Neuropilin 1 is expressed on thymus-derived natural regulatory T cells,but not mucosa-generated induced Foxp3+ T reg cells

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-β. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1^low. We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.The powerful effects of Foxp3⁺ regulatory T cells are illustrated by the devastating inflammatory diseases caused by Foxp3 mutations in mice and humans (Bennett et al., 2001; Brunkow et al., 2001; Wildin et al., 2001). As a consequence, experimental or clinical manipulation of the entire Foxp3⁺ T reg compartment could have catastrophic consequences (Kim et al., 2007).It has been proposed that, because of their different developmental origin and TCR repertoires, Foxp3⁺ nT reg and iT reg cells could have some nonoverlapping regulatory functions in vivo (Bluestone and Abbas, 2003; Curotto de Lafaille and Lafaille, 2009; Haribhai et al., 2009). It was recently shown that to completely prevent mortality and inflammation in Foxp3-deficient mice, both nT reg and iT reg cells were necessary (Bilate and Lafaille, 2011; Haribhai et al., 2011).The nonoverlapping functions of nT reg and iT reg cells raise the possibility of selective intervention strategies that would not affect all T reg cells—only nT reg or iT reg cells, or subsets of them. A major barrier to such an approach is the lack of suitable surface markers that distinguish nT reg and iT reg cell populations. The aforementioned studies addressing the issue of division of labor required very specialized strains of mice. However, these experimental systems cannot be used to identify nT reg and iT reg cells in unmanipulated WT mice. In this study, we show that surface expression of Neuropilin 1 (Nrp1) is preferentially up-regulated by nT reg cells in WT mice, and that, in contrast, iT reg cells generated under several in vivo conditions, including the physiologically relevant mucosal route, express low levels of surface Nrp1.     

Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice

The inhibitory programmed death 1 (PD-1)–programmed death ligand 1 (PD-L1) pathway contributes to the functional down-regulation of T cell responses during persistent systemic and local virus infections. The blockade of PD-1–PD-L1–mediated inhibition is considered as a therapeutic approach to reinvigorate antiviral T cell responses. Yet previous studies reported that PD-L1–deficient mice develop fatal pathology during early systemic lymphocytic choriomeningitis virus (LCMV) infection, suggesting a host protective role of T cell down-regulation. As the exact mechanisms of pathology development remained unclear, we set out to delineate in detail the underlying pathogenesis. Mice deficient in PD-1–PD-L1 signaling or lacking PD-1 signaling in CD8 T cells succumbed to fatal CD8 T cell–mediated immunopathology early after systemic LCMV infection. In the absence of regulation via PD-1, CD8 T cells killed infected vascular endothelial cells via perforin-mediated cytolysis, thereby severely compromising vascular integrity. This resulted in systemic vascular leakage and a consequential collapse of the circulatory system. Our results indicate that the PD-1–PD-L1 pathway protects the vascular system from severe CD8 T cell–mediated damage during early systemic LCMV infection, highlighting a pivotal physiological role of T cell down-regulation and suggesting the potential development of immunopathological side effects when interfering with the PD-1–PD-L1 pathway during systemic virus infections.The inhibitory programmed death 1 (PD-1)–programmed death ligand 1 (PD-L1) pathway was initially described to be involved in the induction and maintenance of peripheral tolerance, as PD-1–PD-L1 KO mice develop spontaneous autoimmune disease at the age of 6 mo (Nishimura et al., 1998, 1999, 2001; Nishimura and Honjo, 2001) and exacerbated induced autoimmunity (Dong et al., 2004; Latchman et al., 2004; Keir and Sharpe, 2005; Grabie et al., 2007; Hamel et al., 2010). Recent studies, however, suggest a novel role of the PD-1–PD-L1 pathway in the functional down-regulation of T cell responses during persistent viral, bacterial, and protozoan infections (Barber et al., 2006; Lázár-Molnár et al., 2010; Bhadra et al., 2011). This role was best studied in HIV infection in humans and in a mouse model of antiviral immunity during persistent systemic virus infections using lymphocytic choriomeningitis virus (LCMV; Wilson and Brooks, 2010).PD-1 is expressed constitutively at high levels on CD4 and CD8 T cells during HIV, SIV, hepatitis C virus (HCV), and persistent LCMV infection and expression levels were shown to correlate with the degree of T cell dysfunction (Barber et al., 2006; Day et al., 2006; D’Souza et al., 2007; Blackburn et al., 2009, 2010; Nakamoto et al., 2009; Velu et al., 2009). This persistently high expression level was observed to be driven by the sustained presence of viral antigen (Bucks et al., 2009; Mueller and Ahmed, 2009) and to significantly contribute to T cell down-regulation, as the antibody-mediated blockade of PD-1–PD-L1 signaling partially restored the function of previously unresponsive T cells (Barber et al., 2006; Day et al., 2006; Blackburn et al., 2008). As viral persistence is supposed to be intimately linked to the down-regulation of antiviral T cell responses, restoring T cell function through the blockade of PD-1 or its ligand PD-L1 is considered as a therapeutic approach to treat HIV and persistent HCV infections in humans (Urbani et al., 2008; Nakamoto et al., 2009; Velu et al., 2009; Chiodi, 2010).However, the increasing number of studies reporting PD-1–PD-L1–mediated down-regulation of T cell responses during persistent bacterial or viral infections suggests a potentially vital role of this inhibitory pathway. A growing body of evidence from mouse model systems indicates that the impairment of the PD-1–PD-L1 pathway can cause aggravated if not lethal pathology during distinct infections (Iwai et al., 2003; Barber et al., 2006, 2011; Lázár-Molnár et al., 2010; Mueller et al., 2010; Phares et al., 2010; Chen et al., 2011). Barber et al. (2006) showed that PD-L1 KO mice succumb to a systemic LCMV infection within 7 d, indicating a protective role of this pathway during the early phase of systemic infection. Likewise, Mueller et al. (2010) described a rapid development of fatal pathology in systemically infected mice that lacked PD-L1 expression on nonhematopoietic cells. Yet the pathophysiological mechanisms that contribute to pathology development under conditions of PD-1–PD-L1 deficiency have remained elusive. It also remained unknown which specific nonhematopoietic cell type required PD-L1 expression to prevent fatal pathology.In this study, we investigated the role of the PD-1–PD-L1 pathway during the early phase of systemic LCMV infection. We determined the impact of impaired PD-1–PD-L1 signaling on early virus-directed immune responses and elucidated the immunological processes that lead to fatality. We found that pathology was driven by virus-specific CD8 T cells and depended on the expression of perforin. During systemic infection, endothelial cells strongly up-regulated PD-L1 expression on their cell surface which inhibited the killing of infected endothelial cells by virus-specific CD8 T cells. PD-1 deficiency or the Ab-mediated blockade of PD-L1 facilitated endothelial cell killing, leading to increased vascular permeability and ultimately to circulatory collapse.     

Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity

The immune response plays an important role in staving off cancer; however, mechanisms of immunosuppression hinder productive anti-tumor immunity. T cell dysfunction or exhaustion in tumor-bearing hosts is one such mechanism. PD-1 has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1–PD-1L interactions has been shown to partially restore T cell function. We have found that T cell immunoglobulin mucin (Tim) 3 is expressed on CD8⁺ tumor-infiltrating lymphocytes (TILs) in mice bearing solid tumors. All Tim-3⁺ TILs coexpress PD-1, and Tim-3⁺PD-1⁺ TILs represent the predominant fraction of T cells infiltrating tumors. Tim-3⁺PD-1⁺ TILs exhibit the most severe exhausted phenotype as defined by failure to proliferate and produce IL-2, TNF, and IFN-γ. We further find that combined targeting of the Tim-3 and PD-1 pathways is more effective in controlling tumor growth than targeting either pathway alone.The importance of the immune system in protection against cancer was originally proposed in the theory of cancer immunosurveillance. This theory holds that the immune system can recognize cancerous cells as they arise and can mount both innate and adaptive immune responses to eliminate them. In support of cancer immunosurveillance is the fact that both immunodeficient or immunosuppressed patients and experimental animals are more susceptible to tumor development (for reviews see Dunn et al., 2004; Zitvogel et al., 2006; Swann and Smyth, 2007). Counter to the role of the immune system in staving off cancer is the ability of tumors to escape the immune system by engendering a state of immunosuppression (for review see Zitvogel et al., 2006). One example of a mechanism of immunosuppression present in tumor-bearing hosts is the promotion of T cell dysfunction or exhaustion.T cell exhaustion describes a state of T cell dysfunction that was initially observed during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice (Zajac et al., 1998). Exhausted T cells fail to proliferate and exert effector functions such as cytotoxicity and cytokine secretion in response to antigen stimulation. Further studies identified that exhausted T cells are characterized by sustained expression of the inhibitory molecule PD-1 (programmed cell death 1) and that blockade of PD-1 and PD-L1 (PD-1 ligand) interactions can reverse T cell exhaustion and restore antigen-specific T cell responses in LCMV-infected mice (Barber et al., 2006). T cell exhaustion also occurs during chronic infections in humans (for review see Klenerman and Hill, 2005). CD8⁺ T cells in humans chronically infected with HIV (Day et al., 2006; Petrovas et al., 2006; Trautmann et al., 2006), hepatitis B virus (Boettler et al., 2006), and hepatitis C virus (HCV; Urbani et al., 2006) express high levels of PD-1, and blocking of PD-1–PD-L interactions can restore T cell function in vitro.Several lines of evidence also implicate the PD-1–PD-L pathway in T cell exhaustion in cancer. First, PD-1 expression is found on tumor-infiltrating CD8⁺ T cells in multiple solid tumors (Blank et al., 2006; Ahmadzadeh et al., 2009; Gehring et al., 2009) and on antigen-specific CD8⁺ T cells in hosts with nonsolid tumors (Yamamoto et al., 2008; Mumprecht et al., 2009). Second, these PD-1⁺ T cells are dysfunctional. Third, PD-L1 is expressed at high levels in several different cancers (Latchman et al., 2001; Dong et al., 2002; Brown et al., 2003), and high expression of PD-L1 on tumors is strongly associated with poor prognosis (Thompson et al., 2006). Fourth, interference with PD-1–PD-L signaling, either through antibody blockade or PD-1 deficiency, has been shown to improve clinical outcome and restore functional T cell responses in several cancers (Blank et al., 2006; Yamamoto et al., 2008; Mumprecht et al., 2009; Zhang et al., 2009). However, targeting the PD-1–PD-L1 pathway does not always result in reversal of T cell exhaustion (Blackburn et al., 2008; Gehring et al., 2009) and PD-1 expression is not always associated with exhausted phenotype (Petrovas et al., 2006; Fourcade et al., 2009), indicating that other molecules are likely involved in T cell exhaustion.A recent study in patients with HIV has shown that the immune regulator T cell immunoglobulin mucin (TIM) 3 is up-regulated on exhausted CD8⁺ T cells (Jones et al., 2008). Tim-3 is a molecule originally identified as being selectively expressed on IFN-γ–secreting Th1 and Tc1 cells (Monney et al., 2002). Interaction of Tim-3 with its ligand, galectin-9, triggers cell death in Tim-3⁺ T cells. Thus, both Tim-3 and PD-1 can function as negative regulators of T cell responses. In HIV patients, TIM-3 and PD-1 mark distinct populations of exhausted cells, with cells positive for both PD-1 and TIM-3 comprising the smallest fraction (Jones et al., 2008) of CD8⁺ T cells. Similarly, another group has shown that TIM-3 is up-regulated on exhausted T cells in patients with HCV (Golden-Mason et al., 2009). In this case, cells that coexpress TIM-3 and PD-1 are the most abundant fraction among HCV-specific CD8⁺ T cells. In both studies, blocking TIM-3 restored T cell proliferation and enhanced cytokine production.Because targeting the PD-1–PD-L pathway alone does not result in complete restoration of T cell function (Blackburn et al., 2008), and in some cancers targeting the PD-1–PD-L pathway does not restore T cell function at all (Gehring et al., 2009), there is a need to identify other molecules and inhibitory pathways that are involved in T cell exhaustion. Indeed, one study has identified LAG-3 as being expressed on exhausted T cells, and although treatment with anti–LAG-3 alone did not restore T cell function in LCMV-infected mice, it synergized with PD-1 blockade to improve T cell responses and reduce viral load (Blackburn et al., 2009). Unfortunately, this study did not identify whether LAG-3 and PD-1 are expressed on distinct or overlapping populations of exhausted T cells. Given these observations, it appears that targeting multiple pathways may prove most effective in reversing T cell exhaustion.We report in this paper the coexpression of Tim-3 and PD-1 on a large fraction of tumor-infiltrating lymphocytes (TILs) in mice bearing solid tumors. TILs that coexpress Tim-3 and PD-1 predominate among CD8⁺ TILs and exhibit the most profound defects in T cell effector function. We further show that combined targeting of the Tim-3 and PD-1 pathways is highly effective in controlling tumor growth.     

