Persistent type I interferon (IFN-I) signaling contributes to immune exhaustion and promotes HIV-1 persistence. While we and others have demonstrated that blocking IFN-I signaling in vivo restores anti-HIV-1 T-cell function and reduces viral reservoirs, the underlying mechanism remains unclear. Here, we showed that in humanized mice (hu-mice) and cells from people living with HIV-1 (PLWH), IFN-I signaling impaired mitochondrial activity in CD8+ T cells during chronic HIV-1 infection with effective antiretroviral therapy. Reprogramming immunometabolism by transient inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) rescued mitochondrial activity, reversed aberrant immune activation, and enhanced CD8+ T-cell activity in HIV-infected hosts, both ex vivo and in vivo. When combined with an HIV-1 reservoir-activating agent, 2-DG reduced the viral reservoir size in hu-mice and suppressed HIV-1 amplification in cells from PLWH. These findings indicate that 2-DG-mediated immunometabolic reprogramming represents a novel strategy to restore host immunity and control HIV-1 reservoirs.

Abstract Introduction Chronic hepatitis B virus (HBV) infection remains a major global health burden. Current antiviral therapies suppress viral replication but fail to eliminate cccDNA or substantially reduce HBsAg, making functional cure rare. Innate immune control of HBV is limited by poor engagement of the STING pathway in hepatocytes due to low endogenous expression and inefficient agonist delivery. We developed a universal STING mimic (uniSTING) delivered via lipid nanoparticles encoding uniSTING mRNA, which drives formation of endomembrane-independent STING polymers and preferential activation of the IRF3/type I interferon axis while minimizing NF-κB signaling. Methods HBV-positive cells were treated with LNP/uniSTING, followed by assessment of type I interferon and interferon-stimulated genes (ISG) induction. In vivo, HBV carrier mice were administered LNP/uniSTING, and serum HBV viremia were monitored alongside HBV-specific innate and adaptive immune responses. Results uniSTING elicited strong IFN-I responses in HBV negative cells. In the HepAD38 cell line where HBV transcription from the integrated HBV genome is suppressed by doxycycline to produce HBV predominantly from HBV cccDNA, uniSTING induced robust IFN-β and ISGs expression, whereas in the Dox- condition, characterized by high-level HBV replication, uniSTING-induced IFN-β and ISG responses were attenuated. In vivo, administration of LNP/uniSTING to naive mice induced robust IFN-β production and ISG expression. In chronic HBV carrier mice, repeated administration of LNP/uniSTING resulted in sustained reductions in serum HBV DNA and HBsAg levels, accompanied by induction of anti-HBs IgG responses. Conclusion These findings demonstrate that uniSTING can break HBV immune tolerance and elicit a functional anti-HBs antibody response in vivo. Collectively, this work highlights uniSTING as a promising immunotherapeutic strategy for chronic HBV infection by simultaneously activating innate antiviral pathways and promoting protective humoral immunity. Funding Source NIH R01 AI181664 Topic Categories Viral Immunology (VIR)

Immune-stimulating antibody conjugates (ISACs) represent an emerging class of immunotherapeutics that combine tumor-targeted antibodies with innate immune agonists. However, current ISAC platforms often rely on heterogeneous conjugation strategies and poorly defined antibody architectures, making it difficult to disentangle how Fc glycosylation, payload presentation, and linker design collectively govern immune activation. Here, we report a dual-enzymatic antibody-editing strategy that integrates Fc glyco-engineering with site-specific payload conjugation to construct structurally defined immune-stimulating antibody conjugates. Using microbial transglutaminase-mediated conjugation at the Fc Q295 residue together with Endo-S2-mediated Fc glycan remodeling at N297, we generated trastuzumab-based ISACs with defined Fc glycoforms and linker architectures. Systematic evaluation reveals that Fc glycosylation state and payload presentation act as orthogonal and synergistic determinants of immune activation. In particular, an intact and afucosylated Fc glycan is essential for robust tumor-cell killing and cytokine induction, while cleavable linkers further enhance innate immune activation through efficient intracellular release of the TLR7/8 agonist. This work establishes a versatile chemical strategy for controlling antibody architecture and immune activation, providing a broadly applicable platform for the rational design of next-generation antibody therapeutics.

