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Macrophages Chase Neutrophils Away From Wounds to Resolve Inflammation - Newswise (press release)

Macrophages Chase Neutrophils Away From Wounds to Resolve Inflammation - Newswise (press release) | Immunology | Scoop.it
Macrophages are best known for their Pac Man–like ability to gobble up cellular debris and pathogens in order to thwart infection.

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Journal of Cell Biology, Vol. 207, No. 5; 13POST16190005; GM074827; HL007899; GM008692

Immunology
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Cardioimmunology: the immune system in cardiac homeostasis and disease

Cardioimmunology: the immune system in cardiac homeostasis and disease | Immunology | Scoop.it
Recent studies have characterized complex interactions between resident and infiltrating immune cells in the heart and cardiac cells, including cardiomyocytes, fibroblasts and endothelial cells. This Review explores the role of immune cells in cardiac development and physiological function, as...
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Researchers find elusive source of most abundant immune cell

Researchers find elusive source of most abundant immune cell | Immunology | Scoop.it
Neutrophils—short-lived, highly mobile and versatile—outnumber all other immune cells circulating through the blood stream. Yet, despite the cells' abundance, the progenitor cell that only gives rise to neutrophils had eluded all efforts to track it down. Now, researchers at La Jolla Institute for Allergy and Immunology identified a progenitor of neutrophils in the bone marrow of mice and humans and tied it to cancer-promoting activities.
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JCI - The role of the complement system in cancer

Review Free access | 10.1172/JCI90962 The role of the complement system in cancer Vahid Afshar-Kharghan First published March 1, 2017 - More info Abstract In addition to being a component of innate immunity and an ancient defense mechanism against invading pathogens, complement activation also participates in the adaptive immune response, inflammation, hemostasis, embryogenesis, and organ repair and development. Activation of the complement system via classical, lectin, or alternative pathways generates anaphylatoxins (C3a and C5a) and membrane attack complex (C5b-9) and opsonizes targeted cells. Complement activation end products and their receptors mediate cell-cell interactions that regulate several biological functions in the extravascular tissue. Signaling of anaphylatoxin receptors or assembly of membrane attack complex promotes cell dedifferentiation, proliferation, and migration in addition to reducing apoptosis. As a result, complement activation in the tumor microenvironment enhances tumor growth and increases metastasis. In this Review, I discuss immune and nonimmune functions of complement proteins and the tumor-promoting effect of complement activation. Introduction The complement system is a cascade of serine proteases encoded by genes originating from the same ancestral genes as coagulation proteins (1). Like the coagulation system, complement activation involves several steps, is tightly regulated, and requires both plasma and membrane proteins (2, 3). Many complement proteins possess dual functions that provide crosstalk between the complement system and other effector and regulatory systems. As a result, the complement system participates in adaptive immunity, hemostasis, neuroprotection and synaptic pruning, and organ development in addition to its role in innate immunity. It is also involved in a diverse array of pathologic conditions, such as thrombotic disorders, autoimmune disorders, schizophrenia, alloimmune responses including allograft rejection and graft-versus-host disease, and cancer. The complement system’s role in fighting invasive pathogens has been extensively studied (4, 5), but recent discoveries provide new perspectives on the complement system’s function in the extravascular and interstitial tissue compartment. These discoveries illustrate an important role for complement proteins in cell-cell and stroma-cell communications. In this Review, I briefly discuss activation, regulation, immune, and nonimmune functions of the complement system to provide a framework for examining the role of complement in cancer. Activation of the complement system The complement system is activated by three major pathways: the classical pathway, via antigen-antibody complexes; the alternative pathway, via any permissive surfaces; and the lectin pathway, via binding of pattern-recognizing mannose-binding lectins (MBLs) to carbohydrate ligands on the surface of pathogens (Figure 1 and refs. 6–9). The convergence point for all complement activation pathways is the formation of the C3 convertase complex on the surface of targeted cells, summarized in Figure 1, A–C. After forming C3 convertase, complement is able to carry out its effector functions. Figure 1 Complement activation. (A) The classical pathway is initiated by a complement-fixing antibody binding to an antigen on targeted cells. C1q binds to the antibody’s Fc domain in the antibody-antigen complex. C1r and C1s assemble on C1q, C1r cleaves and activates C1s, and activated C1s cleaves C4 and C2 into C4b and C2a, respectively. C4b and C2a form the C3 convertase C4bC2a. (B) In the lectin pathway, MBL binds to repetitive sugar moieties such as mannose. MBL and MASP2 then form a C1-like complex. Activated MASP2 in MBL-MASP2 complex cleaves C4 and C2 and generates C3 convertase (C4bC2a). (C) In the alternative pathway, small amounts of hydrolyzed plasma C3 [C3(H2O)] bind to factor B, which forms the C3(H2O)Bb complex with help from factor D. C3(H2O)Bb cleaves additional plasma C3 to generate highly active C3b, which binds to cell the surface. On a complement-activating surface, C3b binds Bb (produced by factor D–mediated cleavage of factor B) and generates C3bBb (the alternative pathway’s C3 convertase). (D) Regardless of the initiation steps, C3 convertase deposits additional C3b molecules and generates C3a. If it remains intact, C3 convertase binds to additional C3b to generate C5 convertase. C5 convertase cleaves C5 to generate C5b. (E) C5b binds to C6, C7, and C8, forming a C5b-8 complex, which polymerizes several C9 molecules, forming the cytolytic MAC. In all three complement activation pathways, C3 convertase complex cleaves C3 molecules to C3a, one of the two major anaphylatoxins, and to C3b, a potent opsonin. Binding of C3b molecules to the surface of cells or cell debris in a process called opsonization marks them for phagocytosis by macrophages. Surface-bound C3b and its degradation products are ligands for complement receptors CR1, CR3, and CR2 that are expressed on myelomonocytic cells, lymphocytes, and follicular dendritic cells. Binding of C3b and its degradation products to correspondent receptors are crucial to cell-cell interactions in the innate and adaptive immune responses and in the removal of complement-coated apoptotic and necrotic cells. Propagation of complement activation by C3 convertase results in the generation of the C5 convertase complex on the cell surface. C5 convertase then cleaves C5 to C5a and C5b. C5a is a potent anaphylatoxin and recruits neutrophils to areas of inflammation and tissue damage. C5b forms a complex with C6 and C7 that may insert into cell membrane, and subsequently be joined by C8 and multiple C9 to form the membrane attack complex (MAC or C5b-9 complex; Figure 1D). Deposition of an adequate number of MACs disrupts the phospholipid bilayer of the cell membrane, leading to massive calcium influx, loss of mitochondrial membrane potential, and cell lysis. However, MAC deposition at sublytic concentrations on cell membrane has a different result, activating intracellular signal transduction and cell proliferation (10). Eukaryotic cells have developed several defense mechanisms to counteract the dire consequences of MAC accumulation at the cell surface, including expression of complement regulatory proteins (CRPs) that disassemble MAC (i.e., CD59, vitronectin, and clusterin), and endocytosis or shedding of MAC from the cell surface. Thus, the three main consequences of complement activation are tagging of cells by C3b degradation products for phagocytosis; chemotaxis of inflammatory cells in response to C3a and C5a; and MAC-mediated cell lysis. As described below, complement activation end products affect tumor growth by altering cancer cell behavior and modulating the immune response to the tumor. Regulation of the complement system The complement system’s ability to cause cellular damage is strictly controlled at several steps, both in the fluid phase and on the cell surface (6). In the classical and lectin pathways, C1 inhibitor (C1INH) binds to and inactivates C1r, C1s, and MBL-associated serine proteases (MASPs). The activities of other CRPs can be