Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection.

Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: 'intrahepatic myeloid-cell aggregates for T cell population expansion') without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.

CLEC5A mediates Zika virus-induced testicular damage.

BACKGROUND: Zika virus (ZIKV) infection is clinically known to induce testicular swelling, termed orchitis, and potentially impact male sterility, but the underlying mechanisms remain unclear. Previous reports suggested that C-type lectins play important roles in mediating virus-induced inflammatory reactions and pathogenesis. We thus investigated whether C-type lectins modulate ZIKV-induced testicular damage. METHODS: C-type lectin domain family 5 member A (CLEC5A) knockout mice were generated in a STAT1-deficient immunocompromised background (denoted clec5a-/-stat1-/-) to enable testing of the role played by CLEC5A after ZIKV infection in a mosquito-to-mouse disease model. Following ZIKV infection, mice were subjected to an array of analyses to evaluate testicular damage, including ZIKV infectivity and neutrophil infiltration estimation via quantitative RT-PCR or histology and immunohistochemistry, inflammatory cytokine and testosterone detection, and spermatozoon counting. Furthermore, DNAX-activating proteins for 12 kDa (DAP12) knockout mice (dap12-/-stat1-/-) were generated and used to evaluate ZIKV infectivity, inflammation, and spermatozoa function in order to investigate the potential mechanisms engaged by CLEC5A. RESULTS: Compared to experiments conducted in ZIKV-infected stat1-/- mice, infected clec5a-/-stat1-/- mice showed reductions in testicular ZIKV titer, local inflammation and apoptosis in testis and epididymis, neutrophil invasion, and sperm count and motility. CLEC5A, a myeloid pattern recognition receptor, therefore appears involved in the pathogenesis of ZIKV-induced orchitis and oligospermia. Furthermore, DAP12 expression was found to be decreased in the testis and epididymis tissues of clec5a-/-stat1-/- mice. As for CLEC5A deficient mice, ZIKV-infected DAP12-deficient mice also showed reductions in testicular ZIKV titer and local inflammation, as well as improved spermatozoa function, as compared to controls. CLEC5A-associated DAP12 signaling appears to in part regulate ZIKV-induced testicular damage. CONCLUSIONS: Our analyses reveal a critical role for CLEC5A in ZIKV-induced proinflammatory responses, as CLEC5A enables leukocytes to infiltrate past the blood-testis barrier and induce testicular and epididymal tissue damage. CLEC5A is thus a potential therapeutic target for the prevention of injuries to male reproductive organs in ZIKV patients.

DUSP6 mediates T cell receptor-engaged glycolysis and restrains TFH cell differentiation.

Activated T cells undergo metabolic reprogramming and effector-cell differentiation but the factors involved are unclear. Utilizing mice lacking DUSP6 (DUSP6-/-), we show that this phosphatase regulates T cell receptor (TCR) signaling to influence follicular helper T (TFH) cell differentiation and T cell metabolism. In vitro, DUSP6-/- CD4+ TFH cells produced elevated IL-21. In vivo, TFH cells were increased in DUSP6-/- mice and in transgenic OTII-DUSP6-/- mice at steady state. After immunization, DUSP6-/- and OTII-DUSP6-/- mice generated more TFH cells and produced more antigen-specific IgG2 than controls. Activated DUSP6-/- T cells showed enhanced JNK and p38 phosphorylation but impaired glycolysis. JNK or p38 inhibitors significantly reduced IL-21 production but did not restore glycolysis. TCR-stimulated DUSP6-/- T cells could not induce phosphofructokinase activity and relied on glucose-independent fueling of mitochondrial respiration. Upon CD28 costimulation, activated DUSP6-/- T cells did not undergo the metabolic commitment to glycolysis pathway to maintain viability. Unexpectedly, inhibition of fatty acid oxidation drastically lowered IL-21 production in DUSP6-/- TFH cells. Our findings suggest that DUSP6 connects TCR signaling to activation-induced metabolic commitment toward glycolysis and restrains TFH cell differentiation via inhibiting IL-21 production.

Immunopathogenesis of Hepatitis B Virus.