Background:Chronic hepatitis B virus (HBV) infection (CHB) affects nearly 300 million individuals globally and remains incurable with current antiviral therapies, which suppress viral replication but rarely achieve functional cure defined by sustained loss of hepatitis B surface antigen (HBsAg). CHB is characterized by profound virus-induced immune tolerance that limits the efficacy of conventional therapeutic vaccination strategies. Objective:To evaluate the therapeutic efficacy and immunological mechanisms of HEPLISAV-B, a CpG-1018-adjuvanted HBsAg vaccine, in breaking immune tolerance and inducing functional cure-like responses in a murine model of CHB. Design:Using the adeno-associated virus-HBV (AAV-HBV) mouse model, mice with high levels of persistent HBV viremia were vaccinated with two doses of HEPLISAV-B. Virological outcomes in the blood and liver, immune responses and mechanisms were assessed. Results:HEPLISAV-B induced rapid and durable HBsAg clearance, markedly reduced circulating and intrahepatic HBV DNA and RNA, and suppressed viral replication without hepatocellular injury. Vaccination elicited robust, sustained anti-HBs IgG1 and IgA responses, enhanced HBsAg-specific T and B cell immunity, reduced CD4 regulatory T cells, and decreased PD-1 expression on CD4 T cells. Therapeutic efficacy was strictly dependent on CD4 T cells and the CD40/CD40L signaling pathway, but independent of CD8 T cells, indicating a CD4-driven, non-cytolytic antiviral mechanism critical for HEPLISAV-B induced HBV control. Conclusion:HEPLISAV-B effectively breaks HBV-induced immune tolerance and restores coordinated antiviral immunity through a CD4 T cell-/CD40L-dependent pathway. The findings support its potential as a therapeutic vaccine in CHB patients. Key messages:What is already known on this topic: Chronic HBV infection is marked by profound virus-induced immune tolerance, current antiviral therapies and vaccines fail to reliably induce HBsAg loss or restore effective antiviral immunity, highlighting the need for immune-based therapeutic strategies.What this study adds: This study demonstrates that the clinically approved vaccine HEPLISAV-B can break HBV immune tolerance in a chronic HBV mouse model, inducing durable HBsAg clearance and anti-HBs immunity, non-cytolytic depletion of intrahepatic HBV DNA, through a mechanism strictly dependent on CD4 T cells and CD40/CD40L signaling.How this study might affect research, practice or policy: These findings defined a CD4 T cell-CD40L/CD40 axis that is critical in CHB functional cure, and support testing HEPLISAV-B as a therapeutic vaccine in CHB patients. Abstract Figure:

Abstract Chimeric antigen receptor (CAR) T cells specific for myeloid-associated antigens expressed on the cell surface of acute myeloid leukemia (AML) can cause depletion of normal myeloid progenitor cells. We developed a CAR specific for an HLA-A∗02:01-restricted peptide of the myeloid-restricted cathepsin-G (CG) protein. CG-specific CAR T cells (CG1.CAR) were further engineered to increase their functional avidity. Specifically, we developed CG1.CAR T cells coexpressing the lymphocyte-specific protein tyrosine kinase (LCK) and duplicated CD3ζ chain, which allows the functional recognition of the CG1 peptide as low as 0.025μM. Optimized CG1.CAR T cells displayed antileukemia effects in vitro and in vivo in patient-derived AML xenotransplant mouse models and did not cause hematopoietic toxicity in colony assays and humanized mice. Mechanistically, LCK overexpression in CG1.CAR T cells caused transcriptional modifications characterized by the overexpression of mitochondrial-encoded electron transport chain components that were correlated with increased mitochondrial mass and improved respiratory capacity. Based on these data, CG1.CAR T cells hold clinical potential for the treatment of AML.

