oncogene c-Rel cooperate in growth and antiviral drug resistance of adult T-cell leukemia/lymphoma (ATLL). To elucidate the target of IRF4 and c-Rel in ATLL, we determined the simultaneous binding sites of IRF4 and c-Rel using ChIP-seq technology. Nine genes were identified within 2 kb of binding sites, including MIR3662. Expression of miR-3662 was regulated by IRF4, and to a lesser extent by c-Rel. Cell proliferation was inhibited by knockdown of miR-3662 and expression of miR-3662 was correlated with antiviral drug resistance in ATLL cell lines. Thus, miR-3662 represents a target for therapies against ATLL.

The Inhibitory Effect of S-777469, a Cannabinoid Type 2 Receptor Agonist, on Skin Inflammation in Mice.

We investigated the effects of S-777469 (1-[[6-Ethyl-1-[4-fluorobenzyl]-5-methyl-2-oxo-1, 2-dihydropyridine-3-carbonyl]amino]-cyclohexanecarboxylic acid), a novel cannabinoid type 2 receptor (CB2) agonist, on 1-fluoro-2,4-dinitrobenzene (DNFB)-induced ear inflammation and mite antigen-induced dermatitis in mice. The oral administration of S-777469 significantly suppressed DNFB-induced ear swelling in a dose-dependent manner. In addition, S-777469 significantly alleviated mite antigen-induced atopic dermatitis-like skin lesions in NC/Nga mice. A histological analysis revealed that S-777469 significantly reduced the epidermal thickness and the number of mast cells infiltrating skin lesions. We demonstrated that S-777469 inhibited mite antigen-induced eosinophil accumulation in skin lesions and an endogenous CB2 ligand, 2-arachidonoylglycerol (2-AG)-induced eosinophil migration in vitro. Moreover, we confirmed that 2-AG levels significantly increased in skin lesions of mite antigen-induced dermatitis model. Together, these results suggest that S-777469 inhibits skin inflammation in mice by blocking the activities of 2-AG.

Potential role of IL-17-producing CD4/CD8 double negative αß T cells in psoriatic skin inflammation in a TPA-induced STAT3C transgenic mouse model.

BACKGROUND: Psoriasis is one of the most common immune-mediated chronic inflammatory skin disorders and is accompanied by erythematous scaly plaques. There is growing evidence that the IL-23/Th17 axis plays a critical role in development of the disease. It was recently shown that in addition to CD4+ Th17 cells, various IL-17-producing cell subsets such as CD8+ Tc17 cells, dermal γδ T cells, and innate lymphoid cells are also involved in the development of psoriatic inflammation in humans. OBJECTIVE: To investigate which subsets of IL-17-producing cells are involved in psoriasis-like skin inflammation in a TPA (tumor promoter 12-O-tetradecanoylphorbol-13-acetate)-induced K14.Stat3C mouse model. METHOD: Skin-infiltrating cells were isolated from inflamed lesions of TPA-treated K14.Stat3C transgenic mice, and analyzed for IL-17 producing cell subsets by flow cytometry. RESULTS: We observed significantly increased numbers of IL-17-producing CD4+ T cells, CD8+ T cells and dermal γδ T cells in TPA-induced skin lesions of K14.Stat3C mice. Additionally, we found that another IL-17-producing T cell subset, αß-TCR+ CD4CD8 double negative T cells (DN αß T cells), was also increased in lesional skin. These IL-17-producing DN αß T cells are NK1.1 negative, suggesting they are not natural killer T cells or mucosal associated invariant T cells. As well as other IL-17-producing cells, DN αß T cells in the inflamed skin can also respond to IL-23 stimulation to produce IL-17. It is also suggested that DN αß T cells may express retinoic acid-related orphan receptor gamma t and CC chemokine receptor 6. CONCLUSION: In TPA-induced lesional skin of K14.Stat3C mice, IL-17-producing CD4+ Th17 cells, CD8+ Tc17 cells, dermal γδ T cells and TCR- cells probably containing ILCs all participated in skin inflammation, which is similar to human clinical psoriatic features. Furthermore, we showed for the first time the possibility that an IL-17-producing DN αß T cell subset is also involved in psoriatic inflammation.

Gamma-interferon-inducible, lysosome/endosome-localized thiolreductase, GILT, has anti-retroviral activity and its expression is counteracted by HIV-1.

