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Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast,T cells with other antigen specificities from these patients,or autoreactive T cells from healthy individuals and disease controls,express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells,Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2,SAP97,ZIP,p56(lck),and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation,they suppress Ca2+-signaling,cytokine production,and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted. View Publication

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation,efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved,while retaining their pluripotency. When added during the reprogramming process,CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus,CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication

Human coronaviruses (HCoVs) enter cells via two distinct pathways: the endosomal pathway using cathepsins to activate spike protein and the cell-surface or early endosome pathway using extracellular proteases such as transmembrane protease serine 2 (TMPRSS2). We previously reported that clinical isolates of HCoV-229E preferred cell-surface TMPRSS2 to endosomal cathepsin for cell entry,and that they acquired the ability to use cathepsin L by repeated passage in cultured cells and were then able to enter cells via the endosomal pathway. Here,we show that clinical isolates of HCoV-OC43 and -HKU1 preferred the cell-surface TMRRSS2 to endosomal cathepsins for cell entry,similar to HCoV-229E. In addition,the cell-culture-adapted HCoV-OC43 lost the ability to infect and replicate in air-liquid interface cultures of human bronchial tracheal epithelial cells. These results suggest that circulating HCoVs in the field generally use cell-surface TMPRSS2 for cell entry,not endosomal cathepsins,in human airway epithelial cells. View Publication

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of textgreater10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb,and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells. View Publication

Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI,while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore,we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H2O2-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed,which were subjected to exosome treatment. The expression of inflammatory factors,heart function,and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H2O2-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition,they promoted heart function,suppressed inflammatory responses,and de-activated TLR4/NF-$\kappa$B signaling pathway in MI mice. In conclusion,miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4. View Publication

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that,in most transplantation patients,variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype,as well as through phenotypic and functional analyses of NK cells,both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells,including patient leukemia blasts. Differently,KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly,this was due to recognition of C2 by KIR2DL2/3,as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably,however,C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether,these results may have important clinical implications for the selection of optimal donors for haplo-HSCT. View Publication

Physical frailty as a sign of accelerated aging is not well characterized in breast cancer (BC) and hematopoietic cell transplant (HCT) survivors and its correlation with outcomes and quality of life (QOL) is not defined. We conducted a prospective study to determine the prevalence of frailty in adult BC and HCT survivors,examine its impact on QOL,and determine its association with p16INK4a,a molecular biomarker for biological aging. The study included 59 BC and 65 HCT survivors. Median age was 60 years (range 27-81),68.5% were female and 49.2% were 18-59 vs. 51.8% ≥60 years old. A total of 71 (57.3%) were “fit” (frailty score 0) vs. 53 (42.7%) were pre-frailty/frail (frailty scores ≥1),and of the latter 17 (32.1%) were BC and 36 (67.9%) HCT patients. On multivariate analysis,patients >60 years were twice as likely to be frail (OR 2.04,95% CI,0.96-4.33; p=0.07),HCT were more likely to be frail compared to BC patients,and female HCT had 2.43 (95% CI,0.92-6.40) and male HCT patients had 3.25 (95% CI,1.37-7.72) times higher risk of frail; p=0.02. Frailty was associated with significant decline in QOL,measured by Medical Outcomes Study (MOS) Short Form 36 (SF-36) Physical Component Summary (PCS) and Mental Component Summary (MCS),and FACT (Functional Assessment of Cancer Therapy) scores. p16INK4a expression was higher in those who were frail,older than 60,and with higher expression in frail vs. fit patients who are 18-59 years. Our study highlights the high prevalence of frailty in survivors with detrimental effects on physical and overall wellbeing,and supports an association between frailty and the senescence marker p16INK4a. View Publication

It is advantageous to culture the ex vivo retina and other tissues at the air-liquid interface to allow for more efficient gas exchange. However,gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery,but typically requires submergence in liquid buffer for electrical current flow. We have developed a submergence-free electroporation technique that incorporates an agarose hydrogel disk between the positive electrode and retina. Inner retinal neurons and Müller glia are transfected with increased propensity toward Müller glia transfection after extended time in culture. We also observed an increase in BrdU incorporation in Müller glia following electrical stimulation,and variation in detection of transfected cells from expression vectors with different promoters. This method advances our ability to use ex vivo retinal tissue for genetic studies and should be adaptable for other tissues cultured at an air-liquid interface. Subject areas: Genetic engineering,Methodology in biological sciences,Bioelectrical engineering View Publication

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells,resulting in reversal of Treg suppression. Importantly,TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression,which may be used as a strategy to advance tumor immunotherapy. View Publication

Abnormal trophoblast self-renewal and differentiation during early gestation is the major cause of miscarriage,yet the underlying regulatory mechanisms remain elusive. Here,we show that trophoblast specific deletion of Kat8,a MYST family histone acetyltransferase,leads to extraembryonic ectoderm abnormalities and embryonic lethality. Employing RNA-seq and CUT&Tag analyses on trophoblast stem cells (TSCs),we further discover that KAT8 regulates the transcriptional activation of the trophoblast stemness marker,CDX2,via acetylating H4K16. Remarkably,CDX2 overexpression partially rescues the defects arising from Kat8 knockout. Moreover,increasing H4K16ac via using deacetylase SIRT1 inhibitor,EX527,restores CDX2 levels and promoted placental development. Clinical analysis shows reduced KAT8,CDX2 and H4K16ac expression are associated with recurrent pregnancy loss (RPL). Trophoblast organoids derived from these patients exhibit impaired TSC self-renewal and growth,which are significantly ameliorated with EX527 treatment. These findings suggest the therapeutic potential of targeting the KAT8-H4K16ac-CDX2 axis for mitigating RPL,shedding light on early gestational abnormalities. Embryo implantation failure is a leading cause of miscarriage,though the mechanisms underlying trophoblast defects are not well understood. Here they show that the histone acetyltransferase KAT8 is essential for proper activation of the trophoblast stemness gene CDX2,and that placental development can be partially rescued by inhibiting histone deacetylase activity. View Publication