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The molecular mechanisms regulating CD8 + cytotoxic T lymphocytes (CTL) are not fully understood. Here,we show that the peroxisome proliferator–activated receptor δ (PPARδ) suppresses CTL cytotoxicity by inhibiting RelA DNA binding. Treatment of Apc Min/+ mice with the PPARδ agonist GW501516 reduced the activation of normal and tumor-associated intestinal CD8 + T cells and increased intestinal adenoma burden. PPARδ knockout or knockdown in CTLs increased their cytotoxicity against colorectal cancer cells,whereas overexpression of PPARδ or agonist treatment decreased it. Correspondingly,perforin,granzyme B,and IFNγ protein and mRNA levels were higher in PPARδ knockout or knockdown CTLs and lower in PPARδ overexpressing or agonist-treated CTLs. Mechanistically,we found that PPARδ binds to RelA,interfering with RelA–p50 heterodimer formation in the nucleus,thereby inhibiting its DNA binding in CTLs. Thus,PPARδ is a critical regulator of CTL effector function. Significance: Here,we provide the first direct evidence that PPARδ plays a critical role in suppressing the immune response against tumors by downregulating RelA DNA-binding activity. This results in decreased expression of perforin,granzyme B,and IFNγ. Thus,PPARδ may serve as a valuable target for developing future cancer immunotherapies. View Publication

Radiotherapy is widely regarded as the primary therapeutic modality for nasopharyngeal cancer (NPC). Studies have shown that cancer cells with high resistance to radiation,known as radioresistant cancer cells,may cause residual illness,which in turn might contribute to the occurrence of cancer recurrence and metastasis. It has been shown that cancer stem-like cells (CSCs) exhibit resistance to radiation therapy. In the present study,fractionated doses of radiation-induced epithelial-mesenchymal transition (EMT) and ALDH+ CSCs phenotype of NPC tumor spheroids. Furthermore,it has been shown that cells with elevated ALDH activity have increased resistance to the effects of fractionated irradiation. Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular antioxidant systems. A large body of evidence suggests that Nrf2 plays a significant role in the development of radioresistance in cancer. The authors’ research revealed that the application of fractionated irradiation resulted in a decline in Nrf2-dependent reactive oxygen species (ROS) levels,thereby mitigating DNA damage in ALDH+ stem-like NPC cells. In addition,immunofluorescence analysis revealed that subsequent to the process of fractionated irradiation of ALDH+ cells,activated Nrf2 was predominantly localized inside the nucleus. Immunofluorescent analysis also revealed that the presence of the nuclear Nrf2+/NQO1+/ALDH1+ axis might potentially serve as an indicator of poor prognosis and resistance to radiotherapy in patients with NPC. Thus,the authors’ findings strongly suggest that the radioresistance of ALDH-positive NPC CSCs to fractionated irradiation is regulated by nuclear Nrf2 accumulation. Nrf2 exerts its effects through the downstream effector NQO1/ALDH1,which depends on ROS attenuation. View Publication

UNLABELLED The effects of oleuropein on the processes of osteoblastogenesis and adipogenesis in mesenchymal stem cells (MSCs) from human bone marrow have been studied. We report that oleuropein,a polyphenol abundant in olive tree products,reduces the expression of peroxisome proliferator-activated receptor gamma (PPAR$\gamma$),inhibits adipocyte differentiation,and enhances differentiation into osteoblast. INTRODUCTION Age-related bone loss is associated with osteoblast insufficiency during continuous bone remodeling. It has been suggested that the formation of osteoblasts in bone marrow is closely associated with adipogenesis,and age-related changes in this relationship could be responsible for the progressive adiposity of bone marrow which occurs with osteoporosis. In addition,the consumption of oleuropein,a major polyphenol in olive leaves and olive oil,has been associated with a reduction in bone loss. METHODS We have analyzed the effects of oleuropein-at concentrations between 10(-6) and 10(-4) M-on the processes of osteoblastogenesis and adipogenesis in MSCs from human bone marrow. RESULTS The results show an increase in osteoblast differentiation and a decrease in adipocyte differentiation when there is oleuropein in the culture media. The gene expression of osteoblastogenesis markers,RUNXII,osterix,collagen type I,osteocalcin,or alkaline phosphatase (ALP),was higher in osteoblast-induced oleuropein-treated cells. Also,the ALP activity and extracellular matrix mineralization were higher when oleuropein was present in the media. Oleuropein in MSCs induced adipocytes to produce a decrease in the expression of the genes involved in adipogenesis,the PPAR$\gamma$,lipoprotein lipase,or fatty acid-binding protein 4,and minor fat accumulation. CONCLUSION Our data suggest that oleuropein,highly abundant in olive tree products included in the traditional Mediterranean diet,could prevent age-related bone loss and osteoporosis. View Publication

