技术资料

Inhibitory receptors (IRs) function as critical regulators of immune responses by tempering T cell activity. In humans,several persisting viruses as well as cancers exploit IR signaling by upregulating IR ligands,resulting in suppression of T cell function (i.e.,exhaustion). This allows escape from immune surveillance and continuation of disease. Here,we report the design,implementation,and results of a phenotypic high-throughput screen for molecules that modulate CD8+ T cell activity. We identify 19 compounds from the ReFRAME drug-repurposing collection that restore cytokine production and enhance the proliferation of exhausted T cells. Analysis of our top hit,ingenol mebutate,a protein kinase C (PKC) inducing diterpene ester,reveals a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on T cells. Collectively,these results demonstrate a disease-relevant methodology for identifying modulators of T cell function and reveal new targets for immunotherapy. 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

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease with no known cause or mechanism. There is an increasing appreciation for the role of immune and metabolic dysfunction in the disease. ME/CFS has historically presented in outbreaks,often has a flu-like onset,and results in inflammatory symptoms. Patients suffer from severe fatigue and post-exertional malaise. There is little known about the metabolism of specific immune cells in ME/CFS patients. To investigate immune metabolism in ME/CFS,we isolated CD4+ and CD8+ T cells from 53 ME/CFS patients and 45 healthy controls. We analyzed glycolysis and mitochondrial respiration in resting and activated T cells,along with markers related to cellular metabolism,and plasma cytokines. We found that ME/CFS CD8+ T cells have reduced mitochondrial membrane potential compared to healthy controls. Both CD4+ and CD8+ T cells from ME/CFS patients had reduced glycolysis at rest,while CD8+ T cells also had reduced glycolysis following activation. ME/CFS patients had significant correlations between measures of T cell metabolism and plasma cytokine abundance that differed from healthy control subjects. Our data indicate that patients have impaired T cell metabolism consistent with ongoing immune alterations in ME/CFS that may illuminate the mechanism behind this disease. View Publication

Drug resistance and relapse remain key challenges in pancreatic cancer. Here,we have used RNA sequencing (RNA-seq),chromatin immunoprecipitation (ChIP)-seq,and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells,highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular,the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (ROR$\gamma$),known to drive inflammation and T cell differentiation,was upregulated during pancreatic cancer progression,and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further,a large-scale retrospective analysis in patients revealed that ROR$\gamma$ expression may predict pancreatic cancer aggressiveness,as it positively correlated with advanced disease and metastasis. Collectively,these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells,suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients. View Publication

The SARS-CoV-2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID-19 pathogenesis,especially host factors facilitating virus infection and replication. SARS-CoV-2 was reported to enter cells via binding to ACE2,followed by its priming by TMPRSS2. Here,we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors,39,778 cells) and in cells derived from subsegmental bronchial branches (four donors,17,521 cells) by single nuclei and single cell RNA sequencing,respectively. While TMPRSS2 is strongly expressed in both tissues,in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly,these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS-CoV-2 infection. Our data provide a rich resource for future investigations of COVID-19 infection and pathogenesis. View Publication

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1),an actin bundling protein,resulted in an ocular commensal overgrowth,characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas,altered inflammatory responses,and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. Cre pos LPLfl/fl ) and S100A8 Cre (S100A8.Cre pos LPLfl/fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast,infected CD11c.Cre pos LPLfl/fl mice did not display elevated susceptibility to infection,implicating the myeloid L-plastin-sufficient cells (e.g.,macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically,the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa,augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively,our data highlight the importance of neutrophils in controlling ocular paucibacteriality,reveal molecular and cellular events involved in the process,and suggest a link between commensal exposure and resistance to infection. View Publication

Historically,it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However,in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research,including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR),which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications,including regenerative medicine,drug sensitivity testing,gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue,and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d,the technique is directly applicable to diagnostic and predictive medicine. Moreover,the epithelial cells can be propagated indefinitely in vitro,yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment. View Publication

