Long-lived intestinal tuft cells serve as colon cancer–initiating cells

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth–sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT–dependent genetic lineage–tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate–induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer.     

Human skin carcinoma arising from kidney transplant–derived tumor cells

Tumor cells with donor genotype have been identified in human skin cancer after allogeneic transplantation; however, the donor contribution to the malignant epithelium has not been established. Kidney transplant recipients have an increased risk of invasive skin squamous cell carcinoma (SCC), which is associated with accumulation of the tumor suppressor p53 and TP53 mutations. In 21 skin SCCs from kidney transplant recipients, we systematically assessed p53 expression and donor/recipient origin in laser-microdissected p53⁺ tumor cells. In one patient, molecular analyses demonstrated that skin tumor cells had the donor genotype and harbored a TP53 mutation in codon 175. In a kidney graft biopsy performed 7 years before the skin SCC diagnosis, we found p53⁺ cells in the renal tubules. We identified the same TP53 mutation in these p53⁺ epithelial cells from the kidney transplant. These findings provide evidence for a donor epithelial cell contribution to the malignant skin epithelium in the recipient in the setting of allogeneic kidney transplantation. This finding has theoretical implications for cancer initiation and progression and clinical implications in the context of prolonged immunosuppression and longer survival of kidney transplant patients.     

The combination of bleomycin with suicide or interferon-β gene transfer is able to efficiently eliminate human melanoma tumor initiating cells

We explored the potential of a chemogene therapy combination to eradicate melanoma tumor initiating cells, key producers of recurrence and metastatic spread. Three new human melanoma cell lines, two obtained from lymph nodes and one from spleen metastasis were established and characterized. They were cultured as monolayers and spheroids and, in both spatial configurations they displayed sensitivity to single treatments with bleomycin (BLM) or human interferon-β (hIFNβ) gene or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) lipofection. However, the combination of bleomycin with SG or hIFNβ gene transfer displayed greater antitumor efficacy. The three cell lines exhibited a proliferative behavior consistent with melan A and gp100 melanoma antigens expression, and BRAF V600E mutation. BLM and both genetic treatments increased the fraction of more differentiated and treatment-sensitive cells. Simultaneously, they significantly decreased the sub-population of tumor initiating cells. There was a significant correlation between the cytotoxicity of treatments with BLM and gene transfer and the fraction of cells exhibiting (i) high proliferation index, and (ii) high intracellular levels of reactive oxygen species. Conversely, the fraction of cells surviving to our treatments closely paralleled their (i) colony and (ii) melanosphere forming capacity. A very significant finding was that the combination of BLM with SG or hIFNβ gene almost abrogated the clonogenic capacity of the surviving cells. Altogether, the results presented here suggest that the combined chemo-gene treatments are able to eradicate tumor initiating cells, encouraging further studies aimed to apply this strategy in the clinic.     

Shank-interacting protein–like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells

Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein–like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell–derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP₃) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.     

Immature myeloid cells directly contribute to skin tumor development by recruiting IL-17–producing CD4+ T cells

