IL-12 and type I interferon prolong the division of activated CD8 T cells by maintaining high-affinity IL-2 signaling in vivo

TCR ligation and co-stimulation induce cellular division; however, optimal accumulation of effector CD8 T cells requires direct inflammatory signaling by signal 3 cytokines, such as IL-12 or type I IFNs. Although in vitro studies suggest that IL-12/type I IFN may enhance T cell survival or early proliferation, the mechanisms underlying optimal accumulation of CD8 T cells in vivo are unknown. In particular, it is unclear if disparate signal 3 cytokines optimize effector CD8 T cell accumulation by the same mechanism and how these inflammatory cytokines, which are transiently produced early after infection, affect T cell accumulation many days later at the peak of the immune response. Here, we show that transient exposure of CD8 T cells to IL-12 or type I IFN does not promote survival or confer an early proliferative advantage in vivo, but rather sustains surface expression of CD25, the high-affinity IL-2 receptor. This prolongs division of CD8 T cells in response to basal IL-2, through activation of the PI3K pathway and expression of FoxM1, a positive regulator of cell cycle progression genes. Thus, signal 3 cytokines use a common pathway to optimize effector CD8 T cell accumulation through a temporally orchestrated sequence of cytokine signals that sustain division rather than survival.The magnitude of the effector CD8 T cell response is critical for eliminating intracellular pathogens and for regulating the size of the memory pool after resolution of infection or vaccination (Hou et al., 1994; Badovinac and Harty, 2006; Schmidt et al., 2008). TCR stimulation by mature DCs expressing cognate antigen in the context of MHC I initiates activation of naive, pathogen-specific CD8 T cells. Additional signals from co-stimulatory molecules amplify the magnitude and/or duration of the TCR signaling, thereby enhancing activation and functionality (Cronin and Penninger, 2007). Although these two signals are sufficient to induce the division of naive CD8 T cells, pathogen-, or adjuvant-induced inflammatory cytokines act as third signals to promote optimal accumulation of effector CD8 T cells (Curtsinger and Mescher, 2010). Because the clearance of intracellular pathogens is often dependent on the total number of responding effector CD8 T cells (Badovinac and Harty, 2006; Hikono et al., 2006; Lefrançois, 2006), it is important to understand how the magnitude of CD8 T cell responses are regulated to effectively control pathogen burden.Using short-term (∼3 d) in vitro experiments, an early study by Curtsinger et al. (1999) clearly established that addition of a specific inflammatory cytokine (IL-12) during T cell activation in response to artificial APCs expressing signal 1 and signal 2 and with exogenous addition of IL-2 increased the accumulation of responding CD8 T cells. The importance of signal 3 cytokines for the optimal accumulation of effector CD8 T cells has also been established in vivo (Gately et al., 1992; Trinchieri, 1998). For example, direct IL-12 signaling is essential for optimal accumulation of antigen-specific CD8 T cells after Listeria monocytogenes (LM) infection (Keppler et al., 2009; Xiao et al., 2009; Keppler et al., 2012). Direct IFN-α/β receptor signaling has also been shown to be critical for the optimal accumulation of CD8 T cells in some in vitro studies (Curtsinger et al., 2005) and for P14 TCR-transgenic CD8 T cells responding to lymphocytic choriomeningitis virus (LCMV) infection (Aichele et al., 2006; Kolumam et al., 2005). Together, these studies highlighted the impact of IL-12 and IFN α/β on the accumulation of activated CD8 T cells. However, a mechanistic understanding of how inflammatory cytokines such as IL-12 and IFN α/β regulate accumulation of effector CD8 T cells in vivo has yet to be determined.Results from short-term in vitro studies provide two models to explain how the signal 3 cytokine IL-12 promotes the optimal accumulation of activated CD8 T cells. The first model suggests that IL-12 stimulation during activation promotes increased accumulation by conferring a survival advantage to responding CD8 T cells (Mitchell et al., 2001; Valenzuela et al., 2005; Curtsinger and Mescher, 2010). This conclusion was drawn from experiments where IL-12 enhanced accumulation of CD8 T cells over the 3-d culture period, without detectable impact on cellular division. However, validated survival pathways regulated by signal 3 cytokines in vivo have not been identified to date. Alternatively, other data suggest that IL-12 increases the accumulation of activated CD8 T cells by transiently increasing the expression of the high-affinity