The mechanism by which type II interferon (IFN) inhibits virus replications remains to be identified. Murine leukemia virus (MLV) replication was significantly restricted by γ-IFN, but not human immunodeficiency virus type 1 (HIV-1) replication. Because MLV enters host cells via endosomes, we speculated that certain cellular factors among γ-IFN-induced, endosome-localized proteins inhibit MLV replication. We found that γ-IFN-inducible lysosomal thiolreductase (GILT) significantly restricts HIV-1 replication as well as MLV replication by its thiolreductase activity. GILT silencing enhanced replication-defective HIV-1 vector infection and virion production in γ-IFN-treated cells, although γ-IFN did not inhibit HIV-1 replication. This result showed that GILT is required for the anti-viral activity of γ-IFN. Interestingly, GILT protein level was increased by γ-IFN in uninfected cells and env-deleted HIV-1-infected cells, but not in full-length HIV-1-infected cells. γ-IFN-induced transcription from the γ-IFN-activation sequence was attenuated by the HIV-1 Env protein. These results suggested that the γ-IFN cannot restrict HIV-1 replication due to the inhibition of γ-IFN signaling by HIV-1 Env. Finally, we found that 4,4'-dithiodipyridine (4-PDS), which inhibits S-S bond formation at acidic pH, significantly suppresses HIV-1 vector infection and virion production, like GILT. In conclusion, this study showed that GILT functions as a host restriction factor against the retroviruses, and a GILT mimic, 4-PDS, is the leading compound for the development of novel concept of anti-viral agents.

S-777469, a novel cannabinoid type 2 receptor agonist, suppresses itch-associated scratching behavior in rodents through inhibition of itch signal transmission.

We have previously reported that S-777469 [1-([6-ethyl-1-(4-fluorobenzyl)-5-methyl-2-oxo-1,2-dihydropyridine-3-carbonyl]amino)-cyclohexanecarboxylic acid], a novel cannabinoid type 2 receptor (CB2) agonist, significantly suppressed compound 48/80-induced scratching behavior in mice in a dose-dependent manner when it was administered orally. Here, we demonstrated that the inhibitory effects of S-777469 on compound 48/80-induced scratching behavior are reversed by pretreatment with SR144528, a CB2-selective antagonist. In addition, we investigated the effects of S-777469 on itch-associated scratching behavior induced by several pruritogenic agents in mice and rats. S-777469 significantly suppressed scratching behavior induced by histamine or substance P in mice or by serotonin in rats. In contrast, the H1-antihistamine fexofenadine clearly inhibited histamine-induced scratching behavior but did not affect scratching behavior induced by substance P or serotonin. Moreover, S-777469 significantly inhibited histamine-induced peripheral nerve firing in mice. In conclusion, these results suggest that S-777469 produces its antipruritic effects by inhibiting itch signal transmission through CB2 agonism.

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis.

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within.

Mechanism of pathogenesis of imiquimod-induced skin inflammation in the mouse: a role for interferon-alpha in dendritic cell activation by imiquimod.

Topical application of imiquimod (IMQ), a Toll-like receptor (TLR)7 ligand, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. In a mouse model of IMQ-induced psoriasis-like skin inflammation, T-helper (Th)17 cells and interleukin (IL)-17/IL-22-producing γδ-T cells have been shown to play a pivotal role. However, the mechanisms of induction of the Th17 pathway and development of psoriasis-like skin inflammation by IMQ treatment remain unclear. In this study, we investigated pathogenic mechanisms of IMQ-induced psoriasis-like skin inflammation in mice. We first confirmed that, together with an increase in IL-17 and IL-22 production, application of IMQ to mouse skin induced the expression of cytokines required for activation of the Th17 pathway, and pro-inflammatory mediators involved in the pathology of psoriasis. Analysis of Tlr7(-/-) mice demonstrated that most of the in vivo effects of IMQ were mediated via TLR7. In an in vitro study using plasmacytoid dendritic cells (DCs), IMQ induced production of interferon (IFN)-α, IL-23, IL-6 and tumor necrosis factor (TNF)-α. Furthermore, when we analyzed in vitro-generated bone marrow-derived DCs with features similar to TNF-α and inducible nitric oxide synthase (iNOS)-producing DCs, IL-23, IL-6, IL-1ß, TNF-α and iNOS/NO production was weakly induced by IMQ alone and further enhanced after co-stimulation with IMQ and IFN-α. These in vitro effects of IMQ were also mediated via TLR7 and the synergistic effect of IMQ, and IFN-α was suggested to be caused by upregulation of TLR7 expression by IFN-α. These results demonstrate part of the mechanism by which the Th17 pathway and psoriasis-like skin inflammation are induced by IMQ and IFN-α in a mouse model.

Impact of TRPV3 on the development of allergic dermatitis as a dendritic cell modulator.

