IFI16-dependent STING signaling is a crucial regulator of anti-HER2 immune response in HER2+ breast cancer.

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2+ breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2+ BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2+, but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2+ BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2+ BC to confer resistance to trastuzumab treatment.

Preclinical Activity of Embryonic Annexin A2-Specific Chimeric Antigen Receptor T Cells Against Ovarian Cancer.

Chimeric antigen receptors (CARs) have found clinical success in B cell malignancies, but a dearth of potential targets limits their wider clinical application, especially in solid tumours. Here, we describe the development of an anti-annexin A2 CAR, CAR(2448), derived from an antibody found to have activity against epithelial ovarian cancer cell lines. The spacer length of CAR(2448) was optimised based on in vitro cytotoxic activity against ovarian cancer (OC) cell lines via a real-time cytotoxicity assay. The longer spacer CAR(2448)L T cells exhibit significant effector activity, inducing inflammatory cytokine release and cytotoxicity against OC cell lines. Furthermore, CAR(2448)L-BBz T cells induced enhanced survival in an in vivo OC xenograft model and reduced tumour volume by 76.6%. Our preclinical studies of CAR(2448) suggest its potential for the unmet need of novel strategies for the treatment of ovarian cancer.

Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection.

OBJECTIVE: HCV infection affects millions of people worldwide, and many patients develop chronic infection leading to liver cancers. For decades, the lack of a small animal model that can recapitulate HCV infection, its immunopathogenesis and disease progression has impeded the development of an effective vaccine and therapeutics. We aim to provide a humanised mouse model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. DESIGN: Recently, we have established human liver cells with a matched human immune system in NOD-scid Il2rg(-/-) (NSG) mice (HIL mice). These mice are infected with HCV by intravenous injection, and the pathologies are investigated. RESULTS: In this study, we demonstrate that HIL mouse is capable of supporting HCV infection and can present some of the clinical symptoms found in HCV-infected patients including hepatitis, robust virus-specific human immune cell and cytokine responses as well as liver fibrosis and cirrhosis. Similar to results obtained from the analysis of patient samples, the human immune cells, particularly T cells and macrophages, play critical roles during the HCV-associated liver disease development in the HIL mice. Furthermore, our model is demonstrated to be able to reproduce the therapeutic effects of human interferon alpha 2a antiviral treatment. CONCLUSIONS: The HIL mouse provides a model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies. It could also serve as a platform for antifibrosis and immune-modulatory drug testing.

IL-37 dampens immunosuppressive functions of MDSCs via metabolic reprogramming in the tumor microenvironment.

Interleukin-37 (IL-37) has been shown to inhibit tumor growth in various cancer types. However, the immune regulatory function of IL-37 in the tumor microenvironment is unclear. Here, we established a human leukocyte antigen-I (HLA-I)-matched humanized patient-derived xenograft hepatocellular carcinoma (HCC) model and three murine orthotopic HCC models to study the function of IL-37 in the tumor microenvironment. We found that IL-37 inhibited HCC growth and promoted T cell activation. Further study revealed that IL-37 impaired the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs). Pretreatment of MDSCs with IL-37 before adoptive transfer attenuated their tumor-promoting function in HCC tumor-bearing mice. Moreover, IL-37 promoted both glycolysis and oxidative phosphorylation in MDSCs, resulting in the upregulation of ATP release, which impaired the immunosuppressive capacity of MDSCs. Collectively, we demonstrated that IL-37 inhibited tumor development through dampening MDSCs' immunosuppressive capacity in the tumor microenvironment via metabolic reprogramming, making it a promising target for future cancer immunotherapy.

IL12/18/21 Preactivation Enhances the Antitumor Efficacy of Expanded γδT Cells and Overcomes Resistance to Anti-PD-L1 Treatment.

γδT cells are promising candidates for cellular immunotherapy due to their immune regulation through cytokine production and MHC-independent direct cytotoxicity against a broad spectrum of tumors. However, current γδT cell-based cancer immunotherapy has limited efficacy, and novel strategies are needed to improve clinical outcomes. Here, we report that cytokine pretreatment with IL12/18, IL12/15/18, IL12/18/21, and IL12/15/18/21 effectively enhanced the activation and cytotoxicity of in vitro-expanded murine and human γδT cells. However, only adoptive transfer of IL12/18/21 preactivated γδT cells significantly inhibited tumor growth in a murine melanoma model and a hepatocellular carcinoma model. Both IL12/18/21 preactivated antibody-expanded and zoledronate-expanded human γδT cells effectively controlled tumor growth in a humanized mouse model. IL12/18/21 preactivation promoted γδT cell proliferation and cytokine production in vivo and enhanced IFNγ production and activation of endogenous CD8+ T cells in a cell-cell contact- and ICAM-1-dependent manner. Furthermore, the adoptive transfer of IL12/18/21 preactivated γδT cells could overcome the resistance to anti-PD-L1 therapy, and the combination therapy had a synergistic effect on the therapeutic outcomes. Moreover, the enhanced antitumor function of adoptively transferred IL12/18/21 preactivated γδT cells was largely diminished in the absence of endogenous CD8+ T cells when administered alone or in combination with anti-PD-L1, suggesting a CD8+ T cell-dependent mechanism. Taken together, IL12/18/21 preactivation can promote γδT cell antitumor function and overcome the resistance to checkpoint blockade therapy, indicating an effective combinational cancer immunotherapeutic strategy.

Comparison of infection and human immune responses of two SARS-CoV-2 strains in a humanized hACE2 NIKO mouse model.

The COVID-19 pandemic has sickened millions, cost lives and has devastated the global economy. Various animal models for experimental infection with SARS-CoV-2 have played a key role in many aspects of COVID-19 research. Here, we describe a humanized hACE2 (adenovirus expressing hACE2) NOD-SCID IL2Rγ-/- (NIKO) mouse model and compare infection with ancestral and mutant (SARS-CoV-2-∆382) strains of SARS-CoV-2. Immune cell infiltration, inflammation, lung damage and pro-inflammatory cytokines and chemokines was observed in humanized hACE2 NIKO mice. Humanized hACE2 NIKO mice infected with the ancestral and mutant SARS-CoV-2 strain had lung inflammation and production of pro-inflammatory cytokines and chemokines. This model can aid in examining the pathological basis of SARS-CoV-2 infection in a human immune environment and evaluation of therapeutic interventions.

A humanized mouse model to study mast cells mediated cutaneous adverse drug reactions.

Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.

Humanized Mouse as a Tool to Predict Immunotoxicity of Human Biologics.

Advancements in science enable researchers to constantly innovate and create novel biologics. However, the use of non-human animal models during the development of biologics impedes identification of precise in vivo interactions between the human immune system and treatments. Due to lack of this understanding, adverse effects are frequently observed in healthy volunteers and patients exposed to potential biologics during clinical trials. In this study, we evaluated and compared the effects of known immunotoxic biologics, Proleukin®/IL-2 and OKT3 in humanized mice (reconstituted with human fetal cells) to published clinical outcomes. We demonstrated that humanized mice were able to recapitulate in vivo pathological changes and human-specific immune responses, such as elevated cytokine levels and modulated lymphocytes and myeloid subsets. Given the high similarities of immunological side effects observed between humanized mice and clinical studies, this model could be used to assess immunotoxicity of biologics at a pre-clinical stage, without placing research participants and/or patients at risk.

CD4+ T Cells Mediate the Development of Liver Fibrosis in High Fat Diet-Induced NAFLD in Humanized Mice.

Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4+ T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4+ central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ). In vivo depletion of human CD4+ T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4+ memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics.

Humanized Mouse Models for the Study of Hepatitis C and Host Interactions.