IL-2 receptor subunit (IL-2Rα; CD25; Pipkin et al., 2010; Valenzuela et al., 2002) and IL-2Rβ (CD122; Valenzuela et al., 2002), providing an early proliferative advantage leading to increased accumulation in short-term in vitro studies (Valenzuela et al., 2002; Curtsinger and Mescher, 2010; Pipkin et al., 2010). Consistent with this notion, the absence of CD25 prevented optimal accumulation of CD8 T cells after LM infection (Obar et al., 2010) or LCMV infection (Williams et al., 2006). However, the IL-12–stimulated increase in CD25 expression in vitro was transient, peaking 2 d after cognate-antigen stimulation (Valenzuela et al., 2002). Thus, mechanistic assessment of signal 3 activities to date are limited to short-term in vitro studies focused on IL-12 and the mechanisms by which IL-12 or other signal 3 cytokines (e.g., type I IFN) regulate CD8 T cell accumulation in vivo are not established. For example, it remains unknown if signal 3 cytokines function by common or distinct mechanisms, if these mechanisms regulate survival pathways in vivo or confer an early proliferative advantage, or if both mechanisms account for signal 3-dependent optimal accumulation of effector CD8 T cells in vivo.In addition, the temporal disconnection between early and transient production of signal 3 cytokines (Pham et al., 2009; Keppler et al., 2012) and optimal accumulation of effector CD8 T cells at the peak of the response, many days later, has not been addressed (Harty and Badovinac, 2008). For example, most in vivo experiments used gene KO mouse strains or TCR-transgenic T cells (Kolumam et al., 2005; Aichele et al., 2006; Keppler et al., 2009; Pham et al., 2011; Keppler et al., 2012) that constitutively lack the receptors for inflammatory cytokines, and most in vitro studies were analyzed within a short window (∼3 d) after CD8 T cell activation (Curtsinger et al., 1999, 2003a, Curtsinger et al., a2005; Valenzuela et al., 2002, 2005). These are important considerations given that acute infections (as well as adjuvant-coupled immunizations) induce transient elevations of inflammatory cytokines, often peaking within hours of stimulation, and then returning to baseline within 1–2 d (Pham et al., 2009; Keppler et al., 2012), whereas corresponding CD8 T cell responses generally peak in numbers between day 7 and 9 after immunization/infection (Harty and Badovinac, 2008). Here, we address this temporal conundrum and-dissect the mechanisms by which signal 3 cytokines IL-12 and type I IFN guide the optimal accumulation of CD8 T cells in response to in vivo activation. To address these issues, we use an immunization model with mature, peptide-loaded DCs, wherein antigen concentrations are fixed but the inflammatory milieu can be manipulated by co-administration of Toll-like receptor ligands (Badovinac et al., 2005; Boyman et al., 2006; Pham et al., 2009). Using this model, we uncover a molecular pathway whereby signal 3 cytokines (both IL-12 and IFN-α/β) promote optimal CD8 T cell accumulation after in vivo activation, not by improving survival or early proliferation, but rather by a common mechanism regulating cytokine signaling pathways that maintain cellular division at late time points.     

Small intestinal eosinophils regulate Th17 cells by producing IL-1 receptor antagonist

Eosinophils play proinflammatory roles in helminth infections and allergic diseases. Under steady-state conditions, eosinophils are abundantly found in the small intestinal lamina propria, but their physiological function is largely unexplored. In this study, we found that small intestinal eosinophils down-regulate Th17 cells. Th17 cells in the small intestine were markedly increased in the ΔdblGATA-1 mice lacking eosinophils, and an inverse correlation was observed between the number of eosinophils and that of Th17 cells in the small intestine of wild-type mice. In addition, small intestinal eosinophils suppressed the in vitro differentiation of Th17 cells, as well as IL-17 production by small intestinal CD4⁺ T cells. Unlike other small intestinal immune cells or circulating eosinophils, we found that small intestinal eosinophils have a unique ability to constitutively secrete high levels of IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1β. Moreover, small intestinal eosinophils isolated from IL-1Ra−deficient mice failed to suppress Th17 cells. Collectively, our results demonstrate that small intestinal eosinophils play a pivotal role in the maintenance of intestinal homeostasis by regulating Th17 cells via production of IL-1Ra.The small intestinal lamina propria (LP) contains a variety of immune cells. These include Th17 cells, a subset of activated CD4⁺ T cells characterized by the production