Trail⁺DX5⁻Eomes⁻ natural killer (NK) cells arise in the mouse fetal liver and persist in the adult liver. Their relationships with Trail⁻DX5⁺ NK cells remain controversial. We generated a novel Eomes-GFP reporter murine model to address this question. We found that Eomes⁻ NK cells are not precursors of classical Eomes⁺ NK cells but rather constitute a distinct lineage of innate lymphoid cells. Eomes⁻ NK cells are strictly dependent on both T-bet and IL-15, similarly to NKT cells. We observed that, in the liver, expression of T-bet in progenitors represses Eomes expression and the development of Eomes⁺ NK cells. Reciprocally, the bone marrow (BM) microenvironment restricts T-bet expression in developing NK cells. Ectopic expression of T-bet forces the development of Eomes⁻ NK cells, demonstrating that repression of T-bet is essential for the development of Eomes⁺ NK cells. Gene profile analyses show that Eomes⁻ NK cells share part of their transcriptional program with NKT cells, including genes involved in liver homing and NK cell receptors. Moreover, Eomes⁻ NK cells produce a broad range of cytokines, including IL-2 and TNF in vitro and in vivo, during immune responses against vaccinia virus. Thus, mutually exclusive expression of T-bet and Eomes drives the development of different NK cell lineages with complementary functions.

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.

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.

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 localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca²⁺ flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7–6.7), whereas anti-TLR3-mediated Ca²⁺ flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15–40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling.

Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50–p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.