categorized into two major groups: (a) decay-accelerating activity, which breaks up the C3 convertase complex, as can be seen in C4-binding protein (C4bp), CR1, decay-accelerating factor (DAF, also known as CD55), and factor H; and (b) membrane cofactor activity, which acts as a cofactor for the factor I–mediated cleavage of C3b or C4b to their inactive degradation products, iC3b and iC4b, respectively. CRPs with membrane cofactor activity include C4bp, CR1, membrane cofactor protein (MCP, or CD46), and factor H. Another important CRP is CD59, which is expressed on many different cell types and prevents assembly of MAC on the cell membrane. The anaphylatoxins C3a and C5a are complement activation products that are rapidly inactivated in plasma by carboxypeptidases, particularly carboxypeptidase N (11). CRPs are overexpressed by many cancer cells and may be used as potential therapeutic targets. Immune function of the complement system The complement system is an ancient defense mechanism preceding adaptive immunity (12). It can be activated by pattern-recognition molecules and natural antibodies (13). Complement system activation and generation of anaphylatoxins orchestrate an inflammatory response to pathogens (12, 14). Anaphylatoxins activate macrophages, neutrophils, mast cells, basophils, and eosinophils, resulting in their degranulation and the production of cytokines, which in turn causes vasodilation, increases vascular permeability, and enhances neutrophil extravasation and chemotaxis (13). The complement system links innate immunity to adaptive immunity. Complement deficiency impairs both B and T cell responses (15). The effect of complement on the B cell response is mediated by CR2 on B cells and follicular dendritic cells. Activation of the classical pathway on the surface of an antigen tags that antigen with C3d, enabling its binding to CR2 on B cells. CR2, CD19, and CD81 form a B cell coreceptor complex, and CR2 engagement with C3d enhances signaling through antigen-encountered B cell receptors and decreases the activation threshold of B cells (12, 15). The interaction between CR2 on follicular dendritic cells and C3d on antigens is important for antigen presentation to naive and primed B cells in the germinal center of lymph nodes, in the maturation of B cells, and in the generation of memory B cells. The role of complement proteins in the cognate interaction between antigen-presenting cells (APCs) and T cells is important in the T cell immune response (16). In addition to systemic production in the liver, complement proteins are also produced locally by T cells and APCs (17–20). The effects of complement proteins on activation, proliferation, and differentiation of T cells are mediated by the local complement activation, by production of C3a and C5a at the interface of T cells and APCs, and through anaphylatoxin receptors on T cells and APCs (17–19, 21, 22). Reducing the number of C3a and C5a receptors (C3aR and C5aR, respectively) on T cells or APCs impairs T cell immunity. Complement proteins and receptors are involved in different stages of the interaction between APCs and T cells. APCs produce C3 and express C3aR and C5aR, both of which are essential for their maturation and differentiation (19) and for effective antigen presentation to T cells (17, 23, 24). C3- or C3aR-deficient APCs are much less potent in inducing a T cell immune response compared with WT APCs (19, 25). After APCs present antigen to T cells, C5aR on the T cells is required for their proliferation. Binding of C5a to C5aR on T cells has both antiapoptotic and pro-proliferative effects (22). Nonimmune function of the complement system Cell-cell and stroma-cell interactions mediated by complement proteins regulate several physiologic processes, such as collective cell migration during embryogenesis (26), synaptic pruning during brain development (27–30), cell proliferation and differentiation during liver regeneration (31) and bone development (32, 33), and hematopoietic stem cell migration and engraftment during hematopoiesis (34). Complement and cancer The surge of interest in cancer immunotherapy is mainly focused on manipulating function or number of cytotoxic T cells. However, two important reasons justify studying the role of complement activation in cancer progression and the effect of complement manipulation in cancer therapy. First, the complement system is an important component of the inflammatory response, and inflammation is involved in various stages of tumorigenesis and cancer progression (35). Second, complement activation regulates adaptive immune response (15) and might have a role in regulating T cell response to tumors. Complement system in inflammation and tumorigenesis. Tumor-promoting inflammation has an important role in carcinogenesis and cancer progression (36–38). A series of elegant experiments established that activation of the complement system is an important component of tumor-promoting inflammation. Bonavita et al. showed that C3-deficient mice were protected against chemical carcinogenesis in mesenchymal and epithelial tissues (39), mainly because of reduced inflammation. Authors identified a humoral component of innate immunity, the long pentraxin PTX3, as an important negative regulator of inflammation and complement activation. PTX3-deficient mice were susceptible to chemical carcinogenesis, displaying an increased number of tumor-associated macrophages with M2 phenotype and increased concentration of CCL2 chemokine inside tumors. The tumor-promoting inflammation induced by PTX3 deficiency was complement-dependent and completely reversed after removal of C3, as manifested by a reduction in the susceptibility of Ptx3–/– C3–/– mice to chemical carcinogenesis. Similarly, treatment with the C5aR antagonist PMX-53 reversed the susceptible phenotype of Ptx3–/– mice without affecting the rate of tumorigenesis in Ptx3+/+ mice. Activation and regulation of complement pathways in tumors. Expression of complement and CRPs is increased in malignant tumors and cancer cell lines (summarized in Table 1). Complement proteins, C3 degradation products, and complement activation products (i.e., C5a, C3a, and C5b-9) are easily detectable in various types of cancer, consistent with complement activation inside these tumors. Table 1 Complement proteins in cancer The main pathway involved in activation of complement inside tumors is unclear, and evidence supports activation of each complement pathway in malignant tumors (40). To make matters more complicated, cancer cell membrane-bound serine proteases can also cleave C5 and generate C5a without complement activation (41). Additionally, complement proteins expressed in tumors might also play a role in cancer progression independent of complement activation, as was shown for C1q in a syngeneic murine model of melanoma, where C1q expression affected angiogenesis, tumor progression, and metastasis (42). In this murine model, C1q was expressed in endothelial cells, spindle-shaped fibroblasts, and tumor-infiltrating myeloid cells independently of C4. Lack of C4 coexpression in C1q-expressing tumors hints at a role for C1q in tumor progression independent of the classical pathway. Expression of CRPs, including both membrane proteins (CD55, CD59, MCP, or CD46) and soluble proteins (factor H and factor H–like proteins), is increased in cancer cells (43), although the overexpression of CRPs is heterogeneous among different cancer types and even between different tumor specimens of the same type of cancer (44). One interpretation of the presence of both complement activation products and CRPs in tumors is that complement activation is a host defense mechanism against cancer, and cancer cells resist complement attack by overexpressing CRPs. However, as discussed later in this Review, several recent studies do not support this interpretation and suggest another scenario in which local complement activation inside tumors enhances tumor growth. Complement activation: antitumor or protumor? Evidence for the effects of complement on malignant transformation of epithelial cells and progression of cancer has evolved based on several recent studies showing complex and sometimes contradictory findings. This complexity is similar to the complex role of inflammation in cancer (45). Although inflammatory cells and cytokines are important in immune surveillance, exemplified by the benefit of bacillus Calmette-Guérin therapy in early stages of bladder cancer, chronic inflammation promotes carcinogenesis and tumor growth. Even immune cells, such as macrophages, can have both pro- and antitumor phenotypes. Despite this multifaceted picture, most evidence points toward a protumor effect of chronic inflammation (45). The long-held view of complement activation as an antitumor defense mechanism is based on two main concepts: first, the complement system’s participation in immune surveillance against malignant cells, and second, complement-dependent