Evidence links chronic inflammation with cancer, but cellular mechanisms involved in this process remain unclear. We have demonstrated that in humans, inflammatory conditions that predispose to development of skin and colon tumors are associated with accumulation in tissues of CD33⁺S100A9⁺ cells, the phenotype typical for myeloid-derived suppressor cells in cancer or immature myeloid cells (IMCs) in tumor-free hosts. To identify the direct role of these cells in tumor development, we used S100A9 transgenic mice to create the conditions for topical accumulation of these cells in the skin in the absence of infection or tissue damage. These mice demonstrated accumulation of granulocytic IMCs in the skin upon topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in a dramatic increase in the formation of papillomas during epidermal carcinogenesis. The effect of IMCs on tumorigenesis was not associated with immune suppression, but with CCL4 (chemokine [C-C motif] ligand 4)-mediated recruitment of IL-17–producing CD4⁺ T cells. This chemokine was released by activated IMCs. Elimination of CD4⁺ T cells or blockade of CCL4 or IL-17 abrogated the increase in tumor formation caused by myeloid cells. Thus, this study implicates accumulation of IMCs as an initial step in facilitation of tumor formation, followed by the recruitment of CD4⁺ T cells.Inflammation develops in response to foreign agents or injuries and is crucial to the healing process. However, persistent unresolved inflammation can contribute to the development of cancer (Coussens et al., 2013). Despite the wealth of information implicating inflammation in tumor development, the specific role of different cells, especially myeloid cells, in this process remains largely unclear. Myeloid cells are an important component of inflammation. There is now ample evidence of abnormalities in the myeloid compartment in cancer, which manifests in inhibition of differentiation of DCs, polarization of macrophages (MΦs) toward M2 functional state, and dramatic expansion of myeloid-derived suppressor cells (MDSCs; Gabrilovich et al., 2012). MDSCs represent a heterogeneous population of pathologically activated myeloid cells that includes precursors of neutrophils (polymorphonuclear neutrophils [PMNs]), MΦs, DCs, and cells at earlier stages of myeloid cell differentiation (Gabrilovich and Nagaraj, 2009; Peranzoni et al., 2010; Youn et al., 2012). In mice, these cells are defined as Gr-1⁺CD11b⁺ cells with the recognition of polymorphonuclear (PMN-MDSC) and mononuclear (M-MDSC) subsets based on the expression of Ly6C and Ly6G markers (Fridlender et al., 2009; Peranzoni et al., 2010; Brandau et al., 2011; Youn et al., 2012). MDSCs are characterized by a potent immune-suppressive activity and the ability to promote tumor angiogenesis, tumor cell invasion, and metastases (Bierie and Moses, 2010; Gabrilovich et al., 2012; Talmadge and Gabrilovich, 2013). In tumor-free mice, cells with the same phenotype represent immature myeloid cells (IMCs) lacking immune-suppressive activity. Expansion of MDSCs is considered a consequence of tumor progression. However, in recent years, it has become clear that the cells with phenotypes and functions attributed to MDSCs are readily detectable in different conditions associated with chronic inflammation not directly linked to cancer (Cuenca et al., 2011; Nagaraj et al., 2013). We hypothesize that these cells can contribute to tumor development associated with inflammation. Understanding the role of specific components of inflammation in tumor development is difficult because of the fact that inflammation is a multicomponent complex process. To address this question, we focused on S100A9 protein. This is the member of the S100 family of Ca²⁺-binding proteins with diverse biological activity, including chemotaxis of myeloid cells, fatty acid transport, production of reactive oxygen species, etc. (Markowitz and Carson, 2013). Expression of S100A9 together with its dimerization partner S100A8 is found predominantly in cells of the myeloid lineage. Differentiation of myelocytes/granulocytes is associated with an increase of S100A8/A9 expression, whereas differentiation of MΦs and DCs is associated with loss of their expression (Leder et al., 1990; Ehrchen et al., 2009; Srivastava et al., 2012; Markowitz and Carson, 2013). Cells of the lymphoid lineage do not express these proteins. We and others have previously found that accumulation of MDSCs and inhibition of DC differentiation in cancer were closely associated with up-regulation of S100A8/A9 (Cheng et al., 2008; Sinha et al., 2008; Ichikawa et al., 2011). The expansion of MDSCs was significantly reduced in S100A9-deficient mice treated with complete Freund’s adjuvant or tumor-bearing mice (Cheng et al., 2008). In contrast, overexpression of S100A9 in mice resulted in accumulation of cells with MDSC phenotype (Chen et al., 2013). Furthermore, these data were consistent with a recent report that DCs derived from S100A9-deficient mice induced stronger response of allogeneic T cells (Shimizu et al., 2011). In recent years, S100A9 was identified as a marker of MDSCs in peripheral blood of cancer patients and tumor-bearing mice (Feng et al., 2012; Källberg et al., 2012; Zhao et al., 2012). We asked whether regulation of S100A9 level in myeloid cells could be used to dissect the possible role of MDSCs during early stages of tumor development. Here, we report that cells with the MDSC phenotype accumulate in tissues of patients with precancerous inflammation and that these cells play a major role in tumor development in mice. Importantly, this effect was not directly mediated by immune-suppressive mechanisms but by the recruitment of IL-17–producing CD4⁺ T cells.     

Special delivery: microRNA-200–containing extracellular vesicles provide metastatic message to distal tumor cells

An emerging view is that breast cancer is a systemic disease that utilizes intrinsic and extrinsic tumor cell processes to support both primary tumor growth and metastatic dissemination into distal tissue. Delineation of factors involved in these processes should facilitate a better understanding for both assessing and preventing disease relapse. In this issue of the JCI, Le et al. investigate whether intrinsic properties of metastatic breast cancer cell growth can be regulated through an extrinsic process — contact with tumor cell–derived extracellular vesicles containing microRNAs of the miR-200 family. The authors provide compelling evidence that miR-200s within extracellular vesicles secreted from highly metastatic tumor cells can be internalized by weakly metastatic cells. Thus, internalization and delivery of this metastatic “donor” cell–derived message provide plausible mechanisms by which oncogenic and regulatory factors confer the capability of tumor growth at metastatic lesions. This study provides a strong rationale for detailed assessment of the prognostic and predictive value of circulating extracellular vesicle–bound miR-200s in breast cancer progression and treatment.     

Cardiac mast cells cause atrial fibrillation through PDGF-A–mediated fibrosis in pressure-overloaded mouse hearts

Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast cell–deficient WBB6F1-Kit^W/W-v mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF α-receptor (PDGFR-α). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-Kit^W/W-v mice. Furthermore, injection of the neutralizing PDGFR-α–specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts.     

