技术资料

The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member,IL-23,shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17-producing T cells. We introduce a novel compound,STA-5326,that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level,and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4+CD45Rbhigh T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon-gamma (IFN-gamma) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326,indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis. View Publication

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus,it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression-based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide,we identified AR-42 (OSU-HDAC42),a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate,but with improved activity at submicromolar concentrations. Here,we report that AR-42 induces NF-κB inhibition,disrupts the ability of Hsp90 to stabilize its oncogenic clients,and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide,the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2-driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials,we expect that our results can be extended to the clinic. View Publication

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76),hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly,cell death is triggered in macrophages cultured with GMME1,while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation,inhibition of p44/42,AKT and STAT3 phosphorylation,and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17,MOG-specific Ab titers,and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2. View Publication

A primary goal of cancer immunotherapy is to improve the naturally occurring,but weak,immune response to tumors. Ineffective responses to cancer vaccines may be caused,in part,by low numbers of self-reactive lymphocytes surviving negative selection. Here,we estimated the frequency of CD8(+) T cells recognizing a self-antigen to be textless0.0001% ( approximately 1 in 1 million CD8(+) T cells),which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity,but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit,most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more,acquire polyfunctionality,and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8(+) T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells,followed by in vivo activation,is a new approach that can be applied to human cancer immunotherapy. Further,precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8(+) T cells. View Publication

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

The regulatory properties of B cells have been studied in autoimmune diseases; however,their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C),an axonal guidance molecule,plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure,with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells,indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19(+)CD138(+) cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c(-/-) CD19(+)CD138(+) cells induced marked pulmonary inflammation,eosinophilia,and increased bronchoalveolar lavage fluid IL-4 and IL-5,whereas adoptive transfer of wild-type CD19(+)CD138(+)IL-10(+) cells dramatically decreased allergic airway inflammation in wild-type and Sema4c(-/-) mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138(+) B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore,we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138(+) B cells. View Publication

Circulating Tumor Cells (CTCs) represent an easy,repeatable and representative access to information regarding solid tumors. However,their detection remains difficult because of their paucity,their short half-life,and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast,sensitive and affordable technique,ideal for rare cells detection. Adapted to CTCs detection (i.e. extremely rare cells),most FC-based techniques require a time-consuming pre-enrichment step,followed by a 2-hours staining procedure,impeding on the efficiency of CTCs detection. We overcame these caveats and reduced the procedure to less than one hour,with minimal manipulation. First,cells were simultaneously fixed,permeabilized,then stained. Second,using low-speed FC acquisition conditions and two discriminators (cell size and pan-cytokeratin expression),we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases,this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses. View Publication

The innate ability of B lymphoma cells to escape control by tumor-reactive T cells must be overcome to develop effective immunotherapies for these diseases. Because signals from both the innate and adaptive immune systems direct the acquisition of strong immunogenicity by professional APCs,the effects of IL-2 and the TLR-7 agonist,S28690,on the immunogenic properties of chronic lymphocytic leukemia (CLL) B cells were studied. IL-2 with S28690 caused CLL cells to proliferate and increased their expression of B7-family members,production of TNF-alpha and IL-10,and levels of tyrosine-phosphorylated STAT-1 and STAT-3 proteins. S28690 increased CD25 expression on CLL cells and sensitized them to IL-2 signaling. However,IL-2 did not change TLR-7 expression or signaling in CLL cells. The ability to stimulate T cell proliferation required additional activation of protein kinase C,which inhibited tumor cell proliferation,switched off" IL-10 production� View Publication

Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However,the mechanisms involved in their in situ development remain unclear. Here,we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria,signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium,TGF-β1 finalizes LC differentiation,and ALK5 is crucial to this process. Moreover,the local microbiota has a major impact on the development of mucosal LCs,whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-β1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota. View Publication

PURPOSE: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore,we compared morphological,immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. METHODS: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days,respectively,in FCS-containing medium (FCS),StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. RESULTS: Serum-free culture of AML-APCs resulted in comparable morphology,relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. CONCLUSION: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological,immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP). View Publication

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy,leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this,short-boosted Ag-specific CD8 T cells continue to contract gradually over time,which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant,functional Ag-specific CD8 T cells that are poised for immediate protection; however,this is at the expense of forming stable long-term memory. View Publication

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast,human T cells,a major anticancer effector cell,only rarely express Siglecs. However,here,we report that the majority of intratumoral,but not peripheral blood,cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity,cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement,which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1,but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted,glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset,while confining T-cell activation to the tumor microenvironment. View Publication