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To achieve a functional cure for HIV,treatment regimens that eradicate latently HIV-infected cells must be established. For this,many groups have attempted to reactivate latently-infected cells to induce cytopathic effects and/or elicit cytotoxic T lymphocyte (CTL)/NK cell-mediated immune responses to kill these cells. We believe that not only the reactivation of latently-infected cells,but also the induction of strong CTL responses,would be required for this. Here,we used typical immune activators that target pattern recognition receptors (PRRs). For our experimental model,we identified eight SIV-infected cynomolgus monkeys that became natural controllers of viremia. Although plasma viral loads were undetectable,we could measure SIV-DNA by qPCR in peripheral blood mononuclear cells (PBMCs). Using these PBMCs,we screened 10 distinct PRR ligands to measure IFN-alpha and IFN-gamma production. Among these,STING ligands,cGAMP and c-di-AMP,and the TLR7/8 agonist R848 markedly increased cytokine levels. Both R848 and STING ligands could reactivate latently-infected cells in both cynomolgus monkeys and human PBMCs in vitro. Furthermore,c-di-AMP increased the frequency of SIV Gag-specific CD8+ T cells including polyfunctional CD8+ T cells,as compared to that in untreated control or R848-treated cells. Together,STING ligands might be candidates for HIV treatment. View Publication

Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely,hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT),the catalytic component of the telomerase complex. To this end,we have derived HEF1 cells,a fibroblast-like cell type,differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity,resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures,including morphology,surface marker and transcription factor expression,telomerase activity,differentiation,and karyotypic stability. In addition,HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth,creating a genotypically homogeneous system for the culture of hESCs. 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

The subset of regulatory T (Treg) cells,with its specific transcription Foxp3,is a unique cell type for the maintenance of immune homeostasis by controlling effector T (Teff) cell responses. Although it is common that a defect in Treg cells with Treg/Teff disorder causes autoimmune diseases; however,the precise mechanisms are not thoroughly revealed. Here,we report that miR-34a could attenuate human and murine Foxp3 gene expression via targeting their 3' untranslated regions (3' UTR). The human miR-34a,increased in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) patients,displayed a positive correlation with some serum markers of inflammation including rheumatoid factor (RF),anti-streptolysin antibody (ASO),erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) as well as Th17 signature gene RORgammat,but inversely correlated with the mRNA expression levels of FOXP3. In addition,murine miR-34a levels were downregulated in TGF-beta-induced Treg cells but upregulated in Th17 cells induced in vitro compared to activated CD4+ T cells. It has also been demonstrated that elevated miR-34a disrupting Treg/Th17 balance in vivo contributed to the progress of pathogenesis of collagen induced arthritis (CIA) mice. Furthermore,IL-6 and TNF-alpha were responsible for the upregulation of miR-34a and downregulation of Foxp3,which was reverted by the addition of NF-kappaB/p65 inhibitor BAY11-7082,thus indicating that NF-kappaB/p65 inhibited Foxp3 expression in an miR-34a-dependent manner. Finally,IL-6 or TNF-alpha-activated p65 could bind to the miR-34a promotor and enhance its activity,resulting in upregulation of its transcription. Taken together,we show that NF-kappaB activated by inflammatory cytokines,such as IL-6 and TNF-alpha,ameliorates Foxp3 levels via regulating miR-34a expression,which provides a new mechanistic and therapeutic insight into the ongoing of autoimmune diseases. View Publication

SCOPE Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants,occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC,but the mechanism underlying such protection is currently unclear. METHODS AND RESULTS By extracting HMOs from pooled human breastmilk,the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results,the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression,decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins,it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. CONCLUSIONS Taken together,the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins. View Publication

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs,monocytes,and endothelial cells,which express CD105,was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes,and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs,monocytes,and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added,which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME,but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells. View Publication

Immunological tolerance removes or inactivates self-reactive B cells,including those that also recognize cross-reactive foreign antigens. Whereas a few microbial pathogens exploit these holes" in the B cell repertoire by mimicking host antigens to evade immune surveillance the extent to which tolerance reduces the B cell repertoire to foreign antigens is unknown. Here we use single-cell cultures to determine the repertoires of human B cell antigen receptors (BCRs) before (transitional B cells) and after (mature B cells) the second B cell tolerance checkpoint in both healthy donors and in patients with systemic lupus erythematosus (SLE) . In healthy donors the majority ({\~{}}70{\%}) of transitional B cells that recognize foreign antigens also bind human self-antigens (foreign+self) and peripheral tolerance halves the frequency of foreign+self-reactive mature B cells. In contrast in SLE patients who are defective in the second tolerance checkpoint frequencies of foreign+self-reactive B cells remain unchanged during maturation of transitional to mature B cells. Patterns of foreign+self-reactivity among mature B cells from healthy donors differ from those of SLE patients. We propose that immune tolerance significantly reduces the scope of the BCR repertoire to microbial pathogens and that cross-reactivity between foreign and self epitopes may be more common than previously appreciated." View Publication