The current highly active antiretroviral therapy (HAART or ART) effectively suppresses de novo HIV-1 infection but fails to eliminate HIV reservoir cells, which leads to rapid viral rebound upon ART cessation. Chimeric antigen receptor (CAR) T cells engineered to target HIV-1 Env⁺ cells offer a promising strategy to eliminate or control these persistent reservoirs and achieve durable control of HIV-1 infection. However, a major challenge is the susceptibility of such CAR-T cells to HIV infection, especially those soluble CD4 (sCD4)-based CAR-T cells. In this study, an sCD4-based CAR incorporating the S85C mutation in the CD4 Ig-like domain 1 (termed D1C) was engineered to enable disulfide bond formation with the HIV-1 envelope glycoprotein (Env), thereby reducing viral entry and conferring protection against HIV infection. D1C/sCD4 CAR-T cells exhibited enhanced T-cell activation and cytotoxicity in response to Env stimulation while demonstrating resistance HIV-1 infection in vitro and in vivo. Furthermore, the herpesvirus entry mediator (HVEM) intracellular domain was identified as an optimal costimulatory domain, enhancing cytokine induction, cytotoxicity, and promoting a favorable central memory phenotype and persistence of CAR-T cells. In humanized mouse models, D1C/sCD4 CAR-T cells demonstrated superior persistence and improved control of HIV rebound following ART interruption compared to wild-type (WT)/sCD4 CAR-T cells. These findings highlight a novel strategy to enhance the efficacy and durability of HIV-targeted CAR-T cell therapy by combining HIV resistance and optimized co-stimulation.

Human dendritic cells (DCs) are classified into three subsets based on their ontogeny, transcriptomes, and functions. During primary human immunodeficiency virus (HIV) infection, DCs in the peripheral tissues capture the HIV-1 particles, migrate to the lymph nodes, transfer the particles to CD4+ T cells, and initiate infection. However, the identity of the DC subset involved is yet elusive. Hitherto, a novel subset (AXL+DCs) has been identified in human blood, which is transcriptomically and functionally distinct from three known subsets. Compared to these, resting AXL+DCs express Siglec1 (CD169), capture HIV-1 particles in a CD169-dependent manner, and mediate transinfection. These results suggested that AXL+ DCs may facilitate HIV-1 transmission and the spread of very early-stage HIV infection in patients. Therapeutic strategies that target AXL+DCs or CD169 interaction with HIV-1 may provide pre-exposure protection during the initial stages of HIV-1 infection.

