CD5 blockade, a novel immune checkpoint inhibitor, enhances T cell anti-tumour immunity and delays tumour growth in mice harbouring poorly immunogenic 4T1 breast tumour homografts.

CD5 is a member of the scavenger receptor cysteine-rich superfamily that is expressed on T cells and a subset of B cells (B1a) cell and can regulate the T cell receptor signaling pathway. Blocking CD5 function may have therapeutic potential in treatment of cancer by enhancing cytotoxic T lymphocyte recognition and ablation of tumour cells. The effect of administering an anti-CD5 antibody to block or reduce CD5 function as an immune checkpoint blockade to enhance T cell anti-tumour activation and function in vivo has not been explored. Here we challenged mice with poorly immunogenic 4T1 breast tumour cells and tested whether treatment with anti-CD5 monoclonal antibodies (MAb) in vivo could enhance non-malignant T cell anti-tumour immunity and reduce tumour growth. Treatment with anti-CD5 MAb resulted in an increased fraction of CD8+ T cells compared to CD4+ T cell in draining lymph nodes and the tumour microenvironment. In addition, it increased activation and effector function of T cells isolated from spleens, draining lymph nodes, and 4T1 tumours. Furthermore, tumour growth was delayed in mice treated with anti-CD5 MAb. These data suggest that use of anti-CD5 MAb as an immune checkpoint blockade can both enhance activation of T cells in response to poorly immunogenic antigens and reduce tumour growth in vivo. Exploration of anti-CD5 therapies in treatment of cancer, alone and in combination with other immune therapeutic drugs, is warranted.

Preventing alloimmune rejection using circular RNA FSCN1-silenced dendritic cells in heart transplantation.

BACKGROUND: While heart transplantation is used as a standard treatment for heart failure, transplant rejection continues to pose a challenge. Recent evidence has shown that circular RNA (circRNA) is a new type of gene regulator in cell development. Our aim was to demonstrate that treatment with tolerogenic dendritic cells (Tol-DCs) generated by circular RNA FSCN1 (circFSCN1) silencing could prevent alloimmune rejection and prolong heart graft survival in heart transplantation. METHODS: Bone marrow-derived DCs were transfected with circFSCN1 siRNA in vitro. The circFSCN1 level was measured by qRT-PCR. DC maturation was determined by flow cytometry. Mixed lymphocyte reactions (MLRs) were conducted to assess the function of DCs to activate T cells and to generate regulatory T cells (Tregs). In situ RNA hybridization and fluorescent microscopy were performed to detect the distribution of circFSCN1 in DCs. A heterotopic allogeneic murine heart transplantation was conducted where recipients were pre-treated with donor derived circFSCN1-silenced Tol-DCs. Heartbeat was monitored to assess immune rejection. RESULTS: Exonic circFSCN1 was highly expressed in the cytoplasm of mature DCs. Knockdown of circFSCN1 using siRNA arrested DCs at an immature state, impaired DC's ability to activate T cells and enhanced Treg generation. Treatment with circFSCN1-silenced Tol-DCs prevented alloimmune rejection, prolonged allograft survival, reduced fibrosis, and induced Tregs in vivo. CONCLUSIONS: Knockdown of circFSCN1 induces Tol-DCs and treatment with these Tol-DCs prevents alloimmune rejection and prolongs allograft survival. This is a promising therapeutic target to combat transplant rejection in heart transplantation and increases our understanding of circRNA in the immune system.

Research progress and application opportunities of nanoparticle-protein corona complexes.

Nanoparticles (NPs) can be used to design for nanomedicines with different chemical surface properties owing to their size advantages and the capacity of specific delivery to targeted sites in organisms. The discovery of the presence of protein corona (PC) has changed our classical view of NPs, stimulating researchers to investigate the in vivo fate of NPs as they enter biological systems. Both NPs and PC have their specificity but complement each other, so they should be considered as a whole. The formation and characterization of NP-PC complexes provide new insights into the design, functionalization, and application of nanocarriers. Based on progress of recent researches, we reviewed the formation, characterization, and composition of the PC, and introduced those critical factors influencing PC, simultaneously expound the effect of PC on the biological function of NPs. Especially we put forward the opportunities and challenges when NP-PC as a novel nano-drug carrier for targeted applications. Furthermore, we discussed the pros versus cons of the PC, as well as how to make better PC in the future application of NPs.