Feedback control of regulatory T cell homeostasis by dendritic cells in vivo

CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells (T reg cells) maintain self-tolerance and suppress autoimmune diseases such as type 1 diabetes and inflammatory bowel disease (IBD). In addition to their effects on T cells, T reg cells are essential for maintaining normal numbers of dendritic cells (DCs): when T reg cells are depleted, there is a compensatory Flt3-dependent increase in DCs. However, little is known about how T reg cell homeostasis is maintained in vivo. We demonstrate the existence of a feedback regulatory loop between DCs and T reg cells. We find that loss of DCs leads to a loss of T reg cells, and that the remaining T reg cells exhibit decreased Foxp3 expression. The DC-dependent loss in T reg cells leads to an increase in the number of T cells producing inflammatory cytokines, such as interferon γ and interleukin 17. Conversely, increasing the number of DCs leads to increased T reg cell division and accumulation by a mechanism that requires major histocompatibility complex II expression on DCs. The increase in T reg cells induced by DC expansion is sufficient to prevent type 1 autoimmune diabetes and IBD, which suggests that interference with this feedback loop will create new opportunities for immune-based therapies.CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells (T reg cells) are essential for maintaining self-tolerance (Kim et al., 2007; Sakaguchi et al., 2008). The loss of these cells leads to a fatal autoimmune syndrome affecting multiple organs (Sakaguchi et al., 1995; Kronenberg and Rudensky, 2005). In addition, these cells interfere with the development of organ-specific autoimmune diseases, such as type 1 diabetes (Salomon et al., 2000; Tarbell et al., 2004; You et al., 2008) and inflammatory bowel disease (IBD), by silencing self-reactive Th1 and Th17 cells (Powrie et al., 1993; Izcue et al., 2006; Korn et al., 2007).Several requirements for T reg cell homeostasis in vivo have been defined. For example, T reg cells are IL-2 dependent and maintained by constant homeostatic division in response to self-antigens (Fisson et al., 2003; von Boehmer, 2003; Setoguchi et al., 2005). In addition to self-antigen recognition, which is essential for their activation and function (Thornton and Shevach, 1998; Samy et al., 2005), T reg cell survival in the periphery also requires co-stimulation through the CD28 and B7 pathway (Salomon et al., 2000). Finally, actively dividing T reg cells appear to be more suppressive than those that are quiescent (Klein et al., 2003). However, it is not known whether antigen-presenting cells (and which ones, if any) are required for maintaining T reg cells in vivo in the steady state (Denning et al., 2007; Yamazaki et al., 2008).DCs are specialized antigen-presenting cells that capture, process, and present antigens to T cells (Banchereau and Steinman, 1998). The outcome of the encounter between these two cell types depends on the activation status of the DC. In the steady state, antigen presentation by DCs leads to tolerance by T cell deletion, induction of anergy, or expansion of antigen-specific T reg cells (Brocker et al., 1997; Hawiger et al., 2001; Steinman and Nussenzweig, 2002; Hawiger et al., 2004; Kretschmer et al., 2005; Luckashenak et al., 2008; Yamazaki et al., 2008). In contrast, antigen presentation by DCs that are activated or matured by Toll-like receptor ligands, CD40 ligation, Fc receptor signaling, or inflammatory cytokines leads to protective T cell immunity (Steinman and Nussenzweig, 2002). Given the importance of DCs for immune activation, it might be expected that the loss of these cells would lead to the absence of immune responses. However, congenital DC deficiency leads instead to a complex generalized lympho- and myeloproliferative syndrome with some features of autoimmune disease, including increased numbers of granulocytes, inflammatory mediators, and possibly T reg cells (Birnberg et al., 2008; Ohnmacht et al., 2009). Additional clues that DCs are involved in T reg cell homeostasis are given in the recent report that Flt3 ligand (FL) can expand T reg cells (Swee et al., 2009), and that loss of T reg cells increases DC division by a FL-dependent mechanism (Liu et al., 2009). Whether these effects on DCs result in direct or indirect feedback changes in T reg cell homeostasis in vivo is not known (Birnberg et al., 2008). In this report, we examined whether DCs are required to maintain T reg cells in vivo and uncovered the existence of a feedback regulatory loop required to maintain physiological numbers of the two cell types in the steady state.     

