Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

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.

CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.

Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here, we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.

Persistent hepatitis B virus and HIV coinfections in dually humanized mice engrafted with human liver and immune system.

Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including human immunodeficiency virus type (HIV) and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV, which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV, which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection, and preclinical testing of novel immunotherapeutics.

Soluble CTLA-4 mutants ameliorate immune-related adverse events but preserve efficacy of CTLA-4- and PD-1-targeted immunotherapy.

Immune checkpoint inhibitors (ICIs), such as nivolumab and ipilimumab, not only elicit antitumor responses in a wide range of human cancers but also cause severe immune-related adverse events (irAEs), including death. A largely unmet medical need is to treat irAEs without abrogating the immunotherapeutic effect of ICIs. Although abatacept has been used to treat irAEs, it risks neutralizing the anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) monoclonal antibodies administered for cancer therapy, thereby reducing the efficacy of anti-CTLA-4 immunotherapy. To avoid this caveat, we compared wild-type abatacept and mutants of CTLA-4-Ig for their binding to clinically approved anti-CTLA-4 antibodies and for their effect on both irAEs and immunotherapy conferred by anti-CTLA-4 and anti-PD-1 antibodies. Here, we report that whereas abatacept neutralized the therapeutic effect of anti-CTLA-4 antibodies, the mutants that bound to B7-1 and B7-2, but not to clinical anti-CTLA-4 antibodies, including clinically used belatacept, abrogated irAEs without affecting cancer immunotherapy. Our data demonstrate that anti-CTLA-4-induced irAEs can be corrected by provision of soluble CTLA-4 variants and that the clinically available belatacept may emerge as a broadly applicable drug to abrogate irAEs while preserving the therapeutic efficacy of CTLA-4-targeting ICIs.

CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.

Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here we probe the decline of B cells in human CTLA-4 knock-in mice by using antihuman CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.

Herpes Virus Entry Mediator Costimulation Signaling Enhances CAR T-cell Efficacy Against Solid Tumors Through Metabolic Reprogramming.

Costimulatory domains (CSD) of 4-1BB and CD28 are most widely used in chimeric antigen receptor (CAR)-engineered T cells. These CAR T cells have shown encouraging efficacy in the treatment of hematologic malignancies but have limited efficacy in solid tumors. The herpes virus entry mediator (HVEM) is a costimulatory molecule with a novel downstream signaling pathway. In response to target cells, CAR T cells with a HVEM CSD (HVEM-CAR T) displayed more robust cytokine release and cytotoxicity than 4-1BB-CAR T or CD28-CAR T in vitro. Furthermore, HVEM-CAR T showed superior therapeutic efficacy in several mouse tumor models. Mechanistically, the HVEM CSD endowed CAR T cells with attenuated exhaustion, improved function and persistence, and enhanced metabolic activities in tumor tissue compared with 4-1BB-based or CD28-based CAR T cells. These studies establish that the HVEM CSD has the potential to improve the therapeutic efficacy of CAR T cells against solid tumors.

Metabolic modeling of single bronchoalveolar macrophages reveals regulators of hyperinflammation in COVID-19.

SARS-CoV-2 infection induces imbalanced immune response such as hyperinflammation in patients with severe COVID-19. Here, we studied the immunometabolic regulatory mechanisms for the pathogenesis of COVID-19. We depicted the metabolic landscape of immune cells, especially macrophages, from bronchoalveolar lavage fluid of patients with COVID-19 at single-cell level. We found that most metabolic processes were upregulated in macrophages from lungs of patients with mild COVID-19 compared to cells from healthy controls, whereas macrophages from severe COVID-19 showed downregulation of most of the core metabolic pathways including glutamate metabolism, fatty acid oxidation, citrate cycle, and oxidative phosphorylation, and upregulation of a few pathways such as glycolysis. Rewiring cellular metabolism by amino acid supplementation, glycolysis inhibition, or PPARγ stimulation reduces inflammation in macrophages stimulated with SARS-CoV-2. Altogether, this study demonstrates that metabolic imbalance of bronchoalveolar macrophages may contribute to hyperinflammation in patients with severe COVID-19 and provides insights into treating COVID-19 by immunometabolic modulation.

