Antigen-specific expansion and differentiation of natural killer cells by alloantigen stimulation

Natural killer (NK) cells provide important host defense against microbial pathogens and can generate a population of long-lived memory NK cells after infection or immunization. Here, we addressed whether NK cells can expand and differentiate after alloantigen stimulation, which may be important in hematopoietic stem cell and solid tissue transplantation. A subset of NK cell in C57BL/6 mice expresses the activating Ly49D receptor that is specific for H-2D^d. These Ly49D⁺ NK cells can preferentially expand and differentiate when challenged with allogeneic H-2D^d cells in the context of an inflammatory environment. H-2D^d is also recognized by the inhibitory Ly49A receptor, which, when coexpressed on Ly49D⁺ NK cells, suppresses the expansion of Ly49D⁺ NK cells. Specificity of the secondary response of alloantigen-primed NK cells was defined by the expression of activating Ly49 receptors and regulated by the inhibitory receptors for MHC class I. Thus, the summation of signals through a repertoire of Ly49 receptors controls the adaptive immune features of NK cells responding to allogeneic cells.NK cells recognize abnormal or allogeneic cells by using a repertoire of NK cell receptors that regulates their activation and effector functions (Lanier, 2005). Although NK cells were considered unable to differentiate into memory cells, accumulating evidence demonstrates that NK cells have adaptive immune features, which include antigen-specific expansion and differentiation into a long-lived memory subset (O’Leary et al., 2006; Cooper et al., 2009; Sun et al., 2009a, 2010; Paust et al., 2010; Min-Oo et al., 2013). In some mouse models, NK cells are activated after exposure to pathogens, antigens, and cytokines, and subsequently differentiate into long-lived memory or memory-like NK cells with augmented effector functions in response to a variety of secondary stimuli, as compared with naive NK cells (O’Leary et al., 2006; Cooper et al., 2009; Sun et al., 2009a). The existence of memory NK cells in humans is supported by the specific expansion and persistence for months of NKG2C^high NK cells after human cytomegalovirus (HCMV) infection (Gumá et al., 2004; Lopez-Vergès et al., 2011; Foley et al., 2012a,b; Min-Oo et al., 2013). We have previously demonstrated that mouse NK cells bearing the activating Ly49H receptor, which specifically recognizes the m157 mouse cytomegalovirus (MCMV) glycoprotein on the infected cells (Arase et al., 2002; Smith et al., 2002), undergo activation, expansion, contraction, differentiation into memory NK cells, and persistence for several months after MCMV infection (Sun et al., 2009a, 2010). These MCMV-specific memory NK cells are capable of mounting a recall response and provide more effective host protection against rechallenge with MCMV than naive NK cells (Sun et al., 2009a). The immunoreceptor tyrosine-based activating motif (ITAM)-containing DAP12 adapter protein, the proinflammatory cytokine IL-12, and the co-stimulatory DNAM-1 receptor are essential not only for optimal expansion of effector Ly49H⁺ NK cells, but also for the generation of long-lived memory Ly49H⁺ NK cells after MCMV infection (Sun et al., 2009a, 2012; Nabekura et al., 2014). However, specific receptors, other than Ly49H, that are able to drive the clonal expansion and differentiation of NK cells have not been identified. Furthermore, the specificity of the secondary responses of memory NK cells bearing multiple activating receptors also remains unknown, because an experimental system that allows NK cells to expand and differentiate into memory NK cells in a defined receptor-ligand specific manner has not been established, except for MCMV infection.Cudkowicz and Stimpfling (1964) observed that in certain strains of mice parental bone marrow grafts are rejected by the F1 recipient, and this was subsequently demonstrated to be mediated by NK cells (Kiessling et al., 1977). The inhibitory Ly49 receptors that recognize polymorphic MHC class I ligands are expressed in a stochastic manner on subsets of NK cells in the host (Lanier, 1998; Anderson et al., 2001). As a consequence, in a F1 host, some of the NK cells will lack an inhibitory Ly49 receptor specific for the parental H-2 haplotype. Because they are not inhibited by the parental H-2 ligands, these NK cells are responsible for rejection of the parental graft. Although most Ly49 receptors function as inhibitory receptors for MHC class I, some members of the Ly49 family are activating receptors that transmit signals through the DAP12 and DAP10 signaling molecules (Orr et al., 2009). In C57BL/6 mice a subset of NK cells expresses the activating Ly49D receptor that recognizes H-2^d alloantigens (George et al., 1999a,b). Some of the Ly49D⁺ NK cells in C57BL/6 mice (H-2^b) coexpress the inhibitory Ly49A receptor that recognizes H-2D^d, which inhibits rejection of allogeneic cells bearing H-2D^d (Karlhofer et al., 1992). Because of the structural and signaling similarities shared by Ly49H and Ly49D, we addressed whether an activating signal through Ly49D would result in the expansion and differentiation of Ly49D⁺ NK cells in response to alloantigens, similar to the generation of memory Ly49H⁺ NK cells during MCMV infection. Here, we established an experimental system for alloantigen-driven expansion and differentiation of Ly49D⁺ NK cells. Using this system, we investigated the roles of activating and inhibitory Ly49 receptors in the generation and recall response of NK cells specific for alloantigens.     

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.     

Dual-reactive B cells are autoreactive and highly enriched in the plasmablast and memory B cell subsets of autoimmune mice

Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG⁺ cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG⁺ cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.While developing in the BM, B cells undergo stochastic rearrangement of Ig heavy (IgH) and Ig light (IgL) chain V(D)J gene segments resulting in the random expression of Ig H and L (κ and λ) chains in the emerging B cell population (Schlissel, 2003; Nemazee, 2006). During V(D)J recombination, allelic and isotypic exclusion at the Ig loci are also established, leading to the expression of a unique H and L chain pair and, therefore, of BCRs with unique specificity in each B cell (Langman and Cohn, 2002; Nemazee, 2006; Vettermann and Schlissel, 2010). These mechanisms ensure that developing B cells expressing BCRs reactive with self-antigens (i.e., autoreactive B cells) undergo tolerance induction, whereas those expressing BCRs specific for a foreign antigen or a peripheral self-antigen proceed in differentiation and selection into the periphery (Burnet, 1959). Autoreactive B cells are silenced by central tolerance in the BM via receptor editing and, less frequently, clonal deletion (Halverson et al., 2004; Ait-Azzouzene et al., 2005), whereas peripheral B cell tolerance proceeds via anergy and clonal deletion (Goodnow et al., 2005; Pelanda and Torres, 2006, 2012; Shlomchik, 2008). Despite these tolerance mechanisms, small numbers of autoreactive B cells are detected in peripheral tissues of healthy mice and humans (Grandien et al., 1994; Wardemann et al., 2003) and their numbers are increased in autoimmunity (Andrews et al., 1978; Izui et al., 1984; Warren et al., 1984; Samuels et al., 2005; Yurasov et al., 2005, 2006; Liang et al., 2009).A small population of dual-reactive B cells expressing two types of L chains (or more rarely H chains) has been observed both in mice and humans (Nossal and Makela, 1962; Pauza et al., 1993; Giachino et al., 1995; Gerdes and Wabl, 2004; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007; Kalinina et al., 2011). These allelically and isotypically (overall haplotype) included B cells are <5% of all peripheral B cells in normal mice (Barreto and Cumano, 2000; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007), but they are more frequent in Ig knockin mice in which newly generated B cells are autoreactive and actively undergo receptor editing (Li et al., 2002a,b; Liu et al., 2005; Huang et al., 2006; Casellas et al., 2007). B cells that coexpress autoreactive and nonautoreactive antibodies can escape at least some of the mechanisms of central and peripheral B cell tolerance and be selected into the mature peripheral B cell population (Kenny et al., 2000; Li et al., 2002a,b; Gerdes and Wabl, 2004; Liu et al., 2005; Huang et al., 2006), sometimes with a preference for the marginal zone (MZ) B cell subset (Li et al., 2002b).Furthermore, dual-reactive B cells observed within a normal polyclonal Ig repertoire exhibit characteristics of cells that develop through the receptor editing process, including delayed kinetics of differentiation and more frequent binding to self-antigens (Casellas et al., 2007). Hence, dual-reactive B cells might play a role in autoantibody generation and autoimmunity. However, the contribution of these B cells to autoimmunity has not yet been established. Our hypothesis is that haplotype-included autoreactive B cells are positively selected within the context of genetic backgrounds that manifest defects in immunological tolerance and contribute to the development of autoimmunity.Until recently, the analysis of dual-reactive B cells was impaired by the inability to detect dual-κ cells, which are the most frequent among haplotype-included B cells (Casellas et al., 2007; Velez et al., 2007). To overcome this issue, we took advantage of Igk^h mice that bear a gene-targeted human Ig Ck allele in the context of a wild-type Ig repertoire (Casellas et al., 2001) and crossed these to MRL-Fas^lpr/lpr (MRL/lpr) and MRL mice that develop an autoimmune pathology with characteristics similar to human lupus (Izui et al., 1984; Rordorf-Adam et al., 1985; Theofilopoulos and Dixon, 1985; Cohen and Eisenberg, 1991; Watanabe-Fukunaga et al., 1992). MRL/lpr mice, moreover, display defects in receptor editing (Li et al., 2002a; Lamoureux et al., 2007; Panigrahi et al., 2008) and reduced tolerance induction (Li et al., 2002a), which could potentially contribute to higher frequency of haplotype-included autoreactive B cells.We found that the frequency of dual-κ cells increased with age and progression of disease in autoimmune-prone mice and independent of the expression of Fas. Dual-κ B cells exhibited higher prevalence of autoreactivity than single-κ B cells and were frequently selected into the antigen-activated cell subsets in MRL/lpr and MRL mice where up to half of the plasmablasts and memory B cells were dual-κ B cells. Moreover, disruption of Fas expression appeared to mediate increased survival of dual-reactive memory B cells. Overall, these data indicate that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in the development of autoimmunity.     

Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium

Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4⁺ T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.Inducing specific immunity and maintaining tolerance requires cells of the mononuclear phagocyte lineage. This lineage is comprised of three closely related cell types: DCs, monocytes, and macrophages (Shortman and Naik, 2007; Geissmann et al., 2010a,b; Liu and Nussenzweig, 2010; Yona and Jung, 2010; Chow et al., 2011). DCs are essential to both immunity and tolerance (Steinman et al., 2003); however, the role monocytes and macrophages play in these processes is not as well defined (Geissmann et al., 2008).In mice, DCs and monocytes arise from the same hematopoietic progenitor, known as the macrophage–DC progenitor (MDP; Fogg et al., 2006). Their development diverges when MDPs become either common DC progenitors (CDPs) that are Flt3L-dependent, or monocytes, which are dependent on CSF1 (M-CSF; Witmer-Pack et al., 1993; McKenna et al., 2000; Fogg et al., 2006; Waskow et al., 2008). CDPs develop into either plasmacytoid DCs or preDCs that leave the bone marrow to seed lymphoid and nonlymphoid tissues, where they further differentiate into conventional DCs (cDCs; Liu et al., 2009). In contrast, monocytes circulate in the blood and through tissues, where they can become activated and develop into several different cell types, including some but not all tissue macrophages (Schulz et al., 2012; Serbina et al., 2008; Yona et al., 2013).Despite their common origin from the MDP, steady-state lymphoid tissue cDCs can be distinguished from monocytes or macrophages by expression of cell surface markers. For example, cDCs in lymphoid tissues express high levels of CD11c and MHCII, but lack the expression of CD115 and F4/80 found in monocytes and macrophages, respectively. However, this distinction is far more difficult in peripheral tissues, like the intestine or lung, or during inflammation when monocytes begin to express many features of DC including high levels of MHCII and CD11c (Serbina et al., 2003; León et al., 2007; Hashimoto et al., 2011).The function of cDCs in immunity and tolerance has been explored extensively using a series of different mutant mice to ablate all or only some subsets of cDCs (Jung et al., 2002; Liu and Nussenzweig, 2010; Chow et al., 2011). In contrast, the methods that are currently available to study the function of monocytes and macrophages in vivo are far more restricted and less specific (Wiktor-Jedrzejczak et al., 1990; Dai et al., 2002; MacDonald et al., 2010; Chow et al., 2011). For example, Ccr2^−/− and Ccr2^DTR mice (Boring et al., 1997; Kuziel et al., 1997; Serbina and Pamer, 2006; Tsou et al., 2007) have been used to study monocytes (Boring et al., 1997; Peters et al., 2004; Hohl et al., 2009; Nakano et al., 2009). However, CCR2 is also expressed on some subsets of cDCs, activated CD4⁺ T cells, and NK cells (Kim et al., 2001; Hohl et al., 2009; Egan et al., 2009; Zhang et al., 2010). Thus, it is challenging to dissect the precise role of monocytes as opposed to other cell types in immune responses in Ccr2^−/− or Ccr2^DTR mice. Inducible DTR expression in CD11c^Cre x CX₃CR1^LsL-DTR mice is far more specific (Diehl et al., 2013), but restricted to a small subset of mononuclear phagocytes.Here, we describe a genetic approach to targeting monocytes and macrophages that spares cDCs and lymphocytes, and we compare the effects of monocyte and macrophage ablation to cDC depletion on the adaptive immune response to intestinal infection with Citrobacter rodentium.     