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8,there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether,and how,ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells,and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically,interferon gamma (IFNgamma) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11,two subunits of the glutamate-cystine antiporter system xc-,impairs the uptake of cystine by tumour cells,and as a consequence,promotes tumour cell lipid peroxidation and ferroptosis. In mouse models,depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated,in cancer patients,with CD8+ T cell signature,IFNgamma expression,and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNgamma and CD8. Thus,T cell-promoted tumour ferroptosis is an anti-tumour mechanism,and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach. View Publication

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases,inflammatory diseases,etc.,due to their pleiotropic properties. However,largely incongruent data were obtained from different MSC-based clinical trials,which may be partially due to functional heterogeneity among MSC. Here,we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP),which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression,proliferation,migration,multipotency,immunomodulatory activity and global gene expression profile. Moreover,the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC),and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly,NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo,as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies. View Publication

We have recently demonstrated the formation of interconnecting canalicular cell processes in bone cells upon contact with basement membrane components. Here we have determined whether growth factors in the reconstituted basement membrane (Matrigel) were active in influencing the cellular network formation. Various growth factors including transforming growth factor beta (TGF-beta),epidermal growth factor (EGF),insulin-like growth factor 1,bovine fibroblast growth factor (bFGF),and platelet-derived growth factor (PDGF) were identified in Matrigel. Exogenous TGF-beta blocked the cellular network formation. Conversely,addition of TGF-beta 1 neutralizing antibodies to Matrigel stimulated the cellular network formation. bFGF,EGF,and PDGF all promoted cellular migration and organization on Matrigel. Addition of bFGF to MC3T3-E1 cells grown on Matrigel overcame the inhibitory effect of TGF-beta. Some TGF-beta remained bound to type IV collagen purified from the Engelbreth-Holm-Swarm tumor matrix. These data demonstrate that reconstituted basement membrane contains growth factors which influence cellular behavior,suggesting caution in the interpretation of experiments on cellular activity related to Matrigel,collagen type IV,and possibly other extracellular matrix components. View Publication

Biochemical and biophysical stimuli of stem cell niches finely regulate the self-renewal/differentiation equilibrium. Replicating this in vitro is technically challenging,making the control of stem cell functions difficult. Cell derived matrices capture certain aspect of niches that influence fate decisions. Here,aligned fibrous matrices synthesized by MC3T3 cells were produced and the role of matrix orientation and stiffness on the maintenance of stem cell characteristics and adipo- or osteo-genic differentiation of murine mesenchymal stem cells (mMSCs) was investigated. Decellularized matrices promoted mMSC proliferation. Fibrillar alignment and matrix stiffness work in concert in defining cell fate. Soft matrices preserve stemness,whereas stiff ones,in presence of biochemical supplements,promptly induce differentiation. Matrix alignment impacts the homogeneity of the cell population,that is,soft aligned matrices ameliorate the spontaneous adipogenic differentiation,whereas stiff aligned matrices reduce cross-differentiation. We infer that mechanical signaling is a dominant factor in mMSC fate decision and the matrix alignment contributes to produce a more homogeneous environment,which results in a uniform response of cells to biophysical environment. Matrix thus produced can be obtained in vitro in a facile and consistent manner and can be used for homogeneous stem cell amplification or for mechanotransduction-related studies. View Publication

Accurate characterization of hematopoietic stem cells (HSC) products is needed to better anticipate the hematopoietic reconstitution and the outcome in patients. Although CD34+ viable cells enumeration is a key predictor of time to correction of aplasia,it does not fully inform about functionality of cells contained in the graft. CFU assay is the gold standard in vitro potency assay to assess clonogenicity of HSC and consists on the count and identification of colonies several days after culture in a semi solid media. Manual count of colonies with optic microscope is the most commonly used method but its important variability and subjectivity hinders the universal implementation of this potency assay. The aim of this study is to validate a standardized method using the STEMvision™ system,the first semi-automated instrument for imaging and scoring hematopoietic colonies,according to French and European recommendations. Results obtained highlight better performance criteria with STEMvision™ system than the manual method. This semi-automatic device tends to reduce the coefficients of variation of repeatability,inter-operator variability and intermediate precision. This newly available platform could represent an interesting option,significantly improving performances of CFU assays used for the characterization of hematopoietic progenitors. View Publication