Hepatitis C virus (HCV) infection is commonly attributed as a major cause of chronic hepatotropic diseases, such as, steatosis, cirrhosis and hepatocellular carcinoma. As HCV infects only humans and primates, its narrow host tropism hampers in vivo studies of HCV-mammalian host interactions and the development of effective therapeutics and vaccines. In this context, we will focus our discussion on humanized mice in HCV research. Here, these humanized mice are defined as animal models that encompass either only human hepatocytes or both human liver and immune cells. Aspects related to immunopathogenesis, anti-viral interventions, drug testing and perspectives of these models for future HCV research will be discussed.

Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network.

Human pluripotent stem cell-derived kidney organoids recapitulate developmental processes and tissue architecture, but intrinsic limitations, such as lack of vasculature and functionality, have greatly hampered their application. Here we establish a versatile protocol for generating vascularized three-dimensional (3D) kidney organoids. We employ dynamic modulation of WNT signaling to control the relative proportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. Single-cell RNA sequencing identifies a subset of nephron progenitor cells as a potential source of renal vasculature. These kidney organoids undergo further structural and functional maturation upon implantation. Using this kidney organoid platform, we establish an in vitro model of autosomal recessive polycystic kidney disease (ARPKD), the cystic phenotype of which can be effectively prevented by gene correction or drug treatment. Our studies provide new avenues for studying human kidney development, modeling disease pathogenesis, and performing patient-specific drug validation.

Humanized Mice as Unique Tools for Human-Specific Studies.

With an increasing human population, medical research is pushed to progress into an era of precision therapy. Humanized mice are at the very heart of this new forefront where it is acutely required to decipher human-specific disease pathogenesis and test an array of novel therapeutics. In this review, "humanized" mice are defined as immunodeficient mouse engrafted with functional human biological systems. Over the past decade, researchers have been conscientiously making improvements on the development of humanized mice as a model to closely recapitulate disease pathogenesis and drug mechanisms in humans. Currently, literature is rife with descriptions of novel and innovative humanized mouse models that hold a significant promise to become a panacea for drug innovations to treat and control conditions such as infectious disease and cancer. This review will focus on the background of humanized mice, diseases, and human-specific therapeutics tested on this platform as well as solutions to improve humanized mice for future clinical use.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R-/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia.

BACKGROUND: Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct. METHODS: Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically. RESULTS: Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34+CD117+ subpopulation, CD34+CD117- subpopulation can acquire CD34+CD117+ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML. CONCLUSIONS: Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

Human CD34(lo)CD133(lo) fetal liver cells support the expansion of human CD34(hi)CD133(hi) hematopoietic stem cells.

We have recently discovered a unique CD34(lo)CD133(lo) cell population in the human fetal liver (FL) that gives rise to cells in the hepatic lineage. In this study, we further characterized the biological functions of FL CD34(lo)CD133(lo) cells. Our findings show that these CD34(lo)CD133(lo) cells express markers of both endodermal and mesodermal lineages and have the capability to differentiate into hepatocyte and mesenchymal lineage cells by ex vivo differentiation assays. Furthermore, we show that CD34(lo)CD133(lo) cells express growth factors that are important for human hematopoietic stem cell (HSC) expansion: stem cell factor (SCF), insulin-like growth factor 2 (IGF2), C-X-C motif chemokine 12 (CXCL12), and factors in the angiopoietin-like protein family. Co-culture of autologous FL HSCs and allogenic HSCs derived from cord blood with CD34(lo)CD133(lo) cells supports and expands both types of HSCs.These findings are not only essential for extending our understanding of the HSC niche during the development of embryonic and fetal hematopoiesis but will also potentially benefit adult stem cell transplantations in clinics because expanded HSCs demonstrate the same capacity as primary cells to reconstitute the human immune system and mediate long-term hematopoiesis in vivo. Together, CD34(lo)CD133(lo) cells not only serve as stem/progenitor cells for liver development but are also an essential component of the HSC niche in the human FL.