SUMMARY MicroRNAs regulate the function of several immune cells but their role in promoting CD8+ T-cell immunity remains unknown. Here we report that miR-155 is required for CD8+ T-cell responses to both virus and cancer. In the absence of miR-155, accumulation of effector CD8+ T cells was severely reduced during acute and chronic viral infections and control of virus replication was impaired. Similarly, Mir155-/- CD8+ T cells were in effective at controlling tumor growth, whereas miR-155 overexpression enhanced the antitumor response. miR-155 deficiency resulted in accumulation of SOCS-1 causing defective cytokine signaling through STAT5. Consistently, enforced expression of SOCS-1 in CD8+ T cells phenocopied the miR-155 deficiency, whereas SOCS-1 silencing augmented tumor destruction. These findings identify miR-155 and its target SOCS-1 as key regulators of effector CD8+ T cells that can be modulated to potentiate immunotherapies for infectious diseases and cancer.

Human TLR10 is an orphan member of the TLR family. Genomic studies indicate that TLR10 is in a locus that also contains TLR1 and TLR6, two receptors known to function as coreceptors for TLR2. We have shown that TLR10 was not only able to homodimerize but also heterodimerized with TLRs 1 and 2. In addition, unlike TLR1 and TLR6, TLR10 was expressed in a highly restricted fashion as a highly N-glycosylated protein, which we detected in B cell lines, B cells from peripheral blood, and plasmacytoid dendritic cells from tonsil. We were also able to detect TLR10 in a CD1a+ DC subset derived from CD34+ progenitor cells which resemble Langerhans cells in the epidermis. Although we were unable to identify a specific ligand for TLR10, by using a recombinant CD4TLR10 molecule we also demonstrated that TLR10 directly associates with MyD88, the common Toll IL-1 receptor domain adapter. Additionally, we have characterized regions in the Toll IL-1 receptor domain of TLR10 that are essential in the activation of promoters from certain inflammatory cytokines. Even though TLR10 expression has not been detected in mice, we have identified a partial genomic sequence of the TLR10 gene that was present but nonfunctional and disrupted by a retroviral insertion in all mouse strains tested. However, a complete TLR10 sequence could be detected in the rat genome, indicating that a functional copy may be preserved in this species.

Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly local- Mammalian Toll-like receptors (TLRs) 7 belong to a family of receptors that recognize pathogen-associated molecular patterns. TLRs play a key role in host defense during pathogen infection by regulating and linking the innate and adaptive immune responses (1-3). TLRs are expressed in dendritic cells (DC), sentinels of the immune system, endowing them with the capacity to sense pathogen-derived products and to alert the immune system (4). Members of the TLR family are also variably expressed on nonhematopoietic cells. TLR-deficient mice and transfected cell lines have been the keys to understanding TLR function. Ligand specificity has been elucidated for most TLRs; thus, TLR2 and TLR4 recognize Gram-positive and Gram-negative bacterial cell wall products, respectively. TLR5 recognizes a structural epitope of bacterial flagellin, and TLR7, TLR8, and TLR9 have been demonstrated to recognize different forms of microbial-derived nucleic acid (5). Host-derived ligands for the TLRs have also been identified; in particular, TLR4 recognizes heat shock proteins and pulmonary surfactant (6), and TLR9 recognizes chromatin-IgG complexes (7).TLR3 has been extensively characterized to be a receptor for poly(I-C), a synthetic double-stranded RNA (dsRNA) mimic (8); recently, it has been shown to mediate responses to West Nile virus (9) as well as dsRNA derived from the helminth parasite Schistosoma (10). Additionally, host-derived mRNA has recently been shown to activate TLR3, suggesting that activation via TLR3 can occur in a variety of situations (11). Whereas most TLRs recruit myeloid differentiation factor 88 (12), with some variation in the signaling profile mediated by the additional recruitment of Toll/interleukin-1 receptor-containing adapter protein and TRIF-related adaptor molecule, TLR3 recruits only TRIF (13-15). TRIF can activate both NF-B through TRAF6 and receptor-interacting protein-1 (16) and interferon-regulatory factor 3 through IB kinase/ TANK-binding kinase 1 (17) and phosphoinositide 3-kinase (18). Although the signaling pathway of TLRs is increasingly well characterized, the parameters controlling interactions between the recept...

We have isolated a novel cell surface molecule, the human homolog of osteoclast-associated receptor (OSCAR). Unlike mouse OSCAR, hOSCAR is widely transcribed in cells of the myeloid lineage. Notably, hOSCAR is expressed on circulating blood monocytes and CD11c ؉ dendritic cells but not on T and B cells. hOSCAR is continually expressed during differentiation of CD14 ؉ monocytes into dendritic cells and maintained after maturation. hOSCAR associates with the FcR␥ as shown by translocation of FcR␥ to the cell surface in presence of hOSCAR and coimmunoprecipitation from transfected cell lines and ex vivo cells. Engagement of hOSCAR with specific mAb leads to Ca 2؉ mobilization and cytokine release, indicators of cellular activation. Endocytosis of the receptor in dendritic cells was observed, followed by passage of the internalized material into Lamp-1 ؉ and HLA-DR ؉ compartments, suggesting a role in antigen uptake and presentation.Dendritic cells were able to stimulate a T-cell clone specific for an epitope of mouse IgG1 after uptake and processing of the hOSCAR-specific antibody, demonstrating the capacity of this receptor to mediate antigen presentation. hOSCAR thus represents a novel class of molecule expressed by dendritic cells involved in the initiation of the immune response.