Self-RNA–antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8

Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid–recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA–LL37 complexes activate TLR7 and, like self-DNA–LL37 complexes, trigger the secretion of IFN-α without inducing maturation or the production of IL-6 and TNF-α. In contrast to self-DNA–LL37 complexes, self-RNA–LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF-α and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA–LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis.Dendritic cells (DCs) sense viral infections through a subset of nucleic acid–recognizing Toll-like receptors (TLRs) expressed in endosomal compartments (Akira et al., 2006). These receptors include TLR3, which detects double-stranded viral RNA (Alexopoulou et al., 2001); TLR7 and TLR8, which recognize guanosine- and uridine-rich single-stranded RNA (ssRNA) (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004); and TLR9, which recognizes the phosphodiester backbone in natural DNA or unmethylated CpG motifs (Hemmi et al., 2000; Haas et al., 2008). TLR7 and TLR9 are selectively expressed by human plasmacytoid dendritic cells (pDCs) (Jarrossay et al., 2001; Kadowaki et al., 2001; Hornung et al., 2002), a subset of dendritic cells specialized in type I IFN production (Colonna et al., 2004; Gilliet et al., 2008). In contrast, classical human myeloid DCs (mDCs), which are potent stimulators of T cell responses by virtue of their inherent capacity to present antigens and migrate from the periphery into secondary lymphoid organs, do not express TLR7 and TLR9, but instead express TLR3 and TLR8 (Jarrossay et al., 2001; Kadowaki et al., 2001; Hornung et al., 2002).Through endosomal TLRs, pDCs and mDCs efficiently sense viral nucleic acids but do not respond to self-nucleic acids released into the extracellular environment by dying host cells. Structural differences such as the high levels of unmethylated CpG motifs in viral DNA and clusters of U or GU-rich sequences in viral RNA have been considered a key factor in the discrimination between viral and self-nucleic acids (Krieg, 2002; Diebold et al., 2004; Heil et al., 2004). However, more recently, it has become clear that this discrimination is mainly achieved by the intracellular localization of these TLRs, which allows recognition of viral nucleic acids released into endosomal compartments by the endocytosed virus (Barton et al., 2006). In contrast, self-nucleic acids are rapidly degraded in the extracellular environment and fail to access endosomal compartments spontaneously (Barton et al., 2006). Self-DNA and self-RNA can, however, become a potent trigger of pDC activation when they are aberrantly transported into TLR-containing endosomes in the context of autoimmunity. In systemic lupus erythematosus (SLE), self-RNA and self-DNA are complexed with autoantibodies against the nucleic acid or nucleoproteins, which deliver the nucleic acids into endosomal compartments of pDCs via FcγRII-mediated endocytosis (Rönnblom et al., 2003; Barrat et al., 2005; Means et al., 2005). As a result, pDCs are continuously activated to produce type I IFNs, which drives the development of autoimmunity and disease formation. This has been demonstrated in mouse models of SLE using TLR7- or TLR9-deficient mice or inhibitors for TLR7 and TLR9 (Leadbetter et al., 2002; Lau et al., 2005; Christensen et al., 2006; Barrat et al., 2007), and by a study showing that TLR7 gene duplication in mice induces SLE-like disease (Deane et al., 2007).In psoriasis, a chronic autoimmune-inflammatory disease of the skin, self-DNA forms complexes with the cationic antimicrobial peptide LL37. Self-DNA–LL37 complexes gain access to endosomal TLR9, leading to an aberrant activation of pDCs to produce IFN-α (Lande et al., 2007). pDC-derived IFN-α initiates the autoimmune-inflammatory cascade in psoriasis, a process characterized by the activation of mDCs and their maturation into DCs that stimulate pathogenic autoimmune T cells (Nestle et al., 2005). However, whether LL37 also interacts with extracellular self-RNA, and whether this pathway is involved in the activation of mDCs in psoriasis, is unknown.Here, we found that LL37 also forms complexes with extracellular self-RNA. These complexes are highly protected from RNase degradation and gain access to the endosomal compartments of both pDCs and mDCs. Self-RNA–LL37 complexes induce TLR7 activation in pDCs and trigger the secretion of IFN-α. Self-RNA–LL37 complexes also trigger the direct activation of mDCs to secrete TNF-α and IL-6 and differentiate into mature DCs. This maturation of mDCs is triggered by endosomal TLR8 and is enhanced by the concomitant activation of pDCs to produce IFN-α. In-vivo, self-RNA–LL37 complexes are specifically found in psoriatic skin and the number of these complexes correlates with the presence of mature mDCs. The current findings identify self-RNA–LL37 complexes as endogenous triggers of TLR7 and TLR8 in human DCs, and provide a new link between the expression of antimicrobial peptides and DC-mediated inflammation in psoriasis.     

Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

Netherton syndrome (NS) is a severe genetic skin disease with constant atopic manifestations that is caused by mutations in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lymphoepithelial Kazal-type–related inhibitor (LEKTI). Lack of LEKTI causes stratum corneum detachment secondary to epidermal proteases hyperactivity. This skin barrier defect favors allergen absorption and is generally regarded as the underlying cause for atopy in NS. We show for the first time that the pro-Th2 cytokine thymic stromal lymphopoietin (TSLP), the thymus and activation-regulated chemokine, and the macrophage-derived chemokine are overexpressed in LEKTI-deficient epidermis. This is part of an original biological cascade in which unregulated kallikrein (KLK) 5 directly activates proteinase-activated receptor 2 and induces nuclear factor κB–mediated overexpression of TSLP, intercellular adhesion molecule 1, tumor necrosis factor α, and IL8. This proinflammatory and proallergic pathway is independent of the primary epithelial failure and is activated under basal conditions in NS keratinocytes. This cell-autonomous process is already established in the epidermis of Spink5^−/− embryos, and the resulting proinflammatory microenvironment leads to eosinophilic and mast cell infiltration in a skin graft model in nude mice. Collectively, these data establish that uncontrolled KLK5 activity in NS epidermis can trigger atopic dermatitis (AD)–like lesions, independently of the environment and the adaptive immune system. They illustrate the crucial role of protease signaling in skin inflammation and point to new therapeutic targets for NS as well as candidate genes for AD and atopy.The epidermis is a stratified epithelium providing a first line of defense against the harsh external environment. The skin also maintains the body''s integrity by sequestering the internal milieu and impeding transcutaneous water loss through the formation of an impermeability barrier. This protective barrier is conferred by the outermost layer of the epidermis, the stratum corneum (SC), which results from a finely regulated terminal cell differentiation process from the basal layer of the epidermis to the granular layers (GRs), through the spinous compartment. The SC consists of dead and keratin-filled cells, corneocytes, which are attached to each other by corneodesmosomes and embedded in a lipid matrix (1–3). Proteolytic degradation of corneodesmosomes by epidermal proteases leads to the shedding of the most superficial corneocytes (4). This desquamation process allows the regulation of the skin thickness. Alterations in these processes, as well as mutations in genes encoding proteins involved in terminal differentiation, can lead to abnormal stratification and keratinization, as seen in ichthyoses (5).In this respect, the filament-aggregating protein FILAGGRIN is a key protein that plays an important role in the formation of the barrier function. Recent genetic studies have shown that loss-of-function mutations in the gene encoding FILAGGRIN underlie ichthyosis vulgaris (6), a very common genetic disorder of keratinization. The same FILAGGRIN mutations have also been identified as a major risk factor for atopic dermatitis (AD) and are strongly associated with asthma in AD patients (7, 8). These results linked for the first time a primary skin disease to systemic allergic manifestations, through defective skin barrier function.AD is a chronic inflammatory skin disease characterized by eczema, pruritus, and cutaneous hyperreactivity to environmental factors that are innocuous to normal nonatopic individuals (9). AD has a complex etiology that results from interactions between environment and several susceptibility genes involved in skin barrier function and systemic and local immunological responses (10, 11). AD is often the initial step in the so-called “atopic march,” which leads to asthma and allergic rhinitis in the majority of afflicted patients (12–14). These conditions are all characterized by elevated serum IgE levels and peripheral eosinophilia (9). Clinically unaffected skin in AD manifests impaired skin barrier function that could favor the penetration of microbes and allergens (15), leading to the development of cutaneous and systemic allergies through activation of antigen-presenting cells such as Langerhans cells (LCs) (16). However, keratinocytes are immunologically active cells. Indeed, traumatic barrier disruption alone stimulates both keratinocyte proliferation and their cytokine and chemokine production (IL-1α, IL-1β, TNF-α, and GM-CSF), characteristic features of skin inflammatory diseases (17, 18). Thus, the skin should be considered as an important organ of innate immunity. In fact, keratinocytes from lesional skin of AD patients express a proallergic cytokine, thymic stromal lymphopoietin (TSLP). Notably, the local increase of TSLP is directly associated with LC activation and migration to skin draining lymph node, where they trigger the differentiation of naive CD4⁺ T cells into proallergic CD4⁺ Th2 cells (19, 20). TSLP overexpression is also sufficient in mouse skin to induce an inflammatory Th2 microenvironment and an AD-like skin phenotype (21). Keratinocytes are therefore important orchestrators of the innate immunity through the secretion of a wide range of proinflammatory and proallergic molecules inducing and supporting cutaneous acute and chronic inflammatory responses.One of the most severe ichthyoses of children and young adults is Netherton syndrome (NS; Online Mendelian Inheritance of Man reference number 256500). NS is a rare (1 in 100,000 newborns) autosomal recessive skin disorder characterized by generalized exfoliative erythroderma, a specific hair shaft defect (trichorrhexis invaginata) and severe atopic manifestations, which distinguish NS from the other ichthyoses (22, 23). Indeed, NS patients suffer from recurrent AD with elevated serum IgE levels, asthma, and multiple food allergies (24, 25). Bacterial infection, hypernatraemic dehydration, hypothermia, and extreme weight loss are frequent complications during the neonatal period, resulting in high postnatal mortality, and are probably favored by the severe alteration of the skin barrier function.We previously identified serine protease inhibitor Kazal-type 5 (SPINK5) as the defective gene in NS (26). SPINK5 encodes the multidomain serine protease inhibitor lymphoepithelial Kazal-type–related inhibitor (LEKTI), whose tissue distribution pattern is restricted to the most differentiated viable layers of stratified epithelial tissues and the Hassall''s corpuscles in the thymus (27). In the epidermis, LEKTI is mainly restricted to the GR. It is expressed as high molecular mass precursors, which are rapidly processed into several proteolytic fragments secreted in the intercellular space (27, 28). It has been shown that LEKTI fragments can efficiently and specifically inhibit the epidermal kallikrein (KLK) 5, KLK7, and KLK14 (28–30).Using Spink5 KO mice (Spink5^−/−), which faithfully reproduce key features of NS (31–33), we deciphered the biological functions of LEKTI and identified cutaneous pathophysiological pathways of the disease. We showed that epidermal LEKTI deficiency results in KLK5 and KLK7 hyperactivity and dysregulation of a new epidermal protease, which is under characterization. These unrestricted protease activities lead to abnormal desmosome cleavage in the upper GR, resulting in accelerated SC shedding and consequent loss of skin barrier function. This work identified LEKTI as a key regulator of epidermal protease activity and skin barrier integrity (32).Because NS patients suffer from severe atopic manifestations, such as AD, genetic association between SPINK5 single nucleotide polymorphisms and severe AD has been tested by several groups (34–41). Positive association was found in several studies in patients with different ethnical background (34, 36). Although the skin barrier defect is considered to be the key event leading to skin inflammation and allergy (42), it is likely that more specific and intrinsic signals could be directly induced by SPINK5^−/− keratinocytes. In this paper, we therefore explored the molecular mechanisms leading to the recruitment of inflammatory cells in NS skin. We show that in LEKTI-deficient keratinocytes, KLK5 hyperactivity activates proinflammatory signaling, leading to the recruitment of eosinophilic and mast cells, which is reminiscent of AD-like skin lesions in NS, independent from skin barrier defect, environmental stimuli, and the adaptive immune system.     

TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in melanoma patients

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen–specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1^–TIGIT⁺, and PD-1⁺TIGIT^– CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand–expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.     

