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HIV-1 Tat-interacting protein of 110 kDa [Tip110; p110(nrb)/SART3/p110] is an RNA binding nuclear protein implicated in regulation of HIV-1 gene and host gene transcription,pre-mRNA splicing,and cancer immunology. Recently,we demonstrated a role for Tip110 in regulation of hematopoiesis. Here,we show that TIP110 is also expressed in human embryonic stem cells (hESCs) and expression was decreased with differentiation of these ESCs. TIP110 was found,through up- and down-modulation of expression of Tip110,to be important in maintaining pluripotent factor (NANOG,OCT4,and SOX2) expression in and pluripotency of hESCs,although the mechanisms involved and whether the Tip110 effects are direct remain to be determined. View Publication

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka,who first generated iPSCs by retroviral transduction of four reprogramming factors,several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However,the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study,three different strategies,based on retroviral vectors,episomal vectors,and Sendai virus vectors,were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells,including the expression of alkaline phosphatase and stemness maker genes,and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion,the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs. View Publication

Among different cancer immunotherapy approaches,bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However,most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study,we describe a bispecific antibody,BiHC (bispecific Her2-CD3 antibody),constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format. In contrast to most IgG-like BsAbs,the two arms in BiHC have different molecular weights,making it easier to separate hetero- or homodimers. BiHC can be expressed in Escherichia coli and purified via Protein A affinity chromatography. The purified BiHC can recruit T cells and induce specific cytotoxicity of Her2-expressing tumor cells in vitro. The BiHC can also efficiently inhibit the tumor growth in vivo. Thus,BiHC is a promising candidate for the treatment of Her2-positive cancers. View Publication

We have developed models of HIV latency using microglia derived from adult human patient brain cortex and transformed with the SV40 T large and hTERT antigens. Latent clones infected by HIV reporter viruses display high levels of spontaneous HIV reactivation in culture. BrainPhys,a medium highly representative of the CNS extracellular environment,containing low glucose and 1{\%} FBS,reduced,but did not prevent,HIV reactivation. We hypothesized that spontaneous HIV reactivation in culture was due to the expression of pro-inflammatory genes,such as TNF-alpha$,taking place in the absence of the natural inhibitory signals from astrocytes and neurons. Indeed,expression and secretion of TNF-alpha$ is strongly reduced in HIV-latently infected microglia compared to the subset of cells that have undergone spontaneous HIV reactivation. Whereas inhibitors of NF-kappa$B or of macrophage activation only had a short-term silencing effect,addition of dexamethasone (DEXA),a glucocorticoid receptor (GR) agonist and mediator of anti-inflammation,silenced the HIV provirus in a long-term,and shRNA-mediated knock-down of GR activated HIV. DEXA also decreased secretion of a number of cytokines,including TNF-alpha$. Chromatin immunoprecipitation analysis revealed that DEXA strongly increased GR occupancy at the HIV promoter,and reduced histone 3 acetylated levels. Moreover,TNF-alpha$ expression inhibitors in combination with DEXA induced further HIV silencing and increased the histone 3 lysine 27 tri-methylated epigenetic mark of repression at the HIV promoter region. We conclude that GR is a critical repressor of HIV transcription in microglia,and a novel potential pharmacological target to restrict HIV expression in the CNS. View Publication

Short-term outcomes of kidney transplantation have improved dramatically,but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless,the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study,we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover,these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype,high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple,brief Treg production strategy in clinical trials of mismatched kidney transplantation. View Publication

