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.     

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.     

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.     

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.The UNAIDS/World Health Organization AIDS epidemic update estimated that 33 million people were living with HIV at the end of 2007. New infections have been occurring in the past few years at a rate of 3.2 million per year, and almost 2 million individuals succumbed to AIDS-related diseases in 2007 (UNAIDS, 2007). Most infections are acquired through sexual transmission during vaginal or anal intercourse, with semen being the major transmission vector for HIV-1 (Miller and Shattock, 2003; Pope and Haase, 2003; Haase, 2005; Lederman et al., 2006).Semen contains three major sources of infectious virus: free virions, spermatozoa-associated virions, and infected leukocytes (Miller and Shattock, 2003; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). The role of each of these sources in sexual transmission of HIV-1 is not well defined. Few studies have addressed this important question. Free virus and seminal infected leukocytes appear to play an important role in sexual transmission of HIV-1 (Miller and Shattock, 2003; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). The role of spermatozoa, however, has been a matter of debate (Mermin et al., 1991; Dussaix et al., 1993; Quayle et al., 1997; Pudney et al., 1998), in spite of the fact that the presence of viral particles and/or nucleic acids in spermatozoa from HIV-1–infected men has been largely demonstrated using a variety of techniques (Baccetti et al., 1994, 1998; Bagasra et al., 1994; Nuovo et al., 1994; Dulioust et al., 1998; Muciaccia et al., 1998, 2007; Barboza et al., 2004).The identity of the receptor for HIV-1 expressed in the spermatozoa remains unclear. On the basis of its ability to recognize the HIV gp120, it has been proposed that mannose receptors (MRs) might function as HIV-1 receptors on the spermatozoa (Bandivdekar et al., 2003; Cardona-Maya et al., 2006; Fanibunda et al., 2008). It has also been shown that the gp120 can be recognized by galactosyl-alkyl-acylglycerol, a glycolipid expressed on the spermatozoa, suggesting that, as described for keratinocytes and epithelial cells, this molecule also contributes to the attachment of HIV-1 to the spermatozoa (Brogi et al., 1996, 1998).After deposition of HIV-1 on the mucosa, the virus must cross the mucosal epithelium to interact with T CD4⁺ lymphocytes, macrophages, and DCs, which are the most important targets of infection (Mermin et al., 1991; Miller and Shattock, 2003; Haase, 2005; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). These cells express the HIV-1 receptors CD4 and coreceptors CCR5 or CXCR4 which are required for infection (Haase, 2005; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). It is now clear that DCs are able to capture HIV-1 at entry sites and transport the virus to draining lymph nodes, where HIV-1 is efficiently transmitted to T CD4⁺ cells, which become the center of viral replication (Geijtenbeek et al., 2000; Gurney et al., 2005; Wilkinson and Cunningham, 2006; Wu and KewalRamani, 2006).The pathways used by HIV-1 to cross the mucosal epithelium are not well defined. The virions may transcytose through the genital epithelium (Gupta and Klasse, 2006; Hladik and McElrath, 2008) or may pass the barrier through genital lesions, either as cell-free or cell-associated virus (Piot and Laga, 1989; Serwadda et al., 2003; Galvin and Cohen, 2004). The latter possibility could account for the association of HIV infections with sexually transmitted diseases (Miller and Shattock, 2003; Haase, 2005; Lederman et al., 2006). It is of note that epithelial microabrasions in the vagina are detected in 60% of healthy women after consensual intercourse (Norvell et al., 1984; Guimarães et al., 1997), suggesting that the presence of genital microlesions represent a frequent scenario for the transmission of HIV-1. Anal intercourse is also often associated with mucosal trauma and, because the rectal epithelium is only one cell layer thick, it provides a low degree of protection against trauma, favoring the access of virus to the underlying target cells (Shattock and Moore, 2003). Moreover, the access of HIV-1 to target cells may be facilitated by other mechanisms such as the binding of HIV-1 to DC projections that extend to the luminal surface (Pope and Haase, 2003; Haase, 2005; Wu and KewalRamani, 2006; Sharkey et al., 2007). Transmission may also be favored by the induction of a local inflammatory response triggered by semen (Pandya and Cohen 1985; Thompson et al., 1992; Berlier et al., 2006; Sharkey et al., 2007).In this paper, we analyze the interaction of HIV-1 with human spermatozoa and the ability of spermatozoa to transmit the virus to immature DCs. We found that spermatozoa capture HIV-1 and efficiently transmit the virus to DCs through a mechanism that requires cell-to-cell contacts. DC–spermatozoa contacts lead to the phenotypic maturation of the DCs and the production of IL-10 (but not IL-12). Acidic values of extracellular pH similar to those found in the vaginal mucosa after sexual intercourse markedly increased both the attachment of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs. Our results suggest that spermatozoa-associated virus may play a central role in the sexual transmission of HIV-1.     