Milk fat globule epidermal growth factor–8 blockade triggers tumor destruction through coordinated cell-autonomous and immune-mediated mechanisms

Carcinogenesis reflects the dynamic interplay of transformed cells and normal host elements, but cancer treatments typically target each compartment separately. Within the tumor microenvironment, the secreted protein milk fat globule epidermal growth factor–8 (MFG-E8) stimulates disease progression through coordinated α_vβ₃ integrin signaling in tumor and host cells. MFG-E8 enhances tumor cell survival, invasion, and angiogenesis, and contributes to local immune suppression. We show that systemic MFG-E8 blockade cooperates with cytotoxic chemotherapy, molecularly targeted therapy, and radiation therapy to induce destruction of various types of established mouse tumors. The combination treatments evoke extensive tumor cell apoptosis that is coupled to efficient dendritic cell cross-presentation of dying tumor cells. This linkage engenders potent antitumor effector T cells but inhibits FoxP3⁺ T reg cells, thereby achieving long-term protective immunity. Collectively, these findings suggest that systemic MFG-E8 blockade might intensify the antitumor activities of existing therapeutic regimens through coordinated cell-autonomous and immune-mediated mechanisms.Cancer pathogenesis involves not only the cell-autonomous defects that arise from alterations in oncogenes and tumor suppressors but also the impact of host antitumor responses (1). Cancer cells that have escaped immune control are selected for the ability to exploit factors present in the tumor microenvironment to further disease progression (2–4). Among this array of soluble moieties, inflammatory cytokines including TNF-α, IL-6, and IL-1β play key roles through triggering NF-κB–, STAT-3–, and MyD88-dependent pathways (5–8).GM-CSF is another cytokine frequently produced in the tumor microenvironment, where it may contribute to either tumor protection or promotion (9). Through studies of GM-CSF–deficient mice, we identified milk fat globule epidermal growth factor–8 (MFG-E8) as a critical determinant of the pro- and antiinflammatory activities of the cytokine (10). MFG-E8 is a secreted phosphatidylserine-binding protein that signals through α_vβ₃ and α_vβ₅ integrins (9–12). Under steady-state conditions, GM-CSF induces MFG-E8 expression in mononuclear phagocytes, enabling the efficient uptake of apoptotic cells, the production of TGF-β and CCL22, and the maintenance of FoxP3⁺ T reg cells (10). Under conditions of cellular stress, however, the ligation of Toll-like receptors dampens MFG-E8 expression, whereupon GM-CSF elicits CD4⁺ and CD8⁺ effector T cells through an MFG-E8–independent pathway. Thus, the levels of MFG-E8 present in the tumor microenvironment might modulate the functions of GM-CSF during carcinogenesis.In malignant melanoma, MFG-E8 expression is increased in tumor cells and/or infiltrating myeloid elements upon progression to the vertical growth phase, the stage in which melanoma cells acquire the competence for invasion and dissemination (12, 13). In a mouse melanoma model, MFG-E8 augmented tumorigenicity and metastatic capability through Akt- and Twist-dependent mechanisms (12). MFG-E8 enhanced melanoma cell resistance to apoptosis, induced an epithelial-to-mesenchymal transition, and stimulated invasion and angiogenesis. MFG-E8 also contributed to local immune suppression by evoking FoxP3⁺ T reg cell infiltrates and suppressing Th1 reactions and NK and CD8⁺ T cell cytotoxicity.Because MFG-E8 is expressed at high levels in diverse tumor types (14, 15), including melanoma, this soluble protein might serve as a general target for cancer therapy. In contrast to most oncologic treatments, which primarily address either the tumor or host separately, MFG-E8 antagonists might affect both compartments. Indeed, shRNA knockdowns of MFG-E8 sensitized tumor cells to cytotoxic agents and small molecule inhibitors of receptor tyrosine kinases in vitro, whereas MFG-E8 blockade with a dominant-negative mutant potentiated tumor immunity generated with irradiated, GM-CSF–secreting tumor cell vaccines (10, 12). Based on these results, we hypothesized that systemic targeting of MFG-E8 might contribute to tumor destruction in several complementary ways. In this paper, we show that antibodies to MFG-E8 cooperate with conventional cancer therapies to effectuate sustained control of established mouse tumors through the coupling of cell-autonomous and host-mediated pathways.     

Interactions between Siglec-7/9 receptors and ligands influence NK cell–dependent tumor immunosurveillance

Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid–binding Ig-like lectins Siglec-7 and -9 are MHC class I–independent inhibitory receptors on human NK cells that recognize sialic acid–containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9⁺ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell–sensitive tumor cells and, unexpectedly, of presumably NK cell–resistant tumor cells to NK cell–mediated cytotoxicity. Together, these observations have direct implications for NK cell–based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.     

TMPRSS4 promotes invasiveness of human gastric cancer cells through activation of NF-κB/MMP-9 signaling

