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Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.

Follicular helper T (T FH ) cells control antibody responses by supporting antibody affinity maturation and memory formation. Inadequate T FH function has been found in individuals with ineffective responses to vaccines, but the mechanism underlying T FH regulation in vaccination is not understood. Here, we report that lower serum levels of the metabolic hormone leptin associate with reduced vaccine responses to influenza or hepatitis B virus vaccines in healthy populations. Leptin promotes mouse and human T FH differentiation and IL-21 production via STAT3 and mTOR pathways. Leptin receptor deficiency impairs T FH generation and antibody responses in immunisation and infection. Similarly, leptin deficiency induced by fasting reduces influenza vaccination-mediated protection for the subsequent infection challenge, which is mostly rescued by leptin replacement. Our results identify leptin as a regulator of T FH cell differentiation and function and indicate low levels of leptin as a risk factor for vaccine failure. T follicular helper (TFH) cell numbers are increased after vaccination and fewer of these cells might result in reduced vaccine responses. Here the authors show in mice and humans that leptin promotes T FH differentiation and that low leptin levels can impair T FH response to vaccines and virus protection in mice.

Background and Aims: Treatment with siRNAs that target HBV has demonstrated robust declines in HBV antigens. This effect is also observed in the AAV-HBV mouse model, which was used to investigate if two cycles of GalNAc-HBV-siRNA treatment could induce deeper declines in HBsAg levels or prevent rebound, and to provide insights into the liver immune microenvironment. Methods: C57Bl/6 mice were transduced with one of two different titers of AAV-HBV for 28 days, resulting in stable levels of HBsAg of about 103 or 105 IU/mL. Mice were treated for 12 weeks (four doses q3wk) per cycle with 3 mg/kg of siRNA-targeting HBV or an irrelevant sequence either once (single treatment) or twice (retreatment) with an 8-week treatment pause in between. Blood was collected to evaluate viral parameters. Nine weeks after the last treatment, liver samples were collected to perform phenotyping, bulk RNA-sequencing, and immunohistochemistry. Results: Independent of HBsAg baseline levels, treatment with HBV-siRNA induced a rapid decline in HBsAg levels, which then plateaued before gradually rebounding 12 weeks after treatment stopped. A second cycle of HBV-siRNA treatment induced a further decline in HBsAg levels in serum and the liver, reaching undetectable levels and preventing rebound when baseline levels were 103 IU/mL. This was accompanied with a significant increase in inflammatory macrophages in the liver and significant upregulation of regulatory T-cells and T-cells expressing immune checkpoint receptors. Conclusions: Retreatment induced an additional decline in HBsAg levels, reaching undetectable levels when baseline HBsAg levels were 3log10 or less. This correlated with T-cell activation and upregulation of Trem2.

Unresolved hepatitis B virus (HBV) infection leads to a progressive state of HBV-specific immune dysfunctionality that characterizes chronic infection. The immune-competent adeno associated virus (AAV)-HBV mouse model is commonly used preclinically, though a comprehensive characterization of the liver immune microenvironment and its translatability to human infection is still lacking. We investigated the intrahepatic immune profile of the AAV-HBV mouse model at a single-cell level and compared with data from CHB patients in immune tolerant (IT) and immune active (IA) clinical stages. Immune exhaustion was profiled through an iterative subclustering approach for cell-typing analyses of single-cell RNA-sequencing data in CHB donors and compared to the AAV-HBV mouse model 4-weeks and 24-weeks post-transduction to assess its translatability. This was confirmed using an exhaustion flow cytometry panel at 4 and 42-weeks post-transduction. Using single-cell RNA-sequencing, CD8 pre-exhausted T-cells with self-renewing capacity ( ), and terminally exhausted CD8 T-cells ( ) were detected in the AAV-HBV model. These terminally exhausted CD8 T-cells (expressing ) were significantly enriched versus control mice and independently identified through flow cytometry. Importantly, comparison to CHB human data showed a similar exhausted CD8 T-cell population in IT and IA donors, but not in uninfected individuals. Long term high titer AAV-HBV mouse liver transduction led to T-cell exhaustion, as evidenced by expression of conventional immune checkpoint markers at mRNA and protein levels. In both IT and IA donors, a similar CD8 exhausted T-cell population was identified, with increased frequency observed in IA donors. These data support the use of the AAV-HBV mouse model to study classical T-cell exhaustion in HBV infection and the effect of immune-based therapeutic interventions.

