Publications by authors named "Leonard D Shultz"

215 Publications

Combined inhibition of XIAP and BCL2 drives maximal therapeutic efficacy in genetically diverse aggressive acute myeloid leukemia.

Nat Cancer 2021 03 18;2(3):340-356. Epub 2021 Mar 18.

Laboratory for Human Disease Models, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.

Aggressive therapy-resistant and refractory acute myeloid leukemia (AML) has an extremely poor outcome. By analyzing a large number of genetically complex and diverse, primary high-risk poor-outcome human AML samples, we identified specific pathways of therapeutic vulnerability. Through drug screens followed by extensive in vivo validation and genomic analyses, we found inhibition of cytosolic and mitochondrial anti-apoptotic proteins XIAP, BCL2 and MCL1, and a key regulator of mitosis, AURKB, as a vulnerability hub based on patient-specific genetic aberrations and transcriptional signatures. Combinatorial therapeutic inhibition of XIAP with an additional patient-specific vulnerability eliminated established AML in vivo in patient-derived xenografts (PDXs) bearing diverse genetic aberrations, with no signs of recurrence during off-treatment follow-up. By integrating genomic profiling and drug-sensitivity testing, this work provides a platform for a precision-medicine approach for treating aggressive AML with high unmet need.
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http://dx.doi.org/10.1038/s43018-021-00177-wDOI Listing
March 2021

Circulating CD34+ cells of primary myelofibrosis patients contribute to myeloid-dominant hematopoiesis and bone marrow fibrosis in immunodeficient mice.

Int J Hematol 2022 Feb 13;115(2):198-207. Epub 2021 Nov 13.

Laboratory for Human Disease Models, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.

Introduction: Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how these aberrant myeloid cells and fibroblasts are produced remains unclear.

Aim And Methods: In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination.

Results: Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms.

Conclusion: A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.
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http://dx.doi.org/10.1007/s12185-021-03239-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8905546PMC
February 2022

Novel phosphatidylserine-binding molecule enhances antitumor T-cell responses by targeting immunosuppressive exosomes in human tumor microenvironments.

J Immunother Cancer 2021 10;9(10)

Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York, USA

Background: The human tumor microenvironment (TME) is a complex and dynamic milieu of diverse acellular and cellular components, creating an immunosuppressive environment, which contributes to tumor progression. We have previously shown that phosphatidylserine (PS) expressed on the surface of exosomes isolated from human TMEs is causally linked to T-cell immunosuppression, representing a potential immunotherapeutic target. In this study, we investigated the effect of ExoBlock, a novel PS-binding molecule, on T-cell responses in the TME.

Methods: We designed and synthesized a new compound, (ZnDPA)-DP-15K, a multivalent PS binder named ExoBlock. The PS-binding avidity of ExoBlock was tested using an in vitro competition assay. The ability of this molecule to reverse exosome-mediated immunosuppression in vitro was tested using human T-cell activation assays. The in vivo therapeutic efficacy of ExoBlock was then tested in two different human tumor xenograft models, the melanoma-based xenomimetic (X-)mouse model, and the ovarian tumor-based omental tumor xenograft (OTX) model.

Results: ExoBlock was able to bind PS with high avidity and was found to consistently and significantly block the immunosuppressive activity of human ovarian tumor and melanoma-associated exosomes in vitro. ExoBlock was also able to significantly enhance T cell-mediated tumor suppression in vivo in both the X-mouse and the OTX model. In the X-mouse model, ExoBlock suppressed tumor recurrence in a T cell-dependent manner. In the OTX model, ExoBlock treatment resulted in an increase in the number as well as function of CD4 and CD8 T cells in the TME, which was associated with a reduction in tumor burden and metastasis, as well as in the number of circulating PS+ exosomes in tumor-bearing mice.

Conclusion: Our results establish that targeting exosomal PS in TMEs with ExoBlock represents a promising strategy to enhance antitumor T-cell responses.
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http://dx.doi.org/10.1136/jitc-2021-003148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8488709PMC
October 2021

Role of MicroRNA in Inflammatory Bowel Disease: Clinical Evidence and the Development of Preclinical Animal Models.

Cells 2021 08 26;10(9). Epub 2021 Aug 26.