Transmembrane protease serine 4 (TMPRSS4) is a type-II transmembrane serine protease that is frequently upregulated in human cancers. However, little is known about the biological roles of TMPRSS4 in gastric cancer. In this study, we examined the effect of TMPRSS4 on gastric cancer cell proliferation, migration, and invasion. The expression and secretion of matrix metalloproteinase-9 (MMP-9) and activation of nuclear factor-κB (NF-κB) were determined. The involvement of NF-κB/MMP-9 signaling was checked. Our data showed that TMPRSS4 silencing significantly (P < 0.05) reduced the migration and invasion of AGS and MKN-45 gastric cancer cells, without affecting cell proliferation. Overexpression of TMPRSS4 significantly promoted cell migration and invasion. The expression and secretion of MMP-9 was significantly (P < 0.05) enhanced in TMPRSS4-overexpressing cells. TMPRSS4-overexpressing cells had a significantly (P < 0.05) lower level of IκBα and higher level of nuclear NF-κB. Luciferase reporter assay confirmed that overexpression of TMPRSS4 resulted in a 3–5-fold increase in NF-κB-dependent luciferase activity. Downregulation of MMP-9 significantly (P < 0.05) reversed the invasiveness of gastric cancer cells induced by TMPRSS4 overexpression. Moreover, pharmacological inhibition of NF-κB attenuated the invasion of TMPRSS4-overexpressing cells and the expression of MMP-9. Upregulation of TMPRSS4 enhances the invasiveness of gastric cancer cells, largely through activation of NF-κB and induction of MMP-9 expression. Our study provides the rationale for targeting TMPRSS4 in the treatment of gastric cancer.     

Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10–secreting Th1 cells

Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)–specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10–secreting CD4⁺ T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4⁺ T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-γ to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN-γ and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.Antigens administered in a tolerogenic form have long been known to result in down-regulation of immune responses. In recent years, the potential of antigen-driven immunotherapy for the treatment of allergic and autoimmune diseases has been investigated in several experimental models. Administration of antigenic peptides via the intranasal (i.n.) route induces tolerance, and thus inhibits the development of both autoimmunity (Metzler and Wraith, 1993; Staines et al., 1996; Tian et al., 1996; Karachunski et al., 1997) and allergy (Hoyne et al., 1993). Possible mechanisms of tolerance induction include elimination of peptide-specific T cells by activation-induced cell death/apoptosis (Critchfield et al., 1994; Chen et al., 1995; Liblau et al., 1996) or modification of their function via induction of anergy (Kearney et al., 1994), TCR/coreceptor down-regulation (Schonrich et al., 1991), immune deviation (Guery et al., 1996), or secretion of immunoregulatory cytokines such as IL-10 and TGF-β (Miller et al., 1992; Sundstedt et al., 1997). Most immune cells, including monocytes, macrophages, DCs, NK cells, B cells, and T cells, are capable of secreting IL-10 under specific circumstances (Moore et al., 2001). Among these, IL-10–secreting CD4⁺ T cells are the best characterized because of their recently recognized role in immune regulation (O''Garra et al., 2004). Two phenotypically distinct CD4⁺ T regulatory (T reg) cell types have been described—naturally occurring FoxP3⁺ T reg cells that form an inherent part of the naive T cell repertoire (Sakaguchi et al., 1995) and induced, FoxP3⁻ IL-10-secreting T reg cells (for review see Roncarolo et al., 2006). Numerous subtypes of induced IL-10–secreting T reg cells with variable cytokine profiles have been generated in both murine and human systems. However, in contrast to T helper cells, the differentiation of induced T reg cells remains poorly defined.i.n. administration of a soluble peptide induces peripheral tolerance in TCR transgenic (Tg4) mice specific for the acetylated N-terminal peptide Ac1-9 of murine myelin basic protein (MBP). Increasing the affinity of the peptide for I-A^u greatly enhances the tolerogenicity of the peptide in the Tg4 mouse (Liu et al., 1995). After a single i.n. dose of a high-affinity analogue of the MBP epitope, Ac1-9[4Y], with a tyrosine substituting the lysine at position four, T cell deletion is only transient and incomplete (Burkhart et al., 1999). Instead, Tg4 CD4⁺ T cells become anergic and exhibit a shift in cytokine secretion profile toward IL-10 after repeated i.n. treatment with peptide (Burkhart et al., 1999). Evidence for the generation of CD4⁺ T cells with a regulatory phenotype in this model stems from both in vitro and in vivo suppression assays (Sundstedt et al., 2003). Thus, i.n. treatment with MBP Ac1-9[4Y] induces active tolerance in the form of IL-10–secreting T reg cells (IL-10 T reg cells) rather than deletion. A role for IL-10 in suppression in vivo and in experimental autoimmune encephalomyelitis protection was demonstrated by anti–IL-10 (Burkhart et al., 1999) and anti–IL-10R (Sundstedt et al., 2003) antibody administration. IL-10 has important immunosuppressive and antiinflammatory effects on immune responses to both foreign and self-antigens (Moore et al., 2001) that are primarily mediated by its inhibitory activities on the function of APCs (de Waal Malefyt et al., 1991). Although the role of IL-10 in suppression of experimental autoimmune encephalomyelitis in the Tg4 model is not known, the effect of IL-10 on antigen presentation and inflammation is a likely mechanism. Naturally occurring FoxP3⁺ T reg cells form a part of the Tg4 CD4⁺ T cell repertoire and may rely on IL-10 to mediate suppression, as previously shown in other inflammatory settings (Asseman et al., 1999). Even so, peptide-induced IL-10 T reg cells were found to be distinct in origin from naturally occurring T reg cells in that they do not express Foxp3 (Vieira et al., 2004). Genetic depletion of FoxP3⁺ T reg cells from the CD4⁺ T cell repertoire in the RAG-deficient Tg4 mouse gives rise to spontaneous EAE. However, the onset of disease can be prevented by repetitive treatment with i.n. peptide, correlating with the generation of IL-10 T reg cells (Nicolson et al., 2006).It has been proposed that induced IL-10 T reg cells arise from fully differentiated T effector cells that have lost the ability to secrete their hallmark cytokines as a result of chronic antigenic stimulation (O''Garra et al., 2004). Alternatively, induced IL-10 T reg cells could arise directly from naive precursors without a T effector phase. In this study, we investigate the ontogeny of induced IL-10 T reg cells generated by repeated i.n. peptide treatment. By following the differentiation pathway taken by CD4⁺ T cells over the course of tolerance induction, we demonstrate that peptide-induced IL-10 T reg cells are of Th1 origin and that IL-10 T reg cells complete the negative feedback loop of pathogenic Th1 responses in autoimmunity.     