cytotoxicity of therapeutic monoclonal antibodies. I will discuss these concepts below, and summarize new information pointing toward a protumor effect of complement activation inside tumors. Complement and immune surveillance. The complement system’s ability to distinguish self from non-self makes it an important part of the innate immune response to invading pathogens (46). Expression of non-self antigens and lack of CRPs on microbes make them optimal targets for complement detection and, later on, complement-mediated elimination. Similarly, expression of danger signals and neoantigens by apoptotic cells and cellular debris optimizes their detection and removal by the complement system. Cancer cells, on the other hand, mostly express the same proteins as their normal epithelial cell counterparts, albeit occasionally with a different density. Furthermore, overexpression of CRPs by cancer cells limits immune surveillance by the complement system (3, 43, 46, 47). Putting these findings together, one can conclude that cell-mediated immunity plays a more important role than humoral immunity in immune surveillance against cancer cells (48, 49), and effectiveness of complement in early detection and elimination of cancer cells is uncertain (50). Complement-dependent cytotoxicity. Complement activation was considered detrimental to cancer cells via complement-dependent cytotoxicity, which causes cancer cell lysis via MAC accumulation or phagocytosis of opsonized cancer cells by macrophages and neutrophils. Complement-dependent cytotoxicity is considered to be the main mechanism for the effectiveness of antitumor monoclonal antibodies. Rituximab, an anti-CD20 antibody against malignant B cells, is among the oldest and most widely used therapeutic monoclonal antibodies. Although in vitro and in vivo studies show that rituximab activates the classical complement pathway (51, 52), the notion that its therapeutic benefits are mainly mediated by induction of complement attack on malignant B cells is questionable. In fact, the antitumor effect of rituximab was inhibited by deposited complement proteins on B cells (53), and was enhanced in complement-deficient mice (54). Therefore, the extent to which complement-dependent cytotoxicity contributes to other immunologic effects of rituximab, i.e., antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis, is unknown. Other studies on the therapeutic mechanism of rituximab also showed a complement-independent, proapoptotic effect mediated by cross-linking of CD20 (55), as well as antiproliferative and antisurvival effects that were mediated by inhibition of B cell receptors (56). Furthermore, many in vitro antitumor effects of complement-fixing antibodies on cancer cell lines were not reproduced in vivo (57). Complement activation promotes tumor growth. Considering that complement is not efficient in immune surveillance against cancer cells and that the main antitumor effect of monoclonal antibodies might not arise from complement activation, the data supporting an antitumor role for complement activation are scant. The question remains: If complement does not attack cancer cells, how does local complement activation and deposition of complement proteins affect tumors? To understand the consequence of complement activation inside tumors, it is helpful to reexamine the biological functions of complement activation products. C3b and its degradation products binding to CR1, CR2, and CR3 provide ligands and receptors for cell-cell and stroma-cell interactions in many physiologic and pathologic conditions. Complement activation generates C3a and C5a and MAC. The anaphylatoxin receptors C3aR and C5aR are G protein–coupled receptors present on many cell types, including lymphocytes, monocytes/macrophages, myeloid cells, hematopoietic stem cells, mesenchymal cells, and epithelial cells, including cancer cells. Anaphylatoxin receptor signaling has been studied extensively (58). Activation of C5aR promotes a range of responses depending on the cell type. Relevant to its role in cancer, C5aR activation generates prosurvival and antiapoptotic responses. For example, C5a binding to C5aR decreases apoptosis in neutrophils (59) and T cells (22), and increases cell proliferation in endothelial (60) and colon cancer cell lines (61). Activation of C3aR plays an important role in guiding collective cell migration (26) and epithelial-mesenchymal transition (62, 63), both important mechanisms in metastasis. In a sublytic density, MAC accumulation on the cell membrane promotes cell proliferation (64) and differentiation, inhibits apoptosis (10, 65), and protects cells against complement-mediated lysis (66). Markiewski et al. showed that the activation of the classical complement pathway inside implanted orthotopic tumors in mice enhanced tumor growth (67). Complement’s progrowth effect on tumors was C5a-dependent and was eliminated in C5aR-deficient mice and in WT mice treated with a C5aR antagonist. C5a modulates the immune response to tumors by acting as a chemotactic factor, increasing infiltration of myeloid-derived suppressor cells (MDSCs) and reducing the number of CD8+ cytotoxic T cells inside tumors. MDSCs are immature myeloid cells that increase in blood, bone marrow, and spleen of tumor-bearing mice and cancer patients (68, 69) and assist tumor cells in evading the antitumor immune response. MDSCs reduce proliferation and increase apoptosis in CD8+ T cells by generating ROS and reactive nitrogen species (70). Depletion of CD8+ T cells in mice eliminated the protective effect of complement deficiency against tumor growth. In summary, this study showed that the immunomodulatory effect of activated classical complement pathway inside tumors enhances tumor growth. The origin of complement proteins was the host, but activation of complement occurred inside the tumor microenvironment, and the final effect on the tumor was an indirect immunomodulatory effect mediated by MDSCs (Figure 2). Figure 2 Effect of complement activation in the tumor microenvironment. Activation of the complement system inside tumors releases C5a and C3a into the tumor microenvironment and promotes tumor growth. C5a attracts myeloid cell, including MDSCs, into the tumor. MDSCs then reduce cytotoxic T cell responses to the tumor by inducing apoptosis and inhibiting CD8+ TILs via generation of ROS and reactive nitrogen species and depletion of arginine. In melanoma, secretion of C3 by CD8+ TILs and complement activation in the vicinity of these cells reduce IL-10 production by TILs and inhibit their function. Some cancer cell types secrete complement proteins into the tumor microenvironment and initiate an autocrine loop that increases cell proliferation and promotes metastasis. The effect of complement activation on MDSCs, TILs, and cancer cells is mediated by the C5a and C3a receptors (C5aR and C3aR) on these cells. In a follow-up study, Nunez-Cruz et al. investigated complement’s role in tumorigenesis in a murine model of spontaneous ovarian cancer (71, 72). C3 or C5aR deficiency in these mice prevented the development of ovarian tumors, permitting no tumors or only small and poorly vascularized tumor formation (71). C3 deficiency was associated with a change in the immune profile of leukocytes infiltrating into the tumors, but C5aR deficiency reduced ovarian tumor size without altering the immune profile of infiltrating leukocytes. This result suggested the existence of a protumor effect of complement that was independent of its immunomodulatory effect. We investigated the effect of complement in murine models of ovarian cancer and confirmed activation of complement in the tumor microenvironment (73). However, complement proteins detected inside ovarian tumors originated not from the host, but from tumor cells themselves. Complement activation products were present even inside tumors implanted in C3-deficient mice lacking a functional complement system. Although orthotopic ovarian cancer tumors in C3-deficient mice reached to the same size as those in WT mice, reducing C3 or C5 production in cancer cells significantly reduced the tumor growth independent of the host’s complement sufficiency status. C3 synthesis can be detected in malignant epithelial cells originating from several different organs, particularly lung and ovary. Inhibiting synthesis of complement proteins in cancer cells altered the immune profile of leukocytes infiltrating into tumors, manifested by an increase in the number of CD8+ T cells and reduction in myeloid cells. However, immunomodulatory effect of complement inhibition was not the main mechanism responsible for the observed reduction in tumor growth. Inhibiting complement protein synthesis in cancer cells implanted in CD8+ T cell–deficient mice reduced tumor growth to the same magnitude as in WT mice. We investigated the possibility of an autocrine stimulation of cancer cells as a result of complement activation. Anaphylatoxin receptors