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Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca-1) cells to reconstitute aged BM and rejuvenate the aged heart,and examined the underlying molecular mechanisms. BM Sca-1+ or Sca-1- cells from young (2-3 months) or aged (18-19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca-1+,young Sca-1-,old Sca-1+,and old Sca-1- . Four months later,expression of rejuvenation-related genes (Bmi1,Cbx8,PNUTS,Sirt1,Sirt2,Sirt6) and proteins (CDK2,CDK4) was increased along with telomerase activity and telomerase-related protein (DNA-PKcs,TRF-2) expression,whereas expression of senescence-related genes (p16INK4a,P19ARF,p27Kip1 ) and proteins (p16INK4a,p27Kip1 ) was decreased in Sca-1+ chimeric hearts,especially in the young group. Host cardiac endothelial cells (GFP- CD31+ ) but not cardiomyocytes were the primary cell type rejuvenated by young Sca-1+ cells as shown by improved proliferation,migration,and tubular formation abilities. C-X-C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP+ ) cells isolated from young Sca-1+ chimeric hearts. Protein expression of Cxcr4,phospho-Akt,and phospho-FoxO3a in endothelial cells derived from the aged chimeric heart was increased,especially in the young Sca-1+ group. Reconstitution of aged BM with young Sca-1+ cells resulted in effective homing of functional stem cells in the aged heart. These young,regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells. View Publication

Both targeted therapy and immunotherapy have been used successfully to treat melanoma,but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles,but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein,we describe the development and characterization of a novel,immunogenic variant of the BrafV600ECdkn2a-/-Pten-/- YUMM1.1 tumor model that expresses the immunogen,ovalbumin (YOVAL1.1). We demonstrate that,unlike parental tumors,YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly,YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK,responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable,immunogenic and sensitive to clinical therapies,making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma. View Publication

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM),a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation,we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast,in ML-DS,clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model,we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically,genetically,and transcriptionally mirrored ML-DS. View Publication

Rapidly proliferating cells are highly sensitive to ionizing radiation and can undergo apoptosis if the oxidative and genotoxic injury exceed the defensive and regenerative capacity of the cell. Our earlier work has established the antiapoptotic action of the growth factor-like lipid mediator lysophosphatidic acid (LPA). Activation of the LPA2 GPCR has been hypothesized to elicit antiapoptotic and regenerative actions of LPA. Based on this hypothesis we developed a novel nonlipid agonist of LPA2,which we designated Radioprotectin-1 (RP-1). We tested RP-1 at the six murine LPA GPCR subtypes using the transforming growth factor alpha shedding assay and found that it had a 25 nM EC50 that is similar to that of LPA18:1 at 32 nM. RP-1 effectively reduced apoptosis induced by gamma-irradiation and the radiomimetic drug Adriamycin only in cells that expressed LPA2 either endogenously or after transfection. RP-1 reduced gamma-H2AX levels in irradiated mouse embryonic fibroblasts transduced with the human LPA2 GPCR but was ineffective in vector transduced MEF control cells and significantly increased clonogenic survival after gamma-irradiation. gamma-Irradiation induced the expression of lpar2 transcripts that was further enhanced by RP-1 exposure within 30 min after irradiation. RP-1 decreased the mortality of C57BL/6 mice in models of the hematopoietic and gastrointestinal acute radiation syndromes. Using Lgr5-EGFP-CreER;Tdtomatoflox transgenic mice,we found that RP-1 increased the survival and growth of intestinal enteroids via the enhanced survival of Lgr5+ intestinal stem cells. Taken together,our results suggest that the LPA2-specific agonist RP-1 exerts its radioprotective and radiomitigative action through specific activation of the upregulated LPA2 GPCR in Lgr5+ stem cells. View Publication

Deregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is found in cancer with STAT5A/B controlling leukemic cell survival and disease progression. As mutations in STAT5B,but not STAT5A,have been frequently described in hematopoietic tumors,we used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The absence of STAT5A decreased cell survival and colony formation. Even more drastic effects were observed in the absence of STAT5B. STAT5B-deficient cells formed BCR/ABL+ colonies or stable cell lines at low frequency. The rarely evolving Stat5b-/- cell lines expressed enhanced levels of BCR/ABL oncoprotein compared to wild-type cells. In line,Stat5b-/- leukemic cells induced leukemia with a significantly prolonged disease onset,whereas Stat5a-/- cells rapidly caused a fatal disease superimposable to wild-type cells. RNA-sequencing (RNA-seq) profiling revealed a marked enhancement of interferon (IFN)-alpha and IFN-gamma signatures in Stat5b-/- cells. Inhibition of IFN responses rescued BCR/ABL+ colony formation of Stat5b-/--deficient cells. A downregulated IFN response was also observed in patients suffering from leukemia carrying STAT5B mutations. Our data define STAT5B as major STAT5 isoform driving BCR/ABL+ leukemia. STAT5B enables transformation by suppressing IFN-alpha/gamma,thereby facilitating leukemogenesis. Our findings might help explain the high frequency of STAT5B mutations in hematopoietic tumors. View Publication

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer,early events of tumorigenesis have already taken place. Similarly,development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article,we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model,human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1),referred to as NPM1c. Following engraftment into immunodeficient mice,transduced HSPCs give rise to human myeloid leukemia,whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML,with CD123+ leukemic stem or initiating cells (LSC),resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics. View Publication

Human pluripotent stem cell (hPSC)-derived intestinal organoids (hIOs) form 3D structures organized into crypt and villus domains,making them an excellent in vitro model system for studying human intestinal development and disease. However,hPSC-derived hIOs still require in vivo maturation to fully recapitulate adult intestine,with the mechanism of maturation remaining elusive. Here,we show that the co-culture with human T lymphocytes induce the in vitro maturation of hIOs,and identify STAT3-activating interleukin-2 (IL-2) as the major factor inducing maturation. hIOs exposed to IL-2 closely mimic the adult intestinal epithelium and have comparable expression levels of mature intestinal markers,as well as increased intestine-specific functional activities. Even after in vivo engraftment,in vitro-matured hIOs retain their maturation status. The results of our study demonstrate that STAT3 signaling can induce the maturation of hIOs in vitro,thereby circumventing the need for animal models and in vivo maturation. View Publication

In the inner ear,endolymph fluid surrounds the organ of Corti,which is important for auditory function; notably,even slight environmental changes mediated by trauma or infection can have significant consequences. However,it is unclear how the immune response is modulated in these tissues. Here,we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C,Cochlin,and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection,the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly,hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss. View Publication

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5,originally isolated from a NOD mouse,has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy,leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge,this work is the first to use a hybrid insulin peptide,or any neoepitope,to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients. View Publication

Background/Aim Although IL-6-mediated activation of the signal transduction and activator of transcription 3 (STAT3) axis is involved in inflammation and cancer,the role of STAT3 in Helicobacter-associated gastric inflammation and carcinogenesis is unclear. This study investigated the role of STAT3 in gastric inflammation and carcinogenesis and examined the molecular mechanism of Helicobacter-induced gastric phenotypes. Methods To evaluate the contribution of STAT3 to gastric inflammation and carcinogenesis,we used wild-type (WT) and gastric epithelial conditional Stat3-knockout (Stat3Deltagec ) mice. Mice were infected with Helicobacter felis and euthanized at 18 months postinfection. Mouse gastric organoids were treated with recombinant IL-6 (rIL-6) or rIL-11 and a JAK inhibitor (JAKi) to assess the role of IL-6/STAT3 signaling in vitro. Results Inflammation and mucous metaplasia were more severe in WT mice than in Stat3Deltagec mice. The epithelial cell proliferation rate and STAT3 activation were increased in WT mice. Application of rIL-6 and rIL-11 induced expression of intestinal metaplasia-associated genes,such as Tff2; this induction was suppressed by JAKi administration. Conclusions Loss of STAT3 signaling in the gastric mucosa leads to decreased epithelial cell proliferation,atrophy,and metaplasia in the setting of Helicobacter infection. Therefore,activation of STAT3 signaling may play a key role in Helicobacter-associated gastric carcinogenesis. View Publication

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis,correlating with a decrease of FOXP3+RORgammat+ T cells proportion,dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly,FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon,despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1-/- mice transferred with na{\{i}}ve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions." View Publication

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis,with controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult,TP53 mutated; NPM1 mutated; KMT2A mutated/rearranged; adult,DDX41 mutated; and pediatric,NUP98 rearranged. Genomic features influenced outcome,with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis and TP53,FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45{\%} of cases and included recurrent mutations of ALK and NTRK1,the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease,and the rationale for testing targeted therapies in this high-risk leukemia. View Publication