BACKGROUND Hypoxia and inflammation tumor microenvironment (TME) play a crucial role in tumor development and progression. Although increased understanding of TME contributed to gastric cancer (GC) progression and prognosis,the direct interaction between macrophage and GC cells was not fully understood. METHODS Hypoxia and normoxia macrophage microarrays of GEO database was analyzed. The peripheral blood mononuclear cell acquired from the healthy volunteers. The expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) in GC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR),western-blot,Elisa and immunofluorescence. Cell proliferation,migration,and invasion were evaluated by cell counting kit 8 (CCK8),colony formation,real-time imaging of cell migration and transwell. Flow Cytometers was applied to identify the source of cytokines. Luciferase reporter assays and chromatin immunoprecipitation were used to identify the interaction between transcription factor and target gene. Especially,a series of truncated and mutation reporter genes were applied to identify precise binding sites. The corresponding functions were verified in the complementation test and in vivo animal experiment. RESULTS Our results revealed that hypoxia triggered macrophage secreted CXCL8,which induced GC invasion and proliferation. This macrophage-induced GC progression was CXCL8 activated C-X-C Motif Chemokine Receptor 1/2 (CXCR1/2) on the GC cell membrane subsequently hyperactivated Janus kinase 1/ Signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway. Then,the transcription factor STAT1 directly led to the overexpression and secretion of Interleukin 10 (IL-10). Correspondingly,IL-10 induced the M2-type polarization of macrophages and continued to increase the expression and secretion of CXCL8. It suggested a positive feedback loop between macrophage and GC. In clinical GC samples,increased CXCL8 predicted a patient's pessimistic outcome. CONCLUSION Our work identified a positive feedback loop governing cancer cells and macrophage in GC that contributed to tumor progression and patient outcome. View Publication

Epicardium,the most outer mesothelium,exerts crucial functions in fetal heart development and adult heart regeneration. Here we use a three-step manipulation of WNT signalling entwined with BMP and RA signalling for generating a self-organized epicardial organoid that highly express with epicardium makers WT1 and TCF21 from human embryonic stem cells. After 8-days treatment of TGF-beta following by bFGF,cells enter into epithelium-mesenchymal transition and give rise to smooth muscle cells. Epicardium could also integrate and invade into mouse heart with SNAI1 expression,and give birth to numerous cardiomyocyte-like cells. Single-cell RNA seq unveils the heterogeneity and multipotency exhibited by epicardium-derived-cells and fetal-like epicardium. Meanwhile,extracellular matrix and growth factors secreted by epicardial organoid mimics the ecology of subepicardial space between the epicardium and cardiomyocytes. As such,this epicardial organoid offers a unique ground for investigating and exploring the potential of epicardium in heart development and regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01339-w. View Publication

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor,Y-27632 can enhance hPSC survival and proliferation at high seeding density,dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result,colonies tend to contain heterogeneous mixtures of both modified and unmodified cells,making it difficult to isolate the desired clone buried within the colony. In this study,we report improved clonal expansion of hPSCs using a retinoic acid analogue,TTNPB. When combined with Y-27632,TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%),whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore,TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype,pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts,in part,by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion,TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications. A retinoic acid analogue,TTNPB,improves clonal expansion in adherent and suspension culture of hPSCs by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. View Publication

Tissue-repair regulatory T cells (trTregs) comprise a specialized cell subset essential for tissue homeostasis and repair. While well-studied in sterile injury models,their role in infection-induced tissue damage and antimicrobial immunity is less understood. We investigated trTreg dynamics during acute Trypanosoma cruzi infection,marked by extensive tissue damage and strong CD8+ immunity. Unlike sterile injury models,trTregs significantly declined in secondary lymphoid organs and non-lymphoid target tissues during infection,correlating with systemic and local tissue damage,and downregulation of function-associated genes in skeletal muscle. This decline was linked to decreased systemic IL-33 levels,a key trTreg growth factor,and promoted by the Th1 cytokine IFN-γ. Early recombinant IL-33 treatment increased trTregs,type 2 innate lymphoid cells,and parasite-specific CD8+ cells at specific time points after infection,leading to reduced tissue damage,lower parasite burden,and improved disease outcome. Our findings not only provide novel insights into trTregs during infection but also highlight the potential of optimizing immune balance by modulating trTreg responses to promote tissue repair while maintaining effective pathogen control during infection-induced injury. Author summaryDuring Chagas’ disease,caused by the protozoan Trypanosoma cruzi,severe organ damage is generated by the interplay between the parasite and the immune response. In our investigation,we examined the role of tissue-repair regulatory T cells (trTregs) during the acute phase of T. cruzi infection in mice. Surprisingly,we observed a reduction in trTregs at the peak of tissue damage,contrary to their usual accumulation after injury in other contexts. This decline aligned with decreased levels of interleukin-33,a critical factor for trTreg survival,and was promoted by the effector cytokine IFN-γ. Administering interleukin-33 at early infection times not only boosted trTregs but also expanded other reparative and antiparasitic immune cells. Consequently,these treated mice exhibited reduced damage and lower parasite levels in tissues. Our findings provide new insights into how trTreg function during infection-related injury,paving the way for strategies that balance the immune response to support tissue repair without weakening the body’s ability to fight the infection. This approach could have broader implications for treating infectious diseases and conditions involving tissue damage. View Publication