are present on ovarian cancer cells, and stimulation of these receptors by C3a or C5a agonist peptides increased proliferation and invasiveness of ovarian cancer cells in vitro. Furthermore, knockdown of these receptors on cancer cells reduced growth of orthotopic ovarian tumors in mice. Our studies showed that local complement activation inside the tumor microenvironment enhances tumor growth via a direct autocrine effect on ovarian cancer cells increasing cell proliferation (Figure 2). In a murine model, Wang et al. reported another mechanism for the progrowth effect of complement activation in melanoma, showing that production of IL-10 by CD8+ tumor-infiltrating lymphocytes (TILs) is constitutively inhibited in an autocrine fashion by C3 originating from CD8+ TILs themselves, acting through C5aR and C3aR on the surface of these lymphocytes (74). C3aR and C5aR antagonists increased IL-10 production and activated CD8+ TILs that in turn reduce tumor growth. The IL-10–dependent antitumor activity of complement inhibitors in melanoma was independent of the PD-1/PD-L1 axis or MDSCs. This study provides evidence that local complement activation in the tumor microenvironment results in suppression of the immune response to melanoma by inhibiting CD8+ TIL function (Figure 2). The studies above describe different mechanisms by which complement activation in the tumor microenvironment can enhance tumor growth: (a) by altering the immune profile of tumor-infiltrating leukocytes, (b) by increasing cancer cell proliferation, and (c) by directly suppressing CD8+ TIL function. It is possible that different cancer types use different mechanisms to take advantage of ectopic complement activation inside tumors. For example, ovarian cancer cells synthesize a significant amount of complement proteins and initiate an autocrine loop resulting in increased cell proliferation by a direct effect of anaphylatoxins on cancer cells. Conversely, melanoma cells do not secrete complement proteins, and complement proteins produced by CD8+ TILs reduce their IL-10 production and antitumor activity. An important question remains whether complement activation has any role in malignant transformation of normal cells or only affects the expansion of already established malignant clones. Most available data are based on orthotopic murine models of ovarian cancer or mice genetically engineered to develop ovarian cancer by overexpression of oncogenes. These studies showed that complement promotes growth and expansion of malignant tumors. Bonavita et al. showed that complement promotes malignant transformation of cells exposed to chronic inflammation induced by chemical carcinogens (39). However, additional studies are required to dissect the effect of early versus late stages of complement activation on various stages of oncogenesis. Overexpression of CRPs on cancer cells If complement activation promotes tumor growth and oncogenesis, why are CRPs overexpressed on cancer cells? One would expect that cancer cells, under selective pressure, downregulate expression of CRPs to benefit from complement activation. To reconcile these seemingly counterintuitive observations, we put forward the following hypothesis: Anaphylatoxins and sublytic concentrations of MAC promote tumor growth, but higher concentrations of MAC have a tumoricidal effect. As a result, cancer cells benefit from early stages of complement activation and production of anaphylatoxins, but actively inhibit generation of MAC. Cancer cells reduce MAC concentration by overexpressing CD59, the most consistently overexpressed CRP on different cancer cells (75) and the most effective membrane regulatory protein against complement-mediated lysis (43, 76, 77), eliminating MAC from the cell surface through membrane vesiculation. Thus, from a therapeutic point of view, interventions that reduce complement activation or promote the generation of MAC inside tumors can be considered as logical options to counter the progrowth effect of complement on cancer. Complement activation in epithelial-mesenchymal transition Epithelial-mesenchymal transition (EMT) occurs in physiologic processes such as embryogenesis and organ development, and in pathologic conditions including tissue fibrosis and metastasis (78, 79). Complement participates in EMT in murine models of renal injury and fibrosis (62, 80, 81). We showed that complement activation inside tumors not only increases tumor growth but also enhances metastasis by promoting EMT in cancer cells. In ovarian cancer cells, the transcription factor TWIST1 upregulates C3 gene expression, generating C3a in the tumor microenvironment, which binds to C3aR on ovarian cancer cells. We further showed that C3aR signaling increases EMT and decreases E-cadherin expression in ovarian cancer cells via a Krüppel-like factor 5–dependent mechanism and promotes EMT and metastasis (63). In addition to promoting metastasis, EMT also induces resistance to complement-dependent cytotoxicity in lung cancer cells by increasing expression of CD59 (82). Inhibition or knockdown of CD59 restored sensitivity of cancer cells to complement-dependent lysis without altering the morphologic features or protein markers of EMT in these cells. Therapeutic potential of targeting complement activation in cancer Our understanding of the role of the complement system in cancer biology is evolving, changing our approach to the therapeutic use of reagents modifying the complement system. Traditional methods targeting cancer cells using antitumor antibodies to promote lysis of cancer cells by MACs require identification of tumor-specific antigens that either are expressed with a higher density on cancer cells than normal epithelial cells, or are only expressed on cancer cells. Antibodies against EGFR and CD20 are among the most successful therapeutic antibodies. Development of therapeutic antibodies was initially complicated by induction of an immune response to polyclonal antibodies developed in nonhuman hosts. Development of monoclonal murine antibodies; later, chimeric human-mouse antibodies; and recently, humanized antibodies (83) helps overcome this problem. However, a more important problem in harvesting complement-dependent cytotoxicity induced by therapeutic antibodies is overexpression of membrane CRPs by cancer cells that let cancer cells evade MAC-mediated cytolysis (43). As a result, blockade of membrane CRPs on cancer cells, alone or in conjunction with use of therapeutic antibodies, has been tried as another potential therapeutic strategy. Blocking CRPs reduces cancer cell proliferation in vitro (43) and tumor growth in mice (76). CD59 blocking antibodies or CD59 siRNA enhanced complement-mediated cytolysis induced by anti-EGFR monoclonal antibodies (trastuzumab and cetuximab) in human lung cancer cell lines (84). rILYd4, a recombinant protein inhibitor of CD59, increased sensitivity of malignant B cells to rituximab in vitro and in orthotopic murine models (85). Membrane CRPs are universally expressed, and an important theoretical complication of blocking CRPs is exposing normal cells to complement-dependent cytotoxicity. For example, CD59 and CD55 protect red blood cells against complement-induced hemolysis, and blocking CD59 might cause hemolysis. Interestingly, administration of rILYd4 in mice was not associated with significant increases in hemolysis (85). More recent studies showed a protumor effect of complement, and inhibition of complement activation in vitro or in murine models of cancer was investigated as a novel way to treat cancer. Understanding of the autocrine and paracrine effects of complement production and activation inside tumors versus its systemic immunomodulatory effect is not complete, but blocking complement activation or inhibiting C5aR and C3aR signaling inside tumors seems a reasonable approach. However, several questions and concerns regarding the therapeutic use of anticomplement reagents have not been addressed and require additional studies: Does systemic complement inhibition affect local production and activation of complement in the tumor microenvironment? Pharmacokinetic studies based on measurements of tissue concentration of various anticomplement reagents may resolve this issue, although the leakiness of tumor vasculature likely provides adequate tissue penetrance of these reagents. Does complement have a protumor effect in many or only in a few types of cancer? Recent studies showed that complement activation enhances growth of ovarian, cervical, and non–small-cell lung cancer and sarcoma, and deposition of complement proteins is detectable in more cancer cell types (Table 1). Which complement pathways are activated in cancer? Evidence supporting activation of classical, lectin, and alternative pathways in cancer exists. It is possible that in different types of cancer different pathways are functional. Therefore, it is more reasonable to target common complement proteins or receptors in antitumor