The transient receptor potential channel vanilloid subfamily V member 3 (TRPV3), which functions as a thermosensor in keratinocytes, plays an important role in the development of allergic and itchy dermatitis in rodents. Although real-time PCR analysis using lesional and non-lesional skin samples from patients with atopic dermatitis showed that TRPV3 was expressed in lesional skin, the role that TRPV3 plays in patients with dermatitis is still relatively obscure. Here, we determined whether TRPV3 was a dendritic cell (DC) modulator using DS-Nh mice with a gain-of-function mutation in TRPV3 (TRPV3Gly573Ser), because increasing skin temperature is associated with the modulation of dermal dendritic cells (DCs). Interestingly, increased responses to haptens by skin and DCs were observed in DS-Nh mice compared with those from DS mice with wild-type TRPV3. Increased thymic stromal lymphopoietin (TSLP) responses were also observed in keratinocytes from DS-Nh mice compared with those from DS mice. Taken together, we propose that the DS-Nh mouse is a good model to use in order to better understand the role of this orphan channel and that TRPV3 may represent a new therapeutic target in certain types of dermatitis through the control of DCs.

Selective CB2 agonists with anti-pruritic activity: discovery of potent and orally available bicyclic 2-pyridones.

The CB2 receptor has emerged as a potential target for the treatment of pruritus as well as pain without CB1-mediated side effects. We previously identified 2-pyridone derivatives 1 and 2 as potent CB2 agonists; however, this series of compounds was found to have unacceptable pharmacokinetic profiles with no significant effect in vivo. To improve these profiles, we performed further structural optimization of 1 and 2, which led to the discovery of bicyclic 2-pyridone 18e with improved CB2 affinity and selectivity over CB1. In a mouse pruritus model, 18e inhibited compound 48/80 induced scratching behavior at a dose of 100 mg/kg. In addition, the docking model of 18e with an active-state CB2 homology model indicated the structural basis of its high affinity and selectivity over CB1.

Design, synthesis, and binding mode prediction of 2-pyridone-based selective CB2 receptor agonists.

Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold and of having a highly lipophilic physicochemical property. To address these two major problems, we designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2. Optimization of 15a led to identification of 39f with high affinity for CB2 and selectivity over CB1. Prediction of the binding mode of 39f in complex with an active-state CB2 homology model provided structural insights into its high affinity for CB2.

Serum starvation activates NF-κB through G protein ß2 subunit-mediated signal.

Several cell stresses induce nuclear factor-kappaB (NF-κB) activation, which include irradiation, oxidation, and UV. Interestingly, serum-starving stress-induced NF-κB activation in COS cells, but not in COS-A717 cells. COS-A717 is a mutant cell line of COS cells that is defective of the NF-κB signaling pathway. We isolated genes with compensating activity for the NF-κB pathway and one gene encoded the G protein ß2 (Gß2). Gß2 is one of the G protein-coupled receptor signaling effectors. In COS-A717 cells, Gß2 expression is significantly reduced. In Gß2 cDNA-transfected COS-A717 cells, the NF-κB activity was increased along with the recovery of Gß2 expression. Furthermore, serum-starving stress induced the NF-κB activity in Gß2-transfected COS-A717 cells. Consistently, the serum-starved COS cells with siRNA-reduced Gß2 protein expression showed decreased NF-κB activity. These results indicate that Gß2 is required for starvation-induced NF-κB activation and constitutive NF-κB activity. We propose that serum contains some molecule(s) that strongly inhibits NF-κB activation mediated through Gß2 signaling.

Discovery of S-444823, a potent CB1/CB2 dual agonist as an antipruritic agent.

The optimization of a series of 3-carbamoyl 2-pyridone derivatives as CB agonists is reported. These efforts resulted in the discovery of 3-(2-(1-(cyclohexylmethyl)-2-oxo-1,2,5,6,7,8,9,10-octahydrocycloocta[b]pyridine-3-carboxamido)thiazol-4-yl)propanoic acid (21), a potent dual CB1/CB2 agonist without CNS side effects induced by CB1 receptor activation. It exhibited strong inhibition of scratching as a 1.0% acetone solution in the pruritic model.

Discovery of S-777469: an orally available CB2 agonist as an antipruritic agent.

The discovery of novel CB2 ligands based on the 3-carbamoyl-2-pyridone derivatives by adjusting the size of side chain at 1-, 5- and 6-position is reported. The structure-activity relationship around this template lead to the identification of S-777469 as a selective CB2 receptor agonist, which exhibited the significant inhibition of scratching induced by Compound 48/80 at 1.0 mg/kg po and 10 mg/kg po (55% and 61%, respectively).

CB 1/2 dual agonists with 3-carbamoyl 2-pyridone derivatives as antipruritics: reduction of CNS side effects by introducing polar functional groups.

Our lead compound 1 showed high affinity for both CB1 and CB2 receptors, suggesting the possibility of inducing psychoactive side effects through the CB1 receptor in the brain. To solve this issue, polar functional groups were introduced at the 3-position of the pyridone core of compound 1 to find CB1/2 dual agonists such as 17 and 20 which did not show any CNS side effects.