The Role of Tumor-Derived Exosomes in the Abscopal Effect and Immunotherapy.

Exosomes are microvesicles that can be secreted by various cells and carry a variety of contents; thus, they play multiple biological functions. For instance, the tumor-derived exosomes (TEXs) have been proven to have the effect of immunostimulatory in addition to immunosuppression, making TEXs attractive in clinical immunotherapy and targeted therapy for cancer patients. In addition, TEXs as biomarkers have important clinical diagnostic and prognostic value. Recently, TEXs have been recognized to play important roles in the abscopal effect (AbE), a newly discovered mechanism by which the distant tumors are effectively targeted and repressed during immunotherapy and radiotherapy. Therefore, TEXs has demonstrated great clinical potential in the diagnosis, prognosis and treatment of cancer patients in the future. This review summarizes and discusses the role of TEXs in clinical therapy and their role in AbE in recent studies.

CRISPR/Cas9 library screening uncovered methylated PKP2 as a critical driver of lung cancer radioresistance by stabilizing ß-catenin.

Radiation resistance is a major cause of lung cancer treatment failure. Armadillo (ARM) superfamily proteins participate in various fundamental cellular processes; however, whether ARM proteins regulate radiation resistance is not fully understood. Here, we used an unbiased CRISPR/Cas9 library screen and identified plakophilin 2 (PKP2), a member of the ARM superfamily of proteins, as a critical driver of radiation resistance in lung cancer. The PKP2 level was significantly higher after radiotherapy than before radiotherapy, and high PKP2 expression after radiotherapy predicted poor overall survival (OS) and postprogression survival (PPS). Mechanistically, mass spectrometry analysis identified that PKP2 was methylated at the arginine site and interacted with protein arginine methyltransferase 1 (PRMT1). Methylation of PKP2 by PRMT1 stabilized ß-catenin by recruiting USP7, further inducing LIG4, a key DNA ligase in nonhomologous end-joining (NHEJ) repair. Concomitantly, PKP2-induced radioresistance depended on facilitating LIG4-mediated NHEJ repair in lung cancer. More strikingly, after exposure to irradiation, treatment with the PRMT1 inhibitor C-7280948 abolished PKP2-induced radioresistance, and C-7280948 is a potential radiosensitizer in lung cancer. In summary, our results demonstrate that targeting the PRMT1/PKP2/ß-catenin/LIG4 pathway is an effective approach to overcome radiation resistance in lung cancer.

HDAC4 promotes nasopharyngeal carcinoma progression and serves as a therapeutic target.

Histone deacetylases (HDACs) are involved in tumor progression, and some have been successfully targeted for cancer therapy. The expression of histone deacetylase 4 (HDAC4), a class IIa HDAC, was upregulated in our previous microarray screen. However, the role of HDAC4 dysregulation and mechanisms underlying tumor growth and metastasis in nasopharyngeal carcinoma (NPC) remain elusive. Here, we first confirmed that the HDAC4 levels in primary and metastatic NPC tissues were significantly increased compared with those in normal nasopharyngeal epithelial tissues and found that high HDAC4 expression predicted a poor overall survival (OS) and progression-free survival (PFS). Functionally, HDAC4 accelerated cell cycle G1/S transition and induced the epithelial-to-mesenchymal transition to promote NPC cell proliferation, migration, and invasion in vitro, as well as tumor growth and lung metastasis in vivo. Intriguingly, knockdown of N-CoR abolished the effects of HDAC4 on the invasion and migration abilities of NPC cells. Mechanistically, HDAC3/4 binds to the E-cadherin promoter to repress E-cadherin transcription. We also showed that the HDAC4 inhibitor tasquinimod suppresses tumor growth in NPC. Thus, HDAC4 may be a potential diagnostic marker and therapeutic target in patients with NPC.