Molecular clones of genetically distinct hepatitis B virus genotypes reveal distinct host and drug treatment responses.

Background & Aims: HBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs. Methods: The replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs. Results: Patient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C. Conclusions: The infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening. Lay summary: The hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

Type I interferons and TGF-ß cooperate to induce liver fibrosis during HIV-1 infection under antiretroviral therapy.

Liver diseases have become a major comorbidity health concern for people living with HIV-1 (PLWH) treated with combination antiretroviral therapy (cART). To investigate if HIV-1 infection and cART interact to lead to liver diseases, humanized mice reconstituted with progenitor cells from human fetal livers were infected with HIV-1 and treated with cART. We report here that chronic HIV-1 infection with cART induced hepatitis and liver fibrosis in humanized mice, associated with accumulation of M2-like macrophages (M2LMs), elevated TGF-ß, and IFN signaling in the liver. Interestingly, IFN-I and TGF-ß cooperatively activated human hepatic stellate cells (HepSCs) in vitro. Mechanistically, IFN-I enhanced TGF-ß-induced SMAD2/3 activation in HepSCs. Finally, blockade of IFN-I signaling reversed HIV/cART-induced liver diseases in humanized mice. Consistent with the findings in humanized mice with HIV-1 and cART, we detected elevated markers of liver injury, M2LMs, and of IFN signaling in blood specimens from PLWH compared with those of healthy individuals. These findings identify the IFN-I/M2LM/HepSC axis in HIV/cART-induced liver diseases and suggest that inhibiting IFN-I signaling or M2LM may provide a novel therapeutic strategy for treating HIV/cART-associated liver diseases in PLWH treated with antiretroviral therapy.

Site-Specific Chemoenzymatic Conjugation of High-Affinity M6P Glycan Ligands to Antibodies for Targeted Protein Degradation.

Lysosome-targeting chimeras (LYTACs) offer an opportunity for the degradation of extracellular and membrane-associated proteins of interest. Here, we report an efficient chemoenzymatic method that enables a single-step and site-specific conjugation of high-affinity mannose-6-phosphate (M6P) glycan ligands to antibodies without the need of protein engineering and conventional click reactions that would introduce "unnatural" moieties, yielding homogeneous antibody-M6P glycan conjugates for targeted degradation of membrane-associated proteins. Using trastuzumab and cetuximab as model antibodies, we showed that the wild-type endoglycosidase S (Endo-S) could efficiently perform the antibody deglycosylation and simultaneous transfer of an M6P-glycan from a synthetic M6P-glycan oxazoline to the deglycosylated antibody in a one-pot manner, giving structurally well-defined antibody-M6P glycan conjugates. A two-step procedure, using wild-type Endo-S2 for deglycosylation followed by transglycosylation with an Endo-S2 mutant (D184M), was also efficient to provide M6P glycan-antibody conjugates. The chemoenzymatic approach was highly specific for Fc glycan remodeling when both Fc and Fab domains were glycosylated, as exemplified by the selective Fc-glycan remodeling of cetuximab. SPR binding analysis indicated that the M6P conjugates possessed a nanomolar range of binding affinities for the cation-independent mannose-6-phosphate receptor (CI-MPR). Preliminary cell-based assays showed that the M6P-trastuzumab and M6P-cetuximab conjugates were able to selectively degrade the membrane-associated HER2 and EGFR, respectively. This modular glycan-remodeling strategy is expected to find wide applications for antibody-based lysosome-targeted degradation of extracellular and membrane proteins.

TWO-SIGMA-G: a new competitive gene set testing framework for scRNA-seq data accounting for inter-gene and cell-cell correlation.