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4+ T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4+ T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4+ T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2ΔCD4Cre) were infected with the protozoan parasite Leishmania major. N1N2ΔCD4Cre mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4+ T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4+ T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Interactions between Notch1 receptors on lymphoid progenitors and Delta-like 4 (DL4) ligands on cortical thymic epithelial cells (cTEC) are essential for T cell lineage commitment, expansion, and maturation in the thymus. Using a novel mAb against DL4, we show that DL4 levels on cTEC are very high in the fetal and neonatal thymus when thymocyte expansion is maximal but decrease dramatically in the adult when steady-state homeostasis is attained. Analysis of mutant mouse strains where thymocyte development is blocked at different stages indicates that lymphostromal interactions (“thymus crosstalk”) are required for DL4 down-regulation on cTEC. Reconstitution of thymocyte development in these mutant mice further suggests that maturation of thymocytes to the CD4+CD8+ stage and concomitant expansion are needed to promote DL4 down-regulation on cTEC. Collectively, our data support a model where thymic crosstalk quantitatively regulates the rate of Notch1-dependent thymopoiesis by controlling DL4 expression levels on cTEC.

We have previously described the human osteoclast associated receptor (hOSCAR), expressed in all cells of the myeloid lineage, and its immune functions. This receptor, which associates with the FcRγ chain to transduce an activating signal, induces calcium flux in monocytes and dendritic cells, and modulates specific responses of dendritic cells. In this study, we have examined the effects of hOSCAR ligation on various proinflammatory responses of monocytes and neutrophils. Monocytes stimulated via hOSCAR ligation released IL-8/CXCL8 and other chemokines such as epithelial neutrophil-activating peptide-78/CXCL5, macrophage-derived chemokine/CCL22, and MCP-1/CCL2 and up-regulated markers involved in cell adhesion and costimulatory functions. Monocytes stimulated via hOSCAR in the absence of survival factors had an increased life span. Although the life span of neutrophils was unaffected, these cells, when stimulated via hOSCAR, rapidly released reactive oxygen intermediates, degranulated lactoferrin, myeloperoxidase, and matrix metalloproteinase-9 and also secreted IL-8/CXCL8. Neutrophils also underwent changes in cell surface molecule expression with the cleavage of CD62L and increased expression of CD11b and CD66b after 2-h stimulations. Finally, we demonstrated synergy between hOSCAR and TLR ligands on both monocytes and neutrophils, with up to 8-fold increases in cytokine secretion when hOSCAR was cross-linked in the presence of LPS or R-848. Overall, our data demonstrate that hOSCAR is a functional receptor on monocytes and neutrophils, involved in the induction of the primary proinflammatory cascade and the initiation of downstream immune responses.

IntroductionDendritic cells (DCs) are potent antigen-presenting cells that have a unique ability to prime naive T cells. 1 In addition, they secrete immune-modulating factors that play a key role in the initiation and regulation of both innate resistance and adaptive immunity. DCs undergo striking changes in function and morphology depending on their maturation state and localization. 1 Immature DCs, located in peripheral tissues, are considered immune sentinels and are able to capture and process antigens. 2 Upon activation and maturation induced by proinflammatory signals, such as pathogen-associated molecular patterns (PAMPs), 3 they migrate to the T-cell areas of secondary lymphoid organs, where they are able to present antigens to naive T cells. 4,5 The outcome of the immune response (either immune priming or tolerance) is directly linked to the maturation status of DCs,6 which is generally believed to be induced by endogenous factors such as proinflammatory cytokines (tumor necrosis factor [TNF] or interleukin-1␤ [IL-1␤]) 7,8 or by exogenous products such as PAMPs (eg, lipopolysaccharide [LPS], lipoteichoic acid [LTA], or viral-RNA mimics such as polyinosinicpolycytidylic acid [poly(I:C)]). 9 DC activity can be regulated by both activating and inhibiting immune receptors that transduce signals through immunoreceptor tyrosine-based activating motifs (ITAM; consensus: D/Ex 7 D/ Ex 2 Yx 2 L/Ix 7 Yx 2 L/I) 10,11 and through immunoreceptor tyrosinebased inhibitory motifs (ITIM; consensus: I/V/L/SxYx 2 L/V), 12,13 respectively. This growing family of receptors was first described in lymphocytes, [14][15][16] but in recent years an increasing number of receptors of the lectin and the immunoglobulin superfamilies (IgSFs) linked to ITAM/ITIM signaling expressed on myeloid cells and DCs has been described. 17,18 These data indicate that cells of the myeloid lineage have a large number and variety of receptors regulating their activity. These receptors signal through activating or inhibiting pathways that regulate the amplitude and the duration of the immune response triggered by pathogenic stimuli. [19][20][21] We recently described human osteoclast-associated receptor (hOSCAR), a novel immune receptor associated with the Fc receptor ␥-chain (FcR␥) and involved in endocytosis and antigen presentation through the major histocompatibility complex (MHC) class II pathway in monocyte-derived dendritic cells (monoDCs). 22 Its association with an ITAM-bearing chain confers to hOSCAR the capacity to activate myeloid cells as shown by its ability to trigger calcium flux and cytokine release. 22 As hOSCAR is expressed by both immature and LPS-matured mono-DCs, we proposed that hOSCAR may have a biologic role on human DCs that is probably different to the previously described activating receptors such as the Fc receptors (FcRs) and triggering receptor expressed on myeloid cells-2 (TREM-2), the expression of which is down-regulated after activation of DCs. In contrast to the human receptor, mouse OSCAR (mOSCAR) is only express...