Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Nirmatrelvir treatment of SARS-CoV-2-infected mice blunts antiviral adaptive immune responses.

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir-an orally available inhibitor of the 3-chymotrypsin-like cysteine protease-has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using mouse models of SARS-CoV-2 infection, we show that nirmatrelvir administration blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.

Discovery and antiviral profile of new sulfamoylbenzamide derivatives as HBV capsid assembly modulators.

Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.

Administration of aerosolized SARS-CoV-2 to K18-hACE2 mice uncouples respiratory infection from fatal neuroinvasion.

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model's usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2­infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression.

Tight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.

Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells.

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity.

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Pathogen-specific B-cell receptors drive chronic lymphocytic leukemia by light-chain-dependent cross-reaction with autoantigens.

Several lines of evidence indirectly suggest that antigenic stimulation through the B-cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self-antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen-specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus-specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus-derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus-specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with one or more self-antigens.

Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV-associated HCC involves both viral and host factors. The latter include a functionally inefficient CD8(+) T-cell response that fails to clear the infection from the liver but sustains a chronic necroinflammatory process that contributes to the development of HCC. According to this scenario, amelioration of immune-mediated chronic liver injury may prevent HCC. Because platelets facilitate immune-mediated liver injury by promoting the hepatic accumulation of virus-specific CD8(+) T cells, we evaluated the long-term consequences of antiplatelet therapy in an HBV transgenic mouse model of chronic immune-mediated necroinflammatory liver disease that progresses to HCC. Treatment with aspirin and clopidogrel during the chronic phase of the disease diminished the number of intrahepatic HBV-specific CD8(+) T cells and HBV-nonspecific inflammatory cells, the severity of liver fibrosis, and the development of HCC. Antiplatelet therapy improved overall survival without causing significant side effects. In contrast, the same antiplatelet regimen had no antitumor effect when HCC was induced nonimmunologically by chronic exposure to a hepatotoxic chemical. The unprecedented observation that antiplatelet therapy inhibits or delays immune-mediated hepatocarcinogenesis suggests that platelets may be key players in the pathogenesis of HBV-associated liver cancer and supports the notion that immune-mediated necroinflammatory reactions are an important cause of hepatocellular transformation during chronic hepatitis.

The selective vitamin D receptor agonist, elocalcitol, reduces endometriosis development in a mouse model by inhibiting peritoneal inflammation.

BACKGROUND: Endometriosis, which is characterized by the growth of endometrial tissue at ectopic locations as well as vascular development and inflammation, is still an unmet clinical need since an optimal drug that allows for both pain and infertility management does not exist. Since both the eutopic and the ectopic endometrium express the vitamin D receptor (VDR), and VDR agonists are endowed with anti-proliferative and anti-inflammatory properties, we evaluated the effect of elocalcitol, a VDR agonist with low calcaemic liability, in a mouse model of experimentally induced endometriosis. METHODS AND RESULTS: Endometriosis was induced by injection of syngeneic endometrial tissue fragments into adult Balb/c female mice. After having confirmed by immunohistochemistry that endometriotic lesions developing in mice expressed VDR, the mice were administered with elocalcitol (100 µg/kg) or vehicle orally, once a day, for various durations of time. In this model, elocalcitol was able to reduce total lesion weight up to 70% upon treatment for 1 week before and 2 weeks after disease induction. Interestingly, a therapeutic effect was also observed on already established lesions. Elocalcitol was shown to reduce the capacity of mouse endometrial cells to adhere to collagen. In addition in treated mice, a decreased state of peritoneal inflammation was demonstrated by the inhibition of macrophage recruitment and inflammatory cytokine secretion. CONCLUSIONS: The VDR agonist elocalcitol inhibits lesion development in a validated mouse model of endometriosis, and exerts a protective effect on both the implantation and organization of transferred endometrial tissue. These preliminary data in mice provide a sound rationale for further testing in primate models and eventually in humans.

Vitamin D reserve is higher in women with endometriosis.

BACKGROUND: An immune-mediated defect in recognition and elimination of endometrial fragments refluxed in the peritoneal cavity has been hypothesized to play a crucial role in endometriosis development. Since vitamin D is an effective modulator of the immune system, we have hypothesized that the vitamin D status may have a role in the pathogenesis of endometriosis. METHODS: Women of reproductive age selected for surgery for gynecological indications were enrolled in this prospective cohort study. Serum levels of 25-hydroxyvitamin-D(3), 1,25-dihydroxyvitamin-D(3) and Ca(2+) were assessed. RESULTS: Eighty-seven women with endometriosis and 53 controls were recruited. Mean (+/- SD) levels of 25-hydroxyvitamin-D(3) in women with and without endometriosis were 24.9 +/- 14.8 ng/ml and 20.4 +/- 11.8, respectively (P = 0.05). The Odds Ratio (95% Confidence Interval) for endometriosis in patients with levels exceeding the 75th percentile of the serum distribution of the molecule (28.2 ng/ml) was 4.8 (1.7-13.5). A positive gradient according to the severity of the disease was also documented. A trend towards higher levels of 1,25-dihydroxyvitamin-D(3) and Ca(2+) was observed in women with endometriosis, but differences did not reach statistical significance. As expected, serum concentrations of 25-hydroxyvitamin-D(3) and 1,25-dihydroxyvitamin-D(3,) but not Ca(2+), are influenced by the season (P < 0.001, P = 0.004, P = 0.57, respectively), while levels of the three molecules did not vary according to the phase of the menstrual cycle. CONCLUSIONS: Endometriosis is associated with higher serum levels of vitamin D.

Thiol antioxidants inhibit the formation of the interleukin-12 heterodimer: a novel mechanism for the inhibition of IL-12 production.

IL-12 is a 75 kDa heterodimeric cytokine composed of two disulfide-linked subunits, p35 and p40, which plays an important role in the regulation of the immune response. We tested the hypothesis that thiol antioxidants might interfere with dimerization of the two IL-12 subunits. We thus studied the effect of reduced glutathione (GSH) and N-acetyl-cysteine (NAC) on IL-12 p75 production by human THP-1 cell stimulated with IFN-gamma and Staphylococcus aureus Cowan strain I (SAC), using ELISAs specific for IL-12 p75 or the p40 subunit. NAC and GSH, but not cystine, at concentrations of 5-10 mM inhibited production of IL-12 p75 but not of the p40 subunit. NAC did not inhibit p40 or p35 mRNA expression in dendritic cells or THP-1 cells, or NF-kappa B activation in THP-1 cells. The effect of NAC was specific for IL-12 p75, as NAC did not affect induction of MHC class II expression by IFN-gamma-stimulated THP-1 cells. IL-12 dimer formation appears to be reduced by NAC also in vivo, because pretreatment with NAC (1 g/kg, orally), before LPS injection in mice, inhibited peak IL-12 p75 serum levels without affecting those of p40. We conclude that thiol levels regulate IL-12 p75 production and that assembly of the heterodimer is a step that might represent a target for pharmacological intervention.