We propose TWO-SIGMA-G, a competitive gene set test for scRNA-seq data. TWO-SIGMA-G uses a mixed-effects regression model based on our previously published TWO-SIGMA to test for differential expression at the gene-level. This regression-based model provides flexibility and rigor at the gene-level in (1) handling complex experimental designs, (2) accounting for the correlation between biological replicates and (3) accommodating the distribution of scRNA-seq data to improve statistical inference. Moreover, TWO-SIGMA-G uses a novel approach to adjust for inter-gene-correlation (IGC) at the set-level to control the set-level false positive rate. Simulations demonstrate that TWO-SIGMA-G preserves type-I error and increases power in the presence of IGC compared with other methods. Application to two datasets identified HIV-associated interferon pathways in xenograft mice and pathways associated with Alzheimer's disease progression in humans.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

Extracellular Microvesicles Released From Brain Endothelial Cells are Detected in Animal Models Of HIV-1 Signifying Unresolved Inflammation.

Treatment of HIV-infected patients with antiretroviral therapy (ART) has effectively suppressed viral replication; however, the central nervous system is still a major target and reservoir of the virus leading to the possible development of HIV-associated neurocognitive disorders (HAND). Furthermore, a hallmark feature of HAND is the disruption of the blood-brain barrier that leads to loss of tight junction protein (TJP) complexes. Extracellular vesicles (EVs), released by every cell type in the body, occur in greater quantities in response to cellular activation or injury. We have found that inflammatory insults activate brain endothelial cells (EC) and induce the release of EVs containing TJPs such as Occludin. We thus hypothesized that HIV infection and unresolved neuroinflammation will result in the release of brain-EC derived EVs. Herein, our results show elevated levels of brain-EC EVs in a humanized mouse model of HIV infection. Furthermore, while ART reduced brain-EC EVs, it was unable to completely resolve increased vesicles detectable in the blood. In addition to inflammatory insults, HIV-1 viral proteins (Tat and gp120) increased the release of Occludin + vesicles from human brain microvasculature ECs. This increase in vesicle release could be prevented by knock-down of the small GTPase ARF6. ARF6 has been shown to regulate EV biogenesis in other cell types, and we provide further evidence for the involvement of ARF6 in brain EC derived EVs. Overall, this study offers insight into the process of brain vascular remodeling (via EVs) in the setting of neuroinflammation and thus provides possibilities for biomarker monitoring and targeting of ARF6.

Review of Lambda Interferons in Hepatitis B Virus Infection: Outcomes and Therapeutic Strategies.

Hepatitis B virus (HBV) chronically infects over 250 million people worldwide and causes nearly 1 million deaths per year due to cirrhosis and liver cancer. Approved treatments for chronic infection include injectable type-I interferons and nucleos(t)ide reverse transcriptase inhibitors. A small minority of patients achieve seroclearance after treatment with type-I interferons, defined as sustained absence of detectable HBV DNA and surface antigen (HBsAg) antigenemia. However, type-I interferons cause significant side effects, are costly, must be administered for months, and most patients have viral rebound or non-response. Nucleos(t)ide reverse transcriptase inhibitors reduce HBV viral load and improve liver-related outcomes, but do not lower HBsAg levels or impart seroclearance. Thus, new therapeutics are urgently needed. Lambda interferons (IFNLs) have been tested as an alternative strategy to stimulate host antiviral pathways to treat HBV infection. IFNLs comprise an evolutionarily conserved innate immune pathway and have cell-type specific activity on hepatocytes, other epithelial cells found at mucosal surfaces, and some immune cells due to restricted cellular expression of the IFNL receptor. This article will review work that examined expression of IFNLs during acute and chronic HBV infection, the impact of IFNLs on HBV replication in vitro and in vivo, the association of polymorphisms in IFNL genes with clinical outcomes, and the therapeutic evaluation of IFNLs for the treatment of chronic HBV infection.

TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice.

Mice reconstituted with a human immune system (humanized mice) provide a robust model to study human immunology, vaccinology, and human infectious diseases. However, the development and function of B cells in humanized mice is impaired. B cells from humanized mice are immature and are impaired in IgM to IgG isotype switch in response to infection or vaccination. In the present study we report that Toll-like receptor 9 (TLR9) agonist CpG-B combined with CD40-targeting vaccination triggered human B cell immunoglobin class-switch from IgM+ to IgG+ B cells in humanized mice. Human B cells from mice vaccinated with CpG-B as adjuvant were more mature in phenotype and produced significant levels of both total IgG and antigen-specific IgG. We found that CpG-B treatment activated human pDCs (plasmacytoid dendritic cells) in vivo to induce interferon-alpha (IFN-α)expression in humanized mice. Pre-depletion of human pDC in vivo abrogated the adjuvant effect of CpG-B. Our results indicate that TLR9 and CD40-targeting vaccination triggers human B cell maturation and immunoglobulin class-switch in a pDC-dependent manner in humanized mice. The findings also shed light on induction of human IgG antibodies in humanized mouse models.