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

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

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.     

B cell depletion reduces the development of atherosclerosis in mice

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell–depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti–oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell–derived IFN-γ secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody–mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.Atherosclerosis-related cardiovascular diseases are the leading cause of mortality worldwide. Immune-mediated reactions initiated in response to multiple potential antigens, including oxidatively modified lipoproteins and phospholipids, play prominent roles in atherosclerotic lesion development, progression, and complications (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Besides the critical requirement for monocytes/macrophages (Smith et al., 1995), adaptive immunity substantially contributes to the perpetuation of the immunoinflammatory response, further promoting vascular inflammation and lesion development (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Mice on a severe combined immunodeficiency or Rag-deficient background show reduced susceptibility to atherosclerosis under moderate cholesterol overload (Dansky et al., 1997; Daugherty et al., 1997; Zhou et al., 2000). Resupplementation of these mice with purified T lymphocytes accelerates lesion development (Zhou et al., 2000), even though it does not fully recapitulate lesion development of the immunocompetent mice. The proatherogenic T cells are related to the Th1 lineage (Gupta et al., 1997; Buono et al., 2005), and are counterregulated by both Th2 (Binder et al., 2004; Miller et al., 2008) and T reg cell responses (Ait-Oufella et al., 2006; Tedgui and Mallat, 2006).The development of atherosclerosis is also associated with signs of B cell activation, particularly manifested by enhanced production of natural IgM type and adaptive IgG type anti–oxidized low-density lipoprotein (oxLDL) autoantibodies (Shaw et al., 2000; Caligiuri et al., 2002). However, in contrast to other immune-mediated diseases, i.e., rheumatoid arthritis and systemic lupus erythematosus, B cells have been assigned a protective role in atherosclerosis (Caligiuri et al., 2002; Major et al., 2002; Binder et al., 2004; Miller et al., 2008). Although IgG type anti-oxLDL antibodies show variable association with vascular risk, circulating levels of IgM type anti-oxLDL antibodies have been more frequently linked with reduced vascular risk in humans (Karvonen et al., 2003; Tsimikas et al., 2007). In mice, IL-5– and IL-33–mediated atheroprotective effects have been indirectly associated with specific B1 cell activation and enhanced production of natural IgM type anti-oxLDL antibodies (Binder et al., 2004; Miller et al., 2008). On the other hand, splenectomy (Caligiuri et al., 2002) or transfer of μMT-deficient (B cell–deficient) bone marrow (Major et al., 2002) into lethally irradiated atherosclerosis-susceptible mice resulted in profound reduction of IgG (Caligiuri et al., 2002) or total (Major et al., 2002) anti-oxLDL antibody production, and was associated with acceleration of lesion development. These studies led to the current paradigm that overall B cell activation is atheroprotective. Surprisingly, however, whether mature B cell depletion accelerates atherosclerotic lesion development in immunocompetent mice, as expected from previous studies, is still unexplored. This is a critical question given the potentially important risk of cardiovascular complications that might arise from the clinical use of B cell–depleting CD20-targeted immune therapy in patients with severe rheumatoid arthritis or systemic lupus erythematosus, who are at particularly high risk of cardiovascular diseases (for review see Roman et al., 2001). We have therefore designed a series of experiment to address this important question.     

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.     

Activating receptors promote NK cell expansion for maintenance,IL-10 production,and CD8 T cell regulation during viral infection

