Proapoptotic Bim regulates antigen-specific NK cell contraction and the generation of the memory NK cell pool after cytomegalovirus infection

Apoptosis is critical for the elimination of activated lymphocytes after viral infection. Proapoptotic factor Bim (Bcl2l11) controls T lymphocyte contraction and the formation of memory T cells after infection. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK cell pool. Despite responding similarly early after infection, Bcl2l11^−/− Ly49H⁺ NK cells show impaired contraction and significantly outnumber wild-type (WT) cells after the expansion phase. The inability to reduce the effector pool leads to a larger Bcl2l11^−/− NK memory subset, which displays a less mature phenotype (CD11b^lo, CD27⁺) and lower levels of NK cell memory-associated markers KLRG1 and Ly6C. Bcl2l11^−/− memory NK cells demonstrate a reduced response to m157-mediated stimulation and do not protect as effectively as WT memory NK cells in an MCMV challenge model. Thus, Bim-mediated apoptosis drives selective contraction of effector NK cells to generate a pool of mature, MCMV-specific memory cells.Although NK cells are traditionally classified as innate cells, recent evidence indicates that they may also acquire immunological memory (Paust and von Andrian, 2011; Min-Oo et al., 2013). Work by several groups has uncovered memory-like properties of NK cells, including antigen-specific recall response to haptens and viral-like particles (Paust et al., 2010), cytokine-induced memory (Cooper et al., 2009), and enhanced secondary response to mouse CMV (MCMV; Sun et al., 2009). An expanded and persistent population of NK cells bearing the NKG2C receptor has been found after infection by human CMV, suggesting the existence of memory in human NK cells (Gumá et al., 2004; Lopez-Vergès et al., 2011). Resistance to MCMV is dependent on the NK cell response and is mediated in C57BL/6 mice by the activating Ly49H receptor (Brown et al., 2001; Lee et al., 2001). NK cells undergo robust expansion upon encountering infected cells expressing m157, the MCMV-encoded ligand for Ly49H. Ly49H⁺ NK cell expansion peaks and is followed by a contraction phase (Sun and Lanier, 2011). A small pool of Ly49H⁺ NK cells persists for >90 d after infection; importantly, these cells show enhanced response to secondary challenge (Sun et al., 2009). A previous study has established an important role for cytokine signaling during the expansion phase (Sun et al., 2012), but no work has examined the mechanism driving contraction.The induction of lymphocyte apoptosis is a key mechanism regulating the immune response after viral infection (Prlic and Bevan, 2008; Kurtulus et al., 2010). Failure to control the number of activated lymphocytes can result in fatal immune-mediated pathology. Apoptosis is stimulated through two distinct pathways: death receptor signaling and mitochondrial apoptosis triggered by BH3-only proteins (Strasser, 2005). Bim, a BH3-only family member (O’Connor et al., 1998), binds the prosurvival molecule Bcl-2 and regulates apoptotic signaling through Bax and Bak (Strasser, 2005). Bim regulates the T cell response by reducing the effector T cell pool, in both acute and latent models of viral infection (Kurtulus et al., 2010).Huntington et al. (2007) described Bim-deficient NK cells to be more mature than WT NK cells, but with no defects in cytotoxicity or cytokine production. After MCMV, Bim-deficient mice had an increased number of NK cells. However, Bcl2l11^−/− mice exhibit hematopoietic abnormalities in leukocyte homeostasis (Bouillet et al., 1999), which might impact host response to infection independently of NK cells. Therefore, we examined the cell-intrinsic effect of Bim deficiency in Ly49H⁺ NK cells on the antigen-specific response to MCMV and the generation of memory NK cells.     

Sustained effector function of IL-12/15/18–preactivated NK cells against established tumors

Natural killer cell (NK cell)–based immunotherapy of cancer is hampered by the transient effector function of NK cells. Recently, mouse IL-12/15/18–preactivated NK cells were shown to persist with sustained effector function in vivo. Our study investigated the antitumor activity of such NK cells. A single injection of syngeneic IL-12/15/18–preactivated NK cells, but neither naive nor IL-15– or IL-2–pretreated NK cells, combined with irradiation substantially reduced growth of established mouse tumors. Radiation therapy (RT) was essential for the antitumor activity of transferred NK cells. IL-12/15/18–preactivated NK cells expressed high levels of IL-2Rα (CD25), and their rapid in vivo proliferation depended on IL-2 produced by CD4⁺ T cells. IL-12/15/18–preactivated NK cells accumulated in the tumor tissue and persisted at high cell numbers with potent effector function that required the presence of CD4⁺ T cells. RT greatly increased numbers and function of transferred NK cells. Human IL-12/15/18–preactivated NK cells also displayed sustained effector function in vitro. Our study provides a better understanding for the rational design of immunotherapies of cancer that incorporate NK cells. Moreover, our results reveal an essential role of CD4⁺ T cell help for sustained antitumor activity by NK cells linking adaptive and innate immunity.NK cells are potent antitumor effector cells (Cerwenka and Lanier, 2001; Ljunggren and Malmberg, 2007; Terme et al., 2008; Vivier et al., 2008). Accordingly, individuals with low NK cell activity display an increased risk to develop cancer (Imai et al., 2000), and high numbers of intratumoral NK cells are often correlated with improved prognosis for cancer patients (Coca et al., 1997; Villegas et al., 2002). Human tumors frequently express low levels of MHC class I molecules that interact with inhibitory NK cell receptors. For instance, alterations in the β2m gene can lead to an almost complete and irreversible lack of MHC class I in melanoma cells (D’Urso et al., 1991). In addition, many tumor cells express high levels of ligands for activating NK cell receptors (Raulet and Guerra, 2009), leading to efficient recognition by NK cells (Vivier et al., 2008; Pegram et al., 2011). So far, NK cell–based therapy was mainly successful in patients suffering from leukemia (Moretta et al., 2011). Acute myeloid leukemia patients that received haploidentical bone marrow grafts from Killer immunoglobulin receptor (KIR)–mismatched donors displayed a significantly increased 5-yr disease-free survival (Ruggeri et al., 2002). In addition, clinical benefits were observed upon infusion of KIR-mismatched NK cells after stem cell transplantation (Passweg et al., 2004; Miller et al., 2005; Geller and Miller, 2011; Geller et al., 2011). However, adoptive transfer of autologous IL-2–activated NK cells in patients suffering from solid tumors such as melanoma or renal cell carcinoma did not result in clinical benefits (Parkhurst et al., 2011). Thus, novel strategies are urgently needed to improve the antitumor activity of transferred NK cells against solid tumors.During certain viral infections (Sun et al., 2009a) and contact hypersensitivity reactions (O’Leary et al., 2006), persistent NK cell subpopulations mounting recall responses were detected, indicating previously unappreciated memory properties of NK cells (Paust and von Andrian, 2011; Sun et al., 2011; Vivier et al., 2011). In addition, NK cells preactivated with IL-12, IL-15, and IL-18 in vitro for 15 h were detectable at high numbers 3 wk after transfer into RAG-1^−/− mice and produced high levels of IFN-γ upon restimulation (Cooper et al., 2009). Much lower cell numbers and IFN-γ production were observed when IL-15–preactivated NK cells were transferred. Thus, the activation of NK cells with certain cytokines resulted in an NK cell population with enhanced effector function upon restimulation, indicating that NK cells are able to retain memory of prior activation.Because IL-12/15/18–preactivated NK cells were shown to persist with sustained effector function after restimulation (Cooper et al., 2009), we investigated whether application of IL-12/15/18–preactivated NK cells improves current protocols of immunotherapy of cancer. Our study reveals that a single injection of IL-12/15/18–preactivated NK cells, but neither naive nor of IL-15– or IL-2–pretreated NK cells, combined with radiation therapy (RT), substantially reduced growth of established mouse tumors. Our results raise the possibilities for the development of novel NK cell–based therapeutic strategies for clinical application.     

TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitis

Intestinal epithelial cells (IECs) produce thymic stromal lymphopoietin (TSLP); however, the in vivo influence of TSLP–TSLP receptor (TSLPR) interactions on immunity and inflammation in the intestine remains unclear. We show that TSLP–TSLPR interactions are critical for immunity to the intestinal pathogen Trichuris. Monoclonal antibody–mediated neutralization of TSLP or deletion of the TSLPR in normally resistant mice resulted in defective expression of Th2 cytokines and persistent infection. Susceptibility was accompanied by elevated expression of interleukin (IL) 12/23p40, interferon (IFN) γ, and IL-17A, and development of severe intestinal inflammation. Critically, neutralization of IFN-γ in Trichuris-infected TSLPR^−/− mice restored Th2 cytokine responses and resulted in worm expulsion, providing the first demonstration of TSLPR-independent pathways for Th2 cytokine production. Additionally, TSLPR^−/− mice displayed elevated production of IL-12/23p40 and IFN-γ, and developed heightened intestinal inflammation upon exposure to dextran sodium sulfate, demonstrating a previously unrecognized immunoregulatory role for TSLP in a mouse model of inflammatory bowel disease.Intestinal epithelial cells (IECs) are a critical cell population that maintains intestinal immune homeostasis through both barrier function and the ability to actively modulate intestinal immune responses (1–3). One IEC-derived cytokine with immunomodulatory properties is thymic stromal lymphopoietin (TSLP) (4). TSLP is a four-helix bundle cytokine that is expressed both in humans and mice. Despite poor sequence homology, human and mouse TSLP exhibit similar biological functions (4). Expression of TSLP is regulated by NF-κB and can be induced by exposure to viral, bacterial, and parasitic pathogens, inflammatory cytokines, and the Th2 cell–associated cytokines IL-4 and IL-13 (3, 5–8). TSLP binds to its high affinity receptor, a heterodimer composed of a unique TSLPRα chain and the IL-7Rα chain, that is expressed on hematopoietic cell lineages, including B cells, T cells, mast cells, and DCs (4, 5, 9–12).In vitro studies demonstrated that TSLP-conditioned human DCs can promote Th2 cell responses (11, 13–15). Mechanistically, TSLP treatment of DCs induces Th2 cell differentiation by inhibiting IL-12 production while simultaneously inducing OX40L expression (14–16). The in vivo functions of TSLP have been most extensively studied in the skin and the lung (11, 13, 17, 18). Transgenic overexpression of TSLP in cutaneous or pulmonary epithelial cells results in the onset of Th2 cytokine–mediated inflammation resembling atopic dermatitis or asthma, respectively (17, 18). Based on these studies, it has been proposed that TSLP is both necessary and sufficient for the initiation of Th2 cytokine–driven inflammation (4, 19, 20). We recently showed that TSLP responsiveness is an important component of early immunity to the intestinal nematode pathogen Trichuris (2). However, the mechanisms and absolute requirements for TSLP–TSLPR interactions in the regulation of intestinal immunity and inflammation in vivo remain undefined.In this study, we identify constitutive TSLP expression in IECs throughout the lower gastrointestinal (GI) tract, with the highest level of expression in the proximal large intestine. When challenged with Trichuris, genetically resistant WT mice in which TSLP was neutralized or TSLPR^−/− mice failed to express protective Th2 cell–associated cytokines and maintained persistent parasites beyond day 34 after infection. Disruption of the TSLP–TSLPR pathway additionally resulted in increased expression of IL-12/23p40, IFN-γ, and IL-17A, and the development of severe infection-induced inflammation. Recombinant TSLP inhibited production of IL-12/23p40 in DCs in vitro, and DCs isolated from infected TSLPR^−/− mice exhibited dysregulated production of IL-12/23p40 ex vivo. Significantly, blockade of IFN-γ in Trichuris-infected TSLPR^−/− mice restored expression of Th2 cytokines and host protective immunity. Additionally, TSLPR^−/− mice exhibited elevated expression of proinflammatory cytokines and early onset of intestinal inflammation in a mouse model of inflammatory bowel disease (IBD). Collectively, these data suggest that within the intestinal microenvironment, one function of TSLP–TSLPR interactions may be to limit proinflammatory cytokine production and inflammation.     