Phenotypic and Functional Study of Human Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) are a distinct lineage of bone-marrow-derived cells that reside mainly in blood and lymphoid organs in the steady state but are also present in sites of infection, inflammation, and cancer. The protocols in this article describes (1) detection and quantification of human pDCs in peripheral blood; (2) isolation of human pDCs by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS); (3) evaluation of human pDC function by stimulation with TLR7 or TLR9 agonists; (4) detection of human pDCs in lymphoid tissues of humanized mice (hu-mice) by flow cytometry; (5) functional study of human pDC in hu-mice in vivo; and (6) specific depletion of human pDCs in vivo in hu-mice using monoclonal antibody targeting human pDCs. These assays thus provide comprehensive methods for phenotypic and functional studies in vitro and for the investigation of human plasmacytoid dendritic cells in hu-mice in vivo. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Analysis of pDCs in human peripheral blood mononuclear cells Basic Protocol 2: pDC separation using MACS beads Alternate Protocol 1: pDC sorting using flow cytometer Basic Protocol 3: Evaluation of human pDC function by stimulation with TLR agonists in vitro Alternate Protocol 2: Intracellular staining of cytokines in pDCs Basic Protocol 4: Phenotypic analysis of human pDCs from lymphoid organs in humanized mice Basic Protocol 5: Functional study of human pDCs in humanized mice during HIV infection Basic Protocol 6: pDC depletion and assessment of pDC depletion in acute HIV-infected in humanized mice.

Multi-omics analyses reveal that HIV-1 alters CD4+ T cell immunometabolism to fuel virus replication.

Individuals infected with human immunodeficiency virus type-1 (HIV-1) show metabolic alterations of CD4+ T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4+ T cells from patients with HIV-1 and revealed that the elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the US Food and Drug Administration-approved drug metformin, which targets mitochondrial respiratory chain complex-I, suppresses HIV-1 replication in human CD4+ T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4+ T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein FASTKD5 to promote expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4+ T cells as a target for HIV-1 therapy.

Preclinical Evaluation of B7-H3-specific Chimeric Antigen Receptor T Cells for the Treatment of Acute Myeloid Leukemia.

PURPOSE: The development of safe and effective chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has largely been limited by the concomitant expression of most AML-associated surface antigens on normal myeloid progenitors and by the potential prolonged disruption of normal hematopoiesis by the immunotargeting of these antigens. The purpose of this study was to evaluate B7-homolog 3 (B7-H3) as a potential target for AML-directed CAR T-cell therapy. B7-H3, a coreceptor belonging to the B7 family of immune checkpoint molecules, is overexpressed on the leukemic blasts of a significant subset of patients with AML and may overcome these limitations as a potential target antigen for AML-directed CAR-T therapy. EXPERIMENTAL DESIGN: B7-H3 expression was evaluated on AML cell lines, primary AML blasts, and normal bone marrow progenitor populations. The antileukemia efficacy of B7-H3-specific CAR-T cells (B7-H3.CAR-T) was evaluated using in vitro coculture models and xenograft models of disseminated AML, including patient-derived xenograft models. The potential hematopoietic toxicity of B7-H3.CAR-Ts was evaluated in vitro using colony formation assays and in vivo in a humanized mouse model. RESULTS: B7-H3 is expressed on monocytic AML cell lines and on primary AML blasts from patients with monocytic AML, but is not significantly expressed on normal bone marrow progenitor populations. B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity. CONCLUSIONS: B7-H3 is a promising target for AML-directed CAR-T therapy. B7-H3.CAR-Ts control AML and have a favorable safety profile in preclinical models.