Chronic hepatitis B virus (HBV) infection is a global public health issue. There are >250 million people chronically infected with HBV, and these chronic carriers are at high risk of developing end-stage liver diseases and hepatocellular carcinoma. Patients with chronic hepatitis B (CHB) usually acquire the virus perinatally, while most patients infected during adulthood develop acute hepatitis B (AHB), which usually results in viral clearance. HBV infection is noncytopathic, and liver injury is mostly contributed by host immune responses. The virus is stealthy, since the infection rarely induces type I interferon response in the early phase. In AHB, viral infection is detected and restrained by the innate immune response, which is followed by a strong and robust adaptive immune response and accompanied by viral clearance. In patients with CHB, both innate and adaptive immune responses are weak and thus rarely lead to viral clearance. Interferon α and nucleos(t)ide analogues are 2 classes of approved antiviral therapies. The former treatment activates nature killer (NK) cells and NK T cells, which partially enhances the innate immune response, while the later treatment suppresses viral replication by inhibiting reverse transcriptase, which may restore the HBV-specific adaptive immune response. However, single or combined treatment are still far from achieving seroclearance of HBV surface antigen. Although the treatment response is unsatisfactory in current clinical trials using several immunomodulators for boosting antiviral immunity, immunotherapy that is able to induce immune surveillance is still the most promising modality for HBV cure in the future.

Identification of Thiostrepton as a Novel Inhibitor for Psoriasis-like Inflammation Induced by TLR7-9.

Activation of TLR7-9 has been linked to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. Thus, therapeutic applications of antagonists of these TLRs for such disorders are being investigated. Bortezomib (Velcade) is a proteasome inhibitor known to suppress activation of these TLRs. To identify novel TLR7-9 inhibitors, we searched the Gene Expression Omnibus database for gene expression profiles of bortezomib-treated cells. These profiles were then used to screen the Connectivity Map database for chemical compounds with similar functions as bortezomib. A natural antibiotic, thiostrepton, was identified for study. Similar to bortezomib, thiostrepton effectively inhibits TLR7-9 activation in cell-based assays and in dendritic cells. In contrast to bortezomib, thiostrepton does not inhibit NF-κB activation induced by TNF-α, IL-1, and other TLRs, and it is less cytotoxic to dendritic cells. Thiostrepton inhibits TLR9 localization in endosomes for activation via two mechanisms, which distinguish it from currently used TLR7-9 inhibitors. One mechanism is similar to the proteasome inhibitory function of bortezomib, whereas the other is through inhibition of endosomal acidification. Accordingly, in different animal models, thiostrepton attenuated LL37- and imiquimod-induced psoriasis-like inflammation. These results indicated that thiostrepton is a novel TLR7-9 inhibitor, and compared with bortezomib, its inhibitory effect is more specific to these TLRs, suggesting the potential therapeutic applications of thiostrepton on immunologic disorders elicited by inappropriate activation of TLR7-9.

The role of hepatic immune regulation in systemic immunity to viral infection.

The liver has particular immune functions attributed by its unique microenvironment and its liver-resident cell populations. During autoimmunity and viral hepatitis, the liver serves as target for effector responses of immune cells. However, skewing of effector T cell functions through tolerogenic liver-resident antigen-presenting cells and through the immune regulatory hepatic microenvironment. Importantly, the liver also participates in shaping systemic antigen-specific immunity. Local antigen-presenting cell populations, in particular liver sinusoidal endothelial cells (LSECs), cross-present soluble, circulating or hepatocyte-derived antigens to naïve CD8 T cells. Upon priming by cross-presenting LSECs, naïve CD8 T cells develop into a unique population of antigen-experienced memory-like T cell population that can be reactivated in an inflammatory context to protect against infection with viruses or bacteria. Furthermore, upon prolonged inflammatory TNF-dependent signaling, the induction of intrahepatic myeloid cell aggregates for T cell population expansion (iMATEs) is observed in liver tissue. iMATEs are formed by inflammatory monocytes developing into dendritic cells and function to attract recently activated CD8 T cells. Those CD8 T cells located within the cocoon-like iMATE structure show strong proliferation initiated by co-stimulatory signaling. Locally expanded CD8 T cells are key to control acute and chronic viral infections. The mechanistic understanding of local hepatic T cell priming and local expansion of effector CD8 T cells will help to develop novel therapeutic vaccination strategies.