Targeting acute myeloid leukemia (AML) with chimeric antigen receptor (CAR)-T cells that recognize conventional myeloid-associated antigens expressed on the cell surface of blastic cells causes depletion of normal myeloid progenitor cells. Cathepsin-G (CG1) is a myeloid-restricted protein normally stored in azurophil granules but broadly expressed in the cytoplasm of leukemic blasts. In leukemic blasts, CG1 is processed, and the CG1-derived peptides are presented by the major histocompatibility complex and recognized by CD8 T cells. We developed a CAR that selectively targets an HLA-A2–restricted CG1 peptide in the CG1 peptide/HLA-A2 complex. Next, we enhanced the CAR to reach a functional avidity approximating that of a conventional alpha-beta T-cell receptor recognizing a peptide/HLA complex. Specifically, CAR-T cells with duplicated CD3ζ ITAM and overexpression of kinase LcK showed recognition of target cells presenting less than 0.025 uM of CG1 peptide. The enhanced CAR-T cells exhibited faster CD3ζ phosphorylation without significant modification of their molecular signature and a more robust metabolic profile than did conventional CAR-T cells. Furthermore, the enhanced CAR-T cells displayed significant antileukemia effects in vitro, in three independent AML PDX models, and in humanized mice without showing hematopoietic toxicity. Our findings demonstrate the clinical potential of CG1-specific CAR-T cells for the treatment of AML. The Leukemia and Lymphoma Society ID:6625-21 Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Purpose of review Recent findings on the critical pathogenic role of inflammatory pDC and type 1 interferons (IFN-I) in HIV-1 pathogenesis in humanized mice and its correlation with inflammatory diseases in people living with HIV-1 (PLWH) suggest that targeting the pDC/IFN-I signaling pathway will reverse HIV-induced inflammation to treat HIV-associated end-organ diseases and rescue anti-HIV immunity to reduce or control HIV-1 reservoirs. Recent findings In both humanized mice and in PBMC from people living with HIV-1 (PLWH), depletion of pDC or inhibition of IFN-I signaling resolves IFN-associated inflammation, rescues anti-HIV T cell functions and reduces HIV-1 reservoir cells. In humanized mice with HIV-1 persistent infection under effective HAART, persistent pDC activation and IFN-I signaling has been shown to induce HIV-associated tissue injury and anti-HIV T cell impairment. in HIV-infected mice with effective HAART, pDC depletion phenocopies what is achieved with blocking IFN-I signaling in reversing HIV-induced inflammation, rescuing anti-HIV T cells and reducing HIV-1 reservoirs. Interestingly, in both humanized mice and in PBMC from PLWH, depletion of pDC or inhibition of IFN-I signaling rescues anti-HIV TCF1+ (T cell factor 1) PD1+ (programmed cell death protein 1) CD8 T cell functions to enhance the effect of PD1 immune checkpoint inhibitor (ICI) to reduce HIV-1 reservoir cells. Summary These findings functionally define the role of the pDC/IFN-I pathway in HIV-associated inflammation, HIV-1 reservoir persistence and end-organ diseases, and suggest that inhibiting pDC or blocking IFN-I signaling will provide a novel therapeutic strategy to reverse inflammation-associated diseases and to rescue anti-HIV immunity that cooperates with PD1 ICI effect to reduce or control HIV-1 reservoirs.

Persistent type I interferon (IFN-I) signaling compromises adaptive anti-HIV-1 T cell immunity and promotes viral reservoir persistence, yet its effects on innate lymphoid cells during chronic infection remain unclear. Through integrated single-cell RNA sequencing and functional validation in HIV-1-infected humanized mice with combination antiretroviral therapy (cART) and IFN-I signaling blockade, we reveal IFN-I-induced dysfunction of natural killer (NK) cells and group 3 innate lymphoid cells (ILC3s). Mechanistically, the IFN-I-CD9 axis drives NK cells toward a decidual NK cell-like phenotype, impairing their cytotoxic activity. Furthermore, IFNAR blockade rescues ILC3 functionality, which is critical for IL-17/IL-22-mediated antimicrobial defense and mucosal barrier maintenance. Our study delineates IFN-I-driven immunosuppression across innate lymphocyte compartments and proposes the targeted modulation of this pathway to enhance antiviral and mucosal immunity in HIV-1 management.

The persistence of HIV-1 reservoirs during combination anti-retroviral therapy (cART) is associated with chronic inflammation and systemic immune activation in people infected with HIV-1 (PWH), leading to a suboptimal immune reconstitution as well as an increased risk of non-AIDS events. In this study, we assessed the effect of CD24-Fc, a fusion protein with anti-inflammatory properties that interacts with danger-associated molecular patterns (DAMPs) and siglec-10, in humanized mice with chronic HIV-1 infection under suppressive cART in vivo and in peripheral blood mononuclear cells (PBMCs) from PWH in vitro. We report that CD24-Fc treatment significantly reduced inflammation and immune hyperactivation in humanized mice with HIV-1 infection and cART. CD24-Fc treatment improved recovery of CD4 T cells, reduced immune hyper-activation, increased functional central memory T cells. Notably, CD24-Fc treatment increased CXCR5 + CD8 central memory T cells (TCM) with increased HIV-specific polyfunctionality in humanized mice and in PBMC from PWH. This enhanced anti-HIV T cell activity was associated with improved control of HIV-1 viral rebound and reduced HIV-1 pathogenesis upon cART cessation. Our findings indicate that CD24-Fc may provide a promising new therapeutic for treating chronic inflammation and associated diseases in PWH.

