Cervical cancer development is linked to the persistent infection by high-risk mucosal human papillomaviruses (HPVs) types. The E6 and E7 major oncoproteins from this dsDNA virus play a key role in the deregulation of the cell cycle, apoptosis, and adaptive immune surveillance. In this study, we show for the first time that HPV type 16 (HPV16), the most carcinogenic type among the high-risk subgroup, interferes with innate immunity by affecting the expression of TLRs. Infection of human primary keratinocytes with HPV16 E6 and E7 recombinant retroviruses inhibits TLR9 transcription and hence functional loss of TLR9-regulated pathways. Similar findings were achieved in HPV16-positive cancer-derived cell lines and primary cervical cancers, demonstrating that this event occurs also in an in vivo context. Interestingly, E6 and E7 from the low-risk HPV type 6 are unable to down-regulate the TLR9 promoter. In addition, E6 and E7 from the high-risk HPV type 18, which are known to persist less competently in the host than HPV16, have reduced efficiency compared with HPV16 in inhibiting TLR9 transcription. Furthermore, a CpG motif derived from the HPV16 E6 DNA sequence activated TLR9, indicating this virus is able to initiate innate responses via the receptor it later down-regulates. This study reveals a novel mechanism used by HPV16 to suppress the host immune response by deregulating the TLR9 transcript, providing evidence that abolishing innate responses may be a crucial step involved in the carcinogenic events mediated by HPVs.

We have previously reported that mouse plasmacytoid dendritic cells (DC) produce high levels of IL-12p70, whereas bone marrow-derived myeloid DC and splenic DC produce substantially lower levels of this cytokine when activated with the TLR-9 ligand CpG. We now show that in response to CpG stimulation, high levels of IL-10 are secreted by macrophages, intermediate levels by myeloid DC, but no detectable IL-10 is secreted by plasmacytoid DC. MyD88-dependent TLR signals (TLR4, 7, 9 ligation), Toll/IL-1 receptor domain-containing adaptor-dependent TLR signals (TLR3, 4 ligation) as well as non-TLR signals (CD40 ligation) induced macrophages and myeloid DC to produce IL-10 in addition to proinflammatory cytokines. IL-12p70 expression in response to CpG was suppressed by endogenous IL-10 in macrophages, in myeloid DC, and to an even greater extent in splenic CD8 alpha(-) and CD8 alpha(+) DC. Although plasmacytoid DC did not produce IL-10 upon stimulation, addition of this cytokine exogenously suppressed their production of IL-12, TNF, and IFN-alpha, showing trans but not autocrine regulation of these cytokines by IL-10 in plasmacytoid DC. The Journal of Immunology, 2006, 177: 7551-7558.

Antiviral immunity requires early and late mechanisms in which IFN-alpha and IL-12 play major roles. However, the initial events leading to their production remain largely unclear. Given the crucial role of TLR in innate recognition, we investigated their role in antiviral immunity in vivo. Upon murine CMV (MCMV) infection, both MyD88-/- and TLR9-/- mice were more susceptible and presented increased viral loads compared with C57BL/6, TLR2-/-, TLR3-/-, or TLR4-/- mice. However, in terms of resistance to infection, IFN-alpha production and in many other parameters of early inflammatory responses, the MyD88-/- mice showed a more defective response than TLR9-/- mice. In the absence of the TLR9/MyD88 signaling pathway, cytokine production was dramatically impaired with a complete abolition of bioactive IL-12p70 serum release contrasting with a high flexibility for IFN-alpha release, which is initially (36 h) plasmacytoid dendritic cell- and MyD88-dependent, and subsequently (44 h) PDC-, MyD88-independent and, most likely, TLR-independent. NK cells from MCMV-infected MyD88-/- and TLR9-/- mice displayed a severely impaired IFN-gamma production, yet retained enhanced cytotoxic activity. In addition, dendritic cell activation and critical inflammatory cell trafficking toward the liver were still effective. In the long term, except for isotype switching to MCMV-specific IgG1, the establishment of Ab responses was not significantly altered. Thus, our results demonstrate a critical requirement of TLR9 in the process of MCMV sensing to assure rapid antiviral responses, coordinated with other TLR-dependent and -independent events that are sufficient to establish adaptive immunity.

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 FcRγ as shown by translocation of FcRγ 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 Ca2+ 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.

DC‐LAMP, a member of the lysosomal‐associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DCmaturation. DC‐LAMP is detected first in activated human DC within MHC class II molecules‐containing compartments just before the translocation of MHC class II‐peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC‐LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC‐LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2‐B4 and 3q26) and conserved organization with the human DC‐LAMP gene. Analysis of mouse DC‐LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo . In conclusion, mouse DC‐LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC‐LAMP in human DC.

Members of the ADAM superfamily of metalloprotease genes are involved in a number of biological processes, including fertilization, neurogenesis, muscle development, and the immune response. These proteins have been classified into several groups. The prototypic ADAM family is comprised of a pro-domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, a transmembrane domain, and a variable cytoplasmic tail. We recently identified a novel member of this superfamily, ADAMDEC1 (decysin). Due to the partial lack of a disintegrin domain and the total lack of a cysteine-rich domain, this protein has been placed in a novel subclass of the ADAM gene family. We have investigated the gene structure of the human and mouse ADAMDEC1 and have revealed a metalloprotease gene cluster on human Chromosome 8p12 comprising ADAMDEC1, ADAM7, and ADAM28. Our results suggest that ADAMDEC1 has arisen by partial gene duplication from an ancestral gene at this locus and has acquired a novel function. ADAMDEC1 is expressed in the immune system, by dendritic cells and macrophages. The relatedness of ADAMDEC1, ADAM7, and ADAM28 suggests that these proteases share a similar function.

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a− DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c− DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcγRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

We have previously characterized IGSF6 (DORA), a novel member of the immunoglobulin superfamily (IGSF) from human and rat expressed in dendritic and myeloid cells. Using a probe from the open reading frame of the rat cDNA, we isolated a cosmid which contains the entire mouse gene. By comparative analysis and reverse transcriptase polymerase chain reaction, we defined the intron/exon structure and the mRNA of the mouse gene and, with respect to human BAC clones, the human gene. The genes span 10 kb (mouse) and 12 kb (human), with six exons arranged in a manner similar to other members of the IGSF. All intron/exon boundaries follow the GT-AG rule. Expression of the mouse Igsf6 gene is restricted to cells of the immune system, particularly macrophages. Northern blot revealed a single mRNA of 2.5 kb, in contrast to the human gene which is expressed as two mRNAs of 1 and 2.5 kb. The human and mouse genes were localized to a locus associated with inflammatory bowel disease. Analysis of the flanking regions of the Igsf6 gene revealed the presence of an unrelated gene, transcribed from the opposite strand of the DNA and oriented such that the Igsf6 gene is encoded entirely within an intron. An identical organization is seen in human. This gene of unknown function is transcribed and processed, contains homologues in Caenorhabditis elegans and prokaryotes, and is expressed in most organs in the mouse.