The Jackson Laboratory, Bar Harbor, ME 04609, USA.

The dysregulation of microRNA (miRNA) is implicated in cancer, inflammation, cardiovascular disorders, drug resistance, and aging. While most researchers study miRNA's role as a biomarker, for example, to distinguish between various sub-forms or stages of a given disease of interest, research is also ongoing to utilize these small nucleic acids as therapeutics. An example of a common pleiotropic disease that could benefit from miRNA-based therapeutics is inflammatory bowel disease (IBD), which is characterized by chronic inflammation of the small and large intestines. Due to complex interactions between multiple factors in the etiology of IBD, development of therapies that effectively maintain remission for this disease is a significant challenge. In this review, we discuss the role of dysregulated miRNA expression in the context of clinical ulcerative colitis (UC) and Crohn's disease (CD)-the two main forms of IBD-and the various preclinical mouse models of IBD utilized to validate the therapeutic potential of targeting these miRNA. Additionally, we highlight advances in the development of genetically engineered animal models that recapitulate clinical miRNA expression and provide powerful preclinical models to assess the diagnostic and therapeutic promise of miRNA in IBD.
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http://dx.doi.org/10.3390/cells10092204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8468560PMC
August 2021

Inactive rhomboid proteins RHBDF1 and RHBDF2 (iRhoms): a decade of research in murine models.

Mamm Genome 2021 12 3;32(6):415-426. Epub 2021 Sep 3.

The Jackson Laboratory, Bar Harbor, ME, USA.

Rhomboid proteases, first discovered in Drosophila, are intramembrane serine proteases. Members of the rhomboid protein family that are catalytically deficient are known as inactive rhomboids (iRhoms). iRhoms have been implicated in wound healing, cancer, and neurological disorders such as Alzheimer's and Parkinson's diseases, inflammation, and skin diseases. The past decade of mouse research has shed new light on two key protein domains of iRhoms-the cytosolic N-terminal domain and the transmembrane dormant peptidase domain-suggesting new ways to target multiple intracellular signaling pathways. This review focuses on recent advances in uncovering the unique functions of iRhom protein domains in normal growth and development, growth factor signaling, and inflammation, with a perspective on future therapeutic opportunities.
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http://dx.doi.org/10.1007/s00335-021-09910-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8580931PMC
December 2021

A novel clinically relevant graft-versus-leukemia model in humanized mice.

J Leukoc Biol 2022 02 31;111(2):427-437. Epub 2021 May 31.

Penn State Cancer Institute, Penn State University College of Medicine, Hershey, Pennsylvania, USA.

The prognosis for acute myeloid leukemia (AML) relapse post allogeneic hematopoietic stem cell transplantation (alloSCT) is dismal. Novel effective treatment is urgently needed. Clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. The mechanisms that mediate immune escape of leukemia (thus causing GVL failure) remain poorly understood. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. Here, using our large, longitudinal clinical tissue bank that include AML cells and G-CSF mobilized donor hematopoietic stem cells (HSCs), we successfully established a novel GVL model in humanized mice. Donor HSCs were injected into immune-deficient NOD-Cg-Prkdc IL2rg /SzJ (NSG) mice to build humanized mice. Immune reconstitution in these mice recapitulated some clinical scenario in the patient who received the corresponding HSCs. Allogeneic but HLA partially matched patient-derived AML cells were successfully engrafted in these humanized mice. Importantly, we observed a significantly reduced (yet incomplete elimination of) leukemia growth in humanized mice compared with that in control NSG mice, demonstrating a functional (but defective) GVL effect. Thus, for the first time, we established a novel humanized mouse model that can be used for studying human GVL responses against human AML cells in vivo. This novel clinically relevant model provides a valuable platform for investigating the mechanisms of human GVL and development of effective leukemia treatments.
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http://dx.doi.org/10.1002/JLB.5AB0820-542RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8922387PMC
February 2022

Targeting critical kinases and anti-apoptotic molecules overcomes steroid resistance in MLL-rearranged leukaemia.

EBioMedicine 2021 Feb 10;64:103235. Epub 2021 Feb 10.