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.     

MARCH1-mediated MHCII ubiquitination promotes dendritic cell selection of natural regulatory T cells

Membrane-associated RING-CH1 (MARCH1) is an E3 ubiquitin ligase that mediates ubiquitination of MHCII in dendritic cells (DCs). MARCH1-mediated MHCII ubiquitination in DCs is known to regulate MHCII surface expression, thereby controlling DC-mediated T cell activation in vitro. However, its role at steady state or in vivo is not clearly understood. Here, we show that MARCH1 deficiency resulted in a substantial reduction in the number of thymus-derived regulatory T cells (T reg cells) in mice. A specific ablation of MHCII ubiquitination also significantly reduced the number of thymic T reg cells. Indeed, DCs deficient in MARCH1 or MHCII ubiquitination both failed to generate antigen-specific T reg cells in vivo and in vitro, although both exhibited an increased capacity for antigen presentation in parallel with the increased surface MHCII. Thus, MARCH1-mediated MHCII ubiquitination in DCs is required for proper production of naturally occurring T reg cells, suggesting a role in balancing immunogenic and regulatory T cell development.Membrane-associated RING-CH1 (MARCH1) is an E3 ubiquitin ligase that mediates ubiquitination of MHCII and CD86 in DCs (Matsuki et al., 2007; Baravalle et al., 2011). This ubiquitination induces MHCII and CD86 endocytosis, lysosomal transport, and degradation (Shin et al., 2006; van Niel et al., 2006; Baravalle et al., 2011). The functional role of MARCH1 has been studied mainly in the context of DC maturation and T cell activation or regulation in vitro. When DCs are exposed to maturation stimuli, MARCH1 is rapidly down-regulated (De Gassart et al., 2008; Walseng et al., 2010). This down-regulation leads to an increase in MHCII and CD86 on the DC surface, which enhances the ability of the cell to stimulate antigen-specific T cells (Baravalle et al., 2011). In contrast, when DCs are exposed to the immune suppressive cytokine IL-10, MARCH1 is up-regulated (Tze et al., 2011; Baravalle et al., 2011). This up-regulation results in a reduction of MHCII and CD86 surface levels, and diminishes the DC’s ability to activate T cells (Baravalle et al., 2011). These studies suggest that MARCH1 plays a regulatory role in T cell activation during immune responses. However, the role of MARCH1 at steady state or in vivo is not well understood although a recent study has suggested that MARCH1 might be involved in splenic DC homeostasis (Ohmura-Hoshino et al., 2009).At steady state, MHCII plays an important role in CD4 T cell development in the thymus (Laufer et al., 1996). Furthermore, MHCII critically impacts the development of natural regulatory T cells (T reg cells), a unique CD4 T cell subset equipped with potent immune suppressive capacity (Hsieh et al., 2012). Co-stimulatory molecules, including CD86, also mediate T reg cell and NKT cell development (Salomon et al., 2000; Williams et al., 2008). Thus, given the function of MARCH1 in controlling MHCII and CD86 expression, together with the role of MHCII and CD86 in T cell development, we hypothesized that MARCH1 might be an important regulator of T cell development in the thymus. To test this hypothesis, we examined MARCH1 expression in the thymus and further examined whether its expression plays an important role in the development of specific T cell subsets.     