Paracrine signaling by glial cell–derived triiodothyronine activates neuronal gene expression in the rodent brain and human cells

Hypothyroidism in humans is characterized by severe neurological consequences that are often irreversible, highlighting the critical role of thyroid hormone (TH) in the brain. Despite this, not much is known about the signaling pathways that control TH action in the brain. What is known is that the prohormone thyroxine (T4) is converted to the active hormone triiodothyronine (T3) by type 2 deiodinase (D2) and that this occurs in astrocytes, while TH receptors and type 3 deiodinase (D3), which inactivates T3, are found in adjacent neurons. Here, we modeled TH action in the brain using an in vitro coculture system of D2-expressing H4 human glioma cells and D3-expressing SK-N-AS human neuroblastoma cells. We found that glial cell D2 activity resulted in increased T3 production, which acted in a paracrine fashion to induce T3-responsive genes, including ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), in the cocultured neurons. D3 activity in the neurons modulated these effects. Furthermore, this paracrine pathway was regulated by signals such as hypoxia, hedgehog signaling, and LPS-induced inflammation, as evidenced both in the in vitro coculture system and in in vivo rat models of brain ischemia and mouse models of inflammation. This study therefore presents what we believe to be the first direct evidence for a paracrine loop linking glial D2 activity to TH receptors in neurons, thereby identifying deiodinases as potential control points for the regulation of TH signaling in the brain during health and disease.     

IL-9–mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation

IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)–deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.The cytokine IL-9 was discovered more than 20 yr ago and described as a T cell and mast cell growth factor produced by T cell clones (Uyttenhove et al., 1988; Hültner et al., 1989; Schmitt et al., 1989). Subsequently, IL-9 was shown to promote the survival of a variety of different cell types in addition to T cells (Hültner et al., 1990; Gounni et al., 2000; Fontaine et al., 2008; Elyaman et al., 2009). Until recently, Th2 cells were thought to be the dominant source of IL-9 and the function of IL-9 was mainly studied in the context of Th2 type responses in airway inflammation and helminth infections (Godfraind et al., 1998; Townsend et al., 2000; McMillan et al., 2002; Temann et al., 2002). IL-9 blocking antibodies were shown to ameliorate lung inflammation (Cheng et al., 2002; Kearley et al., 2011) and are currently in clinical trials for the treatment of patients with asthma (Parker et al., 2011). The paradigm that Th2 cells are the dominant source of IL-9 was challenged when it became apparent that naive CD4⁺ T cells cultured in the presence of TGF-β and IL-4 initiate high IL-9 expression without coexpression of IL-4, suggesting the existence of a dedicated subset of IL-9–producing T cells (Dardalhon et al., 2008; Veldhoen et al., 2008; Angkasekwinai et al., 2010; Chang et al., 2010; Staudt et al., 2010). Subsequently, the generation of an IL-9–specific reporter mouse strain enabled the study of IL-9–producing cell types in vivo and revealed that in a model of lung inflammation IL-9 is produced by innate lymphoid cells (ILCs) and not T cells (Wilhelm et al., 2011). IL-9 production in ILCs was transient but important for the maintenance of IL-5 and IL-13 in ILCs. Such type 2 cytokine-producing ILCs (ILC2s; Spits and Di Santo, 2011) were first described as a population of IL-5– and IL-13–producing non-B/non-T cells (Fort et al., 2001; Hurst et al., 2002; Fallon et al., 2006; Voehringer et al., 2006) and later shown to play a role in helminth infection via IL-13 expression (Moro et al., 2010; Neill et al., 2010; Price et al., 2010; Saenz et al., 2010). In addition, important functions were ascribed to such cells in the context of influenza infection (Chang et al., 2011; Monticelli et al., 2011) and airway hyperactivity in mice (Barlow et al., 2012) and humans (Mjösberg et al., 2011). However, although the contribution of ILC2s to host immunity against helminths in the gut is well established (Moro et al., 2010; Neill et al., 2010; Price et al., 2010; Saenz et al., 2010), the function of ILC2s in helminth-related immune responses in the lung remains unknown. ILC2s are marked by expression of the IL-33R (Moro et al., 2010; Neill et al., 2010; Price et al., 2010), as well as the common γ chain (γ_c) cytokine receptors for IL-2 and IL-7 (Moro et al., 2010; Neill et al., 2010). Interestingly, gene expression array analyses have demonstrated that the receptor for IL-9, another member of the γ_c receptor family, is also expressed in ILC2s and differentiates them from Th2 cells (Price et al., 2010) and ROR-γt⁺ ILCs (Hoyler et al., 2012). However, the function of IL-9R expression for ILC2 biology has not been addressed so far.Here we show that the production of IL-5, IL-13, and amphiregulin during infection with Nippostrongylus brasiliensis in the lung depends on ILC2s and their expression of IL-9R. The ability to signal via the IL-9R was crucial for the survival of ILC2s, but not Th2 cells. The absence of IL-9 signaling in IL-9R–deficient mice resulted in reduced lung ILC2 numbers and, consequently, diminished repair of lung damage in the chronic phase after helminth-induced lung injury despite the presence of an intact Th2 cell response. Thus, we identify IL-9 as a crucial autocrine amplifier of ILC2 function and survival.     