A novel multifunctional gold nanorod-mediated and tumor-targeted gene silencing of GPC-3 synergizes photothermal therapy for liver cancer.

Tumor-specific targeted delivery is a major obstacle to clinical treatment of hepatocellular carcinoma (HCC). Here we have developed a novel multi-functional nanostructure GAL-GNR-siGPC-3, which consists of Galactose (GAL) as the HCC-targeting moiety, golden nanorods (GNR) as a framework to destroy tumor cells under laser irradiation, and siRNA of Glypican-3 (siGPC-3) which induce specifically gene silence of GPC-3 in HCC. Glypican-3 (GPC-3) gene is highly associated with HCC and is a new potential target for HCC therapy. On the other hand, Gal can specifically bind to the asialoglycoprotein receptor which is highly expressed on membrane of hepatoma cells. GAL and siGPC-3 can induce targeted silencing of GPC-3 gene in hepatoma cells. In vivo and in vitro results showed that GAL-GNR-siGPC-3 could significantly induce downregulation of GPC-3 gene and inhibit the progression of HCC. More notably, GAL-GNR-siGPC-3 could induce both GPC-3 gene silencing and photothermal effects, and the synergistic treatment of tumors was more effective than individual treatments. In summary, GAL-GNR-siGPC-3 achieved a synergistic outcome to the treatment of cancer, which opens up a new approach for the development of clinical therapies for HCC.

TdIF1-LSD1 Axis Regulates Epithelial-Mesenchymal Transition and Metastasis via Histone Demethylation of E-Cadherin Promoter in Lung Cancer.

We have previously found that TdT-interacting factor 1 (TdIF1) is a potential oncogene expressed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. However, its exact mechanism is still unclear. The lysine-specific demethylase 1 (LSD1) is a crucial mediator of the epithelial-mesenchymal transition (EMT), an important process triggered during cancer metastasis. Here, we confirm that TdIF1 is highly expressed in NSCLC and related to lymph node metastasis through The Cancer Genome Atlas (TCGA) analysis of clinical samples. Silencing TdIF1 can regulate the expression of EMT-related factors and impair the migration and invasion ability of cancer cells in vitro. An analysis of tumor xenografts in nude mice confirmed that silencing TdIF1 inhibits tumor growth. Furthermore, we determined the interaction between TdIF1 and LSD1 using immunoprecipitation. Chromatin immunoprecipitation (ChIP) revealed that TdIF1 was enriched in the E-cadherin promoter region. The knockdown of TdIF1 repressed the enrichment of LSD1 at the E-cadherin promoter region, thereby regulating the level of promoter histone methylation and modulating E-cadherin transcription activity, ultimately leading to changes in EMT factors and cancer cell migration and invasion ability. The LSD1 inhibitor and TdIF1 knockdown combination showed a synergistic effect in inhibiting the growth, migration, and invasion of NSCLC cells. Taken together, this is the first demonstration that TdIF1 regulates E-cadherin transcription by recruiting LSD1 to the promoter region, thereby promoting EMT and tumor metastasis and highlighting the potential of TdIF1 as a therapeutic target for NSCLC.

Intranasal administration of regulatory dendritic cells is useful for the induction of nasal mucosal tolerance in a mice model of allergic rhinitis.