Induction of ferroptosis,an iron-dependent form of regulated cell death,holds promise as a strategy to overcome tumor resistance to conventional therapies and enhance immunotherapy responses. However,while the susceptibility of tumor cells to ferroptosis is extensively studied,limited data exists on the vulnerability of immune cells to disturbed iron balance and lipid peroxidation. Here,we found that T-cell stimulation rewires iron and redox homeostasis and by increasing levels of reactive oxygen species and labile iron promotes lipid peroxidation and T-cells’ ferroptosis. Upon stimulation,we detected changes in the balance of ferroptosis-suppressive proteins,including decrease of GPX4. Subsequently,we identified GPX4 as a master regulator orchestrating T/CAR-T-cells’ sensitivity to ferroptosis and observed that GPX4 inhibitors impair CAR-T cells’ antitumor functions. Our study demonstrated differential GPX4 expression and diverse susceptibility to ferroptosis between CD4⁺ and CD8⁺ T cells. Among analyzed subsets of naïve,central memory (CM),effector memory (EM),and terminally differentiated effector memory (TEMRA),CD8⁺ EM and CD8⁺ TEMRA cells exhibited the highest sensitivity to ferroptosis. We also showed that ferroptosis limited the anti-tumor efficacy of CAR-T cells,while ferroptosis inhibition improved their therapeutic effect,both in vitro and in vivo. Our findings are not only important to understand vulnerabilities of CAR-T cells but may also hold particular significance for their therapeutic development. In this context,future anticancer therapies should be carefully designed to selectively induce the ferroptosis of tumor cells without impeding cytotoxic cells’ antitumor efficacy. Additionally,we postulate that promoting less differentiated phenotype of CAR-T cells should be exploited therapeutically to create CAR-T products characterized by decreased sensitivity to ferroptosis within tumor microenvironment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04133-w. View Publication

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis and limited therapeutic options. Leukemic stem cells (LSCs),which drive disease progression and confer resistance to therapy,pose a significant challenge to conventional treatment strategies. In this study,we identified and characterized the inhibitory mechanisms of TH37,a small molecule derived from traditional Chinese medicine,which selectively targets AML blasts and LSCs. Our analyses identified peroxiredoxin 1 (PRDX1),an enzyme that catalyzes the breakdown of hydrogen peroxide (a reactive oxygen species),as the primary molecular target of TH37. We demonstrated that TH37 directly interacts with PRDX1,inhibiting its enzymatic activity and thereby elevating intracellular reactive oxygen species levels in AML cells. PRDX1 was found to be overexpressed in AML,and its expression correlated with poor prognosis and the activation of AML- and cancer-associated pathways. Targeting PRDX1,either through lentiviral short-hairpin RNA-mediated silencing or TH37 treatment,induced apoptosis,reduced colony formation,and impaired the engraftment and growth of AML cells in immunodeficient mouse models. Furthermore,TH37 synergized with conventional chemotherapeutic agent to significantly reduce the viability and colony-forming capacity of AML cells. These findings demonstrate the critical role of PRDX1 in AML pathogenesis and highlight its potential as a key therapeutic target to improve clinical outcomes for AML patients. View Publication

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta,that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX 3 CR1 + and CSF1R + source. These bipotent progenitors are proliferative and vasculogenic,contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II,which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally. Subject terms: Angiogenesis,Myelopoiesis,Haematopoietic stem cells View Publication