Augmented TLR9-induced Btk activation in PIR-B–deficient B-1 cells provokes excessive autoantibody production and autoimmunity

Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate immune system. However, excess stimulation of TLR pathways is supposed to be connected with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses TLR9 signaling via Bruton''s tyrosine kinase (Btk) dephosphorylation, which leads to attenuated activation of nuclear factor κB p65RelA but not p38 or Erk, and blocks the production of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb^−/−) mice was further augmented in combination with the Fas^lpr mutation, which might be linked to the development of autoimmune glomerulonephritis. These results show the critical link between TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing pathogenic natural autoantibody production.The emergence of autoimmunity is often coupled with aging, and is suggested to be linked to activation of the innate immune system in individuals suffering from bacterial and viral infections (Baccala et al., 2007; Groom et al., 2007; Krieg and Vollmer 2007; Rothlin et al., 2007). Toll-like receptors (TLRs) expressed in leukocytes of the innate immune system play indispensable roles in the sensing of viral and bacterial invasion through binding pathogen-associated molecular patterns, which leads to efficient T cell–mediated inflammatory responses (Akira et al., 2001; Iwasaki and Medzhitov, 2004). The TLR-mediated priming of inflammation and production of neutralizing antibodies against pathogens should be strictly regulated, otherwise there is the possibility of the development of autoimmune diseases (Marsland and Kopf, 2007). The mechanisms underlying the efficient TLR-mediated activation of the innate and adaptive immune systems with prevention of reactivity to autologous tissues remain elusive.Examples of critical cells that express TLRs and could potentially link the innate and adaptive immune systems are relatively primitive B cells, B-1 cells, found mainly in the peritoneal and pleural cavities. In contrast to recirculating follicular B cells (or conventional B or B-2 cells), B-1 cells are characterized by B220^lowIgM^highCD23⁻CD43⁺IgD^low cells (Berland and Wortis, 2002; Tung and Herzenberg, 2007). Although it has been pointed out by many researchers that innate B-1 cells but not conventional B cells are producers of natural antibodies against pathogens (Ochsenbein et al., 1999), accumulating lines of evidence suggest that a major source of autoantibodies is also those B-1 cells (Baumgarth et al., 2005; Carroll and Holers, 2005), but it has been a matter of debate. By stimulation via different TLRs, the B-1 cell population in the peritoneal cavity has been enlarged and B-1 cell–mediated autoantibody production augmented (Murakami et al., 1995). This could be partly because B-1 cells express a set of TLRs, including TLR4, TLR7, and TLR9 (Gururajan et al., 2007), and are more prone to differentiate into plasma cells than B-2 cells upon TLR-mediated stimulation, although B-2 cells similarly possess a range of TLRs (Genestier et al., 2007). For example, Murakami et al. (1995) have shown, in anti–red blood cell autoantibody transgenic mice, that the susceptibility to autoimmune hemolytic anemia was significantly increased when the mice were transferred from germ-free or specific pathogen-free conditions to conventional conditions or injected with a TLR4 ligand, LPS, with a concomitant increase in the peritoneal B-1 cell population, whereas almost all B-2 cells are constitutively deleted in the transgenic mice. These findings again suggest the importance of the regulation of TLR signaling in B-1 cells, which prevents overstimulation of TLRs so as not to evoke overproduction of natural antibodies, including potentially harmful autoantibodies. Therefore, what mechanisms may regulate the overstimulation of the TLR signal, particularly in B-1 cells?We speculated that paired Ig-like receptor B (PIR-B; Hayami et al., 1997; Kubagawa et al., 1997) could participate in the regulation of B-1 cells. Recruitment of SH2 domain–containing tyrosine phosphatase 1 (SHP-1) to phosphotyrosylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic portion of PIR-B was shown to be critical for PIR-B–mediated inhibitory signaling in general (Ho et al., 1999; Maeda et al., 1999), and this inhibition is achieved, at least in part, via constitutive binding of PIR-B to its ligand, i.e., MHC class I molecules, expressed on the same cell surface (Masuda et al., 2007). Interestingly, in PIR-B–deficient (Pirb^−/−) mice, the peritoneal B-1 cell population significantly increased, particularly with aging, compared with wild-type mice (Ujike et al., 2002). However, it has not been determined how the B-1 cell compartment is physiologically regulated by PIR-B or what the physiological or pathological consequence of the expanded B-1 cells is in the contexts of infection and autoimmunity.In this paper, we show that PIR-B can inhibit TLR9-mediated signaling via regulation of Bruton''s tyrosine kinase (Btk) phosphorylation in peritoneal B-1 cells. We found that TLR9 activation immediately activates an Src family kinase, Lyn, which then phosphorylates PIR-B cytoplasmic ITIMs. In the absence of PIR-B, B-1 cells become hyperproducers of natural IgM antibodies, including anti-IgG Fc autoantibodies, i.e., rheumatoid factor (RF), via unmethylated CpG-B oligodeoxynucleotide (CpG-B) stimulation in vitro and in vivo or upon aging. These phenotypes caused by PIR-B deficiency were further exaggerated in combination with the Fas^lpr mutation, which caused the Pirb^−/−Fas^lpr mutant mice to be short-lived mainly because of autoimmune glomerulonephritis with immune complex depositions. Our findings may provide a novel strategy for preventing autoimmunity by reducing the production of pathogenic autoantibodies by B-1 cells, such as through down-regulation of Btk activation or enhancement of PIR-B–mediated B-1 cell regulation.