of IL-17A, IL-17F, IL-21, and IL-22 (Korn et al., 2009). Th17 cells have the potential to protect or damage the intestinal tissue environment, so their activity must be tightly regulated (O’Connor et al., 2009; Morrison et al., 2011). Several cytokines are known to promote the development of Th17 cells; IL-6 and TGF-β are required for the differentiation of Th17 cells from naive CD4⁺ T cells, and IL-1β and IL-23 are critical for the maintenance of Th17 cells, as well as their differentiation (Zhou et al., 2007; Chung et al., 2009). Commensal bacteria contribute to the generation of small intestinal Th17 cells in the steady state (Atarashi et al., 2008, 2015; Ivanov et al., 2009). In particular, commensal-induced IL-1β production by intestinal macrophages is required for the development of Th17 cells (Shaw et al., 2012).IL-1β is a proinflammatory cytokine primarily produced by activated macrophages and acts as a key mediator in various inflammatory diseases, including inflammatory bowel disease and rheumatoid arthritis (Sims and Smith, 2010). Consequently, mice deficient for IL-1 receptor antagonist (IL-1Ra), which competes with IL-1β for receptor binding, spontaneously develop arthritis with a marked increase in Th17 cells (Nakae et al., 2003; Koenders et al., 2008). In humans, a decrease in the IL-1Ra to IL-1 ratio has been linked to inflammatory bowel disease (Casini-Raggi et al., 1995). IL-1Ra secreted by intestinal epithelial cells upon TLR5 activation reduces tissue damage (Carvalho et al., 2011), and treatment with recombinant IL-1Ra ameliorates intestinal graft-versus-host disease by inhibiting Th17 responses (Jankovic et al., 2013). Thus, the balance between IL-1β and IL-1Ra is critical for controlling Th17 cells and maintaining intestinal immune homeostasis.Eosinophils are commonly known as proinflammatory cells, mediating the host responses against helminth infections, as well as the pathogenesis of various allergic diseases and gastrointestinal disorders (Rothenberg and Hogan, 2006). However, recent studies found that eosinophils also play various roles in maintaining homeostasis, such as supporting glucose homeostasis by sustaining alternatively activated macrophages in adipose tissue (Wu et al., 2011) and promoting the generation and survival of plasma cells (Chu et al., 2014b; Jung et al., 2015). Under steady-state conditions, eosinophils develop in the bone marrow and migrate primarily to the gastrointestinal tract (Mishra et al., 1999; Rothenberg and Hogan, 2006). Small intestinal eosinophils have unique phenotypes and extended life spans (Carlens et al., 2009; Verjan Garcia et al., 2011). However, their function under healthy homeostatic conditions remains to be fully elucidated.In this study, we show that small intestinal eosinophils down-regulate Th17 cells by constitutively secreting a large amount of IL-1Ra. We found a decrease in serum IL-1Ra and a concomitant increase in small intestinal Th17 cells in ΔdblGATA-1 mice, which lack eosinophil-lineage cells (Yu et al., 2002). In WT mice, the number of Th17 cells in the small intestine was inversely correlated with that of eosinophils. Furthermore, eosinophils isolated from the small intestine of WT mice, but not of IL-1Ra–deficient mice, inhibited the Th17 cells. Our findings demonstrate a hitherto unappreciated role of small intestinal eosinophils to regulate intestinal homeostasis by controlling Th17 cells.     

IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.The immunological memory that develops during T cell–dependent (TD) immune responses comprises populations of plasma cells and recirculating antigen-experienced B and T lymphocytes (Tarlinton, 2006). Two compartments of humoral memory, plasma cells and memory B cells, are generated in germinal centers (GCs) that develop within the secondary lymphoid organs during TD responses (Tarlinton, 2006). Although composed primarily of B lymphocytes, GCs contain small numbers of CD4⁺ T cells, dendritic cells, and macrophages and develop in association with antigen localized on the surface of follicular dendritic cells (Haberman and Shlomchik, 2003; Allen et al., 2007). After a period of B cell proliferation, several processes are initiated within the GC that affect affinity maturation whereby the mean binding affinity of antigen-specific antibody increases as a function of time (MacLennan, 1994; Allen et al., 2007). Affinity maturation is driven in large part by the somatic hypermutation (SHM) of the immunoglobulin V genes of proliferating GC B cells, a process which is mediated by the enzyme activation-induced cytidine deaminase (AID). B cells expressing antigen receptors of improved affinity, usually as a result of SHM, are preserved