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.B lymphocytes can respond rapidly to nonself-antigens, yet even at mature stages of development can be rendered tolerant if they encounter self-antigen (Goodnow et al., 2005). How B cells distinguish self from nonself has been explained in part by Bretscher and Cohn’s associative recognition (“two-signal”) hypothesis (Bretscher and Cohn, 1970), which posits that B cells can only achieve activation after a second signal is delivered, the first being recognition of antigen by the BCR. Without this second signal, tolerance is induced. In response to T-dependent antigens, activated helper T cells provide this second signal. In a T-independent type 1 response, the second signal might come from the B cells’ Toll-like receptors (TLRs) recognizing conserved microbial motifs attached to the antigen (e.g., lipopolysaccharide; Coutinho et al., 1974). This model, however, fails to explain how T-independent type 2 (TI-2) responses occur, as TI-2 antigens require neither T cells (Mond et al., 1995) nor recognition by known innate immune receptors (Gavin et al., 2006), and can elicit antibody responses in cultures of single B cells (Nossal and Pike, 1984). Although we do not dispute contributory roles of innate immune receptors, cytokines, or accessory cells in amplifying their responses (Mond et al., 1995; Vos et al., 2000; Hinton et al., 2008), TI-2 antigens appear to have only two surprisingly simple properties, high molecular weight and ≥20 closely spaced BCR epitopes (Dintzis et al., 1976), and are thus unlikely to have innate receptors specialized for their recognition.Alternatively, B cells might be capable of “missing self”–recognition (Parish, 1996; Nemazee and Gavin, 2003) similar to that originally observed in NK cells (Kärre et al., 1986). In NK cell recognition, the decision to lyse a target cell depends on integration of opposing signals from activating and inhibitory receptors (Lanier, 2008). Activating receptors trigger recruitment of tyrosine kinases to immunotyrosine activating motifs of associated adapter molecules but are kept in check by inhibitory receptors recognizing classical MHC I molecules expressed on target cells (Lanier, 2008). Inhibitory receptors carry immunotyrosine inhibitory motifs (ITIMs), which serve as docking sites for phosphatases, such as SHP-1, that counteract activation (Ravetch and Lanier, 2000). Target cells that down-regulate MHC I are lysed owing to unopposed activation, hence missing self–recognition.Extrapolating from this model, we hypothesize that besides their BCR epitopes, self-antigens carry self-markers that can engage inhibitory receptors on B cells, preventing antiself TI-2–like responses and rendering activation dependent on second signals. The concept that self-markers might facilitate self-tolerance was first suggested many years ago by Burnet and Fenner (1949) but has garnered little experimental support with respect to lymphocyte tolerance. According to our model, antigens that simultaneously cross-link the BCRs and inhibitory receptors should prevent or blunt B cell responses. Conversely, antigens that bind only the BCR and not inhibitory receptors are predicted to elicit a TI-2 response, provided that they carry the appropriate number and spacing of epitopes. This missing self–model of self-/nonself-discrimination would explain why B cells constitutively express so many inhibitory receptors that recognize ubiquitous self-components, and why null mutations in those receptors or their signaling machinery can lead to autoantibody formation (Nishimura et al., 1998; Pan et al., 1999; Ravetch and Lanier, 2000; Nemazee and Gavin, 2003).In this study, we chose to test if self-/nonself-discrimination is regulated by self-markers through the roles of the sialic acid–binding Ig-like lectins (siglecs) CD22 and Siglec-G in B cells. The siglec family consists of 9 members in mice and 13 members in humans (for review see Crocker et al., 2007). In mice, mature B cells express CD22 (Siglec 2) and Siglec-G, which bind to host sialic acids carried on glycans of glycoproteins and glycolipids and have properties of inhibitory receptors. They carry ITIMs capable of recruiting the tyrosine phosphatase SHP-1 and attenuating BCR signaling (Campbell and Klinman, 1995; Doody et al., 1995; Cornall et al., 1998). Mice carrying null mutations in either CD22 or Siglecg exhibit B cell hyperactivity, variable responses to T-independent antigens, and a tendency toward autoantibody formation (O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; Cornall et al., 1998; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Mouse CD22 exhibits a strong preference for sialoside ligands with the disaccharide sequence NeuGcα2-6Gal (Collins et al., 2006a; Crocker et al., 2007), whereas Siglec-G, before this study, has had an unknown ligand specificity. Their disaccharide ligands represent terminal sugars commonly carried on N- and O-linked glycans of glycoproteins and are found on virtually all cells, including B cells (Crocker et al., 2007). It is well documented that CD22 binds to glycans on endogenous B cell glycoproteins in cis, and masks the ligand binding site from binding synthetic polymeric ligands (Hanasaki et al., 1995; Razi and Varki, 1998; Razi and Varki, 1999; Collins et al., 2002; Han et al., 2005). Yet, CD22 is able to recognize native ligands on glycoproteins of apposing cells in trans, causing it to redistribute to the site of cell contact (Lanoue et al., 2002; Collins et al., 2004). Although mutations of the ligand binding domain of CD22 (Jin et al., 2002; Poe et al., 2004) and ablation of enzymes involved in the synthesis of its glycan ligands (Hennet et al., 1998; Poe et al., 2004; Collins et al., 2006b; Ghosh et al., 2006; Grewal et al., 2006; Naito et al., 2007; Cariappa et al., 2009) document the importance of siglec ligands in the regulation of CD22 function, a unifying role for CD22 ligand interactions in B cell biology has not yet emerged (Crocker et al., 2007; Walker and Smith, 2008). Although less is known about the specificity of Siglec-G and its interaction with ligands, it is assumed that similar concepts regarding cis and trans ligands will apply to the modulation of Siglec-G function (Hoffmann et al., 2007; Chen et al., 2009).Because siglecs see sialylated glycans that are usually absent from microbes, with the notable exceptions of some pathogenic microbes (Crocker et al., 2007; Carlin et al., 2009a), one possible role of siglecs is to discriminate self from nonself. Though CD22 and Siglec-G have been implicated to play roles in B cell tolerance, evidence has been indirect, inferred from the facts that they possess ITIMs able to recruit SHP-1 and dampen Ca²⁺ signaling (Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Hypomorphic or null alleles of CD22 and SHP-1 (Ptpn6) have been correlated with anti-DNA production and development of lupus erythematosus (Shultz et al., 1993; O’Keefe et al., 1999; Mary et al., 2000). CD22 mutations also lead to increased in vivo B cell proliferation and turnover (Otipoby et al., 1996; Nitschke et al., 1997; Poe et al., 2004; Haas et al., 2006; Onodera et al., 2008). However, studies designed to directly assess tolerance induction in antigen-specific CD22^−/− or SHP-1 mutant B cells found, paradoxically, a more robust tolerance relative to unmutated controls (Cyster and Goodnow, 1995; Cornall et al., 1998; Ferry et al., 2005). This suggests that the autoimmune phenotypes of siglec and SHP-1 null mutants could be caused by abnormal B cell selection and development rather than failure of tolerance. It is generally assumed that physical association of CD22 with the BCR will allow CD22 to exert a maximal inhibitory response (Pezzutto et al., 1987; Doody et al., 1995; Lanoue et al., 2002; Courtney et al., 2009), but evidence to support this has been garnered only from in vitro experiments (Ravetch and Lanier, 2000; Lanoue et al., 2002; Tedder et al., 2005; Courtney et al., 2009). In this paper, we show in wild-type mice with unaltered B cell selection and development that decorating a TI-2 antigen with siglec ligands not only prevents its immunogenicity but can also tolerize B cells to subsequent challenges with the unsialylated, immunogenic form. The results suggest that one function of B cell inhibitory receptors like siglecs is to assist B cells in distinguishing self from nonself.     

Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection

Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)–induced memory Ly49H⁺ NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes, a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H⁺ or Ly49H⁻ NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge.Persistent infections by viruses of the herpesvirus family, such as CMV, EBV, or HSV are extremely common in the human population, with prevalence rates of ∼60–90% (Virgin et al., 2009). Although in healthy individuals herpesvirus replication is efficiently controlled during the acute phase of infection, a clinically silent latent phase is established for the lifetime of the host and represents a carefully controlled balance of viral and host mechanisms. However, viral reactivation can result from immune suppression, such as in AIDS patients and transplant patients treated with immunosuppressive drugs, and leads to clinical pathology (Sinclair and Sissons, 2006). The impact of the latent viral pool on the immune system and subsequent response to other infections remain poorly understood (Virgin et al., 2009; Dreyfus, 2013). NK cells are important early responder cells in the immune response against infection, particularly against herpesviruses (Mossman and Ashkar, 2005); this is particularly evident in humans with primary NK cell deficiencies who often present with uncontrolled herpesvirus infections (Orange, 2002). Previous work has suggested that latent herpesvirus infections can create an enhanced immune environment against challenge with other pathogens and may result in an increased NK cell response to heterologous infection (White et al., 2010).Upon encountering infected cells, NK cells rapidly secrete cytokines and release cytotoxic granules; moreover, they play a key role in antibody-dependent cell-mediated cytotoxicity (Lodoen and Lanier, 2006). Studies have also identified an additional role for NK cells in the regulation of virus-specific T cell responses (Waggoner et al., 2012). NK cells are traditionally classified as innate immune lymphocytes, but recent work has revealed that these cells share more features with T and B lymphocytes than previously appreciated, including the ability to acquire immunological memory (Paust and von Andrian, 2011; Min-Oo et al., 2013). The memory-like properties of NK cells include antigen-specific recall response to haptens and virus-like particles (Paust et al., 2010), cytokine-induced memory (Cooper et al., 2009; Romee et al., 2012), and enhanced secondary response to MCMV (Sun et al., 2009). In humans, the existence of memory NK cells has been suggested by the presence of an expanded and persistent population of NK cells bearing the NKG2C receptor after CMV infection (Gumá et al., 2004; Lopez-Vergès et al., 2011).MCMV infection leads to the production of numerous cytokines, including type I IFNs and IL-12, which trigger cytokine-induced activation of NK cells and leads to the production of IFN-γ (Orange and Biron, 1996a). NK cells in C57BL/6 mice also possess an activating receptor, Ly49H, that specifically recognizes infected cells expressing the MCMV-encoded protein m157 (Arase et al., 2002; Smith et al., 2002) and mediates a protective response against MCMV (Brown et al., 2001; Lee et al., 2001). After early non–antigen-specific activation by cytokines, antigen-specific Ly49H⁺ NK cells undergo robust expansion after encountering m157 (Dokun et al., 2001) and generate a population of long-lived memory NK cells (Sun et al., 2009). Thus, the NK response to acute MCMV infection is governed by a combination of cytokine signals and antigen-specific activation through Ly49H. Although the mechanisms governing the expansion, contraction, and survival of MCMV-memory NK cells are still being defined, recent work has shown a role for inflammatory cytokine signaling and miRNA155 in the expansion of Ly49H⁺ NK cells and subsequent memory formation (Sun et al., 2012; Zawislak et al., 2013). Similarly, expansion and memory formation after MCMV is dependent on signaling through the activating receptor DNAM-1 (Nabekura et al., 2014), whereas pro-apoptotic pathways regulate contraction and the size of the memory pool (Min-Oo et al., 2014). Long-lived memory NK cells show an enhanced response to rechallenge with MCMV, as demonstrated by their ability to reduce viral loads more efficiently than naive NK cells and to protect neonates from lethal MCMV infection better than naive NK cells (Sun et al., 2009; Nabekura et al., 2014) despite a similar capacity of naive Ly49H⁺ NK cells for early expansion. Furthermore, in vitro, MCMV-memory NK cells show an enhanced response to antibody stimulation through Ly49H, compared with naive Ly49H⁺ NK cells (Sun et al., 2009). The antigen specificity of memory NK cells responding to rechallenge with haptens has also been shown in a contact hypersensitivity model (Paust et al., 2010). Whether MCMV-memory NK cells are generally superior to naive Ly49H⁺ cells and, as such, offer cross-protection against heterologous pathogens or, alternatively, mediate enhanced response solely via MCMV-specific stimulation has not been addressed.Intranasal administration of influenza to mice results in an acute infection in which viral replication is limited exclusively to the lung airways (Thangavel and Bouvier, 2014). Influenza infection in the airway induces a strong chemokine and cytokine response, including secretion of chemotactic proteins (RANTES, MCP-1, and MIP1α) and proinflammatory factors (IL-1β, IL-6, and TNF), as well as IFN-α and IFN-β (Julkunen et al., 2001). As a result, infection by influenza virus, particularly of the subtype H1N1, causes a potent systemic immune response that can lead to an uncontrolled cytokine storm and subsequent fatal immune-mediated pathology (Oldstone et al., 2013). Limiting the intensity of the cytokine and chemokine production by the innate immune system ensures a strong antiviral cytotoxic T cell and neutralizing antibody response, while increasing survival (McGill et al., 2009). Pathology and mortality caused by H1N1 infection in humans and animals is associated with higher levels of cytokines rather than differences in viral load (Arankalle et al., 2010; Marcelin et al., 2011). The role of NK cells in response to influenza virus remains controversial; depending on the strain and dose of influenza, NK cells have been shown to have detrimental, beneficial, or no effect on the outcome (Jost and Altfeld, 2013). Intravenous infection of mice with Listeria monocytogenes, a Gram-positive bacterium, results in bacterial replication in the spleen and liver, which are also sites of MCMV replication during acute infection. Listeria, similarly to influenza, induces a strong systemic inflammatory response driven by the innate immune system (Pamer, 2004; Serbina et al., 2012). Here, we investigated the response of MCMV-memory NK cells when challenged with these two distinct heterologous infections, influenza virus and Listeria, as well as MCMV lacking the m157 antigen.     

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA– and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC–deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.In humans, chronic mucocutaneous candidiasis (CMC) is characterized by infections of the skin, nail, digestive, and genital mucosae with Candida species, mainly C. albicans, a commensal of the gastrointestinal tract in healthy individuals (Puel et al., 2012). CMC is frequent in acquired or inherited disorders involving profound T cell defects (Puel et al., 2010b; Vinh, 2011; Lionakis, 2012). Human IL-17 immunity has recently been shown to be essential for mucocutaneous protection against C. albicans (Puel et al., 2010b, 2012; Cypowyj et al., 2012; Engelhardt and Grimbacher, 2012; Huppler et al., 2012; Ling and Puel, 2014). Indeed, patients with primary immunodeficiencies and syndromic CMC have been shown to display impaired IL-17 immunity (Puel et al., 2010b). Most patients with autosomal-dominant (AD) hyper-IgE syndrome (AD-HIES) and STAT3 deficiency (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; Renner et al., 2008; Chandesris et al., 2012) and some patients with invasive fungal infection and autosomal-recessive (AR) CARD9 deficiency (Glocker et al., 2009; Lanternier et al., 2013) or Mendelian susceptibility to mycobacterial diseases (MSMD) and AR IL-12p40 or IL-12Rβ1 deficiency (de Beaucoudrey et al., 2008, 2010; Prando et al., 2013; Ouederni et al., 2014) have low proportions of IL-17A–producing T cells and CMC (Cypowyj et al., 2012; Puel et al., 2012). Patients with AR autoimmune polyendocrine syndrome type 1 (APS-1) and AIRE deficiency display CMC and high levels of neutralizing autoantibodies against IL-17A, IL-17F, and/or IL-22 (Browne and Holland, 2010; Husebye and Anderson, 2010; Kisand et al., 2010, 2011; Puel et al., 2010a).These findings paved the way for the discovery of the first genetic etiologies of CMC disease (CMCD), an inherited condition affecting individuals with none of the aforementioned primary immunodeficiencies (Puel et al., 2011; Casanova and Abel, 2013; Casanova et al., 2013, 2014). AR IL-17RA deficiency, AR ACT1 deficiency, and AD IL-17F deficiency were described, each in a single kindred (Puel et al., 2011; Boisson et al., 2013). A fourth genetic etiology of CMCD, which currently appears to be the most frequent, has also been reported: heterozygous gain-of-function (GOF) mutations of STAT1 impairing the development of IL-17–producing T cells (Liu et al., 2011; Smeekens et al., 2011; van de Veerdonk et al., 2011; Hori et al., 2012; Takezaki et al., 2012; Tóth et al., 2012; Al Rushood et al., 2013; Aldave et al., 2013; Romberg et al., 2013; Sampaio et al., 2013; Soltész et al., 2013; Uzel et al., 2013; Wildbaum et al., 2013; Frans et al., 2014; Kilic et al., 2014; Lee et al., 2014; Mekki et al., 2014; Mizoguchi et al., 2014; Sharfe et al., 2014; Yamazaki et al., 2014). We studied three unrelated patients with CMCD without mutations of IL17F, IL17RA, ACT1, or STAT1. We used a genome-wide approach based on whole-exome sequencing (WES). We found AR complete IL-17RC deficiency in all three patients.     

Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.The Ras-mediated Raf–MEK–ERK activation pathway controls many fundamental cellular processes. Rap1 belongs to the Ras superfamily, and their role in regulating NK cell development and functions is not understood. Rap1a and Rap1b isoforms are able to regulate cell proliferation, differentiation, adhesion, and polarization (Stork and Dillon, 2005). These two share >95% amino acid homology (Rousseau-Merck et al., 1990). Conversion of inactive Rap1-GDP to active Rap1-GTP is regulated by multiple guanine nucleotide exchange factors such as C3G, RasGRP/CalDAG-GEFs, and EPACs (Gotoh et al., 1995; de Rooij et al., 1998; Kawasaki et al., 1998). Rap1 signaling is terminated by GTPase-activating proteins (GAPs) such as SPA-1 and RapGAPs (Rubinfeld et al., 1991; Kurachi et al., 1997).Rap1 regulates diverse downstream effectors including B-Raf and C-Raf, whose phosphorylation depends on active Rap1-GTPase and play a critical role in the sequential activation of MEK1/2 and their substrates ERK1/2 (Jin et al., 2006; Romano et al., 2006). Recent studies have shown that scaffolding proteins such as IQGAP (1, 2, and 3), KSR1, MP1, or Paxillin can function as signal processing centers by bringing together GTPases, kinases, and their substrates (Sacks, 2006). IQGAP1 can bind to both Rap1a and Rap1b (Jeong et al., 2007). IQGAP1 is widely expressed in multiple tissues including lymphocytes. The N-terminal calponin homology domain of IQGAP1 binds to actin and the IQ domain recruits calmodulin (Joyal et al., 1997). The C-terminal end of IQGAP1 engages with Cdc42-GTP (Joyal et al., 1997), Rac1-GTP (Hart et al., 1996), E-cadherin (Kuroda et al., 1998), β-catenin (Li et al., 1999), and APC (Watanabe et al., 2004). IQGAP1 also has the ability to bind to B-Raf (Ren et al., 2007), MEK1/2 (Roy et al., 2005), and ERK1/2 (Roy et al., 2004). Irrespective of these findings, IQGAP1-mediated signalosome formation and its relevance in regulating lymphocyte functions have not been investigated.Recently, we generated Rap1a and Rap1b gene knockout mice (Chrzanowska-Wodnicka et al., 2005; Li et al., 2007). Here, we found that the lack of Rap1a or Rap1b did not alter the development or cytotoxicity of NK cells. However, LFA1 polarization, cell spreading of NK cells, and their ability to home and traffic were significantly reduced in the absence of Rap1b. Lack of Rap1b, but not Rap1a, resulted in severe impairment of NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine generation. Upon activation, Rap1b associated with B-Raf or C-Raf and colocalized with IQGAP1 complex. The absence of Rap1b reduced B-Raf, C-Raf, and ERK1/2 phosphorylation in NK cells. Rap1b helped IQGAP1 to form a large signalosome in the perinuclear region to coordinate the phosphorylation of ERK1/2. Rap1b also colocalized with the MTOC and regulated its size and proper formation. These results reveal a previously unrecognized role of Rap1 in signalosome formation and regulation of effector functions in lymphocytes.     

A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5⁺CD19⁺ B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins (Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific for β-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. β-(1,6)-glucan binding depended on both the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for β-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to β-(1,6)-glucan. This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell lymphomas.B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults in the western world (Jemal et al., 2009), is a clonal expansion of mature CD5⁺CD19⁺ B lymphocytes. Two types of CLL are being distinguished as carrying either unmutated (U-CLL) or somatically mutated Igs (M-CLL), which are associated with unfavorable and favorable prognoses, respectively (Damle et al., 1999; Hamblin et al., 1999). Despite this difference in clinical behavior, U-CLL and M-CLL share a highly similar gene expression profile (Klein et al., 2001).Many studies allude to a role for BCR-derived signals in the pathogenesis of B cell non-Hodgkin’s lymphomas (Küppers, 2005). These signals are either antigen-independent, such as in diffuse large B cell lymphomas harboring activating mutations in CD79a and CD79b (Davis et al., 2010), or antigen-dependent, as proposed for CLL (Chiorazzi and Ferrarini, 2003; Packham and Stevenson, 2010). The Ig heavy chain variable (IGHV) gene repertoire in CLL is biased to frequent usage of IGHV1-69, IGHV3-7, and IGHV4-34 (Fais et al., 1998), and over 30% of CLLs can be grouped based on similarities of the amino acid sequences in the highly variable complementary determining region 3 (CDR3; Ghiotto et al., 2004; Messmer et al., 2004; Widhopf et al., 2004; Bende et al., 2005; Stamatopoulos et al., 2007; Murray et al., 2008). These stereotypic IGHV display biased patterns of somatic hypermutations (Murray et al., 2008) and are often paired with distinct Ig light chains (Stamatopoulos et al., 2005; Widhopf et al., 2008; Hadzidimitriou et al., 2009). Collectively, these observations suggest that distinctive antigens are involved in the development of CLL.The majority of U-CLLs express low-affinity BCRs that are polyreactive, recognizing self- and exo-antigens, such as DNA, LPS, insulin, apoptotic cells, oxidized LDL, and the cytoskeletal antigens myosin and vimentin (Hervé et al., 2005; Catera et al., 2008; Chu et al., 2008; Lanemo Myhrinder et al., 2008; Binder et al., 2010). In contrast, M-CLL BCRs are generally not polyreactive (Hervé et al., 2005; Catera et al., 2008; Lanemo Myhrinder et al., 2008). Recently, two stereotypic subsets of M-CLL with specificity for the Fc-tail of IgG, so-called rheumatoid factors, were identified by us and by others (Hoogeboom et al., 2012; Kostareli et al., 2012); this specificity is commonly found among mucosa-associated lymphoid tissue (MALT)-lymphomas, splenic marginal zone lymphomas, and hepatitis C virus–associated lymphomas (De Re et al., 2000; Bende et al., 2005; Hoogeboom et al., 2010; Kostareli et al., 2012). In general, the specificity of M-CLLs with stereotypic BCRs remained unknown. It has been hypothesized that chronic antigenic stimulation drives CLL development (Chiorazzi and Ferrarini, 2003; Chiorazzi et al., 2005), as was also proposed for MALT lymphomas. For MALT lymphomas, this hypothesis is supported by the observation that Helicobacter pylori–associated MALT lymphomas of the stomach can be eradicated by antibiotic treatment alone (Sugiyama et al., 2001; Liu et al., 2002). Nevertheless, it was demonstrated that gastric MALT lymphoma cells themselves were not specific for H. pylori (Hussell et al., 1996). To our knowledge, expression of BCRs with high-affinity for pathogen-derived antigens has not been reported for any lymphoma entity. In this study, we provide evidence that a subset of somatically mutated lymphomas is selected for an antigenic determinant of a major class of pathogens and that these cognate ligands can drive tumor expansion.     