Plasmacytoid dendritic cells (pDCs) rapidly produce type I interferon (IFN-I) in response to acute virus infections. During chronic HIV-1 infection, persistent activation of pDCs contributes to inflammatory diseases. Combination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and prolongs the life span of people living with HIV-1 (PLWH). The persistence of viral reservoir cells under cART, however, is associated with suboptimal immune reconstitution, impaired anti-HIV immunity, and non-AIDS-defining inflammatory diseases through unclear mechanisms. We report here that pDC depletion in HIV-infected humanized mice with suppressive cART alleviated HIV-associated inflammation, reversed T cell immune exhaustion, enhanced HIV-specific CD8+ T cell responses, and reduced HIV-1 reservoirs in lymphoid and nonlymphoid tissues through CD8+ T cell-dependent mechanisms. Specifically, pDC depletion in the mice led to an increase in TCF-1+PD-1+Tim-3- stem-like memory CD8+ T cells in HIV-infected lymphoid tissues, which correlated with a reduction in the HIV-1 reservoir. We further showed that pDCs suppressed the polyfunctional activity of anti-HIV stem-like memory CD8+ T cells isolated from PLWH in vitro. As in the HIV-infected humanized mice, pDC depletion or IFN-I blockade functionally rescued the stem-like memory CD8+ T cells, enhancing their anti-HIV responses. Combination therapy with PD-1 blockade further improved stem-like memory T cell function both in vitro and in HIV-infected humanized mice. Our findings indicate that HIV-induced inflammatory pDCs impair anti-HIV stem-like memory CD8+ T cell responses and suggest that the combination of pDC depletion with PD-1 immune checkpoint blockade represents a candidate therapeutic approach to treating HIV-1 infection and its associated inflammatory diseases.

Background & Aims HIV accelerates liver fibrosis attributable to multiple etiologies, including HCV, HBV, and fatty liver disease. Evidence also suggests that HIV infection itself is associated with liver fibrogenesis. Recent studies have implicated Yes-Associated Protein 1 (YAP1) and the upstream lysophosphatidic acid (LPA)/PI3K/AKT pathway as critical regulators of hepatic fibrogenesis, and also suggests a connection to HIV-related liver fibrosis. However, the relationship between YAP/PI3K/AKT pathway activation and HIV-related liver fibrosis remains uncertain. Methods qPCR, western blot, IFC, and ELISA (replicates n≥3) was performed in an unbiased humanized mouse model (NRG-hu HSC mice, n=6), the precision cut liver slice ex vivo model, and both traditional in vitro models as well as a 3D spheroid system. Results YAP target gene mRNA and protein levels (ANKRD, CTGF, CYR61) were upregulated across all models exposed to HIV. Humanized mice infected with HIV had significant increases in the percentage of YAP positive nuclei (2.2 fold) and the percentage area of Sirius Red collagen staining (3.3 fold) compared to control mice. Serum concentrations of LPA increased 5.8 fold within people living with HIV (PLWH) compared to healthy controls. Modulation of LPAR1, PI3K, and AKT by either inhibitors or siRNAs abrogated the fibrotic effects of HIV exposure and downregulated YAP target genes within cultured liver cells. Conclusions The LPAR/PI3K/AKT axis is vital for the activation of YAP and hepatic fibrogenesis due to HIV infection. This novel mechanistic insight suggests new pharmacologic targets for treatment of liver fibrosis in persons living with HIV. Impact and implications There are currently no FDA approved treatments for cirrhosis, while liver disease is the second leading cause of mortality among PLWH after AIDS2. Increased LPA concentrations and AKT activation after HIV infection found in recent work suggested that the Hippo pathway may be a key regulator of HIV-related fibrogenesis24,28. By linking LPA signaling, YAP activation, and HIV-related fibrogenesis, this mechanism presents a target for future research into therapeutic interventions for not only HIV but also other liver diesases (NAFLD and ALD).