BACKGROUND: Intranasally administered dendritic cells (DCs) migrate into blood and thymus to induce immune responses. Regulatory dendritic cells (DCs) are also useful agents for allergy control. However, to the best of our knowledge, the effects of intranasal administration of regulatory DCs on allergy have not been reported until now. Therefore, we examined the effects of intranasal route of administration of CD40-silenced DCs on allergic responses and compared these with the effects of other administration routes, based on our previous findings on the inhibitory effects of CD40-silenced DCs on allergic responses. METHODS: Mice with allergic rhinitis were treated intranasally, subcutaneously, intraperitoneally, or intravenously with CD40-silenced ovalbumin (OVA)-pulsed DCs that were transfected with CD40 siRNAs and pulsed with OVA antigen. The effects of these DCs on allergic reactions and symptoms were estimated. RESULTS: Intranasal, subcutaneous, intraperitoneal, or intravenous administration of OVA-pulsed CD40-silenced DCs inhibited allergic responses and symptoms in mice. Furthermore, intranasal administration of OVA-pulsed CD40-silenced DCs significantly reduced allergic symptoms and the number of eosinophils in the nasal mucosa compared with subcutaneous, intraperitoneal, or intravenous administration of these DCs. Intranasal administration of OVA-pulsed CD40-silenced DCs resulted in significantly up-regulated IL-10, IL-35, and Foxp3 expression, and enhanced the percentage of CD11c+CD40- and CD4+CD25+ cells within the cervical lymph nodes compared to subcutaneous, intraperitoneal, or intravenous routes of administration. CONCLUSIONS: We believe that this is the first report to demonstrate that regulatory DCs infiltrate into the cervical lymph nodes after intranasal administration of these cells and that intranasal administration of regulatory DCs is more effective for the induction of tolerance in the nasal mucosa than subcutaneous, intraperitoneal, or intravenous administration.

Tumor-Targeted Gene Silencing IDO Synergizes PTT-Induced Apoptosis and Enhances Anti-tumor Immunity.

Background: Photothermal therapy (PTT) has been demonstrated to be a promising cancer treatment approach because it can be modulated to induce apoptosis instead of necrosis via adjusting irradiation conditions. Recently, an abscopal anti-tumor immunity has been highlighted, in which PTT on the primary tumor also induced repression of distant tumors. In PTT cancer treatments, the mechanism and the role of immune checkpoints to enhance anti-tumor immunity needs to be investigated. Methods: We prepared a multi-functional gold nanorod reagent, GMPF-siIDO, that is composed of gold nanorods (GNRs) that act as the nano-platform and photothermal sensitizer; folic acid (FA) as the tumor-targeting moiety; and IDO-specific RNA (siIDO) as an immune-stimulator functionality for inducing anti-tumor immunity. For this study, we adjusted the irradiation condition of PTT to induce apoptosis and to silence the immune checkpoint indoleamine 2,3 dioxygeonase (IDO), simultaneously. Results: Our studies provide evidence that photothermal effects kill tumor cells mainly via inducing apoptosis, which can significantly improve antitumor immunity when IDO was down-regulated in TME through significant increases of localized CD8+ and CD4+ lymphocytes in tumor tissue, the downregulation of CD8+ and CD4+ lymphocyte apoptosis, and the upregulation of antitumor cytokines, TNF-α and IFN-γ. Conclusion: In this study, we, for the first time, validated the role of IDO as a negative regulator for both PTT-induced tumor cell apoptosis and anti-tumor immunity; IDO is a critical immune checkpoint that impedes PTT while combination of gene knockdown of IDO in TME enhances anti-tumor efficacy of PTT.

Silencing IDO2 in dendritic cells: A novel strategy to strengthen cancer immunotherapy in a murine lung cancer model.

While dendritic cell (DC)­based immunotherapy has achieved satisfactory results in animal models, its effects were not satisfactory as initially expected in clinical applications, despite the safety and varying degrees of effectiveness in various types of cancer. Improving the efficacy of the DC­based vaccine is essential for cancer immunotherapy. The present study aimed to investigate methods with which to amplify and enhance the antitumor immune response of a DC­based tumor vaccine by silencing the expression of indoleamine 2,3­dioxygenase 2 (IDO2), a tryptophan rate­limiting metabolic enzyme in DCs. In vitro experiments revealed that the silencing of IDO2 in DCs did not affect the differentiation of DCs, whereas it increased their expression of costimulatory molecules following stimulation with tumor necrosis factor (TNF)­α and tumor lysate from Lewis lung cancer (LLC) cells. In a mixed co­culture system, the IDO2­silenced DCs promoted the proliferation of T­cells and reduced the induction of regulatory T­cells (Tregs). Further in vivo experiments revealed that the silencing of IDO2 in DCs markedly suppressed the growth of tumor cells. Moreover, treatment with the IDO2­silenced DC­based cancer vaccine enhanced cytotoxic T lymphocyte activity, whereas it decreased T­cell apoptosis and the percentage of CD4+CD25+Foxp3+ Tregs. On the whole, the present study provides evidence that the silencing of the tryptophan rate­limiting metabolic enzyme, IDO2, has the potential to enhance the efficacy of DC­based cancer immunotherapy.