Laboratory for Human Disease Models, RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Electronic address:

Background: Acute lymphoblastic leukaemia with mixed lineage leukaemia gene rearrangement (MLL-ALL) frequently affects infants and is associated with a poor prognosis. Primary refractory and relapsed disease due to resistance to glucocorticoids (GCs) remains a substantial hurdle to improving clinical outcomes. In this study, we aimed to overcome GC resistance of MLL-ALL.

Methods: Using leukaemia patient specimens, we performed bioinformatic analyses to identify target genes/pathways. To test inhibition of target pathways in vivo, we created pre-clinical therapeutic mouse patient-derived xenograft (PDX)-models by transplanting human MLL-ALL leukaemia initiating cells (LIC) into immune-deficient NSG mice. Finally, we conducted B-cell lymphoma-2 (BCL-2) homology domain 3 (BH3) profiling to identify BH3 peptides responsible for treatment resistance in MLL-leukaemia.

Findings: Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathway were over-represented in MLL-ALL cells. PDX-models of infant MLL- ALL recapitulated GC-resistance in vivo but RK-20449, an inhibitor of SFKs and FLT3 eliminated human MLL-ALL cells in vivo, overcoming GC-resistance. Further, we identified BCL-2 dependence as a mechanism of treatment resistance in MLL-ALL through BH3 profiling. Furthermore, MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic BCL-2 protein for their survival. Combined inhibition of SFKs/FLT3 by RK-20449 and of BCL-2 by ABT-199 led to substantial elimination of MLL-ALL cells in vitro and in vivo. Triple treatment combining GCs, RK-20449 and ABT-199 resulted in complete elimination of MLL-ALL cells in vivo.

Interpretation: SFKs/FLT3 signaling pathways are promising targets for treatment of treatment-resistant MLL-ALL. Combined inhibition of these kinase pathways and anti-apoptotic BCL-2 successfully eliminated highly resistant MLL-ALL and demonstrated a new treatment strategy for treatment-resistant poor-outcome MLL-ALL.

Funding: This study was supported by RIKEN (RIKEN President's Discretionary Grant) for FI, Japan Agency for Medical Research and Development (the Basic Science and Platform Technology Program for Innovative Biological Medicine for FI and by NIH CA034196 for LDS. The funders had no role in the study design, data collection, data analysis, interpretation nor writing of the report.
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http://dx.doi.org/10.1016/j.ebiom.2021.103235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878180PMC
February 2021

Preclinical evaluation of cancer immune therapy using patient-derived tumor antigen-specific T cells in a novel xenograft platform.

Clin Transl Immunology 2021 2;10(2):e1246. Epub 2021 Feb 2.

Department of Microbiology and Immunology at the Jacobs School of Medicine and Biomedical Sciences University at Buffalo Buffalo NY USA.

Objectives: With a rapidly growing list of candidate immune-based cancer therapeutics, there is a critical need to generate highly reliable animal models to preclinically evaluate the efficacy of emerging immune-based therapies, facilitating successful clinical translation. Our aim was to design and validate a novel model (called Xenomimetic or 'X' mouse) that allows monitoring of the ability of human tumor-specific T cells to suppress tumor growth following their entry into the tumor.

Methods: Tumor xenografts are established rapidly in the greater omentum of globally immunodeficient NOD- (NSG) mice following an intraperitoneal injection of melanoma target cells expressing tumor neoantigen peptides, as well as green fluorescent protein and/or luciferase. Changes in tumor burden, as well as in the number and phenotype of adoptively transferred patient-derived tumor neoantigen-specific T cells in response to immunotherapy, are measured by imaging to detect fluorescence/luminescence and flow cytometry, respectively.

Results: The tumors progress rapidly and disseminate in the mice unless patient-derived tumor-specific T cells are introduced. An initial T cell-mediated tumor arrest is later followed by a tumor escape, which correlates with the upregulation of the checkpoint molecules programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on T cells. Treatment with immune-based therapies that target these checkpoints, such as anti-PD-1 antibody (nivolumab) or interleukin-12 (IL-12), prevented or delayed the tumor escape. Furthermore, IL-12 treatment suppressed PD-1 and LAG3 upregulation on T cells.

