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Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming,but systematic interrogation of transcription factors,and their interactions with each other,has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells,revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation,with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons,which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition. 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

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However,CTC investigation is hampered by their low number,making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy,and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype,stem-cell like characteristics,angiogenesis potential,an osteomimetic signature and the capacity to escape from the immune system. Moreover,they showed changes in mRNA and protein expression (e.g.,DEFA6,ABCB1 and GAL),whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits,treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time. View Publication

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms,mouse and human cell infection models and human biopsy samples were employed. We show that the 'keystone' pathogen Porphyromonas gingivalis,disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + Tregs through an anti-apoptotic pathway. This pathway,involving pAKT1,pFOXO1,FOXP3,IDO1 and BIM,is activated in humans with CP and in mice orally infected with Mfa1 expressing P. gingivalis strains. Mechanistically,activation of this pathway,demonstrating FOXP3 as a direct FOXO1-target gene,was demonstrated by ChIP-assay in human CP gingiva. Expression of oncogenic but not tumor suppressor markers is consistent with tumor cell proliferation demonstrated in OSCC-P. gingivalis cocultures. Importantly,FimA + P. gingivalis strain MFI invades OSCCs,inducing inflammatory/angiogenic/oncogenic proteins stimulating OSCCs proliferation through CXCR4. Inhibition of CXCR4 abolished Pg-MFI-induced OSCCs proliferation and reduced expression of oncogenic proteins SDF-1/CXCR4,plus pAKT1-pFOXO1. Conclusively,P. gingivalis,through Mfa1 and FimA fimbriae,promotes immunosuppression and oncogenic cell proliferation,respectively,through a two-hit receptor-ligand process involving DC-SIGN+hi/CXCR4+hi,activating a pAKT+hipFOXO1+hiBIM-lowFOXP3+hi and IDO+hi- driven pathway,likely to impact the prognosis of oral cancers in patients with periodontitis. View Publication

While it is now acknowledged that CD4+ T cells expressing CD25 and Foxp3 (Treg cells) regulate immune responses and,consequently,influence the pathogenesis of infectious diseases,the regulatory response mediated by Treg cells upon infection by Trypanosoma cruzi was still poorly characterized. In order to understand the role of Treg cells during infection by this protozoan parasite,we determined in time and space the magnitude of the regulatory response and the phenotypic,functional and transcriptional features of the Treg cell population in infected mice. Contrary to the accumulation of Treg cells reported in most chronic infections in mice and humans,experimental T. cruzi infection was characterized by sustained numbers but decreased relative frequency of Treg cells. The reduction in Treg cell frequency resulted from a massive accumulation of effector immune cells,and inversely correlated with the magnitude of the effector immune response as well as with emergence of acute immunopathology. In order to understand the causes underlying the marked reduction in Treg cell frequency,we evaluated the dynamics of the Treg cell population and found a low proliferation rate and limited accrual of peripheral Treg cells during infection. We also observed that Treg cells became activated and acquired a phenotypic and transcriptional profile consistent with suppression of type 1 inflammatory responses. To assess the biological relevance of the relative reduction in Treg cells frequency observed during T. cruzi infection,we transferred in vitro differentiated Treg cells at early moments,when the deregulation of the ratio between regulatory and conventional T cells becomes significant. Intravenous injection of Treg cells dampened parasite-specific CD8+ T cell immunity and affected parasite control in blood and tissues. Altogether,our results show that limited Treg cell response during the acute phase of T. cruzi infection enables the emergence of protective anti-parasite CD8+ T cell immunity and critically influences host resistance. View Publication

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response,resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation,systemic immune activation,and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic,functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L,CD16,and CD80,consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-$\gamma$ via the production of reactive oxygen species. Furthermore,CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID. View Publication

Protective antibody responses to vaccination or infection depend on affinity maturation,a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind,gather,and present antigen to T follicular helper (Tfh) cells. Here,we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with na{\{i}}ve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of na{\"{i}}ve B cells. Thus intrinsic properties of human GC B cells set thresholds for affinity selection.""" View Publication