Abstract Siglec-H is an immune receptor specifically expressed in plasmacytoid dendritic cells (pDCs) that inhibits IFN-α production and regulates pDC maturation. Although, the role of pDCs in autoimmune disorders is well-known, the effect of Siglec-H expression in pDCs in acute liver injury (ALI) remains elusive. To investigate Siglec-H/pDCs axis in ALI, BDCA2 transgenic and Siglec-H knockout (SH KO) mice were administrated with concanavalin A (ConA) to induce a T cell-mediated ALI in combination of either pDCs depletion with anti-BDCA2 antibody or anti-IL-21R and Stat3 blockade. Here, we report that specific depletion of pDCs in BDCA2 transgenic mice exacerbated ALI. Surprisingly, SH KO mice were resistant to ALI (lower ALT, hyaluronic acid, IL-6 and TNF-α). Th1/IFN-γ response and Stat1 signaling were both inhibited in the liver of SH KO mice while intrahepatic IL-21 and Stat3 signaling pathways were elevated. Mechanistically, we identified that Siglec-H-null pDCs exhibited immunosuppressive phenotypes (CD40LowCCR9Low), resulting in a defective CD4 T cell activation and a promotion of IL-21-producing CD4 T cells in the liver. Finally, IL-21R and Stat3 inhibitions in Siglec-H deficient mice restored ConA-induced ALI. We conclude that, Siglec-H-null pDCs mediate hepatic tolerance via induction of IL-21+CD4 T cells in the liver during ALI. These findings indicate that targeting Siglec-H/pDCs axis may provide a novel approach to modulate CD4 T cell polarization and liver disease.

The development of effective vaccines against infectious diseases remains a critical challenge in global health. Animal models play a crucial role in vaccine development by providing valuable insights into the efficacy, safety, and mechanisms of immune response induction, which guide the design and formulation of vaccines. However, traditional animal models often inadequately recapitulate human immune responses. Humanized mice (hu-mice) models with a functional human immune system have emerged as invaluable tools in bridging the translational gap between preclinical research and clinical trials for human vaccine development. This review summarizes commonly used hu-mice models and advances in optimizing them to improve human immune responses. We review the application of humanized mice for human vaccine development with a focus on HIV-1 vaccines. We also discuss the remaining challenges and improvements needed for the currently available hu-mice models to better facilitate the development and testing of human vaccines for infectious diseases.

BACKGROUND & AIMS:Siglec-H is a receptor specifically expressed in mouse plasmacytoid dendritic cells (pDCs), which functions as a negative regulator of interferon-α production and plays a critical role in pDC maturation to become antigen-presenting cells. The function of pDCs in autoimmune and inflammatory diseases has been reported. However, the effect of Siglec-H expression in pDCs in liver inflammation and diseases remains unclear. METHODS:Using the model of concanavalin A-induced acute liver injury (ALI), we investigated the Siglec-H/pDCs axis during ALI in BDCA2 transgenic mice and Siglec-H-/- mice. Anti-BDCA2 antibody, anti-interleukin (IL)-21R antibody, and Stat3 inhibitor were used to specifically deplete pDCs, block IL21 receptor, and inhibit Stat3 signaling, respectively. Splenocytes and purified naive CD4 T cells and bone marrow FLT3L-derived pDCs were cocultured and stimulated with phorbol myristate acetate/ionomycin and CD3/CD28 beads, respectively. RESULTS:Data showed that specific depletion of pDCs aggravated concanavalin A-induced ALI. Remarkably, alanine aminotransferase, hyaluronic acid, and proinflammatory cytokines IL6 and tumor necrosis factor-α levels were lower in the blood and liver of Siglec-H knockout mice. This was associated with attenuation of both interferon-γ/Th1 response and Stat1 signaling in the liver of Siglec-H knockout mice while intrahepatic IL21 and Stat3 signaling pathways were upregulated. Blocking IL21R or Stat3 signaling in Siglec-H knockout mice restored concanavalin A-induced ALI. Finally, we observed that the Siglec-H-null pDCs exhibited immature and immunosuppressive phenotypes (CCR9LowCD40Low), resulting in reduction of CD4 T-cell activation and promotion of IL21+CD4 T cells in the liver. CONCLUSIONS:During T-cell-mediated ALI, Siglec-H-null pDCs enhance immune tolerance and promote IL21+CD4 T cells in the liver. Targeting Siglec-H/pDC axis may provide a novel approach to modulate liver inflammation and disease.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