Shp1 regulates T cell homeostasis by limiting IL-4 signals

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell–intrinsic role of Shp1, we characterized mice with a T cell–specific Shp1 deletion (Shp1^fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1^fl/fl CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1-deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44^hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.T cells are characterized by their ability to expand dramatically in an antigen-specific manner during an immune challenge. After an initial immune response, a small proportion of responding T cells survive and give rise to memory cells (Bruno et al., 1996). Memory T cells express elevated levels of CD44 and can be divided further into central-memory (CD62L^hi CCR7^hi) and effector-memory (CD62L^lo CCR7^lo) compartments. However, not all T cells that display the phenotype of memory cells are the product of a classical antigen-specific immune response (Sprent and Surh, 2011). For example, such cells are found in unimmunized mice, including those raised in germ-free and antigen-free conditions (Dobber et al., 1992; Vos et al., 1992). The precise ontogeny of such cells remains elusive, although several mechanisms by which naive cells can adopt a memory phenotype have been characterized. Naive T cells introduced into lymphopenic environments adopt a memory phenotype through a process of homeostatic proliferation in response to IL-7 and MHC (Goldrath et al., 2000; Murali-Krishna and Ahmed, 2000). Additionally, increased production of IL-4 has been linked to the development of memory phenotype–innate T cell populations in studies of several knockout mouse models (Lee et al., 2011).The T cell response is tightly regulated by the balance of phosphorylation and dephosphorylation of intracellular signaling molecules. Shp1 (encoded by Ptpn6) is a protein tyrosine phosphatase expressed ubiquitously in hematopoietic cells and has been broadly characterized as a negative regulator of immune cell activation (Pao et al., 2007a; Lorenz, 2009). The physiological relevance of Shp1 as a key negative regulator of the immune response is illustrated by the motheaten (me) and motheaten viable (me^v) mutations, which ablate Shp1 expression or greatly reduce Shp1 activity, respectively (Shultz et al., 1993; Tsui et al., 1993). Homozygous me/me or me^v/me^v mice (hereafter, referred to collectively as me mice) suffer from severe systemic inflammation and autoimmunity, which result in retarded growth, myeloid hyperplasia, hypergammaglobulinemia, skin lesions, interstitial pneumonia, and premature death. More recently, a study has identified a third allele of Ptpn6, named spin, which encodes a hypomorphic form of Shp1 (Croker et al., 2008). Mice homozygous for spin develop a milder autoimmune/inflammatory disease that is ablated in germ-free conditions.Shp1 has been implicated in signaling from many immune cell surface receptors (Zhang et al., 2000; Neel et al., 2003), including the TCR (Plas et al., 1996; Lorenz, 2009), BCR (Cyster and Goodnow, 1995; Pani et al., 1995), NK cell receptors (Burshtyn et al., 1996; Nakamura et al., 1997), chemokine receptors (Kim et al., 1999), FAS (Su et al., 1995; Takayama et al., 1996; Koncz et al., 2008), and integrins (Roach et al., 1998; Burshtyn et al., 2000). Shp1 also has been demonstrated to regulate signaling from multiple cytokine receptors by dephosphorylating various Jak (Klingmüller et al., 1995; Jiao et al., 1996; Minoo et al., 2004) and/or Stat (Kashiwada et al., 2001; Xiao et al., 2009) molecules. Several of these cytokines are pertinent to T cell biology. For example, Stat 5 is an essential mediator of signals from IL-2 and IL-7 (Rochman et al., 2009). IL-4 signaling results in Stat 6 phosphorylation and has potent Th2 skewing effects. Additionally, IL-4 has mitogenic effects on CD8⁺ T cells (Rochman et al., 2009). Notably, mutation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in IL-4Rα results in ablation of Shp1 binding and hypersensitivity to IL-4 stimulation (Kashiwada et al., 2001), implicating Shp1 as a regulator of this cytokine receptor.Although development of the me phenotype does not require T cells (Shultz, 1988; Yu et al., 1996), several aspects of T cell biology reportedly are controlled by Shp1 (Lorenz, 2009). Most previous studies that examined the role of Shp1 in T cells used cells derived from me/me or me^v/me^v mice (Carter et al., 1999; Johnson et al., 1999; Zhang et al., 1999; Su et al., 2001) or cells expressing a dominant-negative allele of Shp1 (Plas et al., 1996, 1999; Zhang et al., 1999). Several such reports have concluded that Shp1 negatively regulates the strength of TCR signaling during thymocyte development and/or peripheral activation (Carter et al., 1999; Johnson et al., 1999; Plas et al., 1999; Zhang et al., 1999; Su et al., 2001). Despite the large number of studies that implicate Shp1 in control of TCR signaling, there is no consensus on which component of the TCR signaling cascade is targeted by the catalytic activity of Shp1. Suggested Shp1 targets downstream of T cell activation include TCR-ζ (Chen et al., 2008), Lck (Lorenz et al., 1996; Stefanová et al., 2003), Fyn (Lorenz et al., 1996), ZAP-70 (Plas et al., 1996; Chen et al., 2008), and SLP-76 (Mizuno et al., 2005). Shp1 also is implicated in signal transduction downstream of several immune inhibitory receptors that negatively regulate T cell activity, such as PD-1 (Chemnitz et al., 2004), IL-10R (Taylor et al., 2007), CEACAM1 (Lee et al., 2008), and CD5 (Perez-Villar et al., 1999).The severe inflammation characteristic of the me phenotype might have confounded studies examining the cell-intrinsic role of Shp1 in various hematopoietic cell types. We previously generated a floxed Shp1 allele that facilitates analysis of the role of Shp1 in various lineages (Pao et al., 2007b). Previous studies have used this approach to study the role of Shp1 in T cells during antiviral and antitumor immune responses, respectively (Fowler et al., 2010; Stromnes et al., 2012). However, a more fundamental analysis of the cell-intrinsic role of Shp1 during T cell development, homeostasis, and activation has not been reported. Here, we provide evidence that a major role for Shp1 in T cells is to maintain normal T cell homeostasis through negative regulation of IL-4 signaling.     

Innate immunity defines the capacity of antiviral T cells to limit persistent infection

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4⁺ and CD8⁺ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.The development of effective therapies to prevent and treat persistent infections is of the highest priority, as they cause considerable clinical challenges and ongoing health care costs. Efforts to improve the treatment and prevention of chronic viral infections, such as those elicited by HIV, hepatitis C virus (HCV), and human cytomegalovirus (HCMV), require a better understanding of the immune responses needed to achieve optimal control of persistent viruses in the long term. Although innate and adaptive immune responses have historically been thought to be nonoverlapping, recent evidence clearly indicates that interplays between components of the immune system occur frequently and form the basis of effective immunity. Infection with murine cytomegalovirus (MCMV) is a well-established experimental system to study host–pathogen interactions and to dissect the relevance of different arms of the immune response. Because of the similarities in structure and biology between HCMV and MCMV, and because MCMV is a natural mouse pathogen, MCMV infection provides a unique model to study a medically important virus in vivo after infection of its biological host. Like HCMV, MCMV causes persistent infection, and thus the model can be exploited to gain a better understanding of the requirements for effective prevention and treatment of chronic viral infections.Early control of MCMV infection is dependent on NK cell responses and is associated with a single dominant locus that encodes the NK cell–activating receptor Ly49H (Brown et al., 2001; Daniels et al., 2001; Lee et al., 2001). In mouse strains like C57BL/6J, NK cells express Ly49H and efficiently limit early viral replication (Bancroft et al., 1981; Shellam et al., 1981; Bukowski et al., 1984; Scalzo et al., 1990). Ly49H binds to the MCMV-encoded MHC class I like glycoprotein m157 to deliver activating signals to NK cells (Arase et al., 2002; Smith et al., 2002). In contrast to C57BL/6J (Ly49H⁺) mice, BALB/c mice, whose NK cells lack the Ly49H-activating receptor, show increased susceptibility to MCMV during the early phase of infection (Allan and Shellam, 1984). In BALB/c (Ly49H⁻) mice, activation of NK cells is limited further by viral interference with the expression of ligands for the NKG2D NK cell–activating receptor (Lodoen et al., 2003; Lodoen et al., 2004; Hasan et al., 2005; Krmpotic et al., 2005), and early control of MCMV infection requires a CD8 T cell–mediated response. The antiviral CD8⁺ T cell response commences within 4 d after infection and is directed predominantly against the nonameric peptide YPHFMPTNL (Del Val et al., 1991), which is derived from the virus immediate early 1 (IE1) nonstructural protein (Volkmer et al., 1987). The IE1 epitope, specifically recognized by BALB/c CD8⁺ T cells in an H-2L^d–restricted manner, represents the best-studied MCMV antigenic determinant. Despite the large body of evidence demonstrating a role for CD8⁺ T cells in limiting CMV, virus-specific CD4⁺ T cells are also important. Evidence from both HCMV and MCMV studies suggests that CD4⁺ T cell responses are a critical component of immunity to these viruses. In humans, virus-specific CD4⁺ T cells are required to control HCMV-induced disease (Hsieh et al., 2001; Gamadia et al., 2003). In MCMV infection, CD4⁺ T cells participate in limiting viral replication in salivary glands (Jonjic et al., 1989).Our recent study demonstrated that interplays between DCs and NK cells during the very early phase of infection are important to achieve maximal control of the virus (Andoniou et al., 2005). Functional interrelationships between DCs and Ly49H⁺ NK cells occur during the late stage of acute MCMV infection in vivo (Andrews et al., 2003). Although Ly49H⁺ NK cells are undoubtedly important for the early control of viral infection in visceral organs (Brown et al., 2001; Daniels et al., 2001; Lee et al., 2001), it has been postulated that they may also regulate ensuing adaptive antiviral immune responses (Dokun et al., 2001; Su et al., 2001; Andrews et al., 2003). Recent studies have shown that NK cells promote early activation of MCMV-specific CD8⁺ T cells through the ability to regulate the production of IFN-αβ (Robbins et al., 2007). These effects however are transient, and it is unclear whether they impact on controlling virus replication. Here, we exploited the differences in NK cell activation that occur in Ly49H⁻ (e.g., BALB/c) versus Ly49H⁺ (e.g., C57BL/6) mice during MCMV infection to investigate the impact of differences in NK cell responses on the generation, maintenance, and in vivo effectiveness of antiviral CD4⁺ and CD8⁺ T cell responses.     

Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

The neutralizing activity of anti–HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4⁺ lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies.HIV-1–infected individuals produce high titers of antibodies against the virus, but only a small fraction of the patients develop a broadly neutralizing serologic activity, generally after 2–4 yr of infection (Sather et al., 2009; Simek et al., 2009; Stamatatos et al., 2009; Walker et al., 2011; McCoy and Weiss, 2013). The serologic anti–HIV-1 activity in some of these individuals can be accounted for by a combination of antibodies targeting different sites on the HIV-1 envelope spike (Scheid et al., 2009; Bonsignori et al., 2012; Klein et al., 2012a; Georgiev et al., 2013) and in others, by a predominant highly expanded clone (Scheid et al., 2011; Walker et al., 2011; Burton et al., 2012; McCoy and Weiss, 2013). Although the presence of broad neutralizing activity does not correlate with a better clinical outcome, passive transfer of broadly neutralizing antibodies (bNAbs) can protect against infection in macaques or in mouse models (Hessell et al., 2009; Pietzsch et al., 2012; McCoy and Weiss, 2013). In addition, bNAbs can suppress viremia in humanized mice (Klein et al., 2012b). Moreover, antibodies against the HIV-1 envelope spike appear to be the unique correlate of protection in the RV144 HIV-1 vaccine trial (Haynes et al., 2012). Therefore, it has been proposed that vaccines that would elicit such antibodies may be protective against the infection in humans.The recent development of efficient methods for cloning of human anti–HIV-1 antibodies from single cells (Scheid et al., 2009) led to the discovery of dozens of new bNAbs and new targets for neutralization (Burton et al., 2012; McCoy and Weiss, 2013). The new antibodies target at least six different sites of vulnerability on the HIV-1 spike. These include the CD4-binding site (VRC01, NIH45-46, 3BNC60/117, and CH103), the glycan-dependent V1/V2 loops (PG16 and PGT145) and V3 loop (PGT121, PGT128, and the 10-1074 family), a conformational epitope on gp120 (3BC176), a domain in the vicinity of the CD4bs (8ANC195), and the gp41 membrane-proximal external region (MPER; 2F5, 4E10, and 10E8; Scheid et al., 2009, 2011; Walker et al., 2011; Wu et al., 2011; Kwong and Mascola, 2012; Mouquet et al., 2012; West et al., 2012; Liao et al., 2013). Some of these antibodies display remarkable antiviral activity with median 50% inhibitory concentrations (IC50s) < 0.2 µg/ml for up to 95% of isolates tested (Diskin et al., 2011; Scheid et al., 2011; Walker et al., 2011; Wu et al., 2011; Burton et al., 2012; Liao et al., 2013).The antiviral activity of bNAbs is typically measured in vitro using cell-free pseudovirus particles and reporter cell lines, such as the HeLa-derived TzMbl cell (Heyndrickx et al., 2012). In these assays, neutralization is mediated by inhibition of free virus binding to cellular receptors and/or by inhibition of viral fusion. Although cell-free HIV-1 is infectious, the virus replicates more efficiently and rapidly through direct contact between cells, and this mode of transmission likely mediates a significant fraction of viral spread and immune evasion in vivo (Dimitrov et al., 1993; Sourisseau et al., 2007; Sattentau, 2011; Murooka et al., 2012; Dale et al., 2013). In addition, this form of dissemination appears to be less susceptible to inhibition by antiretroviral drugs than cell-free virus transmission (Chen et al., 2007; Sigal et al., 2011; Abela et al., 2012).Cell to cell spread of HIV-1 is in large part mediated through virological synapses, where viral particles accumulate at the interface between infected cells and targets (Sattentau, 2011; Dale et al., 2013). Synapse formation involves HIV-1 Env-CD4 coreceptor interactions and requires cytoskeletal rearrangements and adhesion molecules (Sattentau, 2011; Dale et al., 2013).Here, we examined the antiviral activity of a panel of 15 newly identified bNAbs targeting all known sites of vulnerability in conventional neutralization and cell to cell transmission assays. We show that only a subset of the bNAbs that target the CD4-binding site or the glycan/V3 loop efficiently neutralize cell to cell viral transfer in co-cultures of infected T cells with primary lymphocytes. We further characterized the antiviral mechanisms used by the effective antibodies and report that they affect multiple steps of viral cell to cell transfer.     

p53-dependent chemokine production by senescent tumor cells supports NKG2D-dependent tumor elimination by natural killer cells

