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The association between type 2 diabetes (T2D) pathogenesis and immune-mediated tissue damage and insulin resistance suggests that T2D patients might benefit from the suppression of pathogenic inflammation. Foxp3+ Treg cells are crucial suppressors of inflammation,but the differentiation of Foxp3+ Treg cells is not static and is subject to conversion into IL-17-producing Th17-like cells upon receiving external signals. In this study,we examined the production of IL-17 by Treg cells. Compared to non-T2D controls,T2D patients presented significantly higher levels of IL-17-expressing cells in both Foxp3- CD4 T cells and Foxp3+ Treg cells. The frequencies of IL-17-nonexpressing Foxp3+ Treg cells,on the other hand,were not changed. Interestingly,IL-17-expressing Foxp3+ Treg cells were mutually exclusive from IL-10-expressing and TGF-$\beta$-expressing Foxp3+ Treg cells,suggesting that multiple subpopulations exist within the Foxp3+ Treg cells from T2D patients. In T2D patients,the frequencies of IL-17-expressing Foxp3+ Treg cells were positively correlated with the body mass index (BMI) and the HbA1c levels of T2D patients. The frequencies of IL-10-expressing Treg cells,on the other hand,were inversely associated with the BMI of both non-T2D controls and T2D patients. In addition,the suppressive activity of Treg cells was significantly lower in T2D patients than in non-T2D controls. Together,our study uncovered a dysregulation in Foxp3+ Treg cells from T2D patients,characterized by high IL-17 expression and low suppression activity. View Publication

Understanding of the factors governing immune responses in cancer remains incomplete,limiting patient benefit. In this study,we used mass cytometry to define the systemic immune landscape in response to tumor development across five tissues in eight mouse tumor models. Systemic immunity was dramatically altered across models and time,with consistent findings in the peripheral blood of patients with breast cancer. Changes in peripheral tissues differed from those in the tumor microenvironment. Mice with tumor-experienced immune systems mounted dampened responses to orthogonal challenges,including reduced T cell activation during viral or bacterial infection. Antigen-presenting cells (APCs) mounted weaker responses in this context,whereas promoting APC activation rescued T cell activity. Systemic immune changes were reversed with surgical tumor resection,and many were prevented by interleukin-1 or granulocyte colony-stimulating factor blockade,revealing remarkable plasticity in the systemic immune state. These results demonstrate that tumor development dynamically reshapes the composition and function of the immune macroenvironment. View Publication

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset,progression,and treatment of a broad spectrum of inflammatory conditions. In this study,we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form,as well as in its partially demannosylated and partially desialylated form,on HEK293 cells expressing TLR-8,and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells,N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent,i.e.,chloroquine (CQN),that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8,it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold,p {\textless} 0.0001),partially demannosylated (0.3-fold,p {\textless} 0.0001) and partially desialylated (0.4-fold,p {\textless} 0.0001) N-glycans. Furthermore,the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN,the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune,infections or other inflammatory disorders. View Publication

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche,but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor,RET,at the cell surface of HSCs,in mediating sustained cellular growth,resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex,GDNF/GFRa1,show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally,we demonstrate that RET drives a multi-faceted intracellular signalling pathway,including key signalling intermediates AKT,ERK1/2,NFkB and p53,responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions. View Publication

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here,we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP,termed the wing region. One mAb targeting the GP2 wing,MR228,showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb,MR235,to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs,resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection. View Publication

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success,yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however,the administration of IL-12 has been associated with immune-related adverse events. Here we show that,after intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive mouse tumours,CBD-IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12,CBD-IL-12 induced sustained intratumoural levels of interferon-$\gamma$,substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy,eliciting complete regression of CPI-unresponsive breast tumours. Furthermore,CBD-IL-12 potently synergized with CPI to eradicate large established melanomas,induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours. View Publication

Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However,it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-$\kappa$B and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+),34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV-) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced,single spliced,and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p {\textless} 0.05). As Tat is synthesized by multiple splicing,the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects,which could indicate a higher Tat activity in these cells despite their resting state. View Publication

Upon immunogenic challenge,lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments,and with resolution,they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation,YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease. View Publication

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs),leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects,we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens,T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore,MV-NIS treatment significantly (P {\textless} 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly,one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine,increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma,providing a basis for continued exploration of this modality in combination with immune checkpoint blockade. View Publication

Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells,macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR),while decreasing at higher doses over a broad physiological range,AID and Blimp1 expression,CSR,somatic hypermutation and plasma cell differentiation. In human and mouse B cells,butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3'UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors,not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes,these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models. View Publication

PURPOSE Imatinib,the breakpoint cluster region protein (BCR)/Abelson murine leukemia viral oncogene homolog (ABL) inhibitor,is widely used to treat chronic myeloid leukemia (CML). However,imatinib resistance develops in many patients. Therefore,new drugs with improved therapeutic effects are urgently needed. Berberine (BBR) is a potent BCR-ABL inhibitor for imatinib-sensitive and -resistant CML. EXPERIMENTAL DESIGN Protein structure analysis and virtual screening were used to identify BBR targets in CML. Molecular docking analysis,surface plasmon resonance imaging,nuclear magnetic resonance assays,and thermoshift assays were performed to confirm the BBR target. The change in BCR-ABL protein expression after BBR treatment was assessed by Western blotting. The effects of BBR were assessed in vitro in cell lines,in vivo in mice,and in human CML bone marrow cells as a potential strategy to overcome imatinib resistance. RESULTS We discovered that BBR bound to the protein tyrosine kinase domain of BCR-ABL. BBR inhibited the activity of BCR-ABL and BCR-ABL with the T315I mutation,and it also degraded these proteins via the autophagic lysosome pathway by recruiting E3 ubiquitin-protein ligase LRSAM1. BBR inhibited the cell viability and colony formation of CML cells and prolonged survival in CML mouse models with imatinib sensitivity and resistance. CONCLUSIONS The results show that BBR directly binds to and degrades BCR-ABL and BCR-ABL T315I via the autophagic lysosome pathway by recruiting LRSAM1. The use of BBR is a new strategy to improve the treatment of patients with CML with imatinib sensitivity or resistance. View Publication

Collective invasion can be led by breast cancer cells expressing basal epithelial markers,typified by keratin-14 (KRT14). We analyzed gene expression data from The Cancer Genome Atlas and demonstrated a significant correlation between a KRT14+ invasion signature and a stromal-mediated extracellular matrix (ECM) organization module. We then developed a novel coculture model of tumor organoids with autologous stromal cells. Coculture significantly increased KRT14 expression and invasion of organoids from both luminal and basal murine breast cancer models. However,stromal cell conditioned medium induced invasion but not KRT14 expression. Cancer cells released TGF$\beta$ and that signaling pathway was required for stromal cell-induced invasion and KRT14 expression. Mechanistically,TGF$\beta$ induced NOX4 expression in stromal cells and NOX4 inhibition reduced invasion and KRT14 expression. In summary,we developed a novel coculture model and revealed dynamic molecular interactions between stromal cells and cancer cells that regulate both basal gene expression and invasive behavior. IMPLICATIONS: Fibroblasts within mammary tumors can regulate the molecular phenotype and invasive behavior of breast cancer cells. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/11/1615/F1.large.jpg. View Publication