Chimeric mouse models with a humanized liver (Hu-HEP mice) provide a unique tool to study human hepatotropic virus diseases, including viral infection, viral pathogenesis, and anti-viral therapy. Here, we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (FAH) knockout mice repopulated with human hepatocytes (FRG-Hu HEP mice). The procedures include (1) maintenance and genotyping of the FRG mice, (2) intrasplenic injection of primary human hepatocytes (PHH), (3) 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV infection and detection. The method is simple and allows for highly reproducible generation of FRG-Hu HEP mice for HBV infection and therapy investigations.

Plasmacytoid dendritic cells were shown to protect against acute liver failure. The present study demonstrated that this protection was enhanced and mediated by interleukin-21producing CD4 T cells and hyperactivation of Stat3 signaling in the liver of Siglec-H deficient fi cient mice. BACKGROUND & AIMS: Siglec-H is a receptor specifically fi cally expressed in mouse plasmacytoid dendritic cells (pDCs), which functions as a negative regulator of interferon-a a production and plays a critical role in pDC maturation to become antigen- presenting cells. The function of pDCs in autoimmune and infl ammatory diseases has been reported. However, the effect of Siglec-H expression in pDCs in liver inflammation fl ammation and diseases remains unclear. METHODS: Using the model of concanavalin A-induced - induced acute liver injury (ALI), we investigated the Siglec-H/pDCs axis during ALI in BDCA2 transgenic mice and Siglec-H-/-/ BDCA2 antibody, anti-interleukin (IL)-21R antibody, and Stat3 inhibitor were used to specifically fi cally deplete pDCs, block IL21 mice. Anti receptor, and inhibit Stat3 signaling, respectively. Splenocytes and purified fi ed naive CD4 T cells and bone marrow FLT3L-derived pDCs were cocultured and stimulated with phorbol myristate acetate/ionomycin and CD3/CD28 beads, respectively. RESULTS: Data showed that specific fi c depletion of pDCs aggravated concanavalin A-induced - induced ALI. Remarkably, alanine aminotransferase, hyaluronic acid, and proinflammatory fl ammatory cytokines IL6 and tumor necrosis factor-a a levels were lower in the blood and liver of Siglec-H knockout mice. This was associated with attenuation of both interferon-g/Th1 g /Th1 response and Stat1 signaling in the liver of Siglec-H knockout mice while intrahepatic IL21 and Stat3 signaling pathways were upregulated. Blocking IL21R or Stat3 signaling in Siglec-H knockout mice restored concanavalin A-induced - induced ALI. Finally, we observed that the Siglec-H-null pDCs exhibited immature and immunosuppressive phenotypes (CCR9LowCD40Low), Low CD40 Low ), resulting in reduction of CD4 T-cell activation and promotion of IL21 & thorn;CD4 & thorn; CD4 T cells in the liver CONCLUSIONS: During T-cell-mediated ALI, Siglec-H-null pDCs enhance immune tolerance and promote IL21 & thorn;CD4 & thorn; CD4 T cells in the liver. Targeting Siglec-H/pDC axis may provide a novel approach to modulate liver inflammation fl ammation and disease.