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Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study,we show that the anti-inflammatory extracellular matrix protein,thrombospondin-1,promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor,CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+ CD25- T cells to become suppressive. The latter expressed increased amounts of CTLA-4,OX40,GITR,and Foxp3 and inhibited autologous Th0,Th1,and Th2 cells. Their regulatory activity was contact dependent,TGF-beta independent,and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation. View Publication

NK cells play an important role in the innate immune response. We have isolated NK cells from human lymphoid tissues and found that these cells express the CD4 molecule on their surface at levels higher than those found on peripheral blood NK cells. To study the functional role of the CD4 molecule on NK cells,we developed an in vitro system by which we are able to obtain robust CD4 expression on NK cells derived from blood. CD4+ NK cells efficiently mediate NK cell cytotoxicity,and CD4 expression does not appear to alter lytic function. CD4+ NK cells are more likely to produce the cytokines gamma-IFN and TNF-alpha than are CD4- NK cells. Ligation of CD4 further increases the number of NK cells producing these cytokines. NK cells expressing CD4 are also capable of migrating toward the CD4-specific chemotactic factor IL-16,providing another function for the CD4 molecule on NK cells. Thus,the CD4 molecule is present and functional on NK cells and plays a role in innate immune responses as a chemotactic receptor and by increasing cytokine production,in addition to its well-described function on T cells as a coreceptor for Ag responsive cell activation. View Publication

Nonmammalian glycan structures from helminths act as Th2 adjuvants. Some of these structures are also common on plant glycoproteins. We hypothesized that glycan structures present on peanut glycoallergens act as Th2 adjuvants. Peanut Ag (PNAg),but not deglycosylated PNAg,activated monocyte-derived dendritic cells (MDDCs) as measured by MHC/costimulatory molecule up-regulation,and by their ability to drive T cell proliferation. Furthermore,PNAg-activated MDDCs induced 2- to 3-fold more IL-4- and IL-13-secreting Th2 cells than immature or TNF/IL-1-activated MDDCs when cultured with naive CD4+ T cells. Human MDDCs rapidly internalized Ag in a calcium- and glycan-dependent manner consistent with recognition by C-type lectin. Dendritic cell (DC)-specific ICAM-grabbing nonintegrin (DC-SIGN) (CD209) was shown to recognize PNAg by enhanced uptake in transfected cell lines. To identify the DC-SIGN ligand from unfractionated PNAg,we expressed the extracellular portion of DC-SIGN as an Fc-fusion protein and used it to immunoprecipitate PNAg. A single glycoprotein was pulled down in a calcium-dependent manner,and its identity as Ara h 1 was proven by immunolabeling and mass spectrometry. Purified Ara h 1 was found to be sufficient for the induction of MDDCs that prime Th2-skewed T cell responses. Both PNAg and purified Ara h 1 induced Erk 1/2 phosphorylation of MDDCs,consistent with previous reports on the effect of Th2 adjuvants on DCs. View Publication

MSCs have received attention for their therapeutic potential in a number of disease states,including bone formation,diabetes,stem cell engraftment after marrow transplantation,graft-versus-host disease,and heart failure. Despite this diverse interest,the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors,we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts,which do not home after MI. This analysis identified monocyte chemotactic protein-3 (MCP-3) as a potential MSC homing factor. Overexpression of MCP-3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs,cardiac function improved in MCP-3-expressing hearts (88.7%,p textless .001) but not in control hearts (8.6%,p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather,MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP-3 is an MSC homing factor; local overexpression of MCP-3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration. View Publication

Despite considerable success in treating newly diagnosed childhood acute lymphoblastic leukemia (ALL),relapsed disease remains a significant clinical challenge. Using a NOD/SCID mouse xenograft model,we report that immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODNs) stimulate significant immune activity against primary human ALL cells in vivo. The administration of CpG ODNs induced a significant reduction in systemic leukemia burden,mediated continued disease control,and significantly improved survival of mice with established human ALL. The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODNs and correlated with the production of IL-12p70,IFN-alpha,and IFN-gamma by the host. In addition,depletion of natural killer cells by anti-asialo-GM1 treatment significantly reduced the in vivo antileukemic activity of CpG ODN. This antileukemia effect was not limited to the xenograft model because natural killer cell-dependent killing of ALL by human peripheral blood mononuclear cells (PBMCs) was also increased by CpG ODN stimulation. These results suggest that CpG ODNs have potential as therapeutic agents for the treatment of ALL. View Publication

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast,T cells with other antigen specificities from these patients,or autoreactive T cells from healthy individuals and disease controls,express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells,Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2,SAP97,ZIP,p56(lck),and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation,they suppress Ca2+-signaling,cytokine production,and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted. View Publication

Chaetocin,a thiodioxopiperazine natural product previously unreported to have anticancer effects,was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes,normal B cells,and neoplastic B-CLL (chronic lymphocytic leukemia) cells,indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore,chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone,and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically,chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell,its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover,the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but,instead,heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively,chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. View Publication

Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation,in innate cell clearance of microbial pathogens,and in neural cell maintenance. However,the role of autophagy in T lymphocyte development and survival is not known. Here,we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5-/- chimeric mice,we found that Atg5-deficient T lymphocytes underwent full maturation. However,the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery,Atg5-/- CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore,Atg5-/- CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation. View Publication

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors,and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions,yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore,we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1,and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors,leading to target cell death. View Publication

CTLs act as the effector arm of the cell-mediated immune system to kill undesirable cells. Two processes regulate these effector cells to prevent self reactivity: a thymic selection process that eliminates autoreactive clones and a multistage activation or priming process that endows them with a license to kill cognate target cells. Hitherto no subsequent regulatory restrictions have been ascribed for properly primed and activated CTLs that are licensed to kill. In this study we show that CTLs possess a novel postpriming regulatory mechanism(s) that influences the outcome of their encounter with cognate target cells. This mechanism gauges the degree of Ag density,whereupon reaching a certain threshold significant changes occur that induce anergy in the effector T cells. The biological consequences of this Ag-induced postpriming control includes alterations in the expression of cell surface molecules that control immunological synapse activity and cytokine profiles and induce retarded cell proliferation. Most profound is genome-wide microarray analysis that demonstrates changes in the expression of genes related to membrane potential,TCR signal transduction,energy metabolism,and cell cycle control. Thus,a discernible and unique gene expression signature for anergy as a response to high Ag density has been observed. Consequently,activated T cells possess properties of a self-referential sensory organ. These studies identify a new postpriming control mechanism of CTL with anergenic-like properties. This mechanism extends our understanding of the control of immune function and regulation such as peripheral tolerance,viral infections,antitumor immune responses,hypersensitivity,and autoimmunity. View Publication

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM,but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition,immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides,there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM,their role in disease resistance in SM remains unclear. View Publication

IL-12 is an immunoregulatory cytokine,which promotes Th1 cell differentiation and is a major inducer of IFN-gamma. IFN-beta,a Type I IFN used in the treatment of multiple sclerosis,has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10,a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-beta may in part be a result of its ability to suppress IL-12. However,IL-12 and IFN-beta signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-beta by observing the effect of prior exposure to IL-12 or IFN-beta on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-beta increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor beta1 and beta2 expression. However,despite this up-regulation,IFN-beta suppressed IL-12-induced IFN-gamma expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-beta. View Publication