Chronic infection with hepatitis B virus (HBV) develops in millions of patients per year, despite the availability of effective prophylactic vaccines. Patients who resolve acute HBV infection develop HBV-specific polyfunctional T cells accompanied by neutralizing antibodies, while in patients with chronic hepatitis B (CHB), immune cells are dysfunctional and impaired. We describe a lipid nanoparticle (LNP)-formulated mRNA vaccine, optimized for the expression of HBV core, polymerase, and surface (preS2-S) antigens with the aim of inducing an effective immune response in patients with CHB. Prime and prime/boost vaccination with LNP-formulated mRNA encoding for core, pol, and/or preS2-S dosing strategies were compared in naive C57BL/6 and BALB/c mice. Immune responses were assessed by IFN-γ ELISpot, intracellular cytokine staining (ICS), and ELISA for antibody production, whereas anti-viral efficacy was evaluated in the AAV-HBV mouse model. The mRNA vaccine induced strong antigen-specific polyfunctional T cell responses in these mouse models, accompanied by the emergence of anti-HBs and anti-HBe antibodies. After three immunizations, the antigen-specific immune stimulation resulted in up to 1.7 log10 IU/mL reduction in systemic HBV surface antigen (HBsAg), accompanied by a transient drop in systemic HBeAg, and this was observed in 50% of the AAV-HBV-transduced mice in the absence of additional modalities such as adjuvants, HBsAg reducing agents, or checkpoint inhibitors. However, no treatment-related effect on viremia was observed in the liver. These results warrant further optimization and evaluation of this mRNA vaccine as a candidate in a multimodal therapeutic regimen for the treatment of chronic HBV infection.

Background: Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. Methods: AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. Results: GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. Conclusions: This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

Follicular helper T (T FH ) cells provide survival and selection signals to germinal center B cells. Here, Carola Vinuesa and colleagues describe a regulatory T cell subset that co-opts the differentiation program of T FH cells and limits their numbers in vivo . Ablation of these T FH -like, T regulatory cells alters the number of antigen-specific B cells suggesting regulatory T cells modulate germinal center responses. Follicular helper (T FH ) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T FH numbers maintains self tolerance. We describe a population of Foxp3 + Blimp-1 + CD4 + T cells constituting 10–25% of the CXCR5 high PD-1 high CD4 + T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T FR ) cells share phenotypic characteristics with T FH and conventional Foxp3 + regulatory T (T reg ) cells yet are distinct from both. Similar to T FH cells, T FR cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T FR cells originate from thymic-derived Foxp3 + precursors, not naive or T FH cells. T FR cells are suppressive in vitro and limit T FH cell and germinal center B cell numbers in vivo . In the absence of T FR cells, an outgrowth of non–antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T FH differentiation pathway is co-opted by T reg cells to control the germinal center response.

The signals controlling T reg cell homeostasis and survival are still being determined. Liston and colleagues demonstrate that peripheral T reg cells depend critically on IL-2 and the survival factor Mcl-1 but not on Bcl-2. Foxp3 + regulatory T (T reg ) cells are a crucial immunosuppressive population of CD4 + T cells, yet the homeostatic processes and survival programs that maintain the T reg cell pool are poorly understood. Here we report that peripheral T reg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2–dependent and costimulation-dependent process. By contrast, excess T reg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-x L and Bcl-2 were dispensable for survival of T reg cells, whereas Mcl-1 was critical for survival of T reg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which T reg cells maintain homeostasis via critical survival pathways.

Interferon-α is a critical mediator of pathogen-induced thymic involution. Liston and colleagues show that the microRNA miR-29a reduces sensitivity of thymic epithelium to infection signals and protects against thymus involution. Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.