The induction of cellular senescence is an important mechanism by which p53 suppresses tumorigenesis. Using a mouse model of liver carcinoma, where cellular senescence is triggered in vivo by inducible p53 expression, we demonstrated that NK cells participate in the elimination of senescent tumors. The elimination of senescent tumor cells is dependent on NKG2D. Interestingly, p53 restoration neither increases ligand expression nor increases the sensitivity to lysis by NK cells. Instead, p53 restoration caused tumor cells to secrete various chemokines with the potential to recruit NK cells. Antibody-mediated neutralization of CCL2, but not CCL3, CCL4 or CCL5, prevented NK cell recruitment to the senescent tumors and reduced their elimination. Our findings suggest that elimination of senescent tumors by NK cells occurs as a result of the cooperation of signals associated with p53 expression or senescence, which regulate NK cell recruitment, and other signals that induce NKG2D ligand expression on tumor cells.Cellular senescence is an established cellular stress response, primarily acting to limit the proliferative potential of cells (Collado and Serrano, 2010). It can be triggered in many cell types in response to diverse cellular damage (Collado and Serrano, 2010). An important trigger of senescence is oncogenic stress, mediated by activation of p53/p21 and p16/Rb tumor suppressor pathways, which promote senescence by transactivating genes that arrest cell cycle progression and promote the senescent state (Serrano et al., 1997; Narita et al., 2003; Braig et al., 2005; Michaloglou et al., 2005; Ventura et al., 2007). It is believed that senescence is a key mechanism by which p53 suppresses tumorigenesis (Braig and Schmitt, 2006; Collado and Serrano, 2010). The senescent state is associated with several phenotypic alterations, including the secretion of soluble factors involved in the maintenance of the senescent state (e.g., CXCL2 [Acosta et al., 2008], PAI-1 [plasminogen activator inhibitor-1; Kortlever et al., 2006], IGFBP7 [insulin-like growth factor-binding protein 7; Wajapeyee et al., 2008]), and other molecules that regulate the immune response (cytokines and chemokines; Kuilman et al., 2008; Rodier et al., 2009, 2011), angiogenesis (vascular endothelial growth factor), and other processes (Coppé et al., 2006). This so-called senescence-associated secretory phenotype (SASP), as well as the resulting immune responses, could promote or repress cancer progression in a context-dependent manner (Rodier and Campisi, 2011). With respect to immune responses, the senescent state has similarly been associated with alterations that promote tumorigenesis (Krtolica et al., 2001; Bavik et al., 2006; Yang et al., 2006; Liu and Hornsby, 2007) but in other cases with immune-mediated tumor elimination (Xue et al., 2007; Krizhanovsky et al., 2008; Kang et al., 2011).Accumulating evidence suggests that immune-mediated destruction of senescent cells may play a role in tumor surveillance as well as in resolution of fibrotic injury to tissues (Xue et al., 2007; Krizhanovsky et al., 2008; Kang et al., 2011; Lujambio et al., 2013). In some cases, immune cells such as NK cells and other immune effector cells like granulocytes and macrophages have been implicated in mediating these effects (Xue et al., 2007; Krizhanovsky et al., 2008; Lujambio et al., 2013).NK cells are lymphocytes that kill tumor cells and infected cells and secrete various inflammatory cytokines, including IFN-γ and TNF (Vivier et al., 2011). Like other lymphocytes and immune cells, NK cells are recruited to infected or transformed tissue by the action of chemokine gradients (Grégoire et al., 2007). NK cell killing requires engagement of specific ligands on tumor cells by NK receptors. Some NK receptors, specific for MHC I molecules, inhibit NK activity, whereas other receptors activate NK functions (Vivier et al., 2011). Several activating NK receptors have been implicated in the killing of tumor cells. The best characterized such receptor is NKG2D (encoded by the Klrk1 gene), which is expressed by all NK cells. NKG2D binds to each of 5–10 (depending on the individual) different MHC I–related cell surface ligands, including the RAE-1/MULT1/H60 subfamilies of proteins in mice and the MICA/ULBP subfamilies of proteins in humans (Raulet, 2003). The ligands are expressed poorly by normal cells but are often induced on cancer cells as the result of stress pathways or other pathways that are dysregulated in cancer cells (Raulet et al., 2013). NKG2D has been implicated in immune surveillance of tumors using transgenic models of spontaneous cancer as well as subcutaneous tumor transfer models (Cerwenka et al., 2001; Diefenbach et al., 2001; Guerra et al., 2008).A recent paper suggested that senescent tumors are targeted for elimination by NK cells and other innate effector cells (Xue et al., 2007). However, it is unknown how p53-expressing senescent tumors mobilize the natural killer cell response. Nor is it known how NK cells recognize the senescent tumors. In this study, we sought to define how NK cells carry out this function by defining the receptors and ligands involved and the alterations in senescent cells that mobilize the NK cell response. Our results demonstrate that induced expression of p53 in a model of transferred liver tumor cells causes the production of various chemokines, including CCL2, and that CCL2 is essential for robust recruitment of NK cells into the tumor. The NK-dependent component of tumor cell elimination is completely dependent on NKG2D-mediated recognition of RAE-1 proteins expressed on the tumor cells, but RAE-1 expression is not induced by p53 expression because it is robust on the tumor cells even before p53 expression is induced. The results suggest that other types of signals associated with the transformed state induce expression of NKG2D ligands, but p53 expression mobilizes effective NK-dependent tumor elimination by inducing CCL2 expression that recruits NK cells into the tumor.     

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4⁺ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.Within 8 d after peroral infection with Toxoplasma gondii, susceptible C57BL/6 mice develop massive necrosis in the ileum, leading to death (Liesenfeld et al., 1996). T. gondii–induced ileitis is characterized by a CD4 T cell–dependent overproduction of proinflammatory mediators, including IFN-γ, TNF, and NO (Khan et al., 1997; Mennechet et al., 2002). Activation of CD4⁺ T cells by IL-12 and IL-18 is critical for the development of small intestinal pathology (Vossenkämper et al., 2004). Recently, we showed that LPS derived from gut flora via Toll-like receptor (TLR)–4 mediates T. gondii–induced immunopathology (Heimesaat et al., 2006). Thus, the immunopathogenesis of T. gondii–induced small intestinal pathology resembles key features of the inflammatory responses in inflammatory bowel disease (IBD) in humans and in models of experimental colitis in rodents (Liesenfeld, 2002). However, most animal models of IBD assessed inflammatory responses in the large intestine, and models of small intestinal pathology are scarce (Kosiewicz et al., 2001; Strober et al., 2002; Olson et al., 2004; Heimesaat et al., 2006).IL-12 shares the p40 subunit, IL-12Rβ1, and components of the signaling transduction pathways with IL-23 (Parham et al., 2002). There is strong evidence that IL-23, rather than IL-12, is important in the development of colitis (Yen et al., 2006). The association of IL-23R encoding variant Arg381Gln with IBD (Duerr et al., 2006) and the up-regulation of IL-23p19 in colon biopsies from patients with Crohn''s disease (Schmidt et al., 2005) underline the importance of IL-23 in intestinal inflammation. Effector mechanisms of IL-23 include the up-regulation of matrixmetalloproteinases (MMPs; Langowski et al., 2006), a large family of endopeptidases that mediate homeostasis of the extracellular matrix. MMPs were significantly up-regulated in experimental models of colitis (Tarlton et al., 2000; Medina et al., 2003) and in colonic tissues of IBD patients (von Lampe et al., 2000).Studies in mouse models of autoimmune diseases have associated the pathogenic role of IL-23 with the accumulation of CD4⁺ T cells secreting IL-17, termed Th17 cells (Aggarwal et al., 2003; Cua et al., 2003). Moreover, increased IL-17 expression was reported in the intestinal mucosa of patients with IBD (Fujino et al., 2003; Nielsen et al., 2003; Kleinschek et al., 2009).In addition to IL-17, Th17 cells also produce IL-22, a member of the IL-10 family (Dumoutier et al., 2000). IL-22, although secreted by certain immune cell populations, does not have any effects on immune cells in vitro or in vivo but regulates functions of some tissue cells (Wolk et al., 2009). Interestingly, IL-22 has been proposed to possess both protective as well as pathogenic roles. In fact, IL-22 mediated psoriasis-like skin alterations (Zheng et al., 2007; Ma et al., 2008; Wolk et al., 2009). In contrast, IL-22 played a protective role in experimental models of colitis (Satoh-Takayama et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008; Zheng et al., 2008), in a model of Klebsiella pneumoniae infection in the lung (Aujla et al., 2007), and against liver damage caused by concanavalin A administration (Radaeva et al., 2004; Zenewicz et al., 2007). IL-22 has been reported to be produced by CD4⁺ T cells (Wolk et al., 2002; Zheng et al., 2007), γδ cells (Zheng et al., 2007), CD11c⁺ cells (Zheng et al., 2008), and natural killer cells (Cella et al., 2008; Luci et al., 2008; Sanos et al., 2009; Satoh-Takayama et al., 2008; Zheng et al., 2008). The role of IL-22 in small intestinal inflammation remains to be determined.In the present study, we investigated the role of the IL-23–IL-17 axis in T. gondii–induced small intestinal immunopathology. We show that IL-23 is essential in the development of small intestinal immunopathology by inducing local MMP-2 up-regulation that could be inhibited by prophylactic or therapeutic chemical blockage. Interestingly, IL-23–dependent IL-22 production was markedly up-regulated and essential for the development of ileal inflammation, whereas IL-17 production was down-regulated after T. gondii infection. IL-22 was mostly produced by CD4⁺ T cells in the small intestinal lamina propria.     

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.