therapies. What are the effects of early (C3a and C5a) versus late (MAC) complement activation end products on cancer cells? If higher concentrations of C3a or C5a promote, and denser MAC deposit reduces, tumor growth, developing bispecific inhibitory antibodies targeting C5aR (or C3aR) and CD59 simultaneously may increase the potency of the antitumor effect of complement therapy. Complement-dependent cytotoxicity has been considered an important component of the therapeutic benefit of monoclonal antibodies in malignant B cell disorders; however, the effects of complement activation on white blood cell dyscrasia have not been studied. A few reports point to a prognostic significance of expression of complement genes in leukemic blasts (86, 87). More comprehensive studies on the role of complement activation in leukemia and lymphoproliferative disorders might reveal possible therapeutic benefits of anticomplement reagents in these disorders. Eculizumab, a humanized anti-C5 monoclonal antibody, is currently available on the market and is used to treat paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. A single i.v. infusion of eculizumab blocks complement activation in plasma for 2–3 weeks (88, 89), but its potency and half-life in the interstitial tissue are unknown. Eculizumab blocks generation of C5a and MAC, but would not affect synthesis and secretion of complement proteins by cancer cells or C3a generation in the tumor microenvironment. Currently, no ongoing clinical trials are evaluating eculizumab in cancer patients; however, because of the clinical use of this reagent for other indications, we have a relatively clear picture of its side effect profile. Patients on eculizumab are at risk for developing infections with encapsulated microorganisms and should receive meningococcal vaccination before initiation of therapy. Lack of bone marrow suppression with eculizumab is a therapeutic advantage that can be used in designing clinical trials combining this reagent with chemotherapeutic reagents in cancer patients. In a few animal studies, C5aR antagonists, including PMX-53, have been shown to be effective in reducing tumor size in mice (67, 71), but this or similar reagents have not entered into clinical practice yet. Targeting C5aR rather than C5 or C3 might have the potential benefit of leaving opsonization and MAC generation intact. Intact opsonization of bacteria would reduce the risk of infectious complications in individuals undergoing treatment, and the generation of lytic concentrations of MAC might have a tumoricidal effect. On the other hand, targeting C5aR has the disadvantage of leaving other complement effector molecules, such as C3a, uninhibited. A potential advantage of using anticomplement reagents in cancer treatment is that they can be combined with traditional chemotherapies without increasing myelosuppression associated with chemotherapies; and combined with immune checkpoint inhibitors, because they have different targets. While checkpoint inhibitors increase proliferation of cytotoxic T cells, complement inhibitors decrease MDSCs infiltrating into the tumor microenvironment, reduce MDSC-induced T cell suppression, and enhance T cell function. Based on experiences collected with the clinical use of eculizumab, another advantage of complement inhibitors is their relatively few side effects. Any therapeutic use of anticomplement therapies in solid or liquid tumors should be carefully balanced with possible interference of complement inhibition with the efficacy of other antitumor reagents: (a) The outcome of combining anticomplement reagents with monoclonal antibodies (such as cetuximab, rituximab, or trastuzumab) may depend on the importance of complement-dependent cytotoxicity in the function of these antibodies. (b) Chimeric antigen receptor (CAR) T cell therapies depend on in vitro expansion and in vivo proliferation of T cells. Complement inhibition may decrease the proliferation of CAR T cells in vivo and may reduce their efficacy. Conclusions By mediating cell-cell and cell-stroma interactions, complement proteins have several immune and nonimmune functions in both plasma and the extravascular interstitial tissue. Activation of the complement system in the tumor microenvironment enhances tumor growth via different mechanisms. 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Become a clinical immunologist

Become a clinical immunologist | Immunology | Scoop.it
Most immunologists have a dual role as both a pathologist in the laboratory and a clinician. Some work primarily as academics pursuing research careers, while others may be scientists who do not see patients directly.
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An evolutionary perspective on the systems of adaptive immunity - Müller - 2018 - Biological Reviews - Wiley Online Library

An evolutionary perspective on the systems of adaptive immunity - Müller - 2018 - Biological Reviews - Wiley Online Library | Immunology | Scoop.it
IV. THE ORIGIN OF DARWINIAN IMMUNITY IN VERTEBRATES Above, we argued that the origin of Darwinian immunity constitutes a major transition in evolution. We now speculate on how it might have happened in the lineage of vertebrates. We propose that the transition occurred only once, before the split between jawed and jawless vertebrates, and explain why we believe that the transition was limited by a difficult evolutionary innovation, rather than the presence or absence of selection pressure for Darwinian immunity. We offer a hypothesis on the nature of the limiting innovation, and outline possible routes of stepwise evolution once the bottleneck had been passed. (1) A single origin There are good reasons to believe that the Darwinian immune systems of jawless and jawed vertebrates can be traced back to a common root, and thus that the major transition occurred only once, in a common ancestor of the two lineages. Lampreys have three distinct classes of lymphocytes that provide cellular and humoral immunity, resembling both major lineages of T cells and B cells of jawed vertebrates, respectively (Guo et al., 2009; Hirano et al., 2013). The similarities between not only functions, but also gene expression profiles suggest that the three kinds of lymphocytes are homologous between the two groups and pre‐date the divergence of jawed and jawless vertebrates (Flajnik, 2014; Kasahara & Sutoh, 2014). In addition, jawless fish have thymus‐like lympho‐epithelial structures (‘thymoids’) that are thought to serve as the sites of lymphocyte development (Bajoghli et al., 2011), and express the lamprey orthologue of the gene encoding forkhead box N1 (Foxn1) transcription factor, a marker of the thymopoietic microenvironment in jawed vertebrates. Finally, receptor diversity in jawless fish is generated by the action of enzymes that are closely related to the gnathostome activation‐induced cytosine deaminase (AID) (Rogozin et al., 2007), which is active in the diversification of B‐cell receptors. The apparent homology of multiple components of clonal selection‐based immunity between jawed and jawless vertebrates strongly suggests that the roots of the system originated in the common ancestor of all vertebrates. (2) Chance or necessity The apparently unique origin of Darwinian immunity can be explained in two possible ways. Either, the transition involved a difficult (i.e. low‐probability) event that occurred only once, ‘by chance’, in a common ancestor of all vertebrates [classifying this transition as ‘variation‐limited’ (Számadó & Szathmáry, 2006)]; or, the selective forces that favour the emergence of Darwinian immunity appeared first (and only) in vertebrates and have then driven, ‘by necessity’, the stepwise evolution of the system [in the frame of a ‘selection‐limited’ transition (Számadó & Szathmáry, 2006)]. Historically, the discovery of the intricate molecular mechanisms of V(D)J recombination (the only mechanism of somatic receptor diversity then known) led researchers to favour the first alternative, assuming a once‐only low‐probability event for the origin of this system. Marchalonis & Schluter (1990, p. 16) termed this event ‘a “Big Bang” because sophisticated rearranging systems consisting of multiple elements appear in a fully functional form without foreshadowing in the antecedent species’. This notion was further strengthened by the recognition that the molecular machinery of V(D)J recombination likely arose by the integration of recombination‐activating genes (RAGs) into the vertebrate genome by horizontal gene transfer (Bernstein et al., 1996; Fugmann, 2010). However, subsequent discoveries have challenged the key role of V(D)J recombination in the origin of Darwinian immunity. First, the discovery of RAG1/2 in sea urchins (Fugmann et al., 2006) suggested that the original horizontal gene transfer event must have preceded the origin of vertebrates. Second, we now know that RAG‐mediated V(D)J recombination [enhanced with non‐templated