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The CD31(+) subset of human naive CD4(+) T cells is thought to contain the population of cells that have recently emigrated from the thymus,while their CD31(-) counterparts have been proposed to originate from CD31(+) cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4(+) T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31(+) naive CD4(+) T cells from adult peripheral blood compared with the CD31(-) subset. IL-7-driven proliferation did not result in loss of CD31 expression,suggesting that CD31(+) naive CD4(+) T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore,IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together,our data suggest that during adulthood CD31(+) naive CD4(+) T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. View Publication

Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However,whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin,where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6,which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs,purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless,these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall,our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations. 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

Identification of rational therapeutic targets is an important strategy to improve the cure rate of diffuse large B-cell lymphoma (DLBCL). We previously showed that inhibition of the phosphodiesterase 4B (PDE4B) unleashes cyclic-AMP (cAMP) inhibitory effects toward the PI3K/AKT pathway and induces apoptosis. These data raised important considerations as to which upstream regulators mediate cAMP inhibition of PI3K/AKT,and how identifying this signaling route could be translated into clinical initiatives. We found that in normal and malignant B cells,cAMP potently inhibit the phosphorylation and activity of the tyrosine kinase SYK. Using genetic models of gain- and loss-of-function,we demonstrated the essential role for PDE4B in controlling these effects in DLBCL. Furthermore,we used a constitutively active SYK mutant to confirm its central role in transducing cAMP effects to PI3K/AKT. Importantly,given SYK credentials as a therapeutic target in B-cell tumors,we explored the role of PDE4B in these responses. In multiple DLBCL models,we found that genetically,hence specifically,inhibiting PDE4B expression significantly improved the efficacy of SYK inhibitors. Our data defined a hitherto unknown role for cAMP in negatively regulating SYK and indicate that combined inhibition of PDE4B and SYK should be actively pursued. View Publication

Persistent viral infections are a major health concern worldwide. During persistent infection,overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection,and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously,we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However,the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8(+) and CD4(+) memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers,illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen. View Publication

Recent studies involving bone marrow mesenchymal stromal cells (MSCs) demonstrated that interferon (IFN)-gamma stimulation induces major histocompatibility complex (MHC) class II-mediated antigen presentation in MSCs both in vitro and in vivo. Concordantly,we investigated the ability of MSCs to present extracellular antigen through their MHC class I molecules,a process known as cross-presentation. Using an in vitro antigen presentation assay,we demonstrated that murine MSCs can cross-present soluble ovalbumin (OVA) to naive CD8(+) T cells from OT-I mice. Cross-presentation by MSC was proteasome dependent and partly dependent on transporter associated with antigen-processing molecules. Pretreatment of MSC with IFN-gamma increased cross-presentation by up-regulating antigen processing and presentation. However,although the transcription of the transporter associated with antigen processing-1 molecules and the immunoproteasome subunit LMP2 induced by IFN-gamma was inhibited by transforming growth factor-beta,the overall cross-presentation capacity of MSCs remained unchanged after transforming growth factor-beta treatment. These observations were validated in vivo by performing an immune reconstitution assay in beta(2)-microglobulin(-/-) mice and show that OVA cross-presentation by MSCs induces the proliferation of naive OVA-specific CD8(+) T cells. In conclusion,we demonstrate that MSCs can cross-present exogenous antigen and induce an effective CD8(+) T-cell immune response,a property that could be exploited as a therapeutic cell-based immune biopharmaceutic for the treatment of cancer or infectious diseases. View Publication

Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion,which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis,raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs),which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes,which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts,respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. View Publication

Murine CMV (MCMV) establishes a systemic,low-level persistent infection resulting in the accumulation of CD8(+) T cells specific for a subset of viral epitopes,a process called memory inflation. Although replicating virus is rarely detected in chronically infected C57BL/6 mice,these inflationary cells display a phenotype suggestive of repeated Ag stimulation,and they remain functional. CD4(+) T cells have been implicated in maintaining the function and/or number of CD8(+) T cells in other chronic infections. Moreover,CD4(+) T cells are essential for complete control of MCMV. Thus,we wondered whether CD4(+) T cell deficiency would result in impaired MCMV-specific CD8(+) T cell responses. Here we show that CD4(+) T cell deficiency had an epitope-specific impact on CD8(+) T cell memory inflation. Of the three codominant T cell responses during chronic infection,only accumulation of the late-appearing IE3-specific CD8(+) T cells was substantially impaired in CD4(+) T cell-deficient mice. Moreover,the increased viral activity did not drive increased CD8(+) T cell division or substantial dysfunction in any MCMV-specific population that we studied. These data show that CD4(+) T cell help is needed for inflation of a response that develops only during chronic infection but is otherwise dispensable for the steady state maintenance and function of MCMV-specific CD8(+) T cells. 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

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However,the role of PKCiota in lung tumor development has not been investigated. To address this question,we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene,Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore,the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus,Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore,aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment. View Publication

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice,regardless of the route of challenge infection. Remarkably,cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts,cps1-1 strain-elicited CD8(+) immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition,we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively,these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain. View Publication

The activating NK cell receptor DNAX accessory molecule-1 (DNAM-1) contributes to tumor immune surveillance and plays a crucial role in NK cell-mediated recognition of several types of human tumors,including ovarian carcinoma. Here,we have analyzed the receptor repertoire and functional integrity of NK cells in peritoneal effusions from patients with ovarian carcinoma. Relative to autologous peripheral blood NK cells,tumor-associated NK cells expressed reduced levels of the DNAM-1,2B4,and CD16 receptors and were hyporesponsive to HLA class I-deficient K562 cells and to coactivation via DNAM-1 and 2B4. Moreover,tumor-associated NK cells were also refractory to CD16 receptor stimulation,resulting in diminished Ab-dependent cellular cytotoxicity against autologous tumor cells. Coincubation of NK cells with ovarian carcinoma cells expressing the DNAM-1 ligand CD155 led to reduction of DNAM-1 expression. Therefore,NK cell-mediated rejection of ovarian carcinoma may be limited by perturbed DNAM-1 expression on tumor-associated NK cells induced by chronic ligand exposure. Thus,these data support the notion that tumor-induced alterations of activating NK cell receptor expression may hamper immune surveillance and promote tumor progression. View Publication