Extrafollicular B cell activation by marginal zone dendritic cells drives T cell–dependent antibody responses

Dendritic cells (DCs) are best known for their ability to activate naive T cells, and emerging evidence suggests that distinct DC subsets induce specialized T cell responses. However, little is known concerning the role of DC subsets in the initiation of B cell responses. We report that antigen (Ag) delivery to DC-inhibitory receptor 2 (DCIR2) found on marginal zone (MZ)–associated CD8α⁻ DCs in mice leads to robust class-switched antibody (Ab) responses to a T cell–dependent (TD) Ag. DCIR2⁺ DCs induced rapid up-regulation of multiple B cell activation markers and changes in chemokine receptor expression, resulting in accumulation of Ag-specific B cells within extrafollicular splenic bridging channels as early as 24 h after immunization. Ag-specific B cells primed by DCIR2⁺ DCs were remarkably efficient at driving naive CD4 T cell proliferation, yet DCIR2-induced responses failed to form germinal centers or undergo affinity maturation of serum Ab unless toll-like receptor (TLR) 7 or TLR9 agonists were included at the time of immunization. These results demonstrate DCIR2⁺ DCs have a unique capacity to initiate extrafollicular B cell responses to TD Ag, and thus define a novel division of labor among splenic DC subsets for B cell activation during humoral immune responses.Upon recognition of T cell–dependent (TD) or T cell–independent (TI) antigen (Ag), B cells differentiate into short-lived antibody (Ab)-forming cells (AFCs), which are critical for providing frontline protection against the spread of blood-borne pathogens such as Salmonella typhimurium and influenza (Gerhard et al., 1997; Cunningham et al., 2007; Rothaeusler and Baumgarth, 2010). Alternatively, cognate interaction of B cells with CD4⁺ T cells results in the formation of germinal centers (GCs) and selection of high-affinity clones for differentiation to memory B cells and long-lived plasma cells (Jacob et al., 1991).  Although GC responses and affinity maturation have been extensively studied, much less is known concerning the early events that govern B cell activation and how they influence the decision to produce extrafollicular AFC responses versus GC B cell differentiation.The context in which B cells encounter Ag is highly influenced by the size, nature, and form of the Ag itself (Roozendaal et al., 2009). Although direct recognition of small, soluble Ag by the BCR can occur in vivo (Pape et al., 2007), acquisition of membrane-associated Ag is also an efficient means to trigger B cell activation (Carrasco and Batista, 2006; Depoil et al., 2008). Multiple APCs can present Ag to B cells including follicular DCs, subcapsular sinus and marginal zone (MZ) macrophages, and DCs (Wykes and MacPherson, 2000; Huang et al., 2005; Qi et al., 2006; Phan et al., 2009; Roozendaal et al., 2009; Suzuki et al., 2009). Among these, DCs in particular have been shown in vitro to influence a range of B cell processes including proliferation, differentiation, and Ig class-switch recombination (CSR; Dubois et al., 1998; Fayette et al., 1998; Litinskiy et al., 2002; Craxton et al., 2003). DC-mediated presentation of Ag to B cells in vivo has been shown to enhance TI Ab responses to immune complexes internalized by FcγRIIb on splenic DCs, as well as TI responses against Streptococcus pneumoniae mediated by blood-derived DCs (Balázs et al., 2002; Bergtold et al., 2005; Dubois and Caux, 2005).In contrast, it is unclear what, if any, role DC–B cell interactions may have during humoral responses to TD Ag. Some evidence has suggested that DCs directly present Ag to B cells during TD immune responses. Qi et al. (2006) showed that adoptively transferred DCs can transfer hen egg lysozyme (HEL) to Ag-specific B cells in the lymph node; however, neither the DC subset responsible for the Ag presentation nor the subsequent B cell response was evaluated. Earlier studies showed that adoptive transfer of Ag-bearing DCs was sufficient to induce TD Ab responses; however, because adoptive transfer strategies were used, the role of Ag uptake in situ by resident DC subsets remained unclear (Wykes et al., 1998; Berney et al., 1999). More recent studies using mAbs to deliver Ag directly to APCs in vivo demonstrated Ab responses after Ag uptake by several C-type lectin receptors (CLRs) including FIRE (F4/80-like receptor), CIRE (C-type lectin immune receptor), Dectin-1, Clec12a, and Clec9a (Corbett et al., 2005; Caminschi et al., 2008; Lahoud et al., 2009). Because the CLRs targeted in these studies are also expressed on macrophages, plasmacytoid DCs (pDCs), and/or B cells, it is again unclear which APC populations were required for the observed Ab induction. In sum, many questions remain concerning the induction of Ab responses by DCs.Here, we describe a novel mechanism underlying DC-mediated induction of Ab responses after Ag uptake by DC-inhibitory receptor 2 (DCIR2), a CLR found exclusively on a subset of MZ-associated CD8α⁻ DCs (Dudziak et al., 2007). Using mAbs to deliver Ag in vivo, we show that Ag uptake by DCIR2, but not DEC205 found on CD8α⁺ DCs, induces robust IgG1-restricted TD Ab responses without the addition of any adjuvant. Although DCIR2⁺ DCs are known to preferentially present peptide–MHCII complexes to CD4 T cells after Ag delivery to DCIR2 (Dudziak et al., 2007), we found that DCIR2⁺ DCs also present Ag to B cells and facilitate their rapid activation in vivo. As early as 24 h after immunization, activated Ag-specific B cells accumulated in extrafollicular splenic bridging channels, coincident with the location of DCIR2⁺ DCs in situ. Importantly, DC-primed Ag-specific B cells were highly efficient APCs for driving naive CD4 T cell proliferation, suggesting that B cell–mediated Ag presentation is a key component to sustaining CD4 T cell responses after Ag delivery to DCs. In the absence of adjuvants, DCIR2 DCs induced extrafollicular Ab responses that did not involve GC formation or affinity maturation of serum Ab. Instead, inclusion of agonists for toll-like receptor (TLR) 7 or TLR9 (but not TLR3, TLR5, or RIG-I) shifted the B cell differentiation program toward GC formation and affinity maturation. Thus, our data show that DCIR2⁺ MZ-associated DCs are uniquely equipped and/or positioned to prime B cells and initiate extrafollicular Ab responses to TD Ag.     