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.     

RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb), which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, whereas lung dendritic cells express PD-L2. Blockade of the RGMb–PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2–deficient mice showed that PD-L2 expression on non–T cells was critical for respiratory tolerance, but expression on T cells was not required. Because PD-L2 binds to both PD-1, which inhibits antitumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer, and other immune-mediated disorders. Understanding this pathway may provide insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events.Programmed death 1 (PD-l, CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are key inhibitory molecules in immune regulation (Keir et al., 2008; Pardoll, 2012). This pathway provides particularly promising targets for cancer immunotherapy (Topalian et al., 2012). There is considerable evidence that PD-L2 inhibits immunity by binding to the PD-1 co-inhibitory receptor (Latchman et al., 2001; Zhang et al., 2006). However, several studies have shown that PD-L2 can function to stimulate T cell proliferation and cytokine production, even in PD-1–deficient T cells or with PD-L2 mutants that did not bind to PD-1 (Liu et al., 2003; Shin et al., 2003; Wang et al., 2003). These findings suggest that PD-L2 may function through a receptor other than PD-1. Most studies using blocking mAbs show a dominant role for PD-L1 in inhibiting immune responses; however, PD-L2 plays a dominant role in responses such as airway hypersensitivity, experimental allergic conjunctivitis and nematode infection (Ritprajak et al., 2012). In some situations, PD-L2 dominance may be explained by preferential PD-L2 up-regulation by IL-4, but other instances may be explained by the binding of PD-L2 to a receptor other than PD-1.Here, we demonstrate that PD-L2 binds to a second receptor, repulsive guidance molecule b (RGMb). RGMb, also known as DRAGON, is a member of the RGM family which consists of RGMa, RGMb, and RGMc/hemojuvelin (Severyn et al., 2009). RGMs are glycosylphosphatidylinositol-anchored membrane proteins that bind bone morphogenetic proteins (BMPs) and neogenin (Conrad et al., 2010). RGMs do not directly signal but can act as co-receptors that modulate BMP signaling (Samad et al., 2005). RGMb is expressed and functions in the nervous system (Severyn et al., 2009). In addition, RGMb expression is observed in macrophages and other cells of the immune system (Xia et al., 2011). However, the function of RGMb in the immune system is only beginning to emerge (Galligan et al., 2007; Xia et al., 2011). RGMb-deficient mice have an early lethal phenotype (Xia et al., 2011).Here, we characterize RGMb binding to PD-L2 and identify RGMb protein expression in mouse hematopoietic cells and human cancer cell lines. Based on the critical role of PD-L2 in lung immune regulation (Akbari et al., 2010; Singh et al., 2011) and RGMb expression in the lung, we investigated the function of RGMb and PD-L2 in respiratory tolerance. Blockade of PD-L2 and RGMb interaction prevented the development of respiratory tolerance.