preferentially. Iterations of proliferation, mutation, and avidity-based selection improve the mean affinity of the responding B cell population (MacLennan, 1994; Allen et al., 2007).Normally, in an immune response to a protein antigen the vast majority of memory B cells and bone marrow plasma cells arise from the somatically diversified affinity-matured population of GC B cells (Tarlinton, 2006). It is inferred that avidity for antigen is a major determinant in plasma cell differentiation of GC B cells, whereas memory B cell formation is more influenced by survival (Lanzavecchia and Sallusto, 2002; Phan et al., 2006; Tarlinton, 2006). It also appears that both types of post-GC B cell are produced throughout the GC reaction rather than being released into the circulation in a single event (Blink et al., 2005). The persistence and continued activity of GC, which is indicated by the continued production of plasma cells and memory B cells and the increasing frequency of V gene mutation, implies that a proportion of GC B cells remain within the GC and undergo additional rounds of proliferation, mutation, and selection (MacLennan, 1994; Allen et al., 2007). B cells within GC therefore have several possible fates: death, division with or without SHM, or differentiation into either the memory B cell or plasma cell compartments.GC persistence, development, and function absolutely require CD4⁺ T cells. T cells activated by antigen-presenting dendritic cells migrate into the B cell area in part as a result of their up-regulation of CXCR5, a chemokine receptor normally restricted to B cells (Allen et al., 2007). Indeed, the expression of CXCR5 contributes to the definition of what are now called T follicular helper (Tfh) cells (Vinuesa et al., 2005). In addition to CXCR5, Tfh cells are distinguished from other CD4 T cells by their elevated expression of ICOS and CD40L (Vinuesa et al., 2005), both of which are critical for the initiation and maintenance of the GC (Tarlinton, 2006). Intriguingly, up-regulation of many of the molecules that define the Tfh phenotype appears to be mediated by Bcl-6, which is required for their development in a cell-intrinsic manner (Johnston et al., 2009). Tfh cells are also enriched for secretion of IL-21 (Chtanova et al., 2004; Nurieva et al., 2008) and IL-4 (Reinhardt et al., 2009). IL-21 is associated with growth and differentiation of many types of lymphocytes, including B and T cells (Ettinger et al., 2008). The effects of IL-21 on B cells vary depending on the context. In vivo, IL-21R deficiency leads to a state of pan-hypogammaglobulinaemia while promoting high titers of IgE (Ozaki et al., 2002). In vitro, IL-21 has been shown to increase both Blimp-1 and Bcl-6 in B cells (Ozaki et al., 2004; Arguni et al., 2006), suggesting an ability for IL-21 to influence multiple aspects of B cell differentiation. Recent data support the notion that IL-21 has a critical, possibly obligatory, role in the development of Tfh cells and, through this, on the formation of GC (Nurieva et al., 2008; Vogelzang et al., 2008), whereas other data suggest a less universal association (Linterman et al., 2009). IL-21 has also been shown to augment the formation of Th17 cells (Korn et al., 2007; Nurieva et al., 2007; Zhou et al., 2007), which have been shown to both secrete IL-21 and promote GC formation in a mouse model of autoimmunity (Hsu et al., 2008), strengthening the view that the effects of IL-21 on GC activity are T cell mediated. An earlier study, however, using IL-21R–deficient mice reported GC and memory development to be normal (Ozaki et al., 2002), raising uncertainty as to exactly what the requirement of IL-21 may be in the GC reaction, on which cell types it may act, and what its activities might be. This uncertainty has been heightened by recent publications suggesting that IL-4 is a key mediator of Tfh activity (King and Mohrs, 2009; Reinhardt et al., 2009).Given the multitude of potential roles for IL-21 on lymphocyte behavior (Ettinger et al., 2008), we wished to assess the development of a humoral immune response in mice lacking IL-21 or the IL-21R. These experiments confirmed a role for IL-21 in the formation of plasma cells, contradicted a mandatory autocrine role for IL-21 in Tfh development or function, and revealed a previously undefined role for this cytokine in the GC reaction and the regulation of their output. These actions of IL-21 on B cells were direct, as they were replicated by the selective absence of the IL-21R on B cells and not on T cells, suggesting that the major activity of IL-21 in the GC is on B cells and is not to establish or maintain cells of a Tfh phenotype.     

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.     

