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.     

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.     

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

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

Naive tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma

In vitro differentiated CD8⁺ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4⁺ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naive tumor/self-specific CD4⁺ T cells naturally differentiated into T helper type 1 cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8⁺, B, natural killer (NK), and NKT cells. Proper activation of CD4⁺ T cells in vivo was important for tumor clearance, as naive tumor-specific CD4⁺ T cells could not completely treat tumor in lymphopenic common gamma chain (γ_c)–deficient hosts. γ_c signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4⁺ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4⁺ T cells.Adoptive cellular therapy (ACT) of cancer using in vitro differentiated CD8⁺ T cells is a powerful treatment against established cancer in humans and mice. In recent years, great progress has been attained in the understanding of the mechanisms involved in enhancing treatment of large established tumors (Gattinoni et al., 2006). Lymphodepletion before adoptive therapy greatly enhances ACT in humans and mice through the creation of cytokine sinks, removal of regulatory T cells (T reg cells), and the release of toll-like receptor agonists (Gattinoni et al., 2005a; Paulos et al., 2007; Dudley et al., 2008). Recent evidence suggests that irradiation also enhances the expression of ICAM and VCAM in the tumor vasculature allowing tumor-reactive T cells to enter more readily (Quezada et al., 2008). Although CD8⁺ T cells are potent mediators of antitumor immunity, there has been little focus on tumor-specific CD4⁺ T cells. CD4⁺ Th cells are important in immunity because in the absence of help, CD8⁺ T cells can be deleted or lose the capacity to develop into memory CD8⁺ T cells upon rechallenge (Janssen et al., 2003; Antony et al., 2005; Williams et al., 2006). Therefore, the use of tumor/self-reactive CD8⁺ T cells in the adoptive immunotherapy of cancer may face similar fates because T cells must remove tumor antigen in the context of persisting self-antigen, which in some cases leads to autoimmunity (Gattinoni et al., 2006; Rosenberg et al., 2008). Adoptive cell therapies that incorporate CD4⁺ T cells are far superior to therapies that only use CD8⁺ T cell clones (Dudley et al., 2002). Therefore, one theoretical means of improving immunotherapy to self may involve the provision of tumor-reactive or self-reactive CD4⁺ T cells (Nishimura et al., 1999; Marzo et al., 2000; Antony et al., 2005), but a more direct role for CD4⁺ T cells in tumor immunity remains unclear (Ho et al., 2002; Muranski and Restifo, 2009).Recently, adoptive transfer of in vitro differentiated tumor-specific CD4⁺ T cells in humans and mice has shown promise against cancer as a therapy (Nishimura et al., 1999; Perez-Diez et al., 2007; Hunder et al., 2008; Muranski et al., 2008). This has rekindled the idea of using antigen-specific CD4⁺ Th during immunotherapy because CD4⁺ Th cells can mediate the proper signals required in vivo to activate CD8⁺ T cells and other cells of the innate immune system (Kahn et al., 1991; Hung et al., 1998; Nishimura et al., 1999; Antony et al., 2006; Williams et al., 2006). In fact, several preclinical and clinical trials have shown the importance of CD4 help during immunotherapy of cancer (Nishimura et al., 1999; Antony et al., 2006; Dudley et al., 2008). However, isolation of tumor-specific CD4⁺ T cells has been difficult (Wang, 2001) and only a few MHC class II vaccines have been produced as a result of the lack of knowledge of how to generate vaccines that specifically activate Th cells instead of tumor-specific Foxp3⁺ T reg cells (Rosenberg, 2001; Vence et al., 2007). In addition, lack of appropriate mouse models to study tumor-specific CD4⁺ T cell responses to self-antigens has hindered progress in our understanding of the role of CD4⁺ T cells in maintaining immunity to cancer.Now, with a better understanding of CD4⁺ T cell biology, the use of cytokines to differentiate and expand T cells in vitro has led to a panoply of CD4 lineages with specific in vivo functions (Weaver and Rudensky, 2009). For example, in vitro differentiated CD4⁺ Th17 tumor-specific T cells have shown superiority over CD4⁺ Th1 differentiated T cells in the adoptive immunotherapy of cancer in a mouse model of melanoma (Muranski et al., 2008). IL-2 and IL-7 in vitro expanded NY-ESO-1–specific CD4⁺ T cells in humans have also shown clinical promise in one patient who had not received prior lymphodepleting conditioning or a vaccine (Hunder et al., 2008). Although these are promising studies, the mechanisms involved in the direct therapy of cancer by CD4⁺ T cells remain elusive. Likewise, methods for enhancing adoptive immunotherapy without prior in vitro manipulation that may lead to the terminal differentiation of T cells also remain unclear (Gattinoni et al., 2005b, 2009; Klebanoff et al., 2005). Although such manipulations can lead to vaccine independence (Klebanoff et al., 2009), long-term benefits from in vivo differentiation may outweigh in vitro stimulation because the in vivo environment may provide the correct signals that cannot be attained in a culture dish.To test a direct role for CD4⁺ T cells in the immunotherapy of cancer, we used a gp75/tyrosinase-related protein (TRP) 1–specific CD4⁺ TCR transgenic (Tg) mouse that produces class II–restricted T cells that recognize mouse TRP-1 in the context of I-A^b (Muranski et al., 2008). TRP-1 is expressed in malignant melanoma and in the skin and eyes of mice and humans; therefore, this model mimics the human condition as closely as possible. Surprisingly, we found that adoptive transfer of naive TRP-1–specific CD4⁺ T cells into lymphopenic animals bearing large established melanoma caused tumor regression and depigmentation independent of vaccination, cytokine administration, and CD8⁺, B, NK, and NKT cells. This therapy was dependent on common gamma chain (γ_c) signaling in the host for survival and differentiation of CD4⁺ T cells in vivo. These data provide a better understanding for the design of immunotherapies that incorporate tumor/self-reactive CD4⁺ T cells.     