Targeted Gene Silencing BRAF Synergized Photothermal Effect Inhibits Hepatoma Cell Growth Using New GAL-GNR-siBRAF Nanosystem.

Liver cancer is one of the most common malignancies worldwide. The RAF kinase inhibitors are effective in the treatment of hepatocellular carcinoma (HCC); therefore, inhibition of the BRAF/MEK/ERK pathway has become a new therapeutic strategy for novel HCC therapy. However, targeted specific delivery systems for tumors are still significant obstacle to clinical applications. Galactose (GAL) can target the asialoglycoprotein receptor (ASGPR) that is highly expressed on liver cancer cells. In this study, we designed a novel multifunctional nanomaterial GAL-GNR-siBRAF which consists of three parts, GAL as the liver cancer-targeting moiety, golden nanorods (GNR) offering photothermal capability under near infrared light, and siRNA specifically silencing BRAF (siBRAF). The nanocarrier GAL-GNR-siBRAF showed high siRNA loading capacity and inhibited the degradation of siRNA in serum. Compared with naked gold nanorods, GAL-GNR-siBRAF possessed lower biotoxicity and higher efficacy of gene silencing. Treatment with GAL-GNR-siBRAF significantly downregulated the expression of BRAF and impaired proliferation, migration, and invasion of liver cancer cells. Moreover, combinatorial photothermal effects and BRAF knockdown by GAL-GNR-siBRAF effectively given rise to tumor cell death. Therefore, our study developed a new type of targeted multi-functional nanomaterial GAL-GNR-siBRAF for the treatment of liver cancer, which provides ideas for the development of new clinical treatment methods.

Systemic Delivery of siRNA Specific for Silencing TLR4 Gene Expression Reduces Diabetic Cardiomyopathy in a Mouse Model of Streptozotocin-Induced Type 1 Diabetes.

INTRODUCTION: Diabetic cardiomyopathy is a cardiac dysfunction in patients with diabetes which may lead to overt heart failure and death. Toll-like receptor (TLR) signaling triggers diabetic cardiomyopathy through various mechanisms, one of which is the upregulation of TLR4 expression. The aim of this study was to delineate the role of TLR4 in diabetic cardiomyopathy. METHODS: C57BL/6 mice were injected with streptozotocin to induce diabetes. The experimental and control groups were treated with 5 µg of TLR4 small interfering RNA (siRNA) or scrambled siRNA. Cardiac histopathology was evaluated by hematoxylin and eosin, Sirius red, and immunofluorescence staining after treatment with TLR4 siRNA. The myocardial fibrosis and inflammatory factors were detected by quantitative real-time polymerase chain reaction after treatment with TLR4 siRNA. The myocardial function was evaluated by echocardiography after treatment with TLR4 siRNA. RESULTS: Compared with non-diabetic mouse hearts, hypertrophy, fibrosis, inflammation of cardiomyocytes, and myocardial dysfunction were significantly increased in diabetic mice (p < 0.05). Knockdown of TLR4 decreased hypertrophy, fibrosis, inflammation of cardiomyocytes, and myocardial dysfunction (p < 0.05). Cardiomyocytic cross-sectional areas in hearts of TLR4 siRNA-treated diabetic mice were similar to those of the sham-treated mice (p > 0.05). The induction of expression of cardiac fetal genes, beta-myosin heavy chain (ß-MHC) and atrial natriuretic peptide (ANP), which are two markers of cardiac hypertrophy, was significantly reduced in TLR4 siRNA-treated hearts compared with controls (p < 0.05). Moreover, siRNA-mediated silencing of TLR4 reduced diabetes-induced collagen deposition (p < 0.05). Paralleled with changes in collagen deposition and the expression of collagen I and collagen III, knockdown of TLR4 also reduced the expression of transforming growth factor-ß1 (TGFß1) mRNA (p < 0.05). The increased expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) was significantly attenuated by TLR4 siRNA treatment in the hearts of diabetic mice (p < 0.05). Furthermore, both fractional shortening (FS) and ejection fraction (EF) values were preserved in TLR4 siRNA-treated diabetic mice compared with control siRNA-treated mice (31.80% ± 2.82% vs. 28.50% ± 5.83% for FS, p < 0.05) (57.95% ± 6.48% vs. 45.34% ± 4.25% for EF, p < 0.05). CONCLUSION: Our study used siRNA to specifically silence TLR4 gene expression in the diabetic mouse heart in vivo and to investigate the role that TLR4 plays in diabetic cardiomyopathy. It is likely that silencing of the TLR4 gene through siRNA could prevent the development of diabetic cardiomyopathy.

miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy.

Dendritic cell (DC) based immunotherapy is a promising approach to clinical cancer treatment. miRNAs are a class of small non-coding RNA molecules that bind to RNAs to mediate multiple events which are important in diverse biological processes. miRNA mimics and antagomirs may be potent agents to enhance DC-based immunotherapy against cancers. miRNA array analysis was used to identify a representative miR-5119 potentially regulating PD-L1 in DCs. We evaluated levels of ligands of immune cell inhibitory receptors (IRs) and miR-5119 in DCs from immunocompetent mouse breast tumor-bearing mice, and examined the molecular targets of miR-5119. We report that miRNA-5119 was downregulated in spleen DCs from mouse breast cancer-bearing mice. In silico analysis and qPCR data showed that miRNA-5119 targeted mRNAs encoding multiple negative immune regulatory molecules, including ligands of IRs such as PD-L1 and IDO2. DCs engineered to express a miR-5119 mimic downregulated PD-L1 and prevented T cell exhaustion in mice with breast cancer homografts. Moreover, miR-5119 mimic-engineered DCs effectively restored function to exhausted CD8+ T cells in vitro and in vivo, resulting in robust anti-tumor cell immune response, upregulated cytokine production, reduced T cell apoptosis, and exhaustion. Treatment of 4T1 breast tumor-bearing mice with miR-5119 mimic-engineered DC vaccine reduced T cell exhaustion and suppressed mouse breast tumor homograft growth. This study provides evidence supporting a novel therapeutic approach using miRNA-5119 mimic-engineered DC vaccines to regulate inhibitory receptors and enhance anti-tumor immune response in a mouse model of breast cancer. miRNA/DC-based immunotherapy has potential for advancement to the clinic as a new strategy for DC-based anti-breast cancer immunotherapy.

CD5 blockade enhances ex vivo CD8+ T cell activation and tumour cell cytotoxicity.

CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.

Reduced CD5 on CD8+ T Cells in Tumors but Not Lymphoid Organs Is Associated With Increased Activation and Effector Function.

