技术资料

Canonically,immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE),IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs),a type of cell of the immune system linked to viral defense,which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE,followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses. View Publication

We demonstrated previously that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1),but it remains unknown whether they exert any effects on B cells,the other component of the adaptive immune system. In this study,we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that HSCs inhibited the upregulation of activation markers on activated B cells,as well as the proliferation of activated B cells and their cytokine/Ig production in vitro,and that pharmaceutically or genetically blocking the interaction of PD-L1 with programmed cell death protein 1 impaired the ability of HSCs to inhibit B cells. To test the newly discovered B cell-inhibitory activity of HSCs in vivo,we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll,a T cell-independent Ag,significantly suppressed Ag-specific IgM and IgG production in vivo,whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion,in addition to inhibiting T cells,mouse HSCs concurrently inhibit B cells via PD-L1. This direct B cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver's immune homeostasis. View Publication

Recently,we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8,a receptor recognizing viral single strand RNA. In this study,we demonstrate that IFNα,a cytokine that modulates the early innate immune responses toward viral and bacterial infections,potently enhances the production of IL-6 in neutrophils stimulated with R848,a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848,but,rather,it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine,in an autocrine manner,leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPβ transcription factor to the IL-6 promoter. Moreover,we show that neutrophils from SLE patients with active disease state,hence displaying an IFN-induced gene expression signature,produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether,data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils,which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases. View Publication

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies,we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system,which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor,yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures,revealing that the reaction engenders both MBC subsets with different immune effector function and,ultimately,LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity,for vaccine development,and for understanding long-term pathogen resistance. View Publication

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones,light and dark,with coordinated roles,controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells,but its functional role in the GC reaction has not been explored. In this study,we report high expression of LLT1 on GC-associated B cells,early plasmablasts,and GC-derived lymphomas. LLT1 expression was readily induced via BCR,CD40,and CpG stimulation on B cells. Unexpectedly,we found high expression of the LLT1 ligand,CD161,on follicular dendritic cells. Triggering of LLT1 supported B cell activation,CD83 upregulation,and CXCR4 downregulation. Overall,these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans. View Publication

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach,however,is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here,we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT." View Publication

Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease,a chronic inflammatory condition causing high morbidity and mortality. Here,we report that in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD),overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1β,driving systemic and tissue inflammation. In patient-derived monocytes and macrophages,increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species,which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3,thus boosting IL-6 and IL-1β production. Reducing glycolysis,scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence,PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction,oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease. View Publication

CD8(+) T cells have a central role in antitumour immunity,but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1,a key cholesterol esterification enzyme,led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells,which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe,which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile,to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1,an established target for atherosclerosis,is therefore also a potential target for cancer immunotherapy. View Publication

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article,we report that these cells,termed 4BL cells,are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result,activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus,for the first time,to our knowledge,these results indicate that aging affects the function of B1a cells. Upon aging,these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells. View Publication

Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments,these three PBMC isolation techniques were compared for cell recovery and viability,PBMC population composition,and cell functionality,aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors,applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures,with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability,assessed by trypan blue exclusion,was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures,for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities,these data may support selection of a specific PBMC isolation technique for downstream analysis. View Publication

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end,we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells,and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo,while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence,we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase,the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings,two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly,these cocktails were either deprived of thrombopoietin or of stem cell factor,two cytokines known to favor megakaryopoiesis and HPC expansion,respectively. Based on these results,a short resource-efficient two-phase culture protocol for the production of Mks near purity (textgreater95%) from human CD34+ CB cells has been established. View Publication

Natural killer (NK) cells have been originally defined by their naturally occurring" effector function. However� View Publication