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PURPOSE: B-cell receptor signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However,blocking B-cell receptor signaling with dasatinib,an inhibitor of SRC kinase,produced variable results in preclinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. EXPERIMENTAL DESIGN: Fresh CLL B cells were treated with dasatinib,and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of B-cell receptor signaling molecules,as well as with molecular and cytogenetic prognostic factors. RESULTS: Among 50 CLL cases,dasatinib treatment reduced cell viability by 2% to 90%,with an average reduction of 47% on day 4 of culture. The drug induced CLL cell death through the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly,phosphorylation of SRC family kinases was inhibited by dasatinib in good,as well as poor,responders. As opposed to SRC family kinases,activities of two downstream molecules,SYK and phospholipase Cgamma2,correlate well with the apoptotic response of CLL cells to dasatinib. CONCLUSIONS: Thus,SYK inhibition predicts cellular response to dasatinib. SYK,together with phospholipase Cgamma2,may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective,our study suggests the existence of alternative mechanisms or pathways that activate SYK,independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. View Publication

Dysregulation of airway inflammation contributes to lung disease in cystic fibrosis (CF). Inflammation is mediated by inflammatory cytokines,including IL-8,which illustrates an increase in biological half-life and proinflammatory activity when bound to glycosaminoglycans (GAGs). The aim of this project was to compare IL-8 and IL-18 for their relative stability,activity,and interaction with GAGs,including chondroitin sulfate,hyaluronic acid,and heparan sulfate,present in high quantities in the lungs of patients with CF. Bronchoalveolar lavage fluid was collected from patients with CF (n = 28),non-CF controls (n = 14),and patients with chronic obstructive pulmonary disease (n = 12). Increased levels of IL-8 and reduced concentrations of IL-18 were detected in bronchial samples obtained from CF individuals. The low level of IL-18 was not a defect in IL-18 production,as the pro- and mature forms of the molecule were expressed and produced by CF epithelial cells and monocytes. There was,however,a marked competition between IL-8 and IL-18 for binding to GAGs. A pronounced loss of IL-18 binding capacity occurred in the presence of IL-8,which displaced IL-18 from these anionic-matrices,rendering the cytokine susceptible to proteolytic degradation by neutrophil elastase. As a biological consequence of IL-18 degradation,reduced levels of IL-2 were secreted by Jurkat T lymphocytes. In conclusion,a novel mechanism has been identified highlighting the potential of IL-8 to determine the fate of other inflammatory molecules,such as IL-18,within the inflammatory milieu of the CF lung. View Publication

IL-27 is formed by the association of a cytokine subunit,p28,with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27,p28/CLF is produced by dendritic cells and is biologically active on human NK cells,increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells,p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore,p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors. View Publication

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs),high interferon-gamma (IFN-gamma) production,but little cytotoxicity. CD56(dim) NK cells have high KIR expression,produce little IFN-gamma,yet display high cytotoxicity. We hypothesized that,if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype,an intermediary NK cell must exist,which demonstrates more functional overlap than these 2 subsets,and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L,CD2,and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively,our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that,in vivo,human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype. View Publication

RATIONALE: Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However,the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. OBJECTIVES: We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. METHODS: The study was performed in a cohort of 21 subjects with allergic asthma,21 healthy control subjects,and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. MEASUREMENTS AND MAIN RESULTS: Dendritic cells cultured with natural regulatory T cells up-regulated IL-10,down-regulated costimulatory molecules,and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore,this defective function of natural regulatory T cells was associated with their decreased IL-10 expression,disease severity,and could be reversed by oral corticosteroid therapy. CONCLUSIONS: These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma. View Publication

Because IL-1beta plays an important role in inflammation in human and murine arthritis,we investigated the contribution of the inflammasome components ASC,NALP-3,IPAF,and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA,decreased levels of synovial IL-1beta,and diminished serum amyloid A levels. In contrast,mice deficient in NALP-3,IPAF,or caspase-1 did not show any alteration of joint inflammation,thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes,we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro,as was the production of IFN-gamma,whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice,but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion,these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation. View Publication

Small intestinal CD103(+) dendritic cells (DCs) have the selective ability to promote de novo generation of regulatory T cells via the production of retinoic acid (RA). Considering that aldehyde dehydrogenase (ALDH) activity controls the production of RA,we used a flow cytometry-based assay to measure ALDH activity at the single-cell level and to perform a comprehensive analysis of the RA-producing DC populations present in lymphoid and nonlymphoid mouse tissues. RA-producing DCs were primarily of the tissue-derived,migratory DC subtype and can be readily found in the skin and in the lungs as well as in their corresponding draining lymph nodes. The RA-producing skin-derived DCs were capable of triggering the generation of regulatory T cells,a finding demonstrating that the presence of RA-producing,tolerogenic DCs is not restricted to the intestinal tract as previously thought. Unexpectedly,the production of RA by skin DCs was restricted to CD103(-) DCs,indicating that CD103 expression does not constitute a universal" marker for RA-producing mouse DCs. Finally� View Publication

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses,the function of some,like virion protein 11/12 (VP11/12),have not been clearly defined. Previously,we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis,M. J. Wagner,R. C. Doepker,J. M. Maciejko,C. M. Crider,K. R. Jerome,and J. R. Smiley,J. Virol. 82:6098-6108,2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here,we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394),a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However,we were unable to detect an interaction between ICP0 and Lck in T lymphocytes,and we show that ICP0,US3,and UL13 are not strictly required for Lck activation. In contrast,VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus,VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. View Publication