T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice.

BACKGROUND & AIMS: Antiviral agents suppress hepatitis B virus (HBV) replication but do not clear the infection. A strong effector T-cell response is required to eradicate HBV, but this does not occur in patients with chronic infection. T cells might be directed toward virus-infected cells by expressing HBV-specific receptors and thereby clear HBV and help to prevent development of liver cancer. In mice, we studied whether redirected T cells can engraft after adoptive transfer, without prior T-cell depletion, and whether the large amounts of circulating viral antigens inactivate the transferred T cells or lead to uncontrolled immune-mediated damage. METHODS: CD8(+) T cells were isolated from mice and stimulated using an optimized protocol. Chimeric antigen receptors (CARs) that bind HBV envelope proteins (S-CAR) and activate T cells were expressed on the surface of cells using retroviral vectors. S-CAR-expressing CD8(+) T cells, which carried the marker CD45.1, were injected into CD45.2(+) HBV transgenic mice. We compared these mice with mice that received CD8(+) T cells induced by vaccination, cells that express a CAR without a proper signaling domain, or cells that express a CAR that does not bind HBV proteins (controls). RESULTS: CD8(+) T cells that expressed HBV-specific CARs recognized different HBV subtypes and were able to engraft and expand in immune-competent HBV transgenic mice. After adoptive transfer, the S-CAR-expressing T cells localized to and functioned in the liver and rapidly and efficiently controlled HBV replication compared with controls, causing only transient liver damage. The large amount of circulating viral antigen did not impair or overactivate the S-CAR-grafted T cells. CONCLUSIONS: T cells with a CAR specific for HBV envelope proteins localize to the liver in mice to reduce HBV replication, causing only transient liver damage. This immune cell therapy might be developed for patients with chronic hepatitis B, regardless of their HLA type.

Transfer of HBV genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice.

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model.

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20-30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-gene-deficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176-185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-gamma response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence.

Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

Mechanical transmission of dengue virus by Aedes aegypti may influence disease transmission dynamics during outbreaks.

BACKGROUND: Dengue virus outbreaks are increasing in number and severity worldwide. Viral transmission is assumed to require a minimum time period of viral replication within the mosquito midgut. It is unknown if alternative transmission periods not requiring replication are possible. METHODS: We used a mouse model of dengue virus transmission to investigate the potential of mechanical transmission of dengue virus. We investigated minimal viral titres necessary for development of symptoms in bitten mice and used resulting parameters to inform a new model of dengue virus transmission within a susceptible population. FINDINGS: Naïve mice bitten by mosquitoes immediately after they took partial blood meals from dengue infected mice showed symptoms of dengue virus, followed by mortality. Incorporation of mechanical transmission into mathematical models of dengue virus transmission suggest that this supplemental transmission route could result in larger outbreaks which peak sooner. INTERPRETATION: The potential of dengue transmission routes independent of midgut viral replication has implications for vector control strategies that target mosquito lifespan and suggest the possibility of similar mechanical transmission routes in other disease-carrying mosquitoes. FUNDING: This study was funded by grants from the National Health Research Institutes, Taiwan (04D2-MMMOST02), the Human Frontier Science Program (RGP0033/2021), the National Institutes of Health (1R01AI143698-01A1, R01AI151004 and DP2AI152071) and the Ministry of Science and Technology, Taiwan (MOST104-2321-B-400-016).

Rejuvenating hepatic tumor microenvironment immunity with a phosphatidylserine-targeting small molecule drug conjugate.