Conclusion: Together, these results validate the X-mouse model and establish its potential to preclinically evaluate the therapeutic efficacy of immune-based therapies.
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http://dx.doi.org/10.1002/cti2.1246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853904PMC
February 2021

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cell 2020 11;183(5):1219-1233.e18

Transplantation Research Center, Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.
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http://dx.doi.org/10.1016/j.cell.2020.10.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927154PMC
November 2020

Lung mesenchymal cells elicit lipid storage in neutrophils that fuel breast cancer lung metastasis.

Nat Immunol 2020 11 21;21(11):1444-1455. Epub 2020 Sep 21.

The Jackson Laboratory, Bar Harbor, ME, USA.

Acquisition of a lipid-laden phenotype by immune cells has been defined in infectious diseases and atherosclerosis but remains largely uncharacterized in cancer. Here, in breast cancer models, we found that neutrophils are induced to accumulate neutral lipids upon interaction with resident mesenchymal cells in the premetastatic lung. Lung mesenchymal cells elicit this process through repressing the adipose triglyceride lipase (ATGL) activity in neutrophils in prostaglandin E2-dependent and -independent manners. In vivo, neutrophil-specific deletion of genes encoding ATGL or ATGL inhibitory factors altered neutrophil lipid profiles and breast tumor lung metastasis in mice. Mechanistically, lipids stored in lung neutrophils are transported to metastatic tumor cells through a macropinocytosis-lysosome pathway, endowing tumor cells with augmented survival and proliferative capacities. Pharmacological inhibition of macropinocytosis significantly reduced metastatic colonization by breast tumor cells in vivo. Collectively, our work reveals that neutrophils serve as an energy reservoir to fuel breast cancer lung metastasis.
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http://dx.doi.org/10.1038/s41590-020-0783-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584447PMC
November 2020

Dual roles of neutrophils in metastatic colonization are governed by the host NK cell status.

Nat Commun 2020 09 1;11(1):4387. Epub 2020 Sep 1.

The Jackson Laboratory, Bar Harbor, ME, 04609, USA.

The role of neutrophils in solid tumor metastasis remains largely controversial. In preclinical models of solid tumors, both pro-metastatic and anti-metastatic effects of neutrophils have been reported. In this study, using mouse models of breast cancer, we demonstrate that the metastasis-modulating effects of neutrophils are dictated by the status of host natural killer (NK) cells. In NK cell-deficient mice, granulocyte colony-stimulating factor-expanded neutrophils show an inhibitory effect on the metastatic colonization of breast tumor cells in the lung. In contrast, in NK cell-competent mice, neutrophils facilitate metastatic colonization in the same tumor models. In an ex vivo neutrophil-NK cell-tumor cell tri-cell co-culture system, neutrophils are shown to potentially suppress the tumoricidal activity of NK cells, while neutrophils themselves are tumoricidal. Intriguingly, these two modulatory effects by neutrophils are both mediated by reactive oxygen species. Collectively, the absence or presence of NK cells, governs the net tumor-modulatory effects of neutrophils.
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http://dx.doi.org/10.1038/s41467-020-18125-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463263PMC
September 2020

A rapid, sensitive, and reproducible in vivo PBMC humanized murine model for determining therapeutic-related cytokine release syndrome.

FASEB J 2020 09 9;34(9):12963-12975. Epub 2020 Aug 9.

The Jackson Laboratory, Sacramento, CA, USA.

Immunotherapy is a powerful treatment strategy being applied to cancer, autoimmune diseases, allergies, and transplantation. Although therapeutic monoclonal antibodies (mAbs) have demonstrated significant clinical efficacy, there is also the potential for severe adverse events, including cytokine release syndrome (CRS). CRS is characterized by the rapid production of inflammatory cytokines following delivery of therapy, with symptoms ranging from mild fever to life-threating pathology and multi-organ failure. Overall there is a paucity of models to reliably and accurately predict the induction of CRS by immune therapeutics. Here, we describe the development of a humanized mouse model based on the NOD-scid IL2rg (NSG) mouse to study CRS in vivo. PBMC-engrafted NSG, NSG-MHC-DKO, and NSG-SGM3 mice were used to study cytokine release in response to treatment with mAb immunotherapies. Our data show that therapeutic-stimulated cytokine release in these PBMC-based NSG models captures the variation in cytokine release between individual donors, is drug dependent, occurs in the absence of acute xeno-GVHD, highlighting the specificity of the assay, and shows a robust response following treatment with a TGN1412 analog, a CD28 superagonist. Overall our results demonstrate that PBMC-engrafted NSG models are rapid, sensitive, and reproducible platforms to screen novel therapeutics for CRS.
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http://dx.doi.org/10.1096/fj.202001203RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436391PMC
September 2020

Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation.

