技术资料

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

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

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

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB,it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study,we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly,fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation,suggesting that although BCR and FcγRIIB can both recruit SHIP,this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation,but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP,whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk,as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP,but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling. View Publication

Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules,and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past,however,isolation of human monoclonal antibodies was difficult and inefficient. Here,we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines,especially lines secreting neutralizing antibodies. View Publication

Type 1 diabetes (T1D) is characterized by a chronic,progressive autoimmune attack against pancreas-specific antigens,effecting the destruction of insulin-producing β-cells. Here we show interleukin-2 (IL-2) is a non-pancreatic autoimmune target in T1D. Anti-IL-2 autoantibodies,as well as T cells specific for a single orthologous epitope of IL-2,are present in the peripheral blood of non-obese diabetic (NOD) mice and patients with T1D. In NOD mice,the generation of anti-IL-2 autoantibodies is genetically determined and their titre increases with age and disease onset. In T1D patients,circulating IgG memory B cells specific for IL-2 or insulin are present at similar frequencies. Anti-IL-2 autoantibodies cloned from T1D patients demonstrate clonality,a high degree of somatic hypermutation and nanomolar affinities,indicating a germinal centre origin and underscoring the synergy between cognate autoreactive T and B cells leading to defective immune tolerance. View Publication

The aryl hydrocarbon receptor (AhR),a transcription factor known for mediating xenobiotic toxicity,is expressed in B cells,which are known targets for environmental pollutants. However,it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR,FICZ,induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1,showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr(-/-)) B cells proliferate less than AhR-sufficient (Ahr(+/+)) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr(-/-) B cells are outcompeted by Ahr(+/+) cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno),a direct target of AhR,as a top candidate affected by AhR deficiency. View Publication

The control of uptake and utilization of necessary extracellular nutrients-glucose and glutamine-is an important aspect of B cell activation. Let-7 is a family of microRNAs known to be involved in metabolic control. Here,we employed several engineered mouse models,including B cell-specific overexpression of Lin28a or the let-7a-1/let-7d/let-7f-1 cluster (let-7adf) and knockout of individual let-7 clusters to show that let-7adf specifically inhibits T cell-independent (TI) antigen-induced immunoglobulin (Ig)M antibody production. Both overexpression and deletion of let-7 in this cluster leads to altered TI-IgM production. Mechanistically,let-7adf suppresses the acquisition and utilization of key nutrients,including glucose and glutamine,through directly targeting hexokinase 2 (Hk2) and by repressing a glutamine transporter Slc1a5 and a key degradation enzyme,glutaminase (Gls),a mechanism mediated by regulation of c-Myc. Our results suggest a novel role of let-7adf as a metabolic brake" on B cell antibody production." View Publication