技术资料

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals,but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling,we hypothesize that antiviral adenosine analogs,like remdesivir,have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir,we here show that these properties are due to its metabolite,GS-441524,acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics. View Publication

BACKGROUND Posttraumatic stress disorder,a consequence of psychological trauma,is associated with increased inflammation and an elevated risk of developing comorbid inflammatory diseases. However,the mechanistic link between this mental health disorder and inflammation remains elusive. We previously found that S100a8 and S100a9 messenger RNA,genes that encode the protein calprotectin,were significantly upregulated in T lymphocytes and positively correlated with inflammatory gene expression and the mitochondrial redox environment in these cells. Therefore,we hypothesized that genetic deletion of calprotectin would attenuate the inflammatory and redox phenotype displayed after psychological trauma. METHODS We used a preclinical mouse model of posttraumatic stress disorder known as repeated social defeat stress (RSDS) combined with pharmacological and genetic manipulation of S100a9 (which functionally eliminates calprotectin). A total of 186 animals (93 control,93 RSDS) were used in these studies. RESULTS Unexpectedly,we observed worsening of behavioral pathology,inflammation,and the mitochondrial redox environment in mice after RSDS compared with wild-type animals. Furthermore,loss of calprotectin significantly enhanced the metabolic demand on T lymphocytes,suggesting that this protein may play an undescribed role in mitochondrial regulation. This was further supported by single-cell RNA sequencing analysis demonstrating that RSDS and loss of S100a9 primarily altered genes associated with mitochondrial function and oxidative phosphorylation. CONCLUSIONS These data demonstrate that the loss of calprotectin potentiates the RSDS-induced phenotype,which suggests that its observed upregulation after psychological trauma may provide previously unexplored protective functions. View Publication

Phosphoinositide 3-kinase (PI3K) p110$\delta$ signalling negatively regulates the production of mouse IgE. However,there are disparities between the mouse and human IgE biology,and the role of PI3K p110$\delta$ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110$\delta$ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114,a small molecule inhibitor of PI3K p110$\delta$. Using FACS,RT-PCR and ELISA we examined the effect of PI3K p110$\delta$ inhibition on IgE production and determined the mechanisms involved. Unlike in mice,we observed that PI3K p110$\delta$ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However,the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110$\delta$ inhibition. The expression levels of AID,$\epsilon$ and $\gamma$1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However,we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40,specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition,PI3K p110$\delta$ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion,PI3K p110$\delta$ signalling is required for the production of human IgE,which makes it a pharmacological target for the treatment of allergic disease. View Publication

UNLABELLED Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however,all patients ultimately still progress from their disease. Lack of CAR T-cell persistence,impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile,fitness,and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts,higher CD4/CD8 ratio,and expanded na{\{i}}ve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells decreased central memory phenotype and increased checkpoint inhibitory markers compared with HD-derived products which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. SIGNIFICANCE Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately patients still relapse. In this study we propose to use T cells from HD volunteers which have a stronger T-cell fitness higher cancer killing capacity and are ready to be administered when needed." View Publication

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy,reliable,and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol,bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested,and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients,leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation,and the infiltration of AML cells was detected in the blood,bone marrow,spleen,and liver of recipients by flow cytometry,qPCR,and histological analyses. Thus,intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells. View Publication

Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI,while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore,we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H2O2-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed,which were subjected to exosome treatment. The expression of inflammatory factors,heart function,and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H2O2-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition,they promoted heart function,suppressed inflammatory responses,and de-activated TLR4/NF-$\kappa$B signaling pathway in MI mice. In conclusion,miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4. View Publication

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here,we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs,which are the most potent subsets,it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol,please refer to Liang et al. (2020) and Qiu et al. (2021). View Publication

BACKGROUND Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far,the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD,and compare them to blood neutrophils. METHODS Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n??=??32) and a control group of patients with non-PCD,non-cystic fibrosis bronchiectasis (n??=??5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization,production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production,actin polymerization and the expulsion of NETs. In addition,PCD sputum displayed high activity of neutrophil elastase,and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD. View Publication

BACKGROUND There is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However,there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further,it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages. RESULTS We used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell,CD4 T,and CD8 T na{\{i}}ve and memory cells states. In the process of na{\"{i}}ve to memory activation across the three lineages we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further in central to effector memory differentiation our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes. CONCLUSIONS Our data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes." View Publication

Asthmatic women tend to develop severe airway disease in their reproductive years,and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells,or ILC2,are readily detected in allergic conditions,such as rhinosinusitis,in individuals that develop nasal polyps do to allergen exposures,and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33,an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in na{\{i}}ve and ovalbumin (OVA) challenged female BALB/c mice in comparison to male mice following stimulation with IL-33. Here we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-$\kappa$B signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated and had reduced NF-$\kappa$B activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2 and Nfkb1 was found among top-ranked regulatory interactions. Together these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases." View Publication