We report the design, synthesis and evaluation of a class of phosphatidylserine-targeting zinc (II) dipicolylamine drug conjugates and show that conjugate 16b elicits immune cell infiltration and remodels the "cold" hepatic tumor microenvironment to the inflamed "hot" tumor. Structure-property relationship study via linker modifications and subsequent pharmacokinetics profiling were carried out to improve the solubility and stability of the conjugates in vivo. In a spontaneous hepatocellular carcinoma mouse model, we showed that conjugate 16b exhibited better antitumor efficacy than sorafenib. In particular, significant increase of CD8+ T cell infiltration and granzyme B level was observed, providing insights in sensitizing tumors from intrinsic immune suppressive microenvironment. Evaluation of tumor inflammation-related mRNA expression profile revealed that conjugate 16b, through inductions of key gene expressions including STAT1, CXCL9, CCL5, and PD-L1, rejuvenated tumor microenvironment with enhancement in T cell-, macrophage-, NK cell-, chemokines and cytokines'- functions. Our study establishes that an apoptosis-targeting theranostic enables enrichment of multifaceted immune cells into the tumor mass, which provides potential therapeutic strategies in the combination with immune checkpoint blockade treatment.

Novel Paired Cell Lines for the Study of Lipid Metabolism and Cancer Stemness of Hepatocellular Carcinoma.

There are few well-characterized syngeneic murine models for hepatocellular carcinoma (HCC), which limits immunological studies and the development of immunotherapies for HCC. We previously established an oncogene-induced spontaneous HCC mouse model based on transposon-mediated oncogene (AKT and NRASV12) insertion into the genome of hepatocytes to induce tumorigenesis. Two tumor clones with different levels of lipid droplets (LDs) showed similar in vitro growth but distinctive in vivo phenotypes, including divergent proliferative capability and varying induction of myeloid-derived suppressor cells (MDSCs). The two clones showed distinct gene expression related to lipid metabolism, glycolysis, and cancer stemness. Endogenous fatty acid (FA) synthesis and exogenous monounsaturated fatty acid (MUFA) consumption promoted both tumor proliferation and cancer stemness, and upregulated c-Myc in the HCC cell lines. Moreover, the LDhi HCC cell line expressed a higher level of type II IL-4 receptor, which promoted tumor proliferation through binding IL-4 or IL-13. The chromosomal DNA of two tumor clones, NHRI-8-B4 (LDhi) and NHRI-1-E4 (LDlo) showed five identical AKT insertion sites in chromosomes 9, 10, 13, 16 and 18 and two NRAS integration sites in chromosomes 2 and 3. Herein, we describe two novel HCC cell lines with distinct features of lipid metabolism related to cancer stemness and differential interplay with the immune system, and present this syngeneic HCC mouse model as a practical tool for the study of cancer stemness and discovery of new therapies targeting liver cancers.

EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities.

The formation of new blood vessels in solid tumors is regulated by various endothelial trophic factors. We identified that CLEC11A, an extracellular C-type lectin, was over-expressed in lung cancer cell lines harboring mutated EGFR. CLEC11A expression was also frequently elevated in lung adenocarcinoma (LAC) tissues with EGFR mutation. CLEC11A-expressing H1299 cells formed larger tumors in nude mice than did the control cells. The CLEC11A-expressing tumors contained more CD31-positive cells, suggesting that they had a higher angiogenic activity. CLEC11A per se did not induce blood vessel formation, but enhanced angiogenesis triggered by VEGF-A or basic FGF in vivo. Additionally, the expression of small hairpin RNA against CLEC11A (shCLEC11A) in HCC827 LAC cells suppressed their tumorigenic ability. Purified CLEC11A exhibited a chemotactic ability, which is dependent on its integrin-binding RGD and LDT motifs, toward endothelial cells. This chemotactic activity was not affected by the presence of a VEGFR inhibitor. Conditioned medium produced by HCC827-shCLEC11A cells had diminished chemotactic ability toward endothelial cells. CLEC11A treatments increased the levels of active integrin ß1 that were not associated with activation of focal adhesion kinases in endothelial cells. Our results indicated that CLEC11A was a factor of angiogenic potential and was involved in lung cancer tumorigenesis.

Synthesis and Evaluation of Small Molecule Drug Conjugates Harnessing Thioester-Linked Maytansinoids.

