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.     

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.     

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.     

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.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell–dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10– and IL-21–mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21–induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.Long-lived immunological memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T-dependent antigens (Ags) and underlies the success of most currently available vaccines (Ahmed and Gray, 1996; Rajewsky, 1996; Tangye and Tarlinton, 2009; Goodnow et al., 2010). PCs reside in survival niches in bone marrow and secondary lymphoid tissues and constantly produce high titers of neutralizing antibodies (Abs; Tangye and Tarlinton, 2009; Tangye, 2011). In contrast, memory B cells recirculate throughout peripheral blood, secondary lymphoid tissues, and bone marrow. Upon reexposure to Ag, they can proliferate and differentiate into Ab-secreting plasmablasts more rapidly than naive cells, thereby replenishing the PC pool and simultaneously expanding the memory cell population (Ahmed and Gray, 1996; Rajewsky, 1996; Tangye and Tarlinton, 2009).Analysis of gene-targeted mice and humans with monogenic primary immunodeficiencies has identified some of the molecular requirements for memory B cell generation. Thus, mutations in B cell–intrinsic genes (CD19/CD81, CD40, IKBKG, DOCK8, and IL2RG) or genes expressed by CD4⁺ T helper cells (CD40LG, ICOS, and SH2D1A [SAP]) all result in reductions in the frequencies of memory B cells and associated deficiencies in total serum Ig levels or Ag-specific Ab (Tangye and Tarlinton, 2009; Recher et al., 2011; Jabara et al., 2012; Tangye et al., 2012). We also have some understanding of the mechanisms that enable memory B cells to respond more rapidly and vigorously than naive cells to cognate Ag. First, memory B cells are recruited into division significantly earlier and undergo more rounds of division than naive cells (Bernasconi et al., 2002; Tangye et al., 2003a,b; Macallan et al., 2005). Second, memory B cells have higher expression of cell surface receptors, TLRs (TLR7/9/10), CD21, CD27, and TACI, that could enable them to respond more efficiently to co-stimulatory signals (Tangye et al., 1998; Bernasconi et al., 2002, 2003; Darce et al., 2007; Good et al., 2009). Third, memory B cells express heightened levels of CD80 and CD86 (Liu et al., 1995; Tangye et al., 1998; Ellyard et al., 2004; Good et al., 2009), which facilitate soliciting help from T helper cells. Fourth, memory B cells express lower levels of genes that restrict the entry of naive B cells into division, limiting their activation (Good and Tangye, 2007; Horikawa et al., 2007). Lastly, distinct signaling pathways downstream of the B cell receptor expressed by naive (i.e., IgM) or memory (IgG) cells have been identified that preferentially promote responsiveness of memory cells (Martin and Goodnow, 2002; Engels et al., 2009; Davey and Pierce, 2012). However, the requirements for cytokine-mediated regulation of naive and memory B cells remain to be determined.Human B cell differentiation is regulated by the actions of numerous cytokines, with IL-10 and IL-21, produced by T follicular helper cells (Tfh cells), being key factors in promoting proliferation, isotype switching, PC differentiation, and secretion of most Ig isotypes by not only naive B cells, but also memory B cells, including both IgM⁺ and isotype-switched subsets (Banchereau et al., 1994; Arpin et al., 1997; Pène et al., 2004; Ettinger et al., 2005; Bryant et al., 2007; Avery et al., 2008a,b). Although the functions of IL-10 and IL-21 on human B cells are similar, the effects of IL-21 exceed those of IL-10 by 10–100-fold (Bryant et al., 2007). The importance of IL-21 to immune regulation has been validated by the recent identification of IL-21R–deficient humans, who exhibit infectious susceptibility to several pathogens (Kotlarz et al., 2013). The predominance of IL-21 in regulating human B cell function over IL-10 is also indicated by the fact that IL21R mutations result in poor Ab responses after vaccination (Kotlarz et al., 2013), whereas specific Abs are produced at normal levels in individuals with mutations in IL10/IL10R (Kotlarz et al., 2012). IL-10 and IL-21 activate STAT1, STAT3, STAT5, as well as MAPK/ERK and PI3K/Akt pathways (Asao et al., 2001; Zeng et al., 2007; Avery et al., 2008b, 2010; Diehl et al., 2008). Autosomal-dominant hyper-IgE syndrome (AD-HIES) is caused by heterozygous mutations in STAT3 (Holland et al., 2007; Minegishi et al., 2007; Casanova et al., 2012). These mutations operate in a dominant-negative manner, effectively reducing the level of functional STAT3 by 75%. Loss-of-function mutations in STAT1 also underlie several immunodeficiency states, such as those characterized by selective susceptibility to infection with environmental mycobacteria and, depending on the nature of the mutation (i.e., dominant/recessive), some viruses (Boisson-Dupuis et al., 2012; Casanova et al., 2012). By examining these patients, we previously found that functional STAT3 deficiency not only severely compromised the generation of memory (i.e., CD27⁺) B cells in vivo, but prevented IL-10– and IL-21–mediated induction of PRDM1 (Blimp-1 [B lymphocyte induced maturation protein-1]) and XBP1 (X-box binding protein 1) in naive B cells and their subsequent differentiation to the PC lineage in vitro. However, STAT3 mutant (STAT3_MUT) naive B cells could still acquire expression of AICDA (activation-induced cytidine deaminase) and undergo IL-21–induced isotype switching in vitro. In contrast, STAT1 was dispensable for human B cell differentiation in vivo and in vitro (Avery et al., 2010).These findings led us to investigate further the role of STATs in governing human B cell differentiation. We have now discovered that the small number of memory B cells generated in STAT3-deficient patients are unaffected by these mutations; thus, they are capable of differentiating into Ab-secreting cells in response to STAT3-actvating cytokines as efficiently as normal memory cells. These findings demonstrate that the threshold of STAT3 activation required for B cell differentiation is significantly lower in memory compared with naive cells. Consequently, limiting amounts of functional STAT3 are sufficient to mediate memory, but not naive, B cell differentiation, thereby revealing an intrinsic difference in the requirements for activating naive versus memory B cells. The memory B cell deficiency in AD-HIES patients likely contributes to impaired Ag-specific Ab responses characteristic of these individuals. Thus, by targeting the residual population of STAT3-deficient memory B cells to respond to IL-21, it may be possible to improve humoral immunity in AD-HIES.     

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.