Genome Biol 2020 07 9;21(1):168. Epub 2020 Jul 9.

The Jackson Laboratory for Mammalian Genetics, Bar Harbor, ME, USA.

Background: Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies-definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9.

Results: We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes.

Conclusions: Exon skipping is a widespread phenomenon occurring across the genome. These findings have significant implications for the application of genome editing in both basic research and clinical practice.
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http://dx.doi.org/10.1186/s13059-020-02086-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350591PMC
July 2020

Understanding Normal and Malignant Human Hematopoiesis Using Next-Generation Humanized Mice.

Trends Immunol 2020 08 3;41(8):706-720. Epub 2020 Jul 3.

RIKEN Center for Integrative Medical Sciences, Yokohama City, Kanagawa, 230-0045, Japan. Electronic address:

Rodent models for human diseases contribute significantly to understanding human physiology and pathophysiology. However, given the accelerating pace of drug development, there is a crucial need for in vivo preclinical models of human biology and pathology. The humanized mouse is one tool to bridge the gap between traditional animal models and the clinic. The development of immunodeficient mouse strains with high-level engraftment of normal and diseased human immune/hematopoietic cells has made in vivo functional characterization possible. As a patient-derived xenograft (PDX) model, humanized mice functionally correlate putative mechanisms with in vivo behavior and help to reveal pathogenic mechanisms. Combined with single-cell genomics, humanized mice can facilitate functional precision medicine such as risk stratification and individually optimized therapeutic approaches.
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http://dx.doi.org/10.1016/j.it.2020.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395895PMC
August 2020

Dapagliflozin Does Not Directly Affect Human α or β Cells.

Endocrinology 2020 08;161(8)

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.
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http://dx.doi.org/10.1210/endocr/bqaa080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375801PMC
August 2020

Improved mouse models and advanced genetic and genomic technologies for the study of neutrophils.

Drug Discov Today 2020 06 5;25(6):1013-1025. Epub 2020 May 5.

The Jackson Laboratory for Mammalian Genetics, 600 Main Street, Bar Harbor, ME 04609 USA.

Mice have been excellent surrogates for studying neutrophil biology and, furthermore, murine models of human disease have provided fundamental insights into the roles of human neutrophils in innate immunity. The emergence of novel humanized mice and high-diversity mouse populations offers the research community innovative and powerful platforms for better understanding, respectively, the mechanisms by which human neutrophils drive pathogenicity, and how genetic differences underpin the variation in neutrophil biology observed among humans. Here, we review key examples of these new resources. Additionally, we provide an overview of advanced genetic engineering tools available to further improve such murine model systems, of sophisticated neutrophil-profiling technologies, and of multifunctional nanoparticle (NP)-based neutrophil-targeting strategies.
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http://dx.doi.org/10.1016/j.drudis.2020.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329609PMC
June 2020

TEG011 persistence averts extramedullary tumor growth without exerting off-target toxicity against healthy tissues in a humanized HLA-A*24:02 transgenic mice.

J Leukoc Biol 2020 06 5;107(6):1069-1079. Epub 2020 Feb 5.

Department of Hematology and Center for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α-dependent tumor-specific allo-HLA-A*24:02-restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αβT cells from HLA-A*24:02 harboring individuals. αβT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA-A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse model to allow the preparation of a first-in-men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft-versus-host disease-like symptoms and extensive analysis of pathologic changes in NSG-A24:02 mice did not show any off-target toxicity of TEG011. However, loss of persistence of TEG011 in tumor-bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock-treated mice. In conclusion, TEG011 is well tolerated without harming HLA-A*24:02 expressing healthy tissues, and TEG011 persistence seems to be crucial for long-term tumor control in vivo.
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http://dx.doi.org/10.1002/JLB.5MA0120-228RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722393PMC
June 2020

Tacrolimus- and sirolimus-induced human β cell dysfunction is reversible and preventable.