CD160 is essential for NK-mediated IFN-γ production

NK-derived cytokines play important roles for natural killer (NK) function, but how the cytokines are regulated is poorly understood. CD160 is expressed on activated NK or T cells in humans but its function is unknown. We generated CD160-deficient mice to probe its function. Although CD160^−/− mice showed no abnormalities in lymphocyte development, the control of NK-sensitive tumors was severely compromised in CD160^−/− mice. Surprisingly, the cytotoxicity of NK cells was not impaired, but interferon-γ (IFN-γ) secretion by NK cells was markedly reduced in CD160^−/− mice. Functionally targeting CD160 signaling with a soluble CD160-Ig also impaired tumor control and IFN-γ production, suggesting an active role of CD160 signaling. Using reciprocal bone marrow transfer and cell culture, we have identified the intrinsic role of CD160 on NK cells, as well as its receptor on non-NK cells, for regulating cytokine production. To demonstrate sufficiency of the CD160⁺ NK cell subset in controlling NK-dependent tumor growth, intratumoral transfer of the CD160⁺ NK fraction led to tumor regression in CD160^−/− tumor-bearing mice, indicating demonstrable therapeutic potential for controlling early tumors. Therefore, CD160 is not only an important biomarker but also functionally controls cytokine production by NK cells.NK cells play multiple roles during the innate immune response, reacting to a myriad of challenges, including pathogen-infected cells, transplanted allogeneic cells, and tumor cells (Moretta et al., 2002; Lanier, 2005). These responses are tightly regulated through multiple activating and inhibitory receptors. Several structurally distinct receptors have been implicated in activating effector functions, including NKp46, NKG2D, 2B4 (CD244), and CS1 (CRACC; Sentman et al., 2006; Marcenaro et al., 2011). Unlike these ubiquitously expressed NK receptors, the CD160 receptor is selectively expressed on the fraction of NK cells with the highest cytotoxic functions (Maïza et al., 1993).CD160 is an immunoglobulin-like, glycosylphosphatidylinositol-anchored protein with homology to killer-cell immunoglobulin-like receptors (Agrawal et al., 1999). In addition to its association with effector function, CD160 was demonstrated to bind broadly to MHC class I molecules with low affinity, first in humans (Barakonyi et al., 2004) and later in mice (Maeda et al., 2005). A recent study, however, demonstrated that human CD160 binds to herpesvirus entry mediator (HVEM), a TNF family member, with much higher affinity than to MHC class I, and leads to suppressed T cell responses in vitro (Cai et al., 2008). Whether this high-affinity interaction exists in vivo and and what role it plays remains unclear.HVEM has been shown to regulate both the innate and adaptive responses through its multiple binding partners, both as a ligand and as a receptor. Via B and T lymphocyte attenuator (BTLA) on T cells, the delivery of HVEM is largely inhibitory, controlling T cell effector responses (Sedy et al., 2005; Deppong et al., 2006) and the innate response (Sun et al., 2009). In contrast, signaling through HVEM activates T cells by LIGHT/TNFSF14 (Cheung et al., 2005; Cai and Freeman, 2009). However, the nature of the HVEM–class I MHC–CD160 interactions has not been well defined in vivo. To directly address these questions, we generated CD160^−/− mice and soluble CD160 (CD160-Ig) fusion protein and investigated the necessity and sufficiency of CD160 on the effector function of NK cells in vivo and in vitro. We reveal here that CD160 is a functional regulator of cytokine production by NK cells and is important for early control of tumor growth.     

The Src family kinases Hck,Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment

Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell–mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in β₂ integrin–deficient (CD18^−/−) mice, Hck^−/−Fgr^−/−Lyn^−/− neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells.Src family kinases are best known for their role in malignant transformation and tumor progression, as well as signaling through cell surface integrins (Parsons and Parsons, 2004; Playford and Schaller, 2004). Due to their role in cancer development and progression, Src family kinases have become major targets of cancer therapy (Kim et al., 2009; Zhang and Yu, 2012). Src family kinases are also present in immune cells with dominant expression of Lck and Fyn in T cells and NK cells; Lyn, Fyn, and Blk in B cells and mast cells; and Hck, Fgr, and Lyn in myeloid cells such as neutrophils and macrophages (Lowell, 2004).The best known function of Src family kinases in the immune system is their role in integrin signal transduction. Indeed, Hck, Fgr, and Lyn mediate outside-in signaling by β₁ and β₂ integrins in neutrophils and macrophages (Lowell et al., 1996; Meng and Lowell, 1998; Mócsai et al., 1999; Suen et al., 1999; Pereira et al., 2001; Giagulli et al., 2006; Hirahashi et al., 2006), Lck participates in LFA-1–mediated T cell responses (Morgan et al., 2001; Fagerholm et al., 2002; Feigelson et al., 2001; Suzuki et al., 2007), and Src family kinases are required for LFA-1–mediated signal transduction and target cell killing by NK cells (Riteau et al., 2003; Perez et al., 2004).Src family kinases also mediate TCR signal transduction by phosphorylating the TCR-associated immunoreceptor tyrosine-based activation motifs (ITAMs), leading to recruitment and activation of ZAP-70 (van Oers et al., 1996; Zamoyska et al., 2003; Palacios and Weiss, 2004). However, their role in receptor-proximal signaling by the BCR and Fc receptors is rather controversial. Although the combined deficiency of Lyn, Fyn, and Blk results in defective BCR-induced NF-κB activation, receptor-proximal BCR signaling (ITAM phosphorylation) is not affected (Saijo et al., 2003). Genetic deficiency of Lyn, the predominant Src family kinase in B cells, even leads to enhanced BCR signaling and B cell–mediated autoimmunity (Hibbs et al., 1995; Nishizumi et al., 1995; Chan et al., 1997). Similarly, both positive (Hibbs et al., 1995; Nishizumi and Yamamoto, 1997; Parravicini et al., 2002; Gomez et al., 2005; Falanga et al., 2012) and negative (Kawakami et al., 2000; Hernandez-Hansen et al., 2004; Odom et al., 2004; Gomez et al., 2005; Falanga et al., 2012) functions for Fyn and Lyn during Fc receptor signaling in mast cells have been reported. In addition, Hck^−/−Fgr^−/− neutrophils respond normally to IgG immune complex–induced activation (Lowell et al., 1996) and Fc receptor–mediated phagocytosis of IgG-coated red blood cells is delayed but not blocked in Hck^−/−Fgr^−/−Lyn^−/− macrophages (Fitzer-Attas et al., 2000; Lowell, 2004). The differential requirement for Src family kinases in TCR, BCR, and Fc receptor signaling is thought to derive from the fact that Syk, but not ZAP-70, is itself able to phosphorylate ITAM tyrosines (Rolli et al., 2002), making Src family kinases indispensable for signaling by the ZAP-70–coupled TCR but not by the Syk-coupled BCR and Fc receptors.Autoantibody production and immune complex formation is one of the major mechanisms of autoimmunity-induced tissue damage. In vivo models of those processes include the K/B×N serum transfer arthritis (Korganow et al., 1999) and autoantibody-induced blistering skin diseases (Liu et al., 1993; Sitaru et al., 2002, 2005), which mimic important aspects of human rheumatoid arthritis, bullous pemphigoid, and epidermolysis bullosa acquisita. Activation of neutrophils or macrophages (Liu et al., 2000; Wipke and Allen, 2001; Sitaru et al., 2002, 2005; Solomon et al., 2005), recognition of immune complexes by Fcγ receptors (Ji et al., 2002; Sitaru et al., 2002, 2005), and β₂ integrin–mediated leukocyte recruitment (Watts et al., 2005; Liu et al., 2006; Chiriac et al., 2007; Monach et al., 2010; Németh et al., 2010) are indispensable for the development of those in vivo animal models.The role of Src family kinases in β₂ integrin signaling and the requirement for β₂ integrins during autoantibody-induced in vivo inflammation prompted us to test the role of Src family kinases in autoantibody-induced inflammatory disease models. We found that Hck^−/−Fgr^−/−Lyn^−/− mice were completely protected from autoantibody-induced arthritis and inflammatory blistering skin disease. Surprisingly, this was not due to a cell-autonomous defect in β₂ integrin–mediated leukocyte migration but to defective generation of an inflammatory microenvironment, likely due to the role of Src family kinases in immune complex–induced neutrophil and macrophage activation.     

Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves

Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP⁺ cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c⁺ cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8⁺ TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.Inflammation is a component of many vascular disorders such as aortic aneurysm, giant cell arteritis, Takayasu’s disease, and atherosclerosis (1–4). DCs carry out many innate responses and orchestrate adaptive immunity (5). It is therefore important to assess the presence and properties of DCs in major blood vessels.Initially, electron microscopy and labeling for several intracellular markers were used to demonstrate DCs in human aorta, primarily in a subendothelial location (6–9). Ma-Krupa et al. (10) and Pryshchep et al. (11) then used more cell-restricted markers to identify DCs in increased numbers in human arteries, whereas Bobryshev (12) reported increased numbers of cells expressing S100, CD1a, and p55 markers in the intima and adventitia during atherosclerosis. Cells bearing the CD11c integrin, which is characteristically expressed at high levels on DCs, were also identified in the normal aortic intima and in atherosclerosis-prone areas in mice (13, 14). The numbers of aortic CD11c⁺ cells increased with aging and atherosclerosis through a recruitment process involving CX3CR1 chemokine receptor and VCAM-1 (13, 15, 16).Although abundant CD11c expression is a well-known marker for DCs, other cell types can express moderate levels of CD11c, including activated NK cells, some macrophages, and even some T cells (17–21). Therefore, additional criteria are required to identify DCs in the vascular wall in the steady state and ultimately disease, particularly the capacity of DCs to express MHC class II and present antigens to T lymphocytes.In this study, we found that the CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mouse developed by Lindquist et al. (22) is valuable to identify and study CD11c⁺ cells from the normal mouse aorta. We will report on the location, cell surface markers, and antigen-presenting functions of DCs and also describe their abundance in all of the cardiac valves.     

