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Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However,the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic,as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim,in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC),dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However,individually,CYP24A1,MUCL1 and MAP7 for vitD3-tolDC; CD163,CCL18,C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC,constituted good candidate biomarkers for each respective cellular product. In addition,a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways,while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes. 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

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

Emerging evidence has shown that resting quiescent hematopoietic stem cells (HSCs) prefer to utilize anaerobic glycolysis rather than mitochondrial respiration for energy production. Compelling evidence has also revealed that altered metabolic energetics in HSCs underlies the onset of certain blood diseases; however,the mechanisms responsible for energetic reprogramming remain elusive. We recently found that Fanconi anemia (FA) HSCs in their resting state are more dependent on mitochondrial respiration for energy metabolism than on glycolysis. In the present study,we investigated the role of deficient glycolysis in FA HSC maintenance. We observed significantly reduced glucose consumption,lactate production,and ATP production in HSCs but not in the less primitive multipotent progenitors or restricted hematopoietic progenitors of Fanca-/- and Fancc-/- mice compared with that of wild-type mice,which was associated with an overactivated p53 and TP53-induced glycolysis regulator,the TIGAR-mediated metabolic axis. We utilized Fanca-/- HSCs deficient for p53 to show that the p53-TIGAR axis suppressed glycolysis in FA HSCs,leading to enhanced pentose phosphate pathway and cellular antioxidant function and,consequently,reduced DNA damage and attenuated HSC exhaustion. Furthermore,by using Fanca-/- HSCs carrying the separation-of-function mutant p53R172P transgene that selectively impairs the p53 function in apoptosis but not cell-cycle control,we demonstrated that the cell-cycle function of p53 was not required for glycolytic suppression in FA HSCs. Finally,ectopic expression of the glycolytic rate-limiting enzyme PFKFB3 specifically antagonized p53-TIGAR-mediated metabolic reprogramming in FA HSCs. Together,our results suggest that p53-TIGAR metabolic axis-mediated glycolytic suppression may play a compensatory role in attenuating DNA damage and proliferative exhaustion in FA HSCs. Stem Cells 2019;37:937-947. View Publication

BACKGROUND Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits T-cell activation and is expressed on T-regulatory cells. Human CTLA-4 deficiency results in severe immune dysregulation. Abatacept (CTLA-4 Ig) is approved for the treatment of rheumatoid arthritis (RA) and its mechanism of action is attributed to effects on T-cells. It is known that CTLA-4 modulates the expression of its ligands CD80 and CD86 on antigen presenting cells (APC) by transendocytosis. As B-cells express CD80/CD86 and function as APC,we hypothesize that B-cells are a direct target of abatacept. OBJECTIVES To investigate direct effects of abatacept on human B-lymphocytes in vitro and in RA patients. METHODS The effect of abatacept on healthy donor B-cells' phenotype,activation and CD80/CD86 expression was studied in vitro. Nine abatacept-treated RA patients were studied. Seven of these were followed up to 24 months,and two up to 12 months only and treatment response,immunoglobulins,ACPA,RF concentrations,B-cell phenotype and ACPA-specific switched memory B-cell frequency were assessed. RESULTS B-cell development was unaffected by abatacept. Abatacept treatment resulted in a dose-dependent decrease of CD80/CD86 expression on B-cells in vitro,which was due to dynamin-dependent internalization. RA patients treated with abatacept showed a progressive decrease in plasmablasts and serum IgG. While ACPA-titers only moderately declined,the frequency of ACPA-specific switched memory B-cells significantly decreased. CONCLUSIONS Abatacept directly targets B-cells by reducing CD80/CD86 expression. Impairment of antigen presentation and T-cell activation may result in altered B-cell selection,providing a new therapeutic mechanism and a base for abatacept use in B-cell mediated autoimmunity. View Publication

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract,associated with altered patterns of cytokine synthesis,excessive reactive oxygen species (ROS) production,and high levels of the innate immune protein,lipocalin-2 (LCN-2),in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1,which consists of the transmembrane proteins,NOX1 and p22PHOX,and the cytosolic proteins,NOXO1,NOXA1,and Rac1. Here,we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFalpha and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2,and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IkappaBzeta,a master inducer of LCN-2. Furthermore,LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally,analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation,and NOXO1 and LCN-2 expression. Therefore,NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression. View Publication

BACKGROUND Human microRNAs (miRNAs) have diverse functions in biology,and play a role in nearly every biological process. Here we report that miR-520d-5p (520d-5p) causes undifferentiated cancer cells to adopt benign or normal status in vivo in immunodeficient mice via demethylation and P53 upregulation. Further we found that 520-5p causes normal cells to elongate cellular lifetime and mesenchymal stem cell-like status with CD105 positivity. We hypothesized that ectopic 520d-5p expression reduced mutations in undifferentiated type of hepatoma (HLF) cells through synergistic modulation of methylation-related enzymatic expression. METHODS To examine whether there were any changes in mutation status in cells treated with 520d-5p,we performed next generation sequencing (NGS) in HLF cells and human iPSC-derivative cells in pre-mesenchymal stem cell status. We analyzed the data using both genome-wide and individual gene function approaches. RESULTS 520d-5p induced a shift towards a wild type or non-malignant phenotype,which was regulated by nucleotide mutations in both HLF cells and iPSCs. Further,520d-5p reduced mutation levels in both the whole genome and genomic fragment assemblies. CONCLUSIONS Cancer cell genomic mutations cannot be repaired in most contexts. However,these findings suggest that applied development of 520d-5p would allow new approaches to cancer research and improve the quality of iPSCs used in regenerative medicine. View Publication

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However,functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells,pointing out the need of mandatory BCR co-factors in this process. Here,we investigated benefits of several BCR co-stimulatory molecules (IL-2,IL-4,IL-15,IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand,IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition,we established a proliferative advantage for ZAP70 positive CLL cells,associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover,the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies. View Publication

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system,hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1,which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype,stable proliferation rate,and high telomerase activity. Similar to cells cultured on feeders,hES cells maintained under feeder-free conditions expressed OCT-4,hTERT,alkaline phosphatase,and surface markers including SSEA-4,Tra 1-60,and Tra 1-81. In addition,hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus,the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication