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Natural killer (NK) cells play a key role in immunity,but how HLA class I (HLA-I) and killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1) polymorphism impacts disease outcome remains unclear. KIR3DL1 (*001/*005/*015) tetramers were screened for reactivity against a panel of HLA-I molecules. This revealed different and distinct hierarchies of specificity for each KIR3DL1 allotype,with KIR3DL1*005 recognizing the widest array of HLA-I ligands. These differences were further reflected in functional studies using NK clones expressing these specific KIR3DL1 allotypes. Unexpectedly,the Ile/Thr80 dimorphism in the Bw4-motif did not categorically define strong/weak KIR3DL1 recognition. Although the KIR3DL1*001,*005,and *015 polymorphisms are remote from the KIR3DL1-HLA-I interface,the structures of these three KIR3DL1-HLA-I complexes showed that the broader HLA-I specificity of KIR3DL1*005 correlated with an altered KIR3DL1*005 interdomain positioning and increased mobility within its ligand-binding site. Collectively,we provide a generic framework for understanding the impact of KIR3DL1 polymorphism on the recognition of HLA-I allomorphs. View Publication

BACKGROUND Many groups have generated insulin-secreting cells from hESCs/iPSCs in multiple differentiation stages by mimicking the developmental processes. However,these cells do not always secrete glucose responsive insulin,one of the most important characteristics of pancreatic $$ cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic $$ cells. METHODS We established a 6-stage protocol for the differentiation process from hiPSCs to pancreatic $$ cells using defined culture media without feeders or serum. We examined the effect of CHIR99021,the selective inhibitor of GSK-3$$,in the presence of Activin,FGF2,and BMP4 during definitive endodermal induction by immunostaining for SOX17 and FOXA2. We also compared the insulin secretion at the last stage between monolayer culture and spheroid culture conditions. Cultured cells were transplanted under the kidney capsules of STZ-induced diabetic NOD-SCID mice,and blood glucose levels were measured. Immunohistochemical analysis was performed 4 weeks and 12 weeks after transplantation. RESULTS Addition of CHIR99021 in the presence of Activin,FGF2,and BMP4 for 2 days improved the viability of the endodermal cells,keeping the high positive rate of SOX17. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than monolayer culture did. After cell transplantation,diabetic mice showed lowered blood glucose levels,and we detected islet-like structures in vivo. CONCLUSION We generated functional pancreatic $$ cells from human iPS cells. Induction of definitive endoderm and spheroid formation might be key steps for producing them. View Publication

BACKGROUND AND OBJECTIVES Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples,potentially due to different temperatures during cell staining. MATERIALS AND METHODS Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients,and from 10 paediatric patients. After thawing,cells were prepared for analysis within 30 min between thawing and acquisition,at either 4°C or at room temperature. RESULTS For cell products cryopreserved in conventional freezing medium (10% final DMSO),viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed,CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT),followed by CD3,CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO),median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS The post-thaw environment and,potentially,the cryoprotectant impact the outcome of cell enumeration,and results from the analysis tube may not be representative of the cells infused into a patient. View Publication

We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types,such as induced pluripotent stem cells (iPSCs) and fibroblasts,reducing the protocol time by one full day. To preserve cell physiology during gene transfer,we designed a microfluidic strategy,which facilitates significant gene delivery in a short transfection time (textless1 min) for several human cell types. This fast,optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. View Publication

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy,generalized seizures,ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However,the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study,we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs,MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption,elevated reactive oxygen species (ROS) production,reduced growth,and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore,MERRF-CMs or -NPCs contained fragmented mitochondria,as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together,these findings showed that MERRF-hiPSCs and MERRF-CM or -NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure,produced increased ROS levels,and expressed upregulated antioxidant genes. View Publication

Under defined differentiation conditions,human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate,the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening,a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification,and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2,increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus,we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE. View Publication

The neutralization of integrin α4 (Itga4) is currently used as treatment in multiple sclerosis. Although most studies have focused on its function on lymphocyte migration to the CNS,we have uncovered the importance of Itga4 for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell-targeting therapies. View Publication

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work,further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR,SGX94,has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies,this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells,resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels,thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation,IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections. View Publication

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells,but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation,the process by which pathogen Ags are internalized,degraded,and presented by MHC class I,is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article,we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs,dendritic cells and macrophages,and CD4 T cells,three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities,reducing NADPH oxidase 2 activation,and lowering phagolysosomal reactive oxygen species production and pH,which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification,to our knowledge,of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV. View Publication

mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here,we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels,particularly when oxidative phosphorylation is impaired,such as in neurons treated with mitochondrial inhibitors,or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly,in mitochondrially defective neurons,but not neuroprogenitor cells,ribosomal S6 and S6 kinase phosphorylation increased over time,despite activation of AMPK,which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis,a major energy-consuming process,may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration. View Publication

textlessptextgreater Hundreds of textlessitalictextgreaterL1CAMtextless/italictextgreater gene mutations have been shown to be associated with congenital hydrocephalus,severe intellectual disability,aphasia,and motor symptoms. How such mutations impair neuronal function,however,remains unclear. Here,we generated human embryonic stem (ES) cells carrying a conditional textlessitalictextgreaterL1CAMtextless/italictextgreater loss-of-function mutation and produced precisely matching control and textlessitalictextgreaterL1CAMtextless/italictextgreater -deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones,we found that deletion of textlessitalictextgreaterL1CAMtextless/italictextgreater dramatically impaired axonal elongation and,to a lesser extent,dendritic arborization. Unexpectedly,we also detected an ∼20–50% and ∼20–30% decrease,respectively,in the levels of ankyrinG and ankyrinB protein,and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM,but not of the L1CAM point mutants R1166X and S1224L,rescued the decrease in ankyrin levels. Importantly,we found that the textlessitalictextgreaterL1CAMtextless/italictextgreater mutation selectively decreased activity-dependent Na textlesssuptextgreater+textless/suptextgreater -currents,altered neuronal excitability,and caused impairments in action potential (AP) generation. Thus,our results suggest that the clinical presentations of textlessitalictextgreaterL1CAMtextless/italictextgreater mutations in human patients could be accounted for,at least in part,by cell-autonomous changes in the functional development of neurons,such that neurons are unable to develop normal axons and dendrites and to generate normal APs. textless/ptextgreater View Publication

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7,which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment,behavioral abnormalities and nervous system defects,though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS,we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation,not cholesterol deficiency,is critical for SLOS-associated defects. We further identified downregulation of Wnt/$$-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs,suggesting that Wnt signaling may be a promising therapeutic target for SLOS. View Publication