Scientific Resources

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However,the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells,which play a critical role in the development of the autoimmune response,has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs,whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression,induced via STAT3 phosphorylation,mediates IL-1beta and IL-23 down-regulation,while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc),IL-17A,and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc,IL-17A,and IL-23R,but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3,respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS. 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

Mtb influences DC activity and T cell-mediated immune responses. We show that the treatment of immature monocyte-derived DC with Mtb elicited the formation of mature DC,producing TNF-alpha,IL-1beta,IL-6,and IL-23 and instructing CD4(+) cells to secrete IFN-gamma and IL-17. Mtb-induced cytokine release by DC depended on dectin-1 receptor engagement,whereas MR or DC-SIGN stimulation inhibited this process. A selective dectin-1 binding by the receptor agonist glucan was sufficient to enable DC to generate Th1/Th17 lymphocytes,showing features comparable with those induced by Mtb-treated DC. Interestingly,DC-SIGN or MR engagement inhibited Th17 and increased Th1 generation by glucan- or Mtb-treated DC. Our results indicate that Mtb modulates the lymphocyte response by affecting DC maturation and cytokine release. Dectin-1 engagement by Mtb enables DC to promote a Th1/Th17 response,whereas DC-SIGN and MR costimulation limits dectin-1-dependent Th17 generation and favors a Th1 response,probably by interfering with release of cytokines. View Publication

Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL),its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1),the AKT modulator and proto-oncogene,is differentially expressed in CLL and linked to its pathogenesis based on CD5(+) B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes,which further included the kinases LYN,SYK,ZAP70,and PKC. High TCL1 levels were also strongly associated with aggressive disease features,such as advanced clinical stage,higher white blood cell counts,and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie,shorter progression-free survival,P textless .001),regardless of therapy regimen,especially for ZAP70(+) tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1,-2,and -3) signals through the same heterodimeric receptor complex,which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution,the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells,IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition,IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13,IFN-gamma,and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential. View Publication

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells,the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung,gut and liver. In the liver,these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus,T(H)-17 cells,like T(reg) cells,can be selected by self antigens in the thymus. View Publication

The origin and function of human double negative (DN) TCR-alphabeta+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study,we provide evidence that human TCR-alphabeta+ CD4- CD8- DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-alphabeta+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1beta,IL-17,IFN-gamma,CXCL3,and CXCL2. These results indicate that,upon activation,CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. 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

IL-12 is a potent proinflammatory cytokine. The effects of IL-12 are thought to be mediated by IFN-gamma production by NK,NKT,and T cells. In this study,we show that although IL-12 stimulates NK and NK1.1(+) T cells in bulk mouse splenocytes,it does not significantly stimulate purified NK cells,indicating that other cells are required. IL-12 stimulates T cell-deficient spleen cells and those depleted of macrophages. Unexpectedly,the depletion of dendritic cells also has little effect on the stimulation of spleen cells with IL-12. In contrast,B cell depletion almost completely inhibits IL-12-induced IFN-gamma production and B cell-deficient spleen cells are poorly stimulated with IL-12. Furthermore,purified NK cells are stimulated with IL-12 in the presence of purified B cells. Thus,B cells are necessary and also sufficient for the stimulation of purified NK cells with IL-12. Whereas spleen cells from IL-18-deficient mice are not stimulated with IL-12,NK cells purified from IL-18-deficient mice are stimulated with IL-12 in the presence of wild-type (WT) B cells,and WT NK cells are not stimulated with IL-12 in the presence of IL-18-deficient B cells. Cell contact between B and NK cells is also required for IL-12-induced IFN-gamma production. Finally,B cell-deficient mice injected with IL-12 produce significantly less IFN-gamma and IL-18 in the sera than WT mice do. Thus,stimulation of NK cells with IL-12 requires B cell cooperation in vitro as well as in vivo. View Publication

Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication,but these defenses fail to suppress human immunodeficiency virus (HIV) infection,which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here,we examined antiviral defenses in CD4+ cells during virus infection and coinfection,revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression,but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells,which supported robust viral replication,whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections. View Publication

Mesenteric lymph node (mLN) CD103 (alphaE integrin)(+) dendritic cells (DCs) induce regulatory T cells and gut tolerance. However,the function of intestinal CD103(-) DCs remains to be clarified. CD47 is the ligand of signal regulatory protein alpha (SIRPalpha) and promotes SIRPalpha(+) myeloid cell migration. We first show that mucosal CD103(-) DCs selectively express SIRPalpha and that their frequency was augmented in the lamina propria and mLNs of mice that developed Th17-biased colitis in response to trinitrobenzene sulfonic acid. In contrast,the percentage of SIRPalpha(+)CD103(-) DCs and Th17 responses were decreased in CD47-deficient (CD47 knockout [KO]) mice,which remained protected from colitis. We next demonstrate that transferring wild-type (WT),but not CD47 KO,SIRPalpha(+)CD103(-) DCs in CD47 KO mice elicited severe Th17-associated wasting disease. CD47 expression was required on the SIRPalpha(+)CD103(-) DCs for efficient trafficking to mLNs in vivo,whereas it was dispensable on both DCs and T cells for Th17 polarization in vitro. Finally,administration of a CD47-Fc molecule resulted in reduced SIRPalpha(+)CD103(-) DC-mediated Th17 responses and the protection of WT mice from colitis. We thus propose SIRPalpha(+)CD103(-) DCs as a pathogenic DC subset that drives Th17-biased responses and colitis,and the CD47-SIRPalpha axis as a potential therapeutic target for inflammatory bowel disease. View Publication