Ligand-targeting drug conjugates are a class of clinically validated biopharmaceutical drugs constructed by conjugating cytotoxic drugs with specific disease antigen targeting ligands through appropriate linkers. The integrated linker-drug motif embedded within such a system can prevent the premature release during systemic circulation, thereby allowing the targeting ligand to engage with the disease antigen and selective accumulation. We have designed and synthesized new thioester-linked maytansinoid conjugates. By performing in vitro cytotoxicity, targeting ligand binding assay, and in vivo pharmacokinetic studies, we investigated the utility of this new linker-drug moiety in the small molecule drug conjugate (SMDC) system. In particular, we conjugated the thioester-linked maytansinoids to the phosphatidylserine-targeting small molecule zinc dipicolylamine and showed that Zn8_DM1 induced tumor regression in the HCC1806 triple-negative breast cancer xenograft model. Moreover, in a spontaneous sorafenib-resistant liver cancer model, Zn8_DM1 exhibited potent antitumor growth efficacy. From quantitative mRNA analysis of Zn8_DM1 treated-tumor tissues, we observed the elevation of gene expressions associated with a "hot inflamed tumor" state. With the identification and validation of a plethora of cancer-associated antigens in the "omics" era, this work provided the insight that antibody- or small molecule-based targeting ligands can be conjugated similarly to generate new ligand-targeting drug conjugates.

Targeting the Phosphatidylserine-Immune Checkpoint with a Small-Molecule Maytansinoid Conjugate.

Ligand-targeting drug delivery systems have made significant strides for disease treatments with numerous clinical approvals in this era of precision medicine. Herein, we report a class of small molecule-based immune checkpoint-targeting maytansinoid conjugates. From the ligand targeting ability, pharmacokinetics profiling, in vivo anti-pancreatic cancer, triple-negative breast cancer, and sorafenib-resistant liver cancer efficacies with quantitative mRNA analysis of treated-tumor tissues, we demonstrated that conjugate 40a not only induced lasting regression of tumor growth, but it also rejuvenated the once immunosuppressive tumor microenvironment to an "inflamed hot tumor" with significant elevation of gene expressions that were not accessible in the vehicle-treated tumor. In turn, the immune checkpoint-targeting small molecule drug conjugate from this work represents a new pharmacodelivery strategy that can be expanded with combination therapy with existing immune-oncology treatment options.

Serum soluble programmed cell death 1 levels predict spontaneous functional cure in inactive carriers with chronic hepatitis B.

BACKGROUND: Hepatitis B surface antigen (HBsAg) seroclearance is the most important milestone indicating favourable clinical outcomes in patients with chronic hepatitis B (CHB). However, it is difficult to achieve due to the impaired HBV-specific immunity, such as programmed cell death 1 (PD-1)-associated T cell exhaustion. We assessed soluble PD-1 (sPD-1) as a novel seromarker for predicting spontaneous HBsAg loss. METHODS: Serial serum levels of sPD-1 were evaluated in 1046 untreated hepatitis B e antigen (HBeAg)-seronegative individuals who had achieved undetectable serum HBV DNA. Multiple regression analyses were applied to assess associations among baseline and subsequent sPD-1 levels, HBsAg decline during follow-up, and spontaneous HBsAg seroclearance. RESULTS: A total of 390 individuals achieved spontaneous HBsAg seroclearance during 6464.4 person-years of follow-up. Baseline sPD-1 levels were inversely associated with baseline HBsAg levels (qHBsAg) as well as a greater decline in qHBsAg during follow-up. Incidence rates of HBsAg seroclearance were 11.5, 61.7, 96.7 and 151.0 per 1000 person-years for sPD-1 levels of ≥4000, 536-3999, 125-535 and <125 pg/mL, respectively (Ptrend  < 0.0001). Compared with baseline sPD-1 levels ≥4000 pg/mL, the rate ratio (95% CI) of HBsAg seroclearance was 2.1 (1.1-3.9), 3.0 (1.6-5.5) and 5.1 (2.8-9.5), for baseline sPD-1 levels of 536-3999, 125-535 and <125 pg/mL, respectively, after adjustment for sex, age and serum alanine aminotransferase and HBsAg levels. CONCLUSION: sPD-1 level is a novel marker which independently predicts spontaneous HBsAg seroclearance of HBeAg-negative inactive CHB patients with undetectable HBV DNA. (word count: 234, <250).