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages),without quantitatively affecting terminal cell differentiation,whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors,Notch targets,and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points,modest upregulation of Notch1,Notch3,and Hes1 was observed in Jagged1-CD34(+) cells,whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later,myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators,whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and,to a lesser extent,Hes1,Lunatic Fringe,and Numb. Together,the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments,which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages. View Publication

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. View Publication

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However,it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps,we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in beta-hemoglobin gene (HBB) that cause severe beta-thalassemia (beta-Thal),corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting,and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing,we uncovered seven copy number variations,five small insertions/deletions,and 64 single nucleotide variations (SNVs) in beta-Thal iPSCs before the gene targeting step and found a single small copy number variation,19 insertions/deletions,and 340 single nucleotide variations in the final gene-corrected beta-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps,suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. View Publication

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene,and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however,the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL,we found that deletion of both Pik3r1 and Pik3r2,genes encoding class IA PI3K regulatory isoforms,severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes,but the cells were smaller,proliferated more slowly,and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However,they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR,PI-103,was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K,alone or together with mTOR,in the treatment of Ph+ ALL. View Publication

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

BACKGROUND/AIMS The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil),a Rho-kinase (ROCK) inhibitor,on cell growth,collagen production,and collagenase activity in HSCs. METHODS Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation,and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs),extra cellular signal -regulated kinase 1/2 (ERK1/2),c-jun kinase (JNK),and p38 were evaluated by western blot analysis. Type I collagen,matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR,respectively. Collagenase activity (active MMP-1) was also evaluated. RESULTS Fasudil (100 microM) inhibited cell spreading,the formation of stress fibers,and expression of alpha-SMA with concomitant suppression of cell growth,although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2,JNK,and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP,stimulated the production and transcription of MMP-1,and enhanced collagenase activity. CONCLUSION These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity. View Publication

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF),a cell surface C3/C5 convertase regulator,exerts control over T cell responses. Extending these results,we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1(-/-)) APCs produce significantly more IL-12,C5a,and IFN-gamma compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-gamma-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC,demonstrating a link between DAF,local complement activation,IL-12,and T cell-produced IFN-gamma. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-gamma-producing effector T cells. Overall,our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production,this in turn,contributes to directing T cell differentiation toward an IFN-gamma-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity. View Publication

Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research,and in human serum,these antibodies contribute to<90% of the neutralization response; however,detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV,CD27+memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells,0.35% recognized RSV-A2-infected cells,of which 59% were IgA-expressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells,44.5% of IgG-expressing B cells and 56% of IgA-expressing B cells reacted to the F protein,while,unexpectedly,41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However,these processes did not seem to depend on a specific epitope. In conclusion,healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCEHuman RSV remains the most common cause of severe lower respiratory tract disease in premature babies,young infants,the elderly,and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries,RSV lower respiratory tract disease has a high mortality. Without an effective vaccine,only passive immunization with palivizumab is approved for prophylactic treatment. However,highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition,these antibodies could guide further vaccine development. In this study,we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease. View Publication

The lymph node periphery is an important site for many immunological functions,from pathogen containment to the differentiation of helper T cells,yet the cues that position cells in this region are largely undefined. Here,through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate),we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node,predominantly in the medulla,and we found that expression of SPNS2,expression of the S1P receptor S1PR5 on NK cells,and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-$\gamma$ by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla. View Publication