Improvements in the quality and fitness of chimeric antigen receptor (CAR)-engineered T cells, through CAR design or manufacturing optimizations, could enhance the therapeutic potential of CAR-T cells. One parameter influencing the effectiveness of CAR-T cell therapy is the differentiation status of the final product: CAR-T cells that are less differentiated and less exhausted are more therapeutically effective. In the current study, we demonstrate that CAR-T cells expanded in IL15 (CAR-T/IL15) preserve a less-differentiated stem cell memory (Tscm) phenotype, defined by expression of CD62L + CD45RA + CCR7 + , as compared to cells cultured in IL2 (CAR-T/IL2). CAR-T/IL15 cells exhibited reduced expression of exhaustion markers, higher anti-apoptotic properties, and increased proliferative capacity upon antigen challenge. Furthermore, CAR-T/IL15 cells exhibited decreased mTORC1 activity, reduced expression of glycolytic enzymes and improved mitochondrial fitness. CAR-T/IL2 cells cultured in rapamycin (mTORC1 inhibitor) shared phenotypic features with CAR-T/IL15 cells, suggesting that IL15mediated reduction of mTORC1 activity is responsible for preserving the Tscm phenotype. CAR-T/IL15 cells promoted superior antitumor responses in vivo in comparison to CAR-T/IL2 cells.Inclusion of cytokines IL7 and/or IL21 in addition to IL15 reduced the beneficial effects of IL15 on CAR-T phenotype and antitumor potency. Our findings show that IL15 preserves the CAR-T cell Tscm phenotype and improves their metabolic fitness, which results in superior in vivo antitumor activity, thus opening an avenue that may improve future adoptive T cell therapies.

Background: Hepatitis C virus (HCV) induces autophagosomes in its host cells. Results: The HCV RNA replication complex colocalizes with autophagosomes, which are induced by HCV via a Class III PI3K-independent pathway. Conclusion: HCV induces autophagosomes and uses their membranes for its RNA replication. Significance: The perturbation of the autophagic pathway by HCV may have important consequences in HCV pathogenesis.

Hepatitis B virus (HBV) chronically infects 250 million people worldwide, resulting in nearly one million deaths annually. Studies in recent years have significantly improved our knowledge on the mechanisms of HBV persistence. HBV uses multiple pathways to harness host innate immunity to enhance its replication. It can also take advantage of the developing immune system and the not-yet-stabilized gut microbiota of young children to facilitate its persistence, and use maternal viral e antigen to educate immunity of the offspring to support its persistence after vertical transmission. The knowledge gained from these recent studies paves the way for the development of new therapies for the treatment of chronic HBV infection, which has so far been very challenging.

Hepatitis B virus (HBV) is a hepatotropic virus that can cause severe liver diseases, including liver cirrhosis and hepatocellular carcinoma. This virus chronically infects approximately 350 million people in the world, causing significant morbidity and mortality. HBV is a small DNA virus with a partially double-stranded and circular DNA genome that has a length of about 3.2 kb. After the infection of hepatocytes, this DNA is repaired to form a covalently closed circular DNA (cccDNA) molecule, which then directs the transcription of viral mRNAs. The mRNA of the viral core protein is larger than the genome length. This core protein mRNA, which is also termed the pregenomic RNA (pgRNA), is packaged by the core protein to form the viral core particle. It is subsequently converted to the partially double-stranded viral genome by the viral RNA polymerase, which is also packaged in the core particle. The core particle subsequently interacts with the viral envelope proteins for the formation of the mature virion, which is then released from infected cells (for a review, see reference 1).Recently, we demonstrated that HBV can induce autophagy in cell cultures, in the mouse liver, and during natural infection

Summary In contrast to horizontal transmission of hepatitis B virus (HBV) between adults, which often leads to self-limited acute infection, vertical transmission of HBV from mother to child often leads to chronic infection. However, the mechanisms linking vertical transmission with chronic infection are not known. We developed a mouse model to study the effect of maternal HBV infection on HBV persistence in offspring and found that HBV carried by the mother impaired CD8+ T cell responses to HBV in her offspring, resulting in HBV persistence. This impairment of CD8+ T cell responses was mediated by hepatic macrophages, which were predisposed by maternal HBV e antigen (HBeAg) to support HBV persistence by upregulation of inhibitory ligand PD-L1 and altered polarization upon restimulation with HBeAg. Depletion of hepatic macrophages led to CD8+ T cell activation and HBV clearance in the offspring, raising the possibility of targeting macrophages to treat chronic HBV patients.

The role of autophagy in carcinogenesis is controversial and apparently complex. By using mice with hepatocyte-specific knockout of Atg5, a gene essential for autophagy, we longitudinally studied the role of autophagy in hepatocarcinogenesis. We found that impairing autophagy in hepatocytes would induce oxidative stress and DNA damage, followed by the initiation of hepatocarcinogenesis, which could be suppressed by the antioxidant N-acetylcysteine. Interestingly, these mice developed only benign tumors with no hepatocellular carcinoma (HCC), even after the treatment with diethylnitrosamine, which induced HCC in wild-type mice. The inability of mice to develop HCC when autophagy was impaired was associated with the induction of multiple tumor suppressors including p53. Further analysis indicated that the induction of p53 was associated with the DNA-damage response. Tumorigenesis studies using an established liver tumor cell line confirmed a positive role of autophagy in tumorigenesis and a negative role of p53 in this process when autophagy was impaired. Our studies thus demonstrate that autophagy is required to maintain healthy mitochondria and to reduce oxidative stress and DNA damage to prevent the initiation of hepatocarcinogenesis. However, once hepatocarcinogenesis has been initiated, its presence is also required to suppress the expression of tumor suppressors to promote the development of HCC. Autophagy (i.e., macroautophagy) is important for cells to remove protein aggregates and damaged organelles. Its dysfunction can cause a variety of diseases including cancers. 1,2 However, its role in carcinogenesis is apparently complex, as it has been shown in different reports to positively or negatively regulate carcinogenesis. 3,4 Autophagy apparently can function as a tumor suppressor, as the gene encoding Beclin-1, a component of the phosphatidylinositol-3-kinase class III (PI3KC3) complex that is essential for the initiation of autophagy, is often monoallelically deleted or mutated in breast, ovarian and prostate cancers. 5 Frameshift mutations in Atg2B, Atg5, Atg9B and Atg12 autophagy genes are also often found in gastric and colorectal cancers with microsatellite instability. 6 The tumor suppressor role of autophagy is further supported by the studies using mouse models. It has been shown that the monoallelic deletion of the Beclin-1 gene in mice induced tumor lesions in various tissues, 7 Atg4C-knockout (KO) mice had increased susceptibility to carcinogens for the development of fibrosarcomas 8 and the systemic mosaic KO of Atg5 and the liver-specific KO of Atg7 in mice led to the development of benign liver adenomas. 9,10 Autophagy has also been shown to promote tumor growth. It has been shown that autophagy can enhance the survival of tumor cells in the hypoxic regions of solid tumors. 11 It has also been shown that in cells expressing oncogenic Ras, autophagy is required to promote tumorigenesis by maintaining oxidative metabolism or facilitating glycolysis. 12,13 Moreover, it has also been demonstrate...

Hepatitis B virus (HBV) is an important pathogen that chronically infects more men than women. To understand the molecular mechanism of this gender disparity, we analyzed HBV replication in transgenic mice that carried the HBV genome with or without the ability to express the HBV X protein (HBx). We found that gender had no effect on HBV surface antigen (HBsAg), DNA, and RNA levels in mice before puberty, but its effect on HBV after puberty was apparent, with HBV replicating approximately twice more efficiently in male mice than in female mice whether or not HBx was expressed. The castration of male mice resulted in a reduction of HBV HBsAg, DNA, and RNA levels, which could be partially restored by the injection of the androgen agonist R1881, indicating a positive role of androgen in HBV replication. The introduction of HBV genomic DNA and androgen receptor (AR) short hairpin RNA (shRNA) into the liver of naïve mice by hydrodynamic injection revealed that the effect of androgen on HBV was dependent on its receptor, which apparently could also stimulate HBV replication via an androgen-independent pathway. Further studies indicated that the two previously identified androgen response elements (AREs) in the HBV genome could indeed mediate the effect of androgen on HBV RNA transcription and DNA replication in vivo. These effects of androgen and its receptor on HBV thus provide an explanation for why men have a higher risk of HBV infection than women. H epatitis B virus (HBV) is a hepatotropic virus that chronically infects approximately 350 million people in the world.Chronic infection by this virus can lead to severe liver diseases, including liver cirrhosis and hepatocellular carcinoma (HCC) (7). HBV has a small DNA genome of about 3.2 kb. This genome carries four genes: the C, S, P, and X genes. The C gene codes for the viral core protein and a related protein named e antigen, the S gene codes for the three viral envelope proteins known as HBV surface antigens (HBsAgs), the P gene codes for the viral DNA polymerase, and the X gene codes for a 17-kDa regulatory protein. The expression of the HBV genes is controlled by four different promoters and two enhancer elements (for a review, see reference 9).HBV pathogenesis is affected by many factors, such as viral load, viral genotypes, and viral variants (1). In addition, gender has also been found to affect HBV replication and pathogenesis (19). Men are three to seven times more likely to become HBV carriers than women, and male HBV carriers are more likely to develop HCC than are female HBV carriers (19). This effect of gender on HBV replication and carcinogenesis appears to be associated with the male sex hormone, as the incidence of HBVassociated HCC is positively correlated with serum androgen levels and inversely correlated with the number of CAG trinucleotide repeats in the androgen receptor (AR) gene, which inversely affects the activity of the AR (10,18,19). The AR is a member of the nuclear receptor superfamily. It consists of several functional domains respo...

Hepatitis B virus (HBV) is a hepatotropic virus that can cause severe liver diseases. By conducting studies using four different transgenic mouse lines that carry either the wild-type HBV genome or the HBV genome incapable of expressing the X gene, we found that liver injury and regeneration induced by a partial hepatectomy (PHx) could have different effects on HBV replication depending on the mouse lines. Further studies using hydrodynamic injection to introduce different amounts of the HBV genomic DNA into the mouse liver revealed that liver injury and regeneration induced by PHx enhanced HBV replication when viral load was low and suppressed HBV replication when viral load was high. These effects of liver injury and regeneration on HBV were independent of the HBV X protein and apparently due to alpha and beta interferons (IFN-α/β), as the effects could be abolished by the injection of anti-IFN-α/β antibodies. Further analysis indicated that PHx could induce the expression of hepatocyte nuclear factor 3 gamma (HNF3γ) when viral load was low and activate the signal transducer and activator of transcription 3 (Stat3) and suppress the expression of the suppressor of cytokine signaling 3 (SOCS3) irrespective of viral load. As both HNF3γ and Stat3 are required to activate the HBV enhancer I to stimulate HBV gene expression and replication, these results provided an explanation to the viral-load-dependent effect of liver injury and regeneration on HBV replication. Our studies thus revealed a novel interaction between HBV and its host and provided important information for understanding HBV replication and pathogenesis during liver injury.