Autoantibodies against IL-17A,IL-17F,and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I

Most patients with autoimmune polyendocrine syndrome type I (APS-I) display chronic mucocutaneous candidiasis (CMC). We hypothesized that this CMC might result from autoimmunity to interleukin (IL)-17 cytokines. We found high titers of autoantibodies (auto-Abs) against IL-17A, IL-17F, and/or IL-22 in the sera of all 33 patients tested, as detected by multiplex particle-based flow cytometry. The auto-Abs against IL-17A, IL-17F, and IL-22 were specific in the five patients tested, as shown by Western blotting. The auto-Abs against IL-17A were neutralizing in the only patient tested, as shown by bioassays of IL-17A activity. None of the 37 healthy controls and none of the 103 patients with other autoimmune disorders tested had such auto-Abs. None of the patients with APS-I had auto-Abs against cytokines previously shown to cause other well-defined clinical syndromes in other patients (IL-6, interferon [IFN]-γ, or granulocyte/macrophage colony-stimulating factor) or against other cytokines (IL-1β, IL-10, IL-12, IL-18, IL-21, IL-23, IL-26, IFN-β, tumor necrosis factor [α], or transforming growth factor β). These findings suggest that auto-Abs against IL-17A, IL-17F, and IL-22 may cause CMC in patients with APS-I.Autoimmune polyendocrine syndrome type I (APS-I), also known as autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy (Online Mendelian Inheritance in Man no. 240300), is a rare, autosomal recessive primary immunodeficiency first described clinically in 1929 (Notarangelo et al., 2006; Husebye et al., 2009). APS-I is characterized principally by multiple autoimmune endocrinopathies, hypoparathyroidism, and adrenal insufficiency in particular, with some of these symptoms being caused by pathogenic autoantibodies (auto-Abs). A genome-wide mapping approach led to the identification of APS-I–causing mutations in the AIRE gene in 1997 (Finnish-German APECED Consortium, 1997; Nagamine et al., 1997). Autoimmunity in patients with APS-I may be accounted for by the key role of AIRE in tolerance. AIRE is expressed in the thymus, where it contributes to the expression of peripheral antigens (Anderson et al., 2002; Mathis and Benoist, 2009). This gene has also been shown to be expressed in secondary lymphoid organs, where it also contributes to tolerance (Gardner et al., 2008). Surprisingly, most patients with APS-I suffer from chronic mucocutaneous candidiasis (CMC) without displaying any marked susceptibility to any other pathogen. The product of the AIRE gene is not involved in any known cellular pathway governing host defense. The pathogenesis of CMC in patients with APS-I has thus remained both intriguing and elusive.High titers of auto-Abs against some type I IFNs, including IFN-α and -ω in particular, are found in all patients (Meager et al., 2006; Meloni et al., 2008). These auto-Abs are a hallmark of APS-I and are therefore useful for diagnostic purposes (Husebye et al., 2009). However, they confer no particular overt predisposition to viral diseases, perhaps because of the large number of redundant type I IFN species, resulting in incomplete neutralization of the overall antiviral activity of IFNs by the auto-Abs. It has been suggested that these auto-Abs contribute to CMC in APS-I patients (Meager et al., 2006). However, this is now thought unlikely because of the absence of CMC in patients with various forms of STAT1 and TYK2 deficiency and impaired responses to type I IFNs, and in patients with various forms of NEMO, UNC-93B, and TLR3 deficiencies and impaired production of type I IFNs (Minegishi et al., 2006; Zhang et al., 2008; Chapgier et al., 2009). Nevertheless, based on this observation, we hypothesized that CMC in patients with APS-I might result from autoimmunity to cytokines other than type I IFNs potentially involved in protective immunity to Candida albicans in the skin and mucosae.Recent studies in the mouse have suggested that IL-17 cytokines, such as IL-17A, IL-17F, and IL-22 (Korn et al., 2009), may be important in host defense against Candida (Conti et al., 2009; van de Veerdonk et al., 2009). However, it is difficult to assess mucocutaneous immunity to Candida in mice (Netea et al., 2008), in which IL-17 cytokines seem to be important for the control of various other pathogens, particularly in the lungs and gastrointestinal tract (Dubin and Kolls, 2008; Khader et al., 2009). Stimulation with Candida in vitro leads to the preferential generation of IL-17A– and IL-22–producing human T cells (Acosta-Rodriguez et al., 2007; Liu et al., 2009). Furthermore, patients with STAT3 deficiency display a predisposition to CMC (and staphylococcal disease) and lack IL-17–producing T cells (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; Minegishi et al., 2009). Similarly, some patients with IL-12p40 or IL-12Rβ1 deficiency present CMC (together with mycobacteriosis and salmonellosis) and have lower than normal proportions of IL-17–producing T cells (de Beaucoudrey et al., 2008). Finally, patients with rare defects of CARD9, which normally controls the production of IL-17 in response to dectin-1 and -2 stimulation by Candida, suffer from candidiasis (LeibundGut-Landmann et al., 2007; Glocker et al., 2009; Robinson et al., 2009). We therefore hypothesized that autoimmunity to IL-17 cytokines might account for CMC in patients with APS-I.     

