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Receptor type protein tyrosine phosphatase-sigma (PTPsigma) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPsigma is also expressed by hematopoietic stem cells (HSCs). Here,we describe small molecule inhibitors of PTPsigma that promote HSC regeneration in vivo. Systemic administration of the PTPsigma inhibitor,DJ001,or its analog,to irradiated mice promotes HSC regeneration,accelerates hematologic recovery,and improves survival. Similarly,DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPsigma and antagonizes PTPsigma via unique non-competitive,allosteric binding. Mechanistically,DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase,RAC1,and induction of BCL-XL. Furthermore,treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective,small-molecule PTPsigma inhibitors for human hematopoietic regeneration. View Publication

Both anti-tumoral and pro-tumoral effects of mesenchymal stem cells (MSCs) in preclinical treatment of ovarian cancer have been controversially demonstrated. In this study,we profiled the phenotypes of mouse compact bone-derived MSCs (CB-MSCs) and bone marrow-derived MSCs (BM-MSCs) and found that CB-MSCs expressed lower CD90 compared to BM-MSCs. We examined gene expression of immune regulating cytokines of CB-MSCs in 2D and 3D culture and under stimulation with TLR4 agonist LPS or immune activator VIC-008. Our data showed that when CB-MSCs were cultured in simulated in vivo 3D condition,CD90 expression was further decreased. Moreover,gene expressions of immune activating cytokines IL-12,IL-21,IFNgamma and a pro-inflammatory cytokine CXCL10 in CB-MSCs were increased in 3D culture whereas gene expression of anti-inflammatory cytokines IL-10 and CCL5 were downregulated. Stimulation of CB-MSCs by LPS or VIC-008 presented similar profile of the cytokine gene expressions to that in 3D culture which might benefit the anti-tumor efficacy of CD90low MSCs. The anti-tumor effects of CD90low CB-MSCs alone or in combination with VIC-008 were evaluated in a syngeneic orthotopic mouse model of ovarian cancer. Treatment that combines CB-MSCs and VIC-008 significantly decreased tumor growth and prolonged mouse survival. This was associated with the increase of activated anti-tumoral CD4+ and CD8+ T cells and the decrease of Treg cells in the tumor microenvironment. Taken together,our study demonstrates the synergistic anti-tumoral efficacy by application of CB-MSCs combined with immune activator VIC-008 and provides new insight into CD90low MSCs as a new anti-tumor arsenal. View Publication

Human adipose-derived stem cells (ADSCs) can release exosomes; however,their specific functions remain elusive. In this study,we verified that exosomes derived from osteogenically differentiated ADSCs can promote osteogenic differentiation of ADSCs. Furthermore,in order to investigate the importance of exosomal microRNAs (miRNAs) in osteogenic differentiation of ADSCs,we used microarray assays to analyze the expression profiles of exosomal miRNAs derived from undifferentiated as well as osteogenically differentiated ADSCs; 201 miRNAs were upregulated and 33 miRNAs were downregulated between the two types of exosomes. Additionally,bioinformatic analyses,which included gene ontology analyses,pathway analysis,and miRNA-mRNA-network investigations,were performed. The results of these analyses revealed that the differentially expressed exosomal miRNAs participate in multiple biological processes,such as gene expression,synthesis of biomolecules,cell development,differentiation,and signal transduction,among others. Moreover,we found that these differentially expressed exosomal miRNAs connect osteogenic differentiation to processes such as axon guidance,MAPK signaling,and Wnt signaling. To the best of our knowledge,this is the first study to identify and characterize exosomal miRNAs derived from osteogenically differentiated ADSCs. This study confirms that alterations in the expression of exosomal miRNAs can promote osteogenic differentiation of ADSCs,which also provides the foundation for further research on the regulatory functions of exosomal miRNAs in the context of ADSC osteogenesis. View Publication

Subclinical hypothyroidism is associated with cardiovascular diseases,yet the underlying mechanism remains largely unknown. Herein,in a common population (n = 1,103),TSH level was found to be independently correlated with both carotid plaque prevalence and intima-media thickness. Consistently,TSH receptor ablation in ApoE-/- mice attenuated atherogenesis,accompanied by decreased vascular inflammation and macrophage burden in atherosclerotic plaques. These results were also observed in myeloid-specific Tshr-deficient ApoE-/- mice,which indicated macrophages to be a critical target of the proinflammatory and atherogenic effects of TSH. In vitro experiments further revealed that TSH activated MAPKs (ERK1/2,p38alpha,and JNK) and IkappaB/p65 pathways in macrophages and increased inflammatory cytokine production and their recruitment of monocytes. Thus,the present study has elucidated the new mechanisms by which TSH,as an independent risk factor of atherosclerosis,aggravates vascular inflammation and contributes to atherogenesis. View Publication

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model,Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe,brain-targeted,atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3DeltaLysM),which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE,however,their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3DeltaLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically,Socs3-deficient neutrophils exhibit enhanced STAT3 activation,a hyper-activated phenotype in response to G-CSF,and upon G-CSF priming,increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall,our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3DeltaLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress,which may explain the detrimental role of G-CSF in MS patients. 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

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