CD5, a member of the scavenger receptor cysteine-rich superfamily, is a marker for T cells and a subset of B cells (B1a). CD5 associates with T-cell and B-cell receptors and increased CD5 is an indication of B cell activation. In tumor-infiltrating lymphocytes (TILs) isolated from lung cancer patients, CD5 levels were negatively correlated with anti-tumor activity and tumor-mediated activation-induced T cell death, suggesting that CD5 could impair activation of anti-tumor T cells. We determined CD5 levels in T cell subsets in different organs in mice bearing syngeneic 4T1 breast tumor homografts and assessed the relationship between CD5 and increased T cell activation and effector function by flow cytometry. We report that T cell CD5 levels were higher in CD4+ T cells than in CD8+ T cells in 4T1 tumor-bearing mice, and that high CD5 levels on CD4+ T cells were maintained in peripheral organs (spleen and lymph nodes). However, both CD4+ and CD8+ T cells recruited to tumors had reduced CD5 compared to CD4+ and CD8+ T cells in peripheral organs. In addition, CD5high/CD4+ T cells and CD5high/CD8+ T cells from peripheral organs exhibited higher levels of activation and associated effector function compared to CD5low/CD4+ T cell and CD5low/CD8+ T cell from the same organs. Interestingly, CD8+ T cells among TILs and downregulated CD5 were activated to a higher level, with concomitantly increased effector function markers, than CD8+/CD5high TILs. Thus, differential CD5 levels among T cells in tumors and lymphoid organs can be associated with different levels of T cell activation and effector function, suggesting that CD5 may be a therapeutic target for immunotherapeutic activation in cancer therapy.

DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA.

Deleted-in-liver cancer 1 (DLC1) exerts its tumor suppressive function mainly through the Rho-GTPase-activating protein (RhoGAP) domain. When activated, the domain promotes the hydrolysis of RhoA-GTP, leading to reduced cell migration. DLC1 is kept in an inactive state by an intramolecular interaction between its RhoGAP domain and the DLC1 sterile α motif (SAM) domain. We have shown previously that this autoinhibited state of DLC1 may be alleviated by tensin-3 (TNS3) or PTEN. We show here that the TNS3/PTEN-DLC1 interactions are mediated by the C2 domains of the former and the SAM domain of the latter. Intriguingly, the DLC1 SAM domain was capable of binding to specific peptide motifs within the C2 domains. Indeed, peptides containing the binding motifs were highly effective in blocking the C2-SAM domain-domain interaction. Importantly, when fused to the tat protein-transduction sequence and subsequently introduced into cells, the C2 peptides potently promoted the RhoGAP function in DLC1, leading to decreased RhoA activation and reduced tumor cell growth in soft agar and migration in response to growth factor stimulation. To facilitate the development of the C2 peptides as potential therapeutic agents, we created a cyclic version of the TNS3 C2 domain-derived peptide and showed that this peptide readily entered the MDA-MB-231 breast cancer cells and effectively inhibited their migration. Our work shows, for the first time, that the SAM domain is a peptide-binding module and establishes the framework on which to explore DLC1 SAM domain-binding peptides as potential therapeutic agents for cancer treatment.

IDO inhibitor synergized with radiotherapy to delay tumor growth by reversing T cell exhaustion.

Previous studies suggest that radiotherapy (RT) can induce immune activation, which not only reduces the progression of tumors, but also causes the release of tumor antigens. The combination of RT and immune checkpoint blockade, such as the inhibition of programmed cell death 1 (PD­1) and programmed cell death ligand 1 (PD­L1), has been demonstrated to yield impressive response rates. However, a substantial proportion of patients develop resistance such therapies. Previous studies have shown that indoleamine 2,3­dioxygenase (IDO) causes T cell exhaustion and increased formation of regulatory T cells (Tregs), upregulating the expression of inhibitory receptors and ligands. Therefore, the application of IDO inhibitors combined with RT may have a synergistic effect by relieving immunosuppression. Lewis lung cancer cell­bearing mice were treated with the IDO inhibitor 1­methyl­tryptophan (1MT) and/or 10 Gy RT. Tumor size was measured every day, flow cytometry was performed to measure the expression of dendritic cell (DC) maturation markers, inhibitory receptors, ligands, cytotoxic T cells and Treg phenotypic markers. Reverse transcription­quantitative PCR was used to evaluate the mRNA expression levels of IDO, PD­L1, PD­1, T cell immunoglobulin domain and mucin domain 3 (TIM­3), B­ and T­lymphocyte attenuator (BTLA) and galectin­9. Compared with the control group, treatment with 1MT or RT reduced tumor growth, however, the combination therapy was more effective than either treatment alone. Flow cytometry showed the upregulation of CD80, CD86 and the major histocompatibility complex II in spleen DCs and the concurrent downregulation of CD4, CD25 and forkhead box protein P3 in lymphocytes in the treatment groups. Compared with the control group, inhibitory receptors and ligands that affect DCs and T cells were observed, expression levels of PD­L1, PD­1, TIM­3, BTLA and galectin­9 are decreased in treatment group compared with control. IDO inhibition synergized with RT to downregulate Tregs, inhibitory receptors and ligands to prevent T cell exhaustion. By activating DCs and T cells, this combination therapy may strongly enhance antitumor immunity and inhibit tumor progression.

