技术资料

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here,we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes,where they trap T cells by releasing the CCR7 ligand,CCL19,and to inflamed central nervous system,where they locally limit immunopathogenesis through interleukin-10 production,thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment,where proB cells develop,is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition,these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. View Publication

BACKGROUND Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples,making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However,it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology,immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells,according to proposed guidelines published by the European Myeloma Network (EMN) in 2012. METHOD Bone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification,13q14 deletion,17p deletion,t(4;14),and t(14;16)] in CD138(+) cells purified by magnetic cell sorting. RESULTS This test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14) were found in two cases. CONCLUSION This technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition,magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies. View Publication

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here,we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen,while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner,while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells,a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack. View Publication

Viral infection triggers induction of antiviral cytokines and effectors,which are critical mediators of innate antiviral immune response. It has been shown that the processing body-associated protein LSm14A is involved in the induction of antiviral cytokines in cell lines but in vivo evidence is lacking. By generating LSm14A-deficient mice,in this study,we show that LSm14A plays a critical and specific role in the induction of antiviral cytokines in dendritic cells (DCs) but not in macrophages and fibroblasts. Induction of antiviral cytokines triggered by the DNA viruses HSV-1 and murid herpesvirus 68 and the RNA virus vesicular stomatitis virus but not Sendai virus was impaired in Lsm14a(-/-) DCs,which is correlated to the functions of the adaptor protein MITA/STING in the antiviral signaling pathways. LSm14A deficiency specifically downregulated MITA/STING level in DCs by impairing its nuclear mRNA precursor processing and subsequently impaired antiviral innate and adaptive immune responses. Our findings reveal a nuclear mRNA precursor processing and cell-specific regulatory mechanism of antiviral immune responses. View Publication

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases,yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process,inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally,IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally,LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP,a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8,thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion,LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders. View Publication

Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade,including arrest,transmigration,and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2,which couples through G$\alpha$i2- and G$\alpha$i3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in G$\alpha$i2-deficient neutrophils. In this study,we show that G$\alpha$i3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested G$\alpha$i2- or G$\alpha$i3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. G$\alpha$i2-,but not G$\alpha$i3-,deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely,G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that G$\alpha$i2 controls arrest and G$\alpha$i3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils. View Publication

PURPOSE Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors,such as prostate cancers. However,targeting MDSCs proved challenging due to their phenotypic heterogeneity. EXPERIMENTAL DESIGN Myeloid cell populations were evaluated using flow cytometry on blood samples,functional assays,and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects,localized and metastatic castration-resistant prostate cancer patients. RESULTS Here,we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3,which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA,thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1,a downstream STAT3 target gene and a potent T-cell inhibitor. CONCLUSIONS Overall,we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression. View Publication

Acute myeloid leukemia (AML) is the most common type of acute leukemia affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (Gemtuzumab ozogamicin) have been shown to improve overall survival,validating CD33 as a target for antibody-based therapy of AML. Here we report the in vitro efficacy of BI 836858,a fully human,Fc-engineered,anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine,two hypomethylating agents that show efficacy in older patients,did not compromise BI 836858-induced NK cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a ten-day course of DAC (pre-DAC,days 4,11 and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared to pre-DAC treatment. Analysis of ligands (L) to activating receptors (NKG2D showed significantly increased NKG2DL expression in day 28 post-DAC samples compared to pre-DAC samples; when NKG2DL receptor was blocked using antibodies,BI 836858-mediated ADCC was significantly decreased,suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML. View Publication

We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity ofNfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation,the formation of GC structures was severely disrupted in theNfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels,which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 repressesIl-6transcription in Fo B cells,with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription ofIl-6.Collectively,our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation ofIl-6gene expression in Fo B cells. View Publication

The T-cell surface molecule TIGIT is an immune checkpoint molecule that inhibits T-cell responses,but its roles in cancer are little understood. In this study,we evaluated the role TIGIT checkpoint plays in the development and progression of gastric cancer. We show that the percentage of CD8 T cells that are TIGIT+ was increased in gastric cancer patients compared with healthy individuals. These cells showed functional exhaustion with impaired activation,proliferation,cytokine production,and metabolism,all of which were rescued by glucose. In addition,gastric cancer tissue and cell lines expressed CD155,which bound TIGIT receptors and inactivated CD8 T cells. In a T cell-gastric cancer cell coculture system,gastric cancer cells deprived CD8 T cells of glucose and impaired CD8 T-cell effector functions; these effects were neutralized by the additional glucose or by TIGIT blockade. In gastric cancer tumor cells,CD155 silencing increased T-cell metabolism and IFNγ production,whereas CD155 overexpression inhibited T-cell metabolism and IFNγ production; this inhibition was neutralized by TIGIT blockade. Targeting CD155/TIGIT enhanced CD8 T-cell reaction and improved survival in tumor-bearing mice. Combined targeting of TIGIT and PD-1 further enhanced CD8 T-cell activation and improved survival in tumor-bearing mice. Our results suggest that gastric cancer cells inhibit CD8 T-cell metabolism through CD155/TIGIT signaling,which inhibits CD8 T-cell effector functions,resulting in hyporesponsive antitumor immunity. These findings support the candidacy of CD155/TIGIT as a potential therapeutic target in gastric cancer. Cancer Res; 77(22); 6375-88. textcopyright2017 AACR. View Publication

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study,we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs,characterized by transcriptional repression of IL-1α and β,IL-1R1,and vascular endothelial growth factor A,and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs,therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated,at least in part,by increases in EPC/CAC proliferation,by decreases in EPC/CAC apoptosis,and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs,and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis,but not anti-neutrophil cytoplasmic Ab-positive vasculitis,showed this pathway to be operational in vivo,with increased IL-1R antagonist,downregulation of vascular endothelial growth factor A,and glomerular and blood vessel decreased capillary density,compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease. View Publication

The adaptive immune response involves T cell differentiation and migration to sites of inflammation. T cell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings,discovered in neutrophils,facilitate cell rolling at high shear stress. Here,we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1),Th17,and regulatory T (Treg) cells but less in Th2 cells. In vivo,endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1,Th17,and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 T cells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 T cell access to sites of inflammation. View Publication