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141⁺ DC subset. CD141⁺ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c⁺ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141⁺ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8⁺ cytotoxic T lymphocytes than poly I:C–activated CD1c⁺ DCs. Importantly, CD141⁺ DCs, but not CD1c⁺ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141⁺ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α⁺ DC subset. The data demonstrate a role for CD141⁺ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.The essential role of DCs in the induction and regulation of immune responses to pathogens, self-antigens (Ags), and cancers is now well established. All DCs excel at processing and presenting Ag and priming naive T cell responses, but the complexity of DC subsets and their individual specialized functions is just becoming apparent (MacDonald et al., 2002; Villadangos and Schnorrer, 2007; Naik, 2008). Promising DC-based therapeutic vaccines have been described to treat malignancies and infections (Vulink et al., 2008), but the majority of these use in vitro–generated monocyte-derived DC (MoDC), and the physiological standing of this DC subtype is currently unclear. Understanding the emerging complexities of human DC subset biology is therefore essential to develop new vaccines and therapeutics targeting DC.The characterization and function of human DC subsets has been confounded by their rarity, the lack of distinctive markers, and limited access to human tissues. Human blood DCs comprise ∼1% of circulating PBMCs and have been classically defined as Ag-presenting leukocytes that lack other leukocyte lineage markers (CD3, 14, 15, 19, 20, and 56) and express high levels of MHC class II (HLA-DR) molecules (Hart, 1997). These can be broadly categorized into two groups: plasmacytoid CD11c⁻CD123⁺ DC and conventional or myeloid CD11c⁺CD123⁻ DC. We have described three further phenotypically distinct subsets of CD11c⁺ DC, defined by their expression of CD16, CD1c (BDCA-1), and CD141 (BDCA-3; MacDonald et al., 2002). Gene expression profiling and hierarchical clustering data has indicated that plasmacytoid DC and CD16⁺ DC arise from separate precursor cells, whereas the CD1c⁺ DC and CD141⁺ DC subsets appear to have a common origin and represent two different stages of a similar subset (Lindstedt et al., 2005). However, CD1c⁺ and CD141⁺ DCs each have unique gene expression profiles distinct from monocytes and MoDC, and this predicts that they have different functions (Dzionek et al., 2000; MacDonald et al., 2002; Lindstedt et al., 2005).The concept of distinct DC subtypes with unique capabilities to influence immunological outcomes is exemplified by the mouse CD8α⁻ and CD8α⁺ conventional DC subsets that reside in the lymph nodes and spleen (Villadangos and Schnorrer, 2007; Naik, 2008). The CD8α⁻ DC subset appears to be most effective at inducing Th2 responses (Maldonado-López et al., 1999; Pulendran et al., 1999) and processing and presenting Ag to CD4⁺ T cells via the MHC class II pathway (Pooley et al., 2001; Dudziak et al., 2007; Villadangos and Schnorrer, 2007). In contrast, the CD8α⁺ DC subset has a unique ability to take up dead or dying cells and to process and present exogenous Ag on MHC class I molecules to CD8⁺ T cells (i.e., cross-presentation; den Haan et al., 2000; Iyoda et al., 2002; Schnorrer et al., 2006). There is now substantial evidence that the CD8α⁺ DC subset plays a crucial role in the induction of protective CD8⁺ CTL responses that are essential for the eradication of cancers, viruses, and other pathogenic infections (Dudziak et al., 2007; Hildner et al., 2008; López-Bravo and Ardavín, 2008; Naik, 2008). The identification of the human DC subset with similar functional capacity would be a significant advance and would enable translation of mouse DC biology into clinical practice.Correlation of the human and mouse DC subsets has been hampered by differences in their defining markers (human DCs do not express CD8α). Interestingly, computational genome-wide expression profiling clustered human CD141⁺ DC and CD1c⁺ DC with the mouse CD8α⁺ and CD8α⁻ conventional DC subsets, respectively (Robbins et al., 2008). Human CD141⁺ DC and mouse CD8α⁺ DC share a number of phenotypic similarities, including expression of Toll-like receptor (TLR) 3 (Edwards et al., 2003; Lindstedt et al., 2005), the novel surface molecule Necl2 (nectin-like protein 2; Galibert et al., 2005), and the C-type lectin CLEC9A (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). Thus, whether the human CD141⁺ DC subset is the human functional equivalent of the mouse CD8α⁺ DC subset has now become a major question for immunologists.CD141⁺ DCs constitute only ∼0.03% of human PBMCs and, although present in other human tissues, their low proportions and difficulties with aseptic human tissue access mean that they have never been isolated in sufficient quantity to study their function until now. We report the first detailed functional analysis of human CD141⁺ DCs in response to TLR3 stimuli and define their role in the induction of Th1 responses and cross-presentation.     

Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of α4β7 and CCR9 by Peyer’s patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid–dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103⁺ MLN DCs, up-regulate the gut-homing integrin α4β7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.Because efficient trafficking of immune cells to the gastrointestinal (GI) tract is critical for host defense against pathogenic challenge, studying cellular recruitment pathways to the GI tract is the key to developing novel vaccines against mucosally transmitted diseases, including HIV-1 infection.Naive T cells acquire the capacity to migrate to extra-lymphoid tissues once activated by their cognate antigen (Butcher et al., 1999; von Andrian and Mackay, 2000). These antigen-experienced effector T cells migrate preferentially to the tissues where they first encountered the antigen (Kantele et al., 1999; Campbell and Butcher, 2002). For example, early observations demonstrated that cells activated in the GI tract home back to the intestinal effector sites (Cahill et al., 1977; Hall et al., 1977). Integrin α4β7 and chemokine receptor 9 (CCR9) are among the best studied gut-specific homing molecules (Berlin et al., 1995; Zabel et al., 1999). The α4β7 ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates recruitment of T cells to the intestinal lamina propria (Berlin et al., 1995), and the CCR9 ligand TECK, expressed by small intestinal epithelial cells, recruits T cells to the small bowel (Zabel et al., 1999).DCs are well recognized as the initiators of the adaptive immune response (Steinman and Cohn, 1973), as well as mediators of tolerance to self-antigens in steady-state conditions (Hawiger et al., 2001). Additionally, there is increasing evidence regarding the role played by DCs as conductors of immunological traffic to the skin and the GI tract (Johansson-Lindbom et al., 2003, 2005; Mora et al., 2003, 2005; Sigmundsdottir et al., 2007). DCs can imprint T cells to migrate to the tissue in which the T cells were originally activated. For example, gut-associated DCs induce the gut-homing receptors α4β7 and CCR9 on T cells upon activation (Johansson-Lindbom et al., 2003; Mora et al., 2003; Stagg et al., 2002).RA is necessary and sufficient to induce gut-homing receptors on T cells (Iwata et al., 2004). The main pathway of RA biosynthesis in vivo is dependent on the intracellular oxidative metabolism of retinol (Napoli, 1999; Duester, 2000), catalyzed by a family of alcohol dehydrogenases including retinal dehydrogenase (RALDH), a class I aldehyde dehydrogenase that mediates the irreversible oxidation of retinal to RA. RA in turn is thought to induce RALDH-2 in a positive feedback loop (Yokota et al., 2009; Hammerschmidt et al., 2011; Villablanca et al., 2011) and RA levels correlate with the ability of the intestinal DCs to induce gut-tropic T cells. Vitamin A is introduced via dietary or biliary sources (Jaensson-Gyllenbäck et al., 2011). Among the cellular sources of RA in the intestinal mucosa are DCs (Iwata et al., 2004), stromal cells (Hammerschmidt et al., 2008; Molenaar et al., 2009), intestinal epithelial cells (Bhat, 1998; Lampen et al., 2000), and intestinal macrophages (Denning et al., 2007), with the DCs likely playing a key role in the induction of gut-homing phenotype on T cells. Among the intestinal DCs, the CD103⁺ DC subsets express high levels of RALDH-2 and are capable of generating high levels of RA (Johansson-Lindbom et al., 2005; Coombes et al., 2007; Sun et al., 2007; Jaensson et al., 2008). In contrast, the CD103⁻CD11b⁺CX3CR1⁺ macrophage-like population in the intestinal lamina propria expresses RALDH-1 and not RALDH-2 and exhibits a lower RA-producing capacity (Schulz et al., 2009; Denning et al., 2011), and therefore a decreased capacity to induce gut-homing potential on T cells (Jaensson et al., 2008).Collectively, a paradigm has emerged wherein only the intestinal CD103⁺ DCs, which are capable of metabolizing vitamin A, can induce GI-specific homing on T cells (Jaensson et al., 2008). This paradigm however, is difficult to reconcile with reports of GI T cell responses after i.n. delivery of antigens that do not directly target the GI lymphoid tissue. For example, mice infected i.n. with influenza virus show no activated virus-specific CD8⁺ T cells in the mesenteric LNs (MLNs) or Peyer’s patches (PPs), yet flu-specific CD8⁺ cells within the lung-associated tissues express α4β7 and memory CD8⁺ T cells are established within the small intestinal epithelium (Masopust et al., 2010). Similarly, a recent study demonstrates that i.n. challenge with H1N1 influenza results in the accumulation of T_H17 cells within the small intestinal lamina propria (SILP; Esplugues et al., 2011). Furthermore, Ciabattini et al. (2011) have demonstrated that after i.n. immunization, antigen-specific T cells are generated in the mediastinal LN and migrate to the MLN in an α4β7- and CD62L-dependent manner. Additionally, it is known that lungs harbor prominent extrahepatic stores of vitamin A (Okabe et al., 1984; Dirami et al., 2004). Its metabolite, RA, plays an important role in pulmonary alveolar development (Dirami et al., 2004) and has a putative therapeutic role in emphysema (Massaro and Massaro, 1997). Finally, although RA production has been considered to be the forte of gut-resident DCs, other DC populations also express RALDH, particularly lung-resident DCs that express RALDH-2 (Heng and Painter, 2008; Guilliams et al., 2010). However, the ability of lung DCs to induce GI-specific T cell homing has not yet been reported.All of the aforementioned factors led us to hypothesize that lung DCs would up-regulate the expression of gut-homing molecules integrin α4β7 and CCR9 on T cells, which in turn would license the migration of T cells to the GI tract. Our hypothesis was made credible by the concept of a common mucosal immunological system proposed by Bienenstock et al. (1978) more than 30 years ago. Indeed, there is increasing appreciation of the mucosal immune system as an integrated network of tissues, cells, and effector molecules, although the cellular factors that link different mucosal compartments are not well understood (Gill et al., 2010).In this study, we show that lung DCs can imprint expression of the gut-homing integrin α4β7 and CCR9 on co-cultured T cells in vitro and on adoptively transferred cells in vivo, licensing T cells to migrate to the GI lamina propria and confer protective immunity against intestinal pathogens. We define a new pathway of DC-mediated mucosal cross talk and challenge the existing dogma that only GI-resident DCs can recruit antigen-specific T cells back to the gut.     

Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide

Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar^−/−) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar^−/− NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar^−/− NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell–mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar^−/− NK cells into NK cell–deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN–dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.Type I IFNs provide a potent line of antiviral defense through direct and indirect effects on cells of the immune system, leading to their activation and effector function (Biron, 2001; González-Navajas et al., 2012) and resulting in the attenuation of viral replication (Müller et al., 1994). IFN-α and IFN-β are members of the type I IFN family. All members of the type I IFN family signal through a ubiquitously expressed heterodimeric receptor that is composed of the IFN-α receptor 1 (IFNAR1) and IFNAR2 chains. Type I IFNs act directly on NK cells to promote their activation, cell cycle entry, and cytotoxic function during viral infection (Biron et al., 1984; Orange and Biron, 1996; Biron, 2001; Nguyen et al., 2002; Martinez et al., 2008; Baranek et al., 2012; Fortin et al., 2013). However, the experimental systems used in previous studies—direct infection of IFN receptor–deficient mice or WT mice with IFN neutralization—are complicated by potential differences in the degree of inflammation, effects on many cell types, and viral load. Thus, the direct influence of type I IFN on effector and long-lived antiviral NK cell responses, while eliminating pleotropic effects on other cells, has not been investigated previously.Although substantial amounts of type I IFN are produced during viral infection, this cytokine is constitutively present at basal levels and affects the development and homeostasis of various hematopoietic lineages (Honda et al., 2004; Sato et al., 2009; Gough et al., 2012). An indirect effect of type I IFN on NK cell development and maturation has been described recently (Mizutani et al., 2012; Guan et al., 2014). Because the prolific expansion and generation of memory NK cells during mouse cytomegalovirus (MCMV) infection are dependent predominantly on the proinflammatory cytokines IL-12 and IL-18 (Andoniou et al., 2005; Sun et al., 2012; Madera and Sun, 2015), it was of interest to determine whether type I IFNs play a role in these processes. Here, we use NK cells deficient in the IFNAR1 chain (Ifnar^−/−) in an adoptive cotransfer system and mixed bone marrow chimeric mice to investigate the direct influence of type I IFN signaling on NK cells responding against MCMV infection.     

Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC

Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2K^b. Conversely, CpG-ODN–dependent IFN-α production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2K^b ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo.Plasmacytoid DCs (pDCs) are potent antiviral effector cells that were originally identified by their plasma cell–like morphology and localization within the T cell zone of lymphoid tissue (1). Also termed type I IFN-producing cells, pDCs secrete more type I IFN on a per-cell basis than any other cell type (2–4). pDCs are especially important in controlling viral infections, a property highlighted by their selective expression of Toll-like receptor (TLR) 7 and TLR9 (5), which recognize single-stranded RNA and double-stranded DNA, respectively. pDCs do not express TLR2, TLR3, TLR4, and TLR5, explaining why they do not respond to common bacterial products recognized by other APCs.pDCs represent a rare cell type constituting ∼1% of bone marrow or splenic leukocytes and <0.5% of lymph node and peripheral blood leukocytes. However, their frequency varies between mouse strains with 129Sv mice possessing a significantly higher proportion of pDCs than other mouse strains (6). Mouse pDCs do not express the lineage markers CD19, CD3, DX5, CD14, or TER119 (7, 8). In addition to their selective pattern of TLR expression, pDCs and myeloid DCs (mDCs) are dissimilar in various other aspects. Unlike mDCs, pDCs are characterized by a CD11b⁻B220⁺Ly6C⁺ phenotype (7). Like mDCs, pDCs express CD11c but they do so at a lower level (8). Resting pDCs have been referred to as immature APCs because they express only low levels of CD86 and class II MHC, and they display little or no endocytic activity. However, upon TLR stimulation all three of these characteristics are up-regulated to allow pDCs to present antigenic peptides and optimally stimulate CD4⁺ T cell function (7). In addition, pDCs have been implicated in promoting mDC maturation and terminal B cell differentiation to functional antibody-producing plasma cells (1). Five different mAb reagents have been reported to specifically recognize mouse pDCs: 120G8 (6), mouse PDC antigen 1 (mPDCA-1), 440c (9), NS-34 (10), and 2E6 (11). The 440c mAb recognizes Siglec-H, a DAP12-coupled receptor that inhibits pDC function, including IFN-α secretion (12). 120G8 and mPDCA-1 both recognize bone marrow stromal cell antigen 2 (BST2) (13). NS-34 and 2E6 recognize Ly49Q, a member of the type II C-type lectin-like Ly49 family. Interestingly, most other Ly49 family members are exclusively expressed on NK, NKT, and T cell subsets, where they are known to regulate cytokine production and cell-mediated cytotoxicity via interactions with cognate class I MHC–related ligands on target cells.Ly49Q is one of the more distantly related Ly49 family members, yet the receptor itself is highly conserved among three mouse haplotypes (C57BL/6 [B6], 129S6, and BALB/c) (14–16). To date, Ly49Q protein has been detected in all mouse strains tested (17), suggesting an important and conserved function for this receptor. The Ly49q gene defines the centromeric end of the B6, 129S6, and BALB/c Ly49 gene clusters. Interestingly, a homologous segment comprising Ly49q- and Ly49e-like genes is repeated three times in the 129S6 genome because of gene duplication (18). Therefore, in addition to Ly49q₁, the 129-related mouse strains contain Ly49q₂ and Ly49q₃, but the latter two genes are considered pseudogenes because they lack exons 6 and 7, which encode the ligand-binding domain (18).Ly49Q was first reported to be expressed at low levels on a proportion of Gr-1⁺ bone marrow myeloid precursor cells, on peripheral blood neutrophils (Gr1⁺CD11b⁺), and on IFN-γ–activated macrophages (10). However, the function of the receptor on these cell types remains unknown. Ly49Q contains a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM), whereas it lacks a positively charged transmembrane residue, both of which are characteristics of inhibitory Ly49 receptors expressed by NK cells. Furthermore, similar to inhibitory Ly49 NK receptors, the Ly49Q ITIM has been reported to associate with the Src homology phosphatases (SHPs) 1/2 upon antibody-mediated cross-linking of the receptor (10). However, Ly49Q is not expressed by NK cells. Moreover, Ly49Q cross-linking on activated macrophages has been reported to induce cytoskeletal rearrangement leading to formation of polarized filopodia and lamellopodia; this suggests a role for Ly49Q in macrophage migration and phagocytosis (10).In subsequent reports, a population of cells expressing significantly higher levels of Ly49Q than neutrophils and macrophages was identified (11, 17, 19). This cell population was originally defined as CD11c⁺B220⁺Gr1^low and has been confirmed to represent pDCs (1, 20). Virtually all peripheral pDCs and the majority of bone marrow pDCs express Ly49Q. The subset of bone marrow pDCs lacking Ly49Q expression are thought to represent immature cells, such that acquisition of Ly49Q expression is linked to sequential development of functional pDCs (11, 19). These Ly49Q⁻ pDCs do not respond to certain stimuli and are defective in the secretion of some cytokines. Ly49Q levels correlate well with pDC maturation, and receptor acquisition is further up-regulated by various stimuli, including IFN-α (17, 19). In some but not all inbred mouse strains, a subset of mDCs also expresses low levels of Ly49Q (17).We recently identified the classical class I MHC molecule, H-2K^b, as the cognate Ly49Q ligand (21). Using reporter cell analysis, a high-affinity ligand for Ly49Q was detected on tumor and normal ex vivo cells derived from H-2^b haplotype mice (B6 and 129) but not on cells from the other mouse MHC haplotypes tested. Direct MHC surveillance by pDCs and the implications of this interaction for innate immunity remain to be investigated.The current study demonstrates a major role for Ly49Q–H-2K^b interactions in pDC production of IFN-α and, to a lesser extent, IL-12. Remarkably, the function of Ly49Q on pDCs appears to be stimulatory in nature, as revealed by cross-linking experiments despite the presence of a cytoplasmic ITIM. To confirm these in vitro findings and to ascertain the role of Ly49Q during immune responses, Ly49Q-null mice were generated and characterized. Ly49Q-null mice exhibit a severe defect in systemic IFN-α production after challenge with agonists and pathogens recognized by TLR7 and TLR9, which translates into weaker antiviral responses in vivo. We propose that Ly49Q recognition of self-MHC is necessary to regulate pDC cytokine responses to pathogens.