JCI Insight 2020 01 16;5(1). Epub 2020 Jan 16.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, and.

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of β cell mass. Interestingly, these β cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced β cell dysfunction and partially prevented SIR-induced β cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human β cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.
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http://dx.doi.org/10.1172/jci.insight.130770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030815PMC
January 2020

Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

FASEB J 2020 01 10;34(1):1901-1911. Epub 2019 Dec 10.

Diabetes Center of Excellence, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted β, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate β, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived β cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.
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http://dx.doi.org/10.1096/fj.201901022RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972551PMC
January 2020

Human iPSC-derived microglia assume a primary microglia-like state after transplantation into the neonatal mouse brain.

Proc Natl Acad Sci U S A 2019 12 26;116(50):25293-25303. Epub 2019 Nov 26.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142;

Microglia are essential for maintenance of normal brain function, with dysregulation contributing to numerous neurological diseases. Protocols have been developed to derive microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, primary microglia display major differences in morphology and gene expression when grown in culture, including down-regulation of signature microglial genes. Thus, in vitro differentiated microglia may not accurately represent resting primary microglia. To address this issue, we transplanted microglial precursors derived in vitro from hiPSCs into neonatal mouse brains and found that the cells acquired characteristic microglial morphology and gene expression signatures that closely resembled primary human microglia. Single-cell RNA-sequencing analysis of transplanted microglia showed similar cellular heterogeneity as primary human cells. Thus, hiPSCs-derived microglia transplanted into the neonatal mouse brain assume a phenotype and gene expression signature resembling that of resting microglia residing in the human brain, making chimeras a superior tool to study microglia in human disease.
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http://dx.doi.org/10.1073/pnas.1913541116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911218PMC
December 2019

Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells.

Nat Commun 2019 10 18;10(1):4730. Epub 2019 Oct 18.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada.

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.
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http://dx.doi.org/10.1038/s41467-019-12726-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802205PMC
October 2019

Genome-wide Analysis of Salmonella enterica serovar Typhi in Humanized Mice Reveals Key Virulence Features.

Cell Host Microbe 2019 09 22;26(3):426-434.e6. Epub 2019 Aug 22.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Global Health, University of Washington, Seattle, WA 98195, USA; Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address:

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, lipopolysaccharide (LPS), and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island was required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and non-typhoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.
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http://dx.doi.org/10.1016/j.chom.2019.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742556PMC
September 2019

AK002, a Humanized Sialic Acid-Binding Immunoglobulin-Like Lectin-8 Antibody that Induces Antibody-Dependent Cell-Mediated Cytotoxicity against Human Eosinophils and Inhibits Mast Cell-Mediated Anaphylaxis in Mice.

Int Arch Allergy Immunol 2019 9;180(2):91-102. Epub 2019 Aug 9.

Allakos, Inc., Redwood City, California, USA.

Introduction: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells.

Methods: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model.

Results: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition.

Conclusion: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.
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http://dx.doi.org/10.1159/000501637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878738PMC
November 2019

Humanized immune system mouse models: progress, challenges and opportunities.

Nat Immunol 2019 07;20(7):770-774

Division of Allergy, Immunology and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

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http://dx.doi.org/10.1038/s41590-019-0416-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265413PMC
July 2019

Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

J Immunother Cancer 2019 05 22;7(1):131. Epub 2019 May 22.

Rutgers University, New Brunswick, NJ, USA.

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host's response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual's recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019.
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http://dx.doi.org/10.1186/s40425-019-0602-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529999PMC
May 2019

Mesenchymal stem cells: From regeneration to cancer.

Pharmacol Ther 2019 08 16;200:42-54. Epub 2019 Apr 16.

