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Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover,Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands,such as HIV and CpG respectively,turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions,and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection,but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here,we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α,TNF-α,IFN-γ and IL-12,and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations,the addition of NK cells did not promote the release of these mediators,suggesting that once efficiently triggered by the virus,pDCs could not integrate new activating signals delivered by NK cells. However,high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly,we identified the alarmin HMGB1,released at pDC-NK cell synapse,as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover,HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1,HMGB1-specific antibodies,sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether,these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells,and they suggest a novel mechanism of innate control of HIV-1 infection. View Publication

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC,the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming,low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo,as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice,but not in high affinity-primed mice. Mechanistically,low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge,low affinity-primed CD45RB(hi) cells became CD45RB(lo),demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus,these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency,low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge. View Publication

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy,reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative,but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC,comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated,through enrichment analysis,their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally,we report an unprecedented coverage of MSC CD markers,as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. View Publication

Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs),including human embryonic stem cells and induced pluripotent stem cells,is a complex,challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study,we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells,which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion,our study demonstrates that the HepaRG ACM,a hepatic progenitor-specific matrix,plays an important role in the hepatic differentiation of hPSCs. View Publication

The incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma has increased in recent decades,though HPV prevention vaccines may reduce this rise in the future. HPV-related cancers express the viral oncoproteins E6 and E7. The latter inactivates the tumor suppressor protein retinoblastoma (Rb),which leads to the overexpression of p16(INK4) protein,providing unique Ags for therapeutic HPV-specific cancer vaccination. We developed potential adenoviral vaccines that express a fusion protein of HPV-16 E6 and E7 (Ad.E6E7) alone or fused with p16 (Ad.E6E7p16) and also encoding an anti-programmed death (PD)-1 Ab. Human monocyte-derived dendritic cells (DC) transduced with Ad.E6E7 or Ad.E6E7p16 with or without Ad.αPD1 were used to activate autologous CD8 CTL in vitro. CTL responses were tested against naturally HPV-infected head and neck squamous cell carcinoma cells using IFN-γ ELISPOT and [(51)Cr]release assay. Surprisingly,stimulation and antitumor activity of CTL were increased after incubation with Ad.E6E7p16-transduced DC (DC.E6E7p16) compared with Ad.E6E7 (DC.E6E7),a result that may be due to an effect of p16 on cyclin-dependent kinase 4 levels and IL-12 secretion by DC. Moreover,the beneficial effect was most prominent when anti-PD-1 was introduced during the second round of stimulation (after initial priming). These data suggest that careful sequencing of Ad.E6E7.p16 with Ad.αPD1 could improve antitumor immunity against HPV-related tumors and that p16 may enhance the immunogenicity of DC,through cyclin-dependent pathways,Th1 cytokine secretion,and by adding a nonviral Ag highly overexpressed in HPV-induced cancers. View Publication

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which,however,has focused on HDR-based strategies and was proven inefficient. Here,we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs,and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy,integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells,and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. View Publication

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research,regenerative therapies,and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging,expansion,maintenance,and differentiation. In this paper,an unbiased,automated high-content profiling toolkit,StemCellQC,is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy,unhealthy,and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups,and these features were linked to growth,motility,and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis,which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features,cell types,treatments,and differentiating cells. View Publication

SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS),including a high risk of autism spectrum disorder (ASD). We used unbiased,quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit,B56β,due to increased steady-state levels of its kinase,Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency. View Publication

Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson's disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson's Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression,especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies,including in vitro modeling of PD. However,further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs. View Publication

BACKGROUND Protection of cerebral endothelial cells (ECs) from hypoxia/reoxygenation (H/R)-induced injury is an important strategy for treating ischemic stroke. In this study,we investigated whether co-culture with endothelial progenitor cells (EPCs) and neural progenitor cells (NPCs) synergistically protects cerebral ECs against H/R injury and the underlying mechanism. RESULTS EPCs and NPCs were respectively generated from inducible pluripotent stem cells. Human brain ECs were used to produce an in vitro H/R-injury model. Data showed: 1) Co-culture with EPCs and NPCs synergistically inhibited H/R-induced reactive oxygen species (ROS) over-production,apoptosis,and improved the angiogenic and barrier functions (tube formation and permeability) in H/R-injured ECs. 2) Co-culture with NPCs up-regulated the expression of vascular endothelial growth factor receptor 2 (VEGFR2). 3) Co-culture with EPCs and NPCs complementarily increased vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) levels in conditioned medium,and synergistically up-regulated the expression of p-Akt/Akt and p-Flk1/VEGFR2 in H/R-injured ECs. 4) Those effects could be decreased or abolished by inhibition of both VEGFR2 and tyrosine kinase B (TrkB) or phosphatidylinositol-3-kinase (PI3K). CONCLUSIONS Our data demonstrate that EPCs and NPCs synergistically protect cerebral ECs from H/R-injury,via activating the PI3K/Akt pathway which mainly depends on VEGF and BDNF paracrine. View Publication

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach,however,is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here,we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT." View Publication

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones,light and dark,with coordinated roles,controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells,but its functional role in the GC reaction has not been explored. In this study,we report high expression of LLT1 on GC-associated B cells,early plasmablasts,and GC-derived lymphomas. LLT1 expression was readily induced via BCR,CD40,and CpG stimulation on B cells. Unexpectedly,we found high expression of the LLT1 ligand,CD161,on follicular dendritic cells. Triggering of LLT1 supported B cell activation,CD83 upregulation,and CXCR4 downregulation. Overall,these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans. View Publication