nucleotide addition diversity (Kallenbach et al., 1992)] is far from being the only mechanism that can generate somatic receptor diversity. Jawless fish generate receptor diversity by RAG‐independent gene conversion (Nagawa et al., 2007), subsets of immunoglobulin (Ig) genes in some jawed vertebrates (sharks, birds, rabbits, sheep) rely heavily on gene conversion and hypermutation to generate antibody diversity (Flajnik & Kasahara, 2010), and invertebrate systems of shotgun immunity generate somatic diversity by gene conversion, alternative splicing, RNA editing, post‐translational modifications, and possibly even somatic recombination (Ghosh et al., 2011). Mechanisms of somatic diversity have thus evolved multiple times independently, and are unlikely to be a limiting ‘bottleneck’ in the evolution of Darwinian immunity. Although it cannot be ruled out that the evolution of V(D)J recombination in particular might have been triggered by a second (intragenomic) transposition event that inserted the RAG transposon into a variable innate immune receptor gene (Koonin & Krupovic, 2015), this can no longer be regarded as ‘the Big Bang’ of adaptive immunity, but rather as one of several ‘smaller bangs’ (Bartl et al., 2003; Flajnik, 2014). Kasahara (1997, 1998) argued that the triggering event of the ‘Big Bang’ might have been the one or two rounds of whole‐genome duplication (WGD) that occurred close to the origin of vertebrates (Smith et al., 2013; Smith & Keinath, 2015). This event duplicated many genes related to immunity, and it ‘might have provided unique opportunities to create many accessory and effector molecules of the adaptive immune system’ (Kasahara et al., 1997, p. 92). We will return to this idea in Section IV.4, proposing possible scenarios as to how the WGD event might have triggered the origin of Darwinian immunity. In turn, several studies have argued against the key role of a single triggering event. Klein & Nikolaidis (2005, p. 174) [along the lines of an earlier argument by Bartl et al. (2003)] favour gradual evolution that ‘consisted initially of changes unrelated to immune response that were selected to serve other functions’ and that, by chance, attained a combination that integrated the elements into a new function giving rise to adaptive immunity. Litman, Rast & Fugmann (2010) also emphasized co‐option and redirection of pre‐existing systems as the main source of innovation, at the same time perceiving ‘no reason to assume that vertebrates require a complex immune system any more than do complex invertebrates’ (Litman et al., 2010, p. 552). However, if the origin of Darwinian immunity is not dependent on a ‘difficult’ (i.e. low‐probability) transition, then vertebrates must have some specific traits that favour Darwinian immunity in this group, but are absent from others. (3) Selective scenarios: not exclusive to vertebrates Long lifespan (Klein, 1989; Lee, 2006) and slow reproduction (Flajnik, 1998; Lee, 2006; Flajnik & Kasahara, 2010), high metabolic intensity (Rolff, 2007; Sandmeier & Tracy, 2014), efficient closed circulation (van Niekerk, Davis & Engelbrecht, 2015), low population density (Klein, 1989) and large (Klein, 1989; Flajnik & Kasahara, 2010) or morphologically complex (Boehm, 2012) bodies have been invoked as factors favouring (Darwinian) adaptive immunity. However, these traits are not exclusive to vertebrates, and, in fact, the last common chordate ancestor (and therefore also the ancestral vertebrate) was probably a lancelet‐(amphioxus‐)like creature (Lowe et al., 2015): small, not particularly long‐lived, and rather inconspicuous. The most extensive phylogenetic analysis so far estimated that the lineages of jawed vertebrates and jawless fish diverged about 650 million years ago (Blair & Hedges, 2005). While molecular clock estimates might be sensitive to assumptions on the tempo and mode of evolution, fossil evidence of two distinct types of jawless fish dated to around 520 million years ago (Shu et al., 1999) confirms that the split must have occurred before or shortly after the Cambrian Explosion: Darwinian immunity must therefore have provided a selective advantage already in the Precambrian or early Cambrian world of small body sizes and simple body plans. Many extant invertebrates very likely surpass the last common ancestor of vertebrates in both size and life expectancy, and yet (to our current knowledge) lack Darwinian immunity. Cephalopods can have large bodies and long lifespan, but Darwinian immunity (clonal selection acting on heritable somatic receptor diversity) has not been found in the group (Castellanos‐Martínez & Gestal, 2013). It must nonetheless be noted that the species investigated so far have been octopuses that have short lifespans; studies of immunity in Nautilus species that can live for several decades (Saunders, 1984) are much awaited. Some further ancestral traits of vertebrates might also have facilitated or favoured the evolution of Darwinian immunity. A closed circulatory system, which seems to be an ancestral chordate character (Stach, 2008), may well be a prerequisite of effective immune surveillance by lymphocytes; however, cephalopods also have a closed circulation. Filter feeding seems to be an ancestral trait for deuterostomes (Gans & Northcutt, 1983; Yu & Holland, 2009; Lowe et al., 2015), and is present in echinoderms (sea urchins, sea cucumbers), tunicates (sea squirts), and also cephalochordates (amphioxus), which are thought to most closely resemble the common ancestor of vertebrates (Gans & Northcutt, 1983; Yu & Holland, 2009). The evolution of this lifestyle probably generated selection pressure for improved immunity (to fight pathogens, and to avoid unnecessary or harmful responses to the myriad harmless microorganisms in the filtrate). Echinoderms (Hibino et al., 2006), amphioxus (Huang et al., 2008) and, independently, also mussels (Gerdol & Venier, 2015) and sponges (Degnan, 2015), the most ancient group of filter‐feeding organisms, took the path of expanding their repertoire of innate pattern‐recognition receptors. While expanded innate receptors indeed imply selection pressure for improved immunity, the defining traits of Darwinian immunity have not been found in any of these groups to date. It has also been noted that vertebrates harbour more complex microbiomes than invertebrates, which tend to have either relatively simple microbial communities or rely on microbial partners that are shielded from immunity within the cells or in compartments enclosed in physical barriers (McFall‐Ngai, 2007). Managing a complex microbiome has been invoked as a selection pressure that may have driven the evolution of Darwinian immunity specifically in vertebrates (Pancer & Cooper, 2006; Weaver & Hatton, 2009; Lee & Mazmanian, 2010; Boehm, 2012). However, this explanation only leads one step back, to another question: why would vertebrates be special in terms of needing a complex microbiome? We find it more plausible that Darwinian immunity evolved for another reason (a rare event that opened up a difficult evolutionary path), and could then enable the acquisition of a more complex microbiome – which then might have provided an evolutionary edge to vertebrates. A further hypothesis has been proposed by Pancer & Cooper (2006, p. 512), who posited that novel selection pressure might have arisen at the origin of vertebrates because a large arsenal of innate receptors ‘presented serious autoimmunity problems at a time of rapid developmental and morphologic innovation’, and rapid changes in the endosymbiotic communities might also have occurred. As a consequence, the complexity of the innate immune system might have been reduced, creating increased selection pressure for the evolution of an alternative system. However, innate receptors, even those belonging to complex families, tend to target classes of molecules that are not present in the host, and the complexity of the vertebrate body plan increased not so much by expanding the set of molecular building blocks, but rather by regulatory and organizational complexity (Heimberg et al., 2008; Lowe et al., 2011). Such an evolutionary trajectory would not have raised the risk of autoimmunity by innate recognition. It is also unclear why the evolution of vertebrate characteristics would have generated a selection pressure for rapid shifts in the microbiome, sufficiently strong to compensate for drastically reduced (innate) immune defence against pathogens. The discoverer of clonal selection, Burnet himself entertained the idea that it might have been the increased developmental flexibility of vertebrates that created the selection pressure for adaptive immunity (Burnet, 1968). He argued that flexible development resulted in an increased risk of cancer, and the threat from the ‘modified self’ of tumours called for a mechanism that was itself variable and adaptable. However, Darwinian immunity requires reliable mechanisms of immune tolerance to be