Improving Therapeutic Vaccination against Hepatitis B-Insights from Preclinical Models of Immune Therapy against Persistent Hepatitis B Virus Infection.

Chronic hepatitis B affects more than 250 million individuals worldwide, putting them at risk of developing liver cirrhosis and liver cancer. While antiviral immune responses are key to eliminating hepatitis B virus (HBV) infections, insufficient antiviral immunity characterized by failure to eliminate HBV-infected hepatocytes is associated with chronic hepatitis B. Prophylactic vaccination against hepatitis B successfully established protective immunity against infection with the hepatitis B virus and has been instrumental in controlling hepatitis B. However, prophylactic vaccination schemes have not been successful in mounting protective immunity to eliminate HBV infections in patients with chronic hepatitis B. Here, we discuss the current knowledge on the development and efficacy of therapeutic vaccination strategies against chronic hepatitis B with particular emphasis on the pathogenetic understanding of dysfunctional anti-viral immunity. We explore the development of additional immune stimulation measures within tissues, in particular activation of immunogenic myeloid cell populations, and their use for combination with therapeutic vaccination strategies to improve the efficacy of therapeutic vaccination against chronic hepatitis B.

Adjuvant Effect of Toll-Like Receptor 9 Activation on Cancer Immunotherapy Using Checkpoint Blockade.

Immunotherapy using checkpoint blockade has revolutionized cancer treatment, improving patient survival and quality of life. Nevertheless, the clinical outcomes of such immunotherapy are highly heterogeneous between patients. Depending on the cancer type, the patient response rates to this immunotherapy are limited to 20-30%. Based on the mechanism underlying the antitumor immune response, new therapeutic strategies have been designed with the aim of increasing the effectiveness and specificity of the antitumor immune response elicited by checkpoint blockade agents. The activation of toll-like receptor 9 (TLR9) by its synthetic agonists induces the antitumor response within the innate immunity arm, generating adjuvant effects and priming the adaptive immune response elicited by checkpoint blockade during the effector phase of tumor-cell killing. This review first describes the underlying mechanisms of action and current status of monotherapy using TLR9 agonists and immune checkpoint inhibitors for cancer immunotherapy. The rationale for combining these two agents is discussed, and evidence indicating the current status of such combination therapy as a novel cancer treatment strategy is presented.

Epigenetic Silencing of Ubiquitin Specific Protease 4 by Snail1 Contributes to Macrophage-Dependent Inflammation and Therapeutic Resistance in Lung Cancer.

There is a positive feedback loop driving tumorigenesis and tumor growth through coordinated regulation of epigenetics, inflammation, and stemness. Nevertheless, the molecular mechanism linking these processes is not well understood. In this study, we analyzed the correlation of de-ubiquitinases (DUBs) expression with survival data from the OncoLnc database. Among the DUBs analyzed, ubiquitin specific protease 4 (USP4) had the lowest negative Cox coefficient. Low expression of USP4 was associated with poor survival among lung cancer patients and was inversely correlated with expression of stemness and inflammation markers. Expression of USP4 were reduced at more advanced stages of lung cancer. Mechanistically, expression of USP4 was downregulated in snail1-overexpressing and stemness-enriched lung cancer cells. Snail1 was induced in lung cancer cells by interaction with macrophages, and epigenetically suppressed USP4 expression by promoter methylation. Stable knockdown of USP4 in lung cancer cells enhanced inflammatory responses, stemness properties, chemotherapy resistance, and the expression of molecules allowing escape from immunosurveillance. Further, mice injected with USP4 knockdown lung cancer cells demonstrated enhanced tumorigenesis and tumor growth. These results reveal that the Snail1-mediated suppression of USP4 is a potential mechanism to orchestrate epigenetic regulation, inflammation and stemness for macrophage-promoted tumor progression.