Anti-CD3 therapy permits regulatory T cells to surmount T cell receptor–specified peripheral niche constraints

Treatment with anti-CD3 is a promising therapeutic approach for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3⁺ regulatory T (T reg) cells may be involved, but the evidence has been conflicting. We investigated this issue in mice derived from the NOD model, which were engineered so that T reg populations were perturbed, or could be manipulated by acute ablation or transfer. The data highlighted the involvement of Foxp3⁺ cells in anti-CD3 action. Rather than a generic influence on all T reg cells, the therapeutic effect seemed to involve an ∼50–60-fold expansion of previously constrained T reg cell populations; this expansion occurred not through conversion from Foxp3⁻ conventional T (T conv) cells, but from a proliferative expansion. We found that T reg cells are normally constrained by TCR-specific niches in secondary lymphoid organs, and that intraclonal competition restrains their possibility for conversion and expansion in the spleen and lymph nodes, much as niche competition limits their selection in the thymus. The strong perturbations induced by anti-CD3 overcame these niche limitations, in a process dependent on receptors for interleukin-2 (IL-2) and IL-7.Treatment with an antibody targeting CD3 is one of the more promising avenues currently being pursued for the therapy of organ-specific autoimmune diseases. Following the precedents from rodent models (Herold et al., 1992; Vallera et al., 1992; Hayward and Shriber, 1992; Chatenoud et al., 1994), administration of anti-CD3 to patients with recently diagnosed diabetes has yielded favorable results in two clinical trials, with a stabilization of disease progression (Herold et al., 2002; Keymeulen et al., 2005). In both mice and humans, anti-CD3 treatment resulted in long-lasting effects that persisted long after clearance of the antibody. However, the mechanism of action is not clear. TCR blockade and internalization, induction of anergy, and perturbation of the T helper (Th) 1/Th2 balance have all been invoked (Hayward and Shriber, 1992; Alegre et al., 1995; Smith et al., 1997). Some studies have suggested an important role for immunosuppression by TGFβ, although conflicting cytokine sources have been proposed (Belghith et al., 2003; Chen et al., 2008; Perruche et al., 2008). More recently, several investigators have suggested that anti-CD3 therapy may elicit an increase in cells with immunoregulatory properties, in particular Foxp3⁺ regulatory T (T reg) cells of the CD4⁺ (You et al., 2007) or CD8⁺ (Ablamunits and Herold, 2008) lineages.Foxp3⁺ T reg cells are the best characterized lymphocyte subset with a regulatory phenotype, playing an important role in the control of antiinfectious, antitumor, and autoimmune responses (Belkaid and Rouse, 2005; Roncarolo and Battaglia, 2007; Dougan and Dranoff, 2009). These regulatory activities are manifest via one or more molecular mechanisms (Vignali et al., 2008). The homeostasis of T reg populations is critical to their potency, but is poorly understood. Although cytokines whose receptors use the common γ chain (γc), as well as other molecules, have been shown to influence the number of peripheral T reg cells, several issues remain unclear: e.g., whether these elements are required purely for peripheral homeostasis or are also involved in thymic differentiation of T reg cells; whether they are involved in proliferation and/or survival; or whether they are implicated only under specific conditions, such as lymphopenia or inflammation.Some studies on anti–CD3-treated mice have variably shown modifications of T reg cells, sometimes present but quantitatively modest (Belghith et al., 2003; Bresson et al., 2006), sometimes absent (Chen et al., 2008), sometimes restricted to particular anatomical locations (Belghith et al., 2003; Kohm et al., 2005) or involving cells of an unusual CD25^low phenotype (You et al., 2007). Certain of the disparate results may have stemmed from the use of CD25 for the identification of T reg cells. This is an issue because NOD mice have an unusually high proportion of the CD25-negative T reg component (Feuerer et al., 2007), which in most other strains constitutes only a minority of Foxp3⁺ cells (Fontenot et al., 2005b).In this context, we thought it worthwhile to reexamine the impact of anti-CD3 treatment on Foxp3⁺ T reg cells, using some powerful new reagents: mice genetically devoid of T reg cells, mice in which T reg cells can be acutely ablated, and mice in which T reg cell detection is facilitated by fluorescent reporters. The results point in an unexpected direction: anti-CD3 appeared to act by lifting niche limitations on the size (and activity) of particular T reg cell clonotypes, through a striking and selective burst of amplification.     

CD4+ T helper cells use CD154–CD40 interactions to counteract T reg cell–mediated suppression of CD8+ T cell responses to influenza