miR-149-3p reverses CD8+ T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells.

Blockade of inhibitory receptors (IRs) is one of the most effective immunotherapeutic approaches to treat cancer. Dysfunction of miRNAs is a major cause of aberrant expression of IRs and contributes to the immune escape of cancer cells. How miRNAs regulate immune checkpoint proteins in breast cancer remains largely unknown. In this study, downregulation of miRNAs was observed in PD-1-overexpressing CD8+ T cells using miRNA array analysis of mouse breast cancer homografts. The data reveal that miR-149-3p was predicted to bind the 3'UTRs of mRNAs encoding T-cell inhibitor receptors PD-1, TIM-3, BTLA and Foxp1. Treatment of CD8+ T cells with an miR-149-3p mimic reduced apoptosis, attenuated changes in mRNA markers of T-cell exhaustion and downregulated mRNAs encoding PD-1, TIM-3, BTLA and Foxp1. On the other hand, T-cell proliferation and secretion of effector cytokines indicative of increased T-cell activation (IL-2, TNF-α, IFN-γ) were upregulated after miR-149-3p mimic treatment. Moreover, the treatment with a miR-149-3p mimic promoted the capacity of CD8+ T cells to kill targeted 4T1 mouse breast tumour cells. Collectively, these data show that miR-149-3p can reverse CD8+ T-cell exhaustion and reveal it to be a potential antitumour immunotherapeutic agent in breast cancer.

CD38 Deficiency Downregulates the Onset and Pathogenesis of Collagen-Induced Arthritis through the NF-κB Pathway.

AIM: The RelB gene plays an important role in guiding the progression of arthritis. We have previously demonstrated that the expression of the RelB gene is decreased significantly in bone marrow DCs of CD38-/- mice. In this study, we demonstrate that the cluster of the differentiation (CD38) gene could be a potentially therapeutic target for autoimmune arthritis. METHOD: Collagen-induced arthritis (CIA) models were generated with both the wild-type (WT) C57BL/6 and CD38-/- mice. The expression of the RelB gene and maturation of bone marrow-derived dendritic cells (DCs) from the WT and CD38-/- mice were detected. Antigen-specific T cell responses, joint damage, and expression of proinflammatory cytokines were assessed. The effects of the Nuclear Factor Kappa B (NF-κB) transcription factor and its mechanisms were characterized. RESULTS: We demonstrated that in CD38-/- mice, the expression of the RelB gene and major histocompatibility complex II (MHC II) was decreased, accompanied with the inhibited T cell reaction in a mixed lymphocyte reaction (MLR) in bone marrow-derived DCs. Compared to the serious degeneration of the cartilage and the enlarged gap of the cavum articular in WT CIA mice, joint pathological changes of the CD38-/- CIA mice revealed marked attenuation, while the joint structures were well preserved. The preserved effects were observed by the inhibition of proinflammatory cytokines and promotion of anti-inflammatory cytokines. Furthermore, decreased phosphorylation of NF-κB was also observed in CD38-/- CIA mice. CONCLUSION: We demonstrate that CD38 could regulate CIA through NF-κB and this regulatory molecule could be a novel target for the treatment of autoimmune inflammatory joint disease.