able to target patterns that are similar to those found in the host self. As we will explain in later sections, it is likely to have started targeting motifs that showed relatively small similarity to host motifs, and could expand to riskier targets only as gradual evolution improved the specificity of targeting and the capacity for antigen‐specific tolerance. Distinguishing tumours from normal self is likely to be the most challenging task for Darwinian immunity that could only be added at advanced stages of its evolution – it cannot have been the initial trigger. Finally, we note that extant vertebrates encompass huge diversity in terms of lifestyles, body size (from shrews to the blue whale) and lifespan (from weeks to >100 years), and while some species have lost or simplified elements of adaptive immunity, the presence of clonal selection‐based Darwinian immunity seems ubiquitous across this dizzying diversity of size and form. (Moreover, the species with reduced adaptive immunity do not seem to follow any discernible pattern of size or lifestyle: these examples may simply reflect stochastic loss in some lineages). Considering that most components of vertebrate Darwinian immunity appear to be scalable in terms of diversity, and a higher diversity of innate immune recognition would probably be quite straightforward to re‐evolve [indeed Atlantic cod (Gadus morhua) have lost MHC class II and have expanded their innate Toll‐like receptor (TLR) repertoire (Star et al., 2011)], the ubiquitous maintenance of Darwinian immunity in vertebrates suggests that this type of adaptive immune defence provides benefits across a very wide range of life‐history parameters. It is hard to see how this wide range also would not cover the lifestyles of a large number of invertebrate species. To conclude, while a number of life‐history traits likely exerted selection pressure on the ancestral vertebrate to develop sophisticated immunity, and some features of the vertebrate body plan might have acted as necessary pre‐adaptations, none of these selection pressures and physical traits seem to be exclusive to this group, and Darwinian immunity would likely be beneficial for many invertebrates as well. We therefore argue that a key piece of the puzzle is still missing: there must have been a difficult evolutionary innovation that emerged, as far as we know, only in vertebrates. (4) Immunological Big Bang 2.0 What had to be invented for the transition from the invertebrate immunity of an amphioxus‐like ancestor to Darwinian vertebrate immunity? The necessary components for the somatic generation of receptor diversity were all in place: amphioxus has RAG1 (Huang et al., 2014; Zhang et al., 2014) and proto‐MHC (Abi‐Rached et al., 2002); sea urchins have RAG1/2 (Fugmann et al., 2006); and the presence of orthologous ancestral genes in both jawed and jawless vertebrates indicates that the vertebrate ancestor had both BCR/TCR and VLR precursors (Flajnik & Kasahara, 2010). In addition, lymphocyte‐like cells have been found in amphioxus (Huang et al., 2007), along with homologues of several genes that are active in immune signalling in the Darwinian immunity of vertebrates (Yu et al., 2005), and recently discovered innate lymphoid cells in mammals perform many functions associated with T cells without expressing T‐cell receptors (Walker, Barlow & McKenzie, 2013). These cells can be induced by microbial products, and NK cells that bear germline‐encoded antigen receptors (specific, e.g. for conserved structures of viruses) establish immune memory by the survival of an amplified cell population (O'Sullivan et al., 2015), possibly constituting a system of proto‐Darwinian immunity. Similar lymphocyte‐like cells bearing germline‐encoded receptors might have existed in the ancestral vertebrate [innate lymphoid cells might be present in jawless fish, as well (Eberl, Di Santo & Vivier, 2015)], and might already have possessed both the genetic circuitry required for pathogen‐induced proliferation and antimicrobial effector mechanisms. In addition to these pre‐existing components, clonal selection‐based Darwinian immunity requires two key properties (Du Pasquier, 2006). First, as recognized very early by Burnet (1970), monoallelic (or at most oligoallelic) expression of the somatically generated, clonally heritable antigen receptors is needed to allow for specific amplification (clonal selection) of an appropriate response. Stable expression and clonal heritability are required to maintain targeting specificity over time and across cell divisions; monoallelic expression is necessary to prevent the simultaneous presence of useful and useless or harmful receptors on the same cell, which would greatly abrogate the efficiency of clonal selection. Second, antigen‐specific immune tolerance is needed to avoid autoimmunity when a somatically generated receptor responds to a molecular pattern of the host (‘self’). We propose that the evolution of antigen‐specific immune tolerance is a difficult (low‐probability) transition that requires major innovations in gene regulation, and therefore imposes a critical bottleneck in the evolution of Darwinian immunity. We argue that in the evolution of vertebrates this transition was made possible by an abrupt increase in regulatory complexity [precipitated by a WGD event and a series of segmental genome duplications (Smith et al., 2013; Smith & Keinath, 2015)] before the divergence of jawless and jawed vertebrates, and once this difficult transition had been achieved, pre‐existing mechanisms of somatic receptor diversity could quickly be co‐opted for clonal selection. We term this concept the ‘Immunological Big Bang 2.0’, and below provide further arguments in its support. Of the two components of the transition, monoallelic expression of receptor genes does not seem to be particularly difficult to evolve. In addition to the antigen receptors of lymphocytes in jawed and jawless vertebrates (Pancer et al., 2004), monoallelic expression occurs in many mammalian genes not associated with immunity (Nag et al., 2013), while inhibitory receptors on mammalian NK cells (Cichocki, Miller & Anderson, 2011) are characterized by the stochastic expression of a subset of receptor genes from a larger germline‐encoded repertoire, and the 185/333 immune‐response genes expressed in sea urchin coelomocytes (a type of immune cell) display near‐monoallelic expression from a set of germline‐encoded alleles (Majeske et al., 2014). However, amplifying lymphocytes with ‘random’ (i.e. somatically generated) receptors carries the risk of autoimmunity – which brings us to the necessity of antigen‐specific tolerance for clonal selection‐based Darwinian immunity. Whereas an autoreactive response without amplification inflicts damage analogous to a fixed dose of a toxic substance, an amplifiable response is analogous to an infectious agent that can multiply and do great harm even at a very low initial dose. As soon as clonal amplification extends to immune recognition motifs that can potentially target self patterns, protective mechanisms are needed to neutralize effector cells based on their self‐reactive targeting specificity. Two main mechanisms operate in jawed vertebrates: clonal deletion (‘negative selection’) removes autoreactive cells during the maturation of lymphocytes (Palmer, 2003) to enable ‘recessive tolerance’ (tolerance by the absence of autoreactivity); by contrast, regulatory T cells (Tregs) enable ‘dominant tolerance’ by actively downregulating autoreactive immune responses in the targeted tissues (Coutinho et al., 2001; Sakaguchi, 2004). Both mechanisms are based on intricate gene regulation mechanisms that are likely to be difficult to evolve, and the (near) simultaneous appearance of both systems is highly unlikely. We propose that the ‘Big Bang’ of vertebrate immunity might have been triggered by the evolution of Treg‐mediated dominant tolerance, facilitated by the greatly increased potential for regulatory complexity following the WGD event that gave rise to vertebrates. Below we explain why dominant, rather than recessive tolerance might have been the key innovation, and show that its main genomic components probably originated at or near the WGD event. We argue that reliable immune tolerance can be achieved by Treg‐mediated dominant tolerance, but not by negative selection alone. Both mechanisms are necessarily imperfect (and must have been even less efficient in the beginning), but there is an important difference in the way the two mechanisms can ‘fail’. Imperfect negative selection is imperfect in terms of coverage: some auto‐reactive clones escape selection; imperfect dominant tolerance is imperfect in terms of degree: all autoimmune reactions are affected, but the degree of control is limited. In the former case, a single escaped clone could wreak havoc without additional control by Tregs in the peripheries, because repeated rounds of clonal expansion would induce exponential