CD4⁺ T cells promote CD8⁺ T cell priming by licensing dendritic cells (DCs) via CD40–CD154 interactions. However, the initial requirement for CD40 signaling may be replaced by the direct activation of DCs by pathogen-derived signals. Nevertheless, CD40–CD154 interactions are often required for optimal CD8⁺ T cell responses to pathogens for unknown reasons. Here we show that CD40 signaling is required to prevent the premature contraction of the influenza-specific CD8⁺ T cell response. CD40 is required on DCs but not on B cells or T cells, whereas CD154 is required on CD4⁺ T cells but not CD8⁺ T cells, NKT cells, or DCs. Paradoxically, even though CD154-expressing CD4⁺ T cells are required for robust CD8⁺ T cell responses, primary CD8⁺ T cell responses are apparently normal in the absence of CD4⁺ T cells. We resolved this paradox by showing that the interaction of CD40-bearing DCs with CD154-expressing CD4⁺ T cells precludes regulatory T cell (T reg cell)–mediated suppression and prevents premature contraction of the influenza-specific CD8⁺ T cell response. Thus, CD4⁺ T helper cells are not required for robust CD8⁺ T cell responses to influenza when T reg cells are absent.Primary CD8⁺ T cell responses often require help from CD4⁺ T cells, which produce cytokines and provide co-stimulation, including the engagement of CD40 by its ligand CD154 (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). In one model, CD4⁺ T cells engage CD40 on DCs and license them to become efficient antigen-presenting cells for naive CD8⁺ T cells (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). However, other models suggest that CD4⁺ T cells provide help to CD8⁺ T cells by activating B cells and promoting CD40-dependent antibody responses (Bachmann et al., 2004) or that they engage CD40 on CD8⁺ T cells (Bourgeois et al., 2002) and directly promote CD8⁺ T cell activation or survival.Interestingly, CD4⁺ T cell help is not required to prime all CD8⁺ T cells responses. Whereas CD8⁺ T cell responses to noninflammatory antigens are impaired in the absence of CD4⁺ T cells or CD40 signaling (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Feau et al., 2011), primary responses to some pathogens occur independently of CD4⁺ T cells or CD40 signaling (Whitmire et al., 1996, 1999; Shedlock and Shen, 2003; Shedlock et al., 2003; Sun and Bevan, 2003), possibly because of the direct activation of DCs through pathogen recognition receptors (Hamilton et al., 2001). Curiously, primary CD8⁺ T cell responses to influenza virus require CD40 signaling (Lee et al., 2003a) but not CD4⁺ T cells (Belz et al., 2002), suggesting that other cell types may express CD154 and license CD40-expressing targets in the absence of CD4⁺ T cells. Consistent with this view, activated CD8⁺ T cells (Hernandez et al., 2007; Wong et al., 2008) and natural killer T cells (NKT) express CD154 (Tomura et al., 1999) and may license DCs (Hernandez et al., 2007, 2008; Wong et al., 2008) and help B cells (Chang et al., 2012) in the absence of CD4⁺ T cells. In addition, CD154 is expressed on activated DCs (Johnson et al., 2009) and may directly activate CD40-expressing CD8⁺ T cells. However, the actual role of CD40 signaling and the cellular basis of CD40-mediated help to CD8⁺ T cells help are not fully understood.Whereas helper CD4⁺ T cells promote T and B cell responses, FoxP3-expressing CD4⁺ regulatory T cells (T reg cells) suppress them (Kim et al., 2007; Campbell and Koch, 2011; Chung et al., 2011; Dietze et al., 2011; Linterman et al., 2011). Although the potent suppressive activity of T reg cells is neutralized during infection to allow robust immune responses to pathogens, T reg cells are also involved in the late stages of immune responses to resolve inflammation and curtail immunopathology (Suvas et al., 2003; Fulton et al., 2010; McNally et al., 2011). However, the relationship between CD40-mediated CD4⁺ T cell help and the immunosuppressive activity of T reg cells in CD8⁺ T cell responses to pathogens remains unexplored.Here we determined what cells use CD40–CD154 interactions and how CD40 signaling promotes CD8⁺ T cell responses to influenza. We found that CD4⁺ T cells were the only cells to functionally express CD154 and that DCs were the only cells that required CD40 for optimal CD8⁺ T cell responses to influenza. However, rather than licensing DCs to prime naive CD8⁺ T cells, CD40 signaling was required to prevent the early contraction of the CD8⁺ T cell response. Despite the necessity for CD154 on CD4⁺ T cells, we also observed apparently normal CD8⁺ T cell responses in the absence of CD4⁺ T cells. Finally, we showed that CD8⁺ T cell responses were normal or even enhanced when T reg cells were depleted and that additional CD40 blockade did not change the CD8⁺ T cell response. Thus, our data demonstrate that CD154-expressing CD4⁺ T cells stimulate DCs through CD40 to counteract T reg cell–mediated suppression of the CD8⁺ T cell response during the contraction phase of the immune response.     

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.     

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.