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49H''s role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h^−/−, perforin 1 (Prf1)^−/−, and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h^−/−Prf1^−/−), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses.Classical (non–T) NK cells are generally found at low frequencies in leukocyte populations (Biron et al., 1999). They have the potential to mediate antiviral and immunoregulatory functions through a variety of mechanisms (Orange et al., 1995; Su et al., 2001; Lee et al., 2007; Robbins et al., 2007; Strowig et al., 2008). By altering cell availability, in vivo conditions changing NK cell numbers may indirectly influence all of their effects. Activating receptors on NK cells are linked to stimulatory pathways overlapping with those used by TCRs to drive cell expansions (Murali-Krishna et al., 1998; Pitcher et al., 2003; French et al., 2006; MacFarlane and Campbell, 2006; Biron and Sen, 2007; Lee et al., 2007) and can induce NK cell proliferation (Dokun et al., 2001; French et al., 2006). Although particular activating receptors have been reported to recognize microbial products (Lanier, 1998; Vidal and Lanier, 2006; Jonjic et al., 2008), dramatic NK cell expansion has not been observed during infections. Except under rare experimental conditions (Caligiuri et al., 1991; Yamada et al., 1996; Fehniger et al., 2001; Huntington et al., 2007a; Sun et al., 2009), NK cell division is generally induced for limited periods of time as a consequence of transient innate cytokine exposure (Biron et al., 1984; Biron et al., 1999; Dokun et al., 2001; Nguyen et al., 2002; Yokoyama et al., 2004). Increasing proportions of NK cell subsets with activating receptors recognizing particular viral ligands can be detected during certain infections (Dokun et al., 2001; Gumá et al., 2006), but this is observed without dramatic increases in overall NK cell numbers, and many viral infections induce striking reductions in NK cell functions, frequencies, and yields (Biron et al., 1999; Tarazona et al., 2002; Lehoux et al., 2004; Reed et al., 2004; Azzoni et al., 2005; Vossen et al., 2005; Morishima et al., 2006). Thus, particular conditions of viral challenges must result in differential regulation of NK cell proportions and numbers, with consequences for the delivery of NK cell functions.An NK cell activating receptor in the mouse is Ly49H (Lanier, 1998; Gosselin et al., 1999; Smith et al., 2000; Vidal and Lanier, 2006). This molecule recognizes a mouse CMV (MCMV) ligand (Arase et al., 2002; Smith et al., 2002), is expressed on NK cell subsets in strains of particular genetic backgrounds, including C57BL/6 (B6) mice, and is reported to be an exclusive marker for the classical NK cell subset (Smith et al., 2000). Through an associated molecule, Ly49H stimulates using signaling pathways overlapping with those used by the TCR (MacFarlane and Campbell, 2006; Biron and Sen, 2007). Additional markers for all NK cells include CD49b, expressed on other activated cell types (Arase et al., 2001); NKp46, selectively expressed on classical NK cells (Gazit et al., 2006; Walzer et al., 2007a; Walzer et al., 2007b); CD122, the IL-2Rβ chain, expressed on all NK cells and activated T cells (Huntington et al., 2007b); and NK1.1, exclusively expressed on C57BL6 (B6) NK and NKT cells (Lian and Kumar, 2002; MacDonald, 2002; Yokoyama et al., 2004; Huntington et al., 2007b). The TCR with associated CD3 molecules is not expressed on their cell surfaces (Biron et al., 1999). The mechanisms for NK cell–enhanced resistance to MCMV infection are incompletely characterized (Lee et al., 2007), but Ly49H contributes to their protective effects (Scalzo et al., 1990; Brown et al., 2001; Daniels et al., 2001; Lee et al., 2001; Lee et al., 2003). Engagement of the Ly49H receptor can lead to killing of target cells (Arase et al., 2002; Smith et al., 2002), and the correlation of increases in viral burdens resulting from the absence of Ly49H (Scalzo et al., 1990) as compared with those resulting from defects in cytotoxicity functions, such as mutation of the membrane pore-forming protein perforin 1 (Prf1; Tay and Welsh, 1997; Loh et al., 2005; van Dommelen et al., 2006), supports a role for Ly49H-dependent killing of virus-infected cells in the delivery of NK cell antiviral effects. The receptor may have other functions associated with its ability to stimulate proliferation, however, as the proportions of NK cells expressing Ly49H are increased during MCMV infection (Dokun et al., 2001).The studies presented in this paper were undertaken to dissect the proliferative from the cytotoxic functions accessed through Ly49H, and to define the contribution of the regulation of NK cell numbers to protection during infection. To carry out the work, responses were evaluated, during MCMV infections, in wild-type (wt) B6 mice and mice deficient in Ly49h (Fodil-Cornu et al., 2008), Prf1 (Kägi et al., 1994), or both. As expected, single Ly49h^−/− and Prf1^−/− mice had profoundly increased viral burdens, but significant differences in NK cell expansion were discovered. The NK cell populations were decreasing in infected Ly49h^−/− mice, whereas infected Prf1^−/− mice had an unexpected dramatic proliferation of NK cells uniformly expressing Ly49H. The expansion was proven to be dependent on Ly49H. During uncontrolled infection in the absence of Prf1, Ly49H beneficially promoted effects for survival, because the sustained NK cells produced IL-10 to control the magnitude of the CD8 T cell response and limit immunopathology. The data suggest that Ly49H-dependent cytotoxicity acts to control viral infection and NK cell expansion, but that in the absence of the killing function, Ly49H promotes a continued NK cell expansion critical for supporting life over death because the NK cells are available to regulate adaptive immune responses.