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand,foot,and mouth disease (HFMD). In our previous study,intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity,while immunization with the inactivated CA16 vaccine did not. In this report,we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different,but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-kappaB pathway signaling molecules,which were identified as being capable of activating DCs,ILCs,and T cells,were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly,ILC colocalization decreased over time,while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore,neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100{\%} protected against wild-type EV71 or CA16 viral challenge. Together,our results provide new insights into the development of vaccines for HFMD. View Publication

The cancer stem cell (CSC) model suggests that a subpopulation of cells within the tumor,the CSCs,is responsible for cancer relapse and metastasis formation. CSCs hold unique characteristics,such as self-renewal,differentiation abilities,and resistance to chemotherapy,raising the need for discovering drugs that target CSCs. Previously we have found that the antihypertensive drug spironolactone impairs DNA damage response in cancer cells. Here we show that spironolactone,apart from inhibiting cancerous cell growth,is also highly toxic to CSCs. Notably,we demonstrate that CSCs have high basal levels of DNA double-strand breaks (DSBs). Mechanistically,we reveal that spironolactone does not damage the DNA but impairs DSB repair and induces apoptosis in cancer cells and CSCs while sparing healthy cells. In vivo,spironolactone treatment reduced the size and CSC content of tumors. Overall,we suggest spironolactone as an anticancer reagent,toxic to both cancer cells and,particularly to,CSCs. View Publication

Type 1 diabetes (T1D) is an autoimmune disease in which a strong inflammatory response causes the death of insulin-producing pancreatic beta-cells,while inefficient regulatory mechanisms allow that response to become chronic. Ethyl pyruvate (EP),a stable pyruvate derivate and certified inhibitor of an alarmin-high mobility group box 1 (HMGB1),exerts anti-oxidant and anti-inflammatory properties in animal models of rheumatoid arthritis and encephalomyelitis. To test its therapeutic potential in T1D,EP was administered intraperitoneally to C57BL/6 mice with multiple low-dose streptozotocin (MLDS)-induced T1D. EP treatment decreased T1D incidence,reduced the infiltration of cells into the pancreatic islets and preserved beta-cell function. Apart from reducing HMGB1 expression,EP treatment successfully interfered with the inflammatory response within the local pancreatic lymph nodes and in the pancreas. Its effect was restricted to boosting the regulatory arm of the immune response through up-regulation of tolerogenic dendritic cells (CD11c+CD11b-CD103+) within the pancreatic infiltrates and through the enhancement of regulatory T cell (Treg) levels (CD4+CD25highFoxP3+). These EP-stimulated Treg displayed enhanced suppressive capacity reflected in increased levels of CTLA-4,secreted TGF-beta,and IL-10 and in the more efficient inhibition of effector T cell proliferation compared to Treg from diabetic animals. Higher levels of Treg were a result of increased differentiation and proliferation (Ki67+ cells),but also of the heightened potency for migration due to increased expression of adhesion molecules (CD11a and CD62L) and CXCR3 chemokine receptor. Treg isolated from EP-treated mice had the activated phenotype and T-bet expression more frequently,suggesting that they readily suppressed IFN-gamma-producing cells. The effect of EP on Treg was also reproduced in vitro. Overall,our results show that EP treatment reduced T1D incidence in C57BL/6 mice predominantly by enhancing Treg differentiation,proliferation,their suppressive capacity,and recruitment into the pancreas. View Publication

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress,with activation of the IRE1alpha-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however,the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress,mainly affecting neutrophils,monocytes,and macrophages. An overactive IRE1alpha-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state,with increased glycolytic rates and mitochondrial function,associated with exaggerated production of TNF and IL-6. This hyper-metabolic state,seen in CF macrophages,is reversed by inhibiting the RNase domain of IRE1alpha,thereby decreasing the increased glycolic rates,mitochondrial function and inflammation. Altogether,our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover,the IRE1alpha-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages,which is associated with the exaggerated inflammatory response. Modulating ER stress,metabolism and inflammation,by targeting IRE1alpha,may improve the metabolic fitness of macrophages,and other immune cells in CF and other immune-related disorders. View Publication