IL-15 trans-presentation by pulmonary dendritic cells promotes effector CD8 T cell survival during influenza virus infection

We have recently demonstrated that peripheral CD8 T cells require two separate activation hits to accumulate to high numbers in the lungs after influenza virus infection: a primary interaction with mature, antigen-bearing dendritic cells (DCs) in the lymph node, and a second, previously unrecognized interaction with MHC I–viral antigen–bearing pulmonary DCs in the lungs. We demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis in the lungs; however, reconstitution with pulmonary plasmacytoid DCs and CD8α⁺ DCs promotes increased T cell survival and accumulation in the lungs. Further, our results show that the absence of DCs after influenza virus infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC–mediated rescue of virus-specific CD8 T cell responses in the lungs requires trans-presentation of IL-15 via DC-expressed IL-15Rα. This study demonstrates a key, novel requirement for DC trans-presented IL-15 in promoting effector CD8 T cell survival in the respiratory tract after virus infection, and suggests that this trans-presentation could be an important target for the development of unique antiviral therapies and more effective vaccine strategies.Clearance of a primary influenza A virus (IAV) infection is known to require killing of virus-infected host cells by activated, antigen-specific CD8 T cells in the lungs (Topham et al., 1997). Until recently, antigen-specific CD8 T cells were thought to undergo programmed activation, whereby a single, brief interaction with a mature, antigen-bearing DC in the LN was sufficient to induce a full program of activation, division, and differentiation from naive to mature, cytotoxic CD8 T cells (Kaech and Ahmed, 2001; Wong and Pamer, 2001). Increasing evidence has suggested, however, that activation of antigen-specific CD8 T cells is not as simple as previously thought, and multiple factors, including cytokine signals such as IL-2 (Wong and Pamer, 2004), IFN-α (Marrack et al., 1999; Price et al., 2000; Kolumam et al., 2005), and IL-12 (Curtsinger et al., 2003a,b; Trinchieri, 2003), and late co-stimulatory signals such as CD70 (Dolfi and Katsikis, 2007) and 4-1BBL (Bertram et al., 2002; Lin et al., 2009), can regulate and fine tune the magnitude and duration of the effector response, as well as the nature of the ensuing memory T cell population.We have recently demonstrated in a model of IAV infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDCs) and CD8α⁺ DCs, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells (McGill et al., 2008). Reconstitution of the lungs with physiological numbers of pDCs or CD8α⁺ DCs is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent on direct DC–T cell interactions, DC-expressed MHC I, and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DCs (aDCs) or alveolar macrophages (aMϕs) was unable to rescue the virus-specific CD8 T cell response (McGill et al., 2008). After IAV infection there is an abundance of IAV antigen– and MHC I–expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8α⁺ DC–mediated rescue of the T cell response in the lungs. Further, it remained unclear what mechanism was contributing to decreased numbers of IAV-specific CD8 T cells in the lungs of aDC-depleted mice: impaired DC migration from the lungs to the LN, impaired CD8 T cell proliferation within the lungs, or impaired CD8 T cell survival within the lungs. It was also unclear what mechanism pulmonary DC subsets were using to rescue this defect.The cytokine IL-15 has been demonstrated to play a key role in promoting lymphoid homeostasis, particularly with respect to CD8 T cells (Budagian et al., 2006; Kim et al., 2008). IL-15 was initially thought to signal similar to IL-2, whereby IL-15Rα formed a heterotrimeric complex with IL-2/IL15Rβ and common γ for high affinity signaling. Although this model appears to hold true in certain situations, recent reports have demonstrated a unique, alternative signaling mechanism, termed trans-presentation. In this model, IL-15Rα is required for the processing and presentation of active IL-15 in trans to cells expressing the IL-2/IL15Rβ–common γ chain complex (Sandau et al., 2004; Schluns et al., 2004; Kobayashi et al., 2005). At this time, it is unclear which cell types serve as the primary trans-presenting cells during an immune response; however, several lines of evidence have indicated that DCs may play an important role (Burkett et al., 2003, 2004). It is known that DCs express protein for both IL-15 and IL-15Rα, and that stimulation by IFN-αβ (Mattei et al., 2001) or IFN-γ (Doherty et al., 1996; Musso et al., 1999), or exposure to viral infection leads to further up-regulation of these molecules (Liu et al., 2000; Dubois et al., 2005; Budagian et al., 2006; Mattei et al., 2009). Interestingly, DCs matured in the presence of IL-15 have been demonstrated to promote enhanced antigen-specific CD8 T cell proliferation (Jinushi et al., 2003; Mattei et al., 2009) and a robust Th1 skewing in vivo (Pulendran et al., 2004).IL-15 has been best characterized for its role in maintaining memory CD8 T cell