Stem cell therapy is widely employed in treating osteoarthritis (OA),and bone marrow-derived mesenchymal stem cells (BMSCs) has gradually become the most attractive new method for treating OA due to the benefit for cartilage tissue repair. However,the apoptosis in the neural stem cell transplantation severely decreases repairing efficacy. Icariin has been reported to exert multiple effects on BMSCs,including its proliferation,osteogenic,and chondrogenic differentiation. However,its effects on the injury induced by oxygen,glucose and serum deprivation (OGD) remains unknown. We prospectively investigated the role of ICA on rabbit BMSCs under conditions of OGD. Firstly,BMSCs were cultured under conditions of OGD,ICA relieved OGD-induced cell damage by promoting cell proliferation and suppressing apoptosis. Secondly,Markers of endoplasmic reticulum stress (ERs),ER stress IRE-1 pathway,and autophagy were both inhibited by ICA via inhibition of phosphor-extracellular regulated protein kinases (p-ERKs),p-P38,p-c-Jun N-terminal kinase (p-JNK) or si-MAPK. Finally,decrease of ERs marker levels enhanced protective effect of ICA against OGD-induced injury by limiting apoptosis and autophagy. Moreover,an autophagy inhibitor (3-methyladenine: 3-MA) contributed to a synergistic effect in conjunction with ICA,in promoting cell proliferation,suggesting that ICA exerts anti-ERs and anti-autophagy effects in OGD-treated BMSCs. Therefore,ICA protected rabbit BMSCs from OGD-induced apoptosis through inhibitory regulation of ERs-mediated autophagy related to the MAPK signaling pathway,which provided insights for a potential therapeutic strategy in OA. View Publication

S100A12 is a calcium-binding protein of the S100 subfamily of myeloid-related proteins that acts as an alarmin to induce a pro-inflammatory innate immune response. It has been linked to several chronic inflammatory diseases,however its role in the common oral immunopathology periodontitis is largely unknown. Previous in vitro monoculture experiments indicate that S100A12 production decreases during monocyte differentiation stages,while the regulation within tissue is poorly defined. This study evaluated S100A12 expression in monocyte subsets,during monocyte-to-macrophage differentiation and following polarization,both in monoculture and in a tissue context,utilizing a three-dimensional co-culture oral tissue model. Further,we explored the involvement of S100A12 in periodontitis by analyzing its expression in peripheral circulation and gingival tissue,as well as in saliva. We found that S100A12 expression was higher in classical than in non-classical monocytes. S100A12 expression and protein secretion declined significantly during monocyte-to-macrophage differentiation,while polarization of monocyte-derived macrophages had no effect on either. Peripheral monocytes from periodontitis patients had higher S100A12 expression than monocytes from controls,a difference particularly observed in the intermediate and non-classical monocyte subsets. Further,monocytes from periodontitis patients displayed an increased secretion of S100A12 compared with monocytes from controls. In oral tissue cultures,monocyte differentiation resulted in increased S100A12 secretion over time,which further increased after inflammatory stimuli. Likewise,S100A12 expression was higher in gingival tissue from periodontitis patients where monocyte-derived cells exhibited higher expression of S100A12 in comparison to non-periodontitis tissue. In line with our findings,patients with severe periodontitis had significantly higher levels of S100A12 in saliva compared to non-periodontitis patients,and the levels correlated to clinical periodontal parameters. Taken together,S100A12 is predominantly secreted by monocytes rather than by monocyte-derived cells. Moreover,S100A12 is increased in inflamed tissue cultures,potentially as a result of enhanced production by monocyte-derived cells. This study implicates the involvement of S100A12 in periodontitis pathogenesis,as evidenced by increased S100A12 expression in inflamed gingival tissue,which may be due to altered circulatory monocytes in periodontitis. View Publication

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of miRNAs in regulating drug resistance and leukemic stem cell (LSCs) fate,we performed global transcriptome profiling in treatment-na{\{i}}ve chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor; its restored expression impaired survival of drug-resistant cells sensitized them to TKIs in vitro and markedly eliminated long-term repopulating LSCs and infiltrating blast cells conferring a survival advantage in pre-clinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185 and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity sensitizing therapy-resistant cells to TKIs. Thus miR-185 presents as a potential predictive biomarker and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL may provide a valuable strategy for overcoming drug resistance in patients." View Publication

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy,but the overall metabolic properties of HSCs remain elusive. Using combined approaches,including single-cell RNA sequencing (RNA-seq),we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed,but not quiescent,HSCs relied readily on glycolysis. Notably,in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant. View Publication

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here,we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFN$\gamma$/TNF-$\alpha$+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN,whereas without oil,antigen is distributed in the medullary region. Following oil immunization,CXCL10-producing inflammatory monocytes accumulate in the IFR,which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment,CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy. View Publication