技术资料

BACKGROUND {\&} AIMS RNase H2 is a holoenzyme,composed of 3 subunits (ribonuclease H2 subunits A,B,and C),that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2b$\Delta$IEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53$\Delta$IEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells,generated intestinal organoids,and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53$\Delta$IEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients,measured levels of ribonuclease H2 subunit B,and associated these with patient survival times and transcriptome data. RESULTS The H2b$\Delta$IEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53$\Delta$IEC mice. However,H2b/p53$\Delta$IEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T{\textgreater}G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients,we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53$\Delta$IEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis. View Publication

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

Alveolar macrophages (AM) perform a primary defense mechanism in the lung through phagocytosis of inhaled particles and microorganisms. AM are known to be relatively immunosuppressive consistent with the aim to limit alveolar inflammation and maintain effective gas exchange in the face of these constant challenges. How AM respond to T cell derived cytokine signals,which are critical to the defense against inhaled pathogens,is less well understood. For example,successful containment of Mycobacterium tuberculosis (Mtb) in lung macrophages is highly dependent on IFN-$\gamma$ secreted by Th-1 lymphocytes,however,the proteomic IFN-$\gamma$ response profile in AM remains mostly unknown. In this study,we measured IFN-$\gamma$ induced protein abundance changes in human AM and autologous blood monocytes (MN). AM cells were activated by IFN-$\gamma$ stimulation resulting in STAT1 phosphorylation and production of MIG/CXCL9 chemokine. However,the global proteomic response to IFN-$\gamma$ in AM was dramatically limited in comparison to that of MN (9 AM vs 89 MN differentially abundant proteins). AM hypo-responsiveness was not explained by reduced JAK-STAT1 signaling nor increased SOCS1 expression. These findings suggest that AM have a tightly regulated response to IFN-$\gamma$ which may prevent excessive pulmonary inflammation but may also provide a niche for the initial survival and growth of Mtb and other intracellular pathogens in the lung. View Publication

SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically,SLC15A4 is critical for Toll-like receptors (TLRs) 7-9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably,SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential,the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study,we used an integrated chemical proteomics approach to develop a suite of chemical tools,including first-in-class functional inhibitors,for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions. 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

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article,we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE,combined with single-molecule,real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV),D (IGHD),and J (IGHJ) gene usage,complementarity-determining region 3 length,and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together,these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs,purified B cells,and whole blood,which recapitulated results generated by commonly used methods,while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide,for the first time,to our knowledge,simultaneous single-molecule characterization of IGHV,IGHD,IGHJ,and IGHC region genes and alleles,allele-resolved subisotype definition,and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes,FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles,28 (87%) of which were previously uncharacterized. Together,these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV,IGHD,IGHJ,and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date. View Publication

UNLABELLED As part of the Multiple Myeloma Research Foundation (MMRF) immune atlas pilot project,we compared immune cells of multiple myeloma bone marrow samples from 18 patients assessed by single-cell RNA sequencing (scRNA-seq),mass cytometry (CyTOF),and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to understand the concordance of measurements among single-cell techniques. Cell type abundances are relatively consistent across the three approaches,while variations are observed in T cells,macrophages,and monocytes. Concordance and correlation analysis of cell type marker gene expression across different modalities highlighted the importance of choosing cell type marker genes best suited to particular modalities. By integrating data from these three assays,we found International Staging System stage 3 patients exhibited decreased CD4+ T/CD8+ T cells ratio. Moreover,we observed upregulation of RAC2 and PSMB9,in natural killer cells of fast progressors compared with those of nonprogressors,as revealed by both scRNA-seq and CITE-seq RNA measurement. This detailed examination of the immune microenvironment in multiple myeloma using multiple single-cell technologies revealed markers associated with multiple myeloma rapid progression which will be further characterized by the full-scale immune atlas project. SIGNIFICANCE scRNA-seq,CyTOF,and CITE-seq are increasingly used for evaluating cellular heterogeneity. Understanding their concordances is of great interest. To date,this study is the most comprehensive examination of the measurement of the immune microenvironment in multiple myeloma using the three techniques. Moreover,we identified markers predicted to be significantly associated with multiple myeloma rapid progression. 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

Pre-existent cardiovascular disease is a risk factor for weak anti-viral immunity,but underlying mechanisms remain undefined. Here,we report that patients with coronary artery disease (CAD) have macrophages (M??) that actively suppress the induction of helper T cells reactive to two viral antigens: the SARS-CoV2 Spike protein and the Epstein-Barr virus (EBV) glycoprotein 350. CAD M?? overexpressed the methyltransferase METTL3,promoting the accumulation of N�?-methyladenosine (m6A) in Poliovirus receptor (CD155) mRNA. m6A modifications of positions 1635 and 3103 in the 3'UTR of CD155 mRNA stabilized the transcript and enhanced CD155 surface expression. As a result,the patients' M?? abundantly expressed the immunoinhibitory ligand CD155 and delivered negative signals to CD4+ T cells expressing CD96 and/or TIGIT receptors. Compromised antigen-presenting function of METTL3hi CD155hi M?? diminished anti-viral T cell responses in vitro and in vivo. LDL and its oxidized form induced the immunosuppressive M?? phenotype. Undifferentiated CAD monocytes had hypermethylated CD155 mRNA,implicating post-transcriptional RNA modifications in the bone-marrow in shaping anti-viral immunity in CAD. 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