homeostasis, primarily through promoting enhanced basal proliferation (Becker et al., 2002; Goldrath et al., 2002; Schluns et al., 2002). More recently, however, there is accumulating evidence that IL-15 is also important for promoting primary effector CD8 T cell responses (Akbar et al., 1996; Bulfone-Paus et al., 1997; Vella et al., 1998; Schluns et al., 2002; Rausch et al., 2006; Yajima et al., 2006). Surface expression of both IL-15Rα and IL-2/IL15-Rβ is up-regulated after TCR activation (Vella et al., 1998), and IL-15 has been proposed to enhance activated CD8 T cell survival after challenge with staphylococcal enterotoxin A (Vella et al., 1998), Mycobacterium tuberculosis (Rausch et al., 2006), and vesicular stomatitis virus infection (Sandau et al., 2004). Collectively, these studies suggest a particularly important role for IL-15 in the generation and maintenance of an appropriate immune response; however, it remains unclear what role IL-15 plays during the effector phase of the immune response or in what context IL-15 contributes to activated CD8 T cell survival in vivo.In this study, we demonstrate a previously unrecognized role for pulmonary DC–mediated IL-15 trans-presentation in regulating virus-specific CD8 T cell responses in the lungs after IAV infection. The reduction in T cell numbers observed in the lungs of aDC-depleted mice after IAV challenge results not from impaired proliferation within the lungs but is caused by significantly increased levels of apoptosis of virus-specific CD8 T cells compared with nondepleted controls. Further, reconstitution with purified pDCs or CD8α⁺ DCs rescues the IAV-specific CD8 T cell response by promoting increased CD8 T cell survival in the lungs of aDC-depleted mice. Additionally, our results show that IAV infection induces up-regulation of both IL-15 mRNA and protein in the lungs and that depletion of aDCs at 48 h post infection (p.i.) results in a significant reduction in pulmonary IL-15 expression. Finally, this study demonstrates that pulmonary DCs prevent virus-specific CD8 T cell apoptosis through trans-presentation of IL-15, as blockade of IL-15 or IL-15Rα on the surface of pulmonary DCs before adoptive transfer, or transfer of IL-15^−/− pulmonary DC subsets ablates the rescue of the virus-specific CD8 T cell response in the lungs of aDC-depleted mice.     

Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A

Many solid cancers display cellular hierarchies with self-renewing, tumorigenic stemlike cells, or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies, they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs, we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays, but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines, including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion, and targeting IL-17A signaling impaired CIC growth. Notably, IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer.Colorectal cancer is the third leading cause of cancer-related death in the United States, with ∼141,210 new cases and 49,380 deaths in 2011 (American Cancer Society, 2011). Despite clinical advances, 50% of stage III and 95% of stage IV colorectal cancer patients will die from their disease (American Cancer Society, 2011). Improving survival for patients afflicted with colorectal cancer will require more effective and durable responses to adjuvant chemotherapy. Advances in the genetics of colorectal cancers have defined molecular targets altered during the development and progression of colorectal cancers, but have translated into targeted therapeutics with only modest efficacy. Tumor suppressor pathways account for most common genetic lesions, but these have proven difficult to target pharmacologically. Molecularly targeted therapies, like the anti–epidermal growth factor receptor (EGFR) agents cetuximab and panitumumab augment the activity of conventional chemotherapy but are not curative (Arnold and Seufferlein, 2010). Resistance to chemotherapy may be associated with the outgrowth of clones harboring advantageous genetic lesions, but cellular diversity derived from nongenetic sources also contributes to recurrent tumor growth (Weaver et al., 2002; Matsunaga et al., 2003; Bissell and Labarge, 2005). Cancers exist as complex systems composed of multiple cell types that collectively support and maintain tumor growth. Nontransformed elements may display relatively few genomic lesions and be more likely to display sustained responses to therapy, suggesting advantages to their use as therapeutic targets (Shaked et al., 2006, 2008; Yamauchi et al., 2008; Gilbert and Hemann., 2010; Hao et al., 2011; Shree et al., 2011; Straussman et al., 2012; Gilbert and Hemann., 2011; Acharyya et al., 2012; Nakasone et al., 2012; Hölzel et al., 2013; Bruchard et al., 2013). Indeed, the microenvironment has become a major focus in modeling the growth of cancer and therapeutic response. Inhibition of tumor vasculature through blockade of endothelial proliferation signals has clinical benefit, leading to the development of bevacizumab, a humanized anti–vascular endothelial growth factor (VEGF) antibody (Winder and Lenz, 2010). Another important compartment of tumor stroma is