The Jackson Laboratory, Bar Harbor, ME 04609, USA. Electronic address:

Mesenchymal stem cells (MSCs) are multipotent tissue stem cells that differentiate into a number of mesodermal tissue types, including osteoblasts, adipocytes, chondrocytes and myofibroblasts. MSCs were originally identified in the bone marrow (BM) of humans and other mammals, but recent studies have shown that they are multilineage progenitors in various adult organs and tissues. MSCs that localize at perivascular sites function to rapidly respond to external stimuli and coordinate with the vascular and immune systems to accomplish the wound healing process. Cancer, considered as wounds that never heal, is also accompanied by changes in MSCs that parallels the wound healing response. MSCs are now recognized as key players at distinct steps of tumorigenesis. In this review, we provide an overview of the function of MSCs in wound healing and cancer progression with the goal of providing insight into the development of novel MSC-manipulating strategies for clinical cancer treatment.
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http://dx.doi.org/10.1016/j.pharmthera.2019.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626571PMC
August 2019

Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice.

Life Sci Alliance 2019 04 1;2(2). Epub 2019 Apr 1.

Laboratory for Human Disease Models, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.
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http://dx.doi.org/10.26508/lsa.201800195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445396PMC
April 2019

Humanized mouse models of immunological diseases and precision medicine.

Mamm Genome 2019 06 7;30(5-6):123-142. Epub 2019 Mar 7.

Diabetes Center of Excellence, The University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA, 01605, USA.

With the increase in knowledge resulting from the sequencing of the human genome, the genetic basis for the underlying differences in individuals, their diseases, and how they respond to therapies is starting to be understood. This has formed the foundation for the era of precision medicine in many human diseases that is beginning to be implemented in the clinic, particularly in cancer. However, preclinical testing of therapeutic approaches based on individual biology will need to be validated in animal models prior to translation into patients. Although animal models, particularly murine models, have provided significant information on the basic biology underlying immune responses in various diseases and the response to therapy, murine and human immune systems differ markedly. These fundamental differences may be the underlying reason why many of the positive therapeutic responses observed in mice have not translated directly into the clinic. There is a critical need for preclinical animal models in which human immune responses can be investigated. For this, many investigators are using humanized mice, i.e., immunodeficient mice engrafted with functional human cells, tissues, and immune systems. We will briefly review the history of humanized mice, the remaining limitations, approaches to overcome them and how humanized mouse models are being used as a preclinical bridge in precision medicine for evaluation of human therapies prior to their implementation in the clinic.
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http://dx.doi.org/10.1007/s00335-019-09796-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610695PMC
June 2019

Co-activation of macrophages and T cells contribute to chronic GVHD in human IL-6 transgenic humanised mouse model.

EBioMedicine 2019 Mar 13;41:584-596. Epub 2019 Feb 13.

Laboratory for Human Disease Models, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan. Electronic address:

Background: Graft-versus host disease (GVHD) is a complication of stem cell transplantation associated with significant morbidity and mortality. Non-specific immune-suppression, the mainstay of treatment, may result in immune-surveillance dysfunction and disease recurrence.

Methods: We created humanised mice model for chronic GVHD (cGVHD) by injecting cord blood (CB)-derived human CD34CD38CD45RA haematopoietic stem/progenitor cells (HSPCs) into hIL-6 transgenic NOD/SCID/Il2rgKO (NSG) newborns, and compared GVHD progression with NSG newborns receiving CB CD34 cells mimicking acute GVHD. We characterised human immune cell subsets, target organ infiltration, T-cell repertoire (TCR) and transcriptome in the humanised mice.

Findings: In cGVHD humanised mice, we found activation of T cells in the spleen, lung, liver, and skin, activation of macrophages in lung and liver, and loss of appendages in skin, obstruction of bronchioles in lung and portal fibrosis in liver recapitulating cGVHD. Acute GVHD humanised mice showed activation of T cells with skewed TCR repertoire without significant macrophage activation.

Interpretation: Using humanised mouse models, we demonstrated distinct immune mechanisms contributing acute and chronic GVHD. In cGVHD model, co-activation of human HSPC-derived macrophages and T cells educated in the recipient thymus contributed to delayed onset, multi-organ disease. In acute GVHD model, mature human T cells contained in the graft resulted in rapid disease progression. These humanised mouse models may facilitate future development of new molecular medicine targeting GVHD.
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http://dx.doi.org/10.1016/j.ebiom.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441951PMC
March 2019
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