cancer-associated fibroblasts (CAFs). CAFs originate from heterogeneous cell types, including bone marrow–derived progenitor cells, smooth muscle cells, preadipocytes, fibroblasts, and myofibroblasts (Orimo and Weinberg, 2007; Worthley et al., 2010; Gonda et al., 2010). CAFs support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion (Gonda et al., 2010; Worthley et al., 2010). They are also an important player in therapeutic resistance (Crawford et al., 2009; Porter et al., 2012), and fibroblasts can serve as a source for cytokines released in the cancer-initiating cell (CIC) microenvironment (Vermeulen et al., 2010). Furthermore, irradiated CAFs have been previously reported to promote tumor growth in breast (Barcellos-Hoff and Ravani, 2000) and lung cancers (Hellevik et al., 2013). It is thus logical that disruption of CAFs in the tumor microenvironment would influence clinical tumor behavior.Cancers are maintained over the long term by a subpopulation of cancer cells, the CICs (Barker et al., 2009; Ricci-Vitiani et al., 2009; Blanpain, 2013). Like tissue-specific stem cells, the identification and characterization of CICs is evolving: the current definition is based on functional assays focused on recapitulation of the parental tumor upon xenotransplantation. The features of self-renewal, differentiation, and sustained proliferation are inherent within the regeneration of the tumor organ system (Magee et al., 2012). Interpatient variation in the genetics and epigenetics of colorectal cancers is so divergent that no identical mutational signatures have been reported for patients (Sanchez et al., 2009; Ogino et al., 2012; Sadanandam et al., 2013). It is therefore not surprising that markers to distinguish CICs from more differentiated progeny have not been absolutely informative across all tumors. Further, most CIC enrichment markers mediate interactions between a cell and its microenvironment, suggesting that the information associated with that marker may be lost after removal from the tumor microenvironment. Whereas CD133 (Prominin-1) had been reported by some groups to selectively identify colorectal CICs (O’Brien et al., 2007; Ricci-Vitiani et al., 2007; Elsaba et al., 2010; Fang et al., 2010), Shmelkov et al. (2008) reported that CD133 failed to inform identification of the CICs. Other groups have reported that CD44 (Dalerba et al., 2007; Du et al., 2008; Yeung et al., 2010; Ohata et al., 2012), CD166 (Dalerba et al., 2007; Vermeulen et al., 2008), CD66c (Gemei et al., 2013), Lgr5 (Barker et al., 2007; Vermeulen et al., 2008; Takahashi et al., 2011), or aldehyde dehydrogenase (ALDH; Huang et al., 2009; Deng et al., 2010) inform CIC characteristics. Regardless of the marker used, CICs are enriched for tumorigenic potential, indicating that these subgroups of tumor cells drive colorectal cancer maintenance and must be targeted to inhibit tumor growth.CICs do not exist in isolation, but rather reside in an interactive niche with multiple cell types, including fibroblasts (Vermeulen et al., 2010; Medema and Vermeulen, 2011), endothelial cells (Lu et al., 2013), and immune cells (Hölzel et al., 2013). Each component contributes to the overall function and maintenance of the tumor and has potential roles in CIC resistance and recurrence. Mechanisms driving CIC maintenance and resistance are still being defined, but cell–cell interactions mediated through numerous molecular mechanisms, including cytokines and chemokines, are critical (Todaro et al., 2007; Vermeulen et al., 2010; Li et al., 2012). Cytokines and chemokines have the capacity to function as both paracrine and autocrine factors, supporting these secreted molecules as ideal mediators of interactions between the cellular hierarchy and other tumor cellular components. Indeed, we have described IL-6 as a key cue derived from more differentiated tumor cells to maintain glioblastoma CICs, which express IL-6 receptors (Wang et al., 2009). Mesenchymal stem cells and tumor-associated macrophages secrete IL-6 and CXCL7 in breast cancer to stimulate CIC growth and dispersal (Liu et al., 2011). These interactions are reciprocal, as CICs create supportive niches for stroma through the recruitment of mesenchymal stem cells via IL-1 secretion. In return, mesenchymal stem cells secrete IL-6 and IL-8 to promote CIC maintenance (Li et al., 2012).Here, we first confirm that chemotherapy preferentially targets non-CICs due to cell autonomous resistance of CICs, but furthermore uncover a novel negative impact of chemotherapy in the stimulation of CAFs to create a chemoresistant niche by releasing cytokines, including IL-17A, as a CIC maintenance factor. These results have important clinical implications as most chemosensitizing approaches focus on disrupting cell autonomous molecular mechanisms without consideration of the interplay with the microenvironment that may display differential molecular dependence and temporal course, suggesting more complex therapeutic paradigms may be required to improve patient outcomes.     

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.     

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

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