growth of the autoimmune reaction. By contrast, imperfect dominant tolerance can afford mistakes, because a self‐reactive clone activated by a stochastic glitch in tolerance could still be brought under control later: negative selection has one chance to act, dominant tolerance has many. To suffice alone, negative selection should be perfect; dominant tolerance just needs to be ‘good enough’ to have a statistically high chance of bringing self‐reactive clones under control before they can do too much damage. We therefore argue (in agreement with Janeway, 2001) that the evolution of regulatory T cells (dominant tolerance) was probably necessary for the emergence of Darwinian immunity. Once dominant tolerance jumpstarted the evolution of Darwinian immunity, the evolution of mechanisms for negative selection against major self‐antigens could provide an economical advantage, removing highly autoreactive cells before they had their first chance to expand. If dominant immune tolerance was a necessary innovation to achieve Darwinian immunity, it certainly cannot have been an easy one. Foxp3 acts as a central switch: it forms complexes with hundreds of genes (Rudra et al., 2012), and affects the expression of more than 2000 genes in mouse T cells (Xie et al., 2015). The task is indeed not trivial. Foxp3+ Tregs often have to respond in the opposite reaction compared with conventional (non‐regulatory) effector T cells: TCR signalling (with co‐stimulation) induces effector functions in conventional T cells, but repressor functions acting on neighbouring T cells in Tregs. In addition, regulatory activity must strike a delicate balance between too little regulation resulting in runaway autoimmunity, and too much, which could downregulate useful responses against pathogens (self antigens are also presented in the vicinity of pathogen invasion). To achieve this complex functionality, Foxp3 acts not only as a repressor of activation‐associated genes, but also upregulates a large number of genes (Zheng et al., 2007), and is likely to operate a bistable autoregulatory loop to maintain a stable identity of regulatory cell clones (Rubtsov et al., 2010). The complexity and difficulty of the task supports the notion that Treg‐mediated tolerance might indeed constitute the major bottleneck towards Darwinian immunity that, in vertebrates, could only be passed by a rare burst of regulatory complexity. Phylogenetic evidence is compatible with the origin of Treg‐mediated dominant tolerance in the vertebrate common ancestor. Foxp3, the key regulatory gene for the development of regulatory T cells (Hori, Nomura & Sakaguchi, 2003), belongs to the ancient eukaryotic family of Forkhead box (Fox) transcription factors. Remarkably, the Foxp class of the family has a single orthologue in invertebrates (including sea urchin), but four members in most vertebrates (Andersen, Nissen & Betz, 2012), which is consistent with the origin of the class at the WGD event [followed by segmental duplication involving Foxp loci (Smith & Keinath, 2015)]. The analysis of the sea lamprey (Petromyzon marinus) genome identified homologues of Foxp1, 2 and 4, but did not find Foxp3 (Smith et al., 2013). However, Foxp3 is most closely related to Foxp4, and both were created by the last gene duplication in the family (Santos et al., 2011). The Foxp4 ortholog identified in lamprey might therefore be homologous to the common ancestor of Foxp3 and Foxp4 [a situation with known precedents among duplicated transcription factors (Kasahara & Sutoh, 2014)], and might perform the regulatory role of Foxp3 in jawless fish. While Foxp3 is at the top of the regulatory cascade of dominant tolerance, the evolution of this complex regulatory function likely required the involvement of a whole suite of regulatory genes – which may have depended on the sudden availability of duplicated genes in the ancestral vertebrate. Of note, the transcription factors Helios and GATA‐3, which are key interacting partners of Foxp3 in the orchestration of the regulatory phenotype (Rudra et al., 2012; Kim et al., 2015), both belong to gene families that were duplicated in the WGD event (Gillis et al., 2009; John, Yoong & Ward, 2009). Another member of the Foxp class, Foxp1 is involved in the regulation of B‐ and T‐cell development and homeostasis (Hu et al., 2006; Feng et al., 2010), and further classes of duplicated regulatory genes might also have contributed to the expanding genetic circuitry of immune cell fates (Rothenberg & Pant, 2004; John et al., 2009). In addition to duplicated transcription factors, the increased regulatory complexity of vertebrates arose partly from a massive increase in microRNAs (miRNAs) in the stem lineage of vertebrates (preceding the split between jawless and jawed vertebrates), both due to genome duplication and to the acquisition of new miRNA families (Heimberg et al., 2008, 2010). miRNAs play multiple complex roles in the development and control of vertebrate adaptive immunity (Xiao & Rajewsky, 2009; Mehta & Baltimore, 2016), including mechanisms of both central and peripheral tolerance (reviewed in Simpson & Ansel, 2015). In particular, the selective disruption of miRNAs in Tregs results in autoimmune pathology closely resembling that caused by deficiency in Foxp3 (Zhou et al., 2008), while the selective knockout of miRNAs in thymic epithelial cells compromises promiscuous gene expression (Ucar et al., 2013) that is crucial for the thymic induction of tolerance against peripheral self‐antigens. By contrast, V(D)J recombination does not seem to require miRNA control (Xiao & Rajewsky, 2009). Compatible with our scenario, the operation of specific immunological tolerance depends on regulatory complexity acquired at the origin of vertebrates, but the generation of receptor diversity does not. Thus many components of the genetic circuitry (transcription factors, miRNAs) seem to have appeared in the series of genomic duplications that occurred at the root of the vertebrate lineage. Since duplicated genes tend to get inactivated then lost unless they acquire new functions, the integration of a large number of elements, duplicated within a short time frame, is consistent with a rapid, ‘Big Bang’ like episode of evolution. Conversely, the construction of the highly complex genetic circuitry of dominant tolerance might have depended on the simultaneous presence of a large number of recently duplicated elements. The analysis of the gene regulatory networks (Martinez‐Sanchez et al., 2015) might eventually elucidate how the duplicated regulatory elements might have triggered the evolution of a Treg cell phenotype. We have thus argued that dominant immune tolerance might be a necessary condition for Darwinian immunity, that the regulatory circuitry required for this function might be very difficult to evolve, and that in vertebrates the origin of the involved genetic machinery apparently goes back to the rare burst of genomic innovation that gave rise to the lineage. Thus, while Burnet (1968) believed that the greater flexibility of development in vertebrates created the selection pressure for adaptive (Darwinian) immunity, we suggest that it created not the need, but the opportunity. However, we note that the presence of Tregs in jawless fish still needs to be demonstrated, and while it is plausible to assume a crucial role of dominant tolerance in Darwinian immunity, the evidence is not unequivocal. We argued that both somatic receptor diversity and clonal selection might have had pre‐existing components, and it was the linking of the two that required a difficult evolutionary innovation: specific (and probably dominant) immune tolerance. However, while the existence of multiple mechanisms of somatic diversity has clearly been demonstrated, clonal selection (amplification) has not been described in any invertebrate to date. It is possible that the machinery for clonal amplification by itself is difficult to evolve, and we cannot exclude that it was this step that imposed a bottleneck for the evolution of Darwinian immunity (L. Du Pasquier, personal communication) that could only be passed by the increased regulatory complexity of vertebrates. We also note that the ‘burst of regulatory complexity’ at the root of the lineage is not quite straightforward to explain. WGDs have occurred rarely, but still multiple times in animals, and much more frequently in plants (Otto & Whitton, 2000). However, while some of these events have given rise to successful new clades and/or duplicated regulatory factors, the origin of vertebrates appears to be unique with respect to the number of regulatory elements retained, and the abrupt increase in regulatory complexity and developmental flexibility that accompanied it. It remains to be elucidated what additional factors (selection pressures, pre‐adaptations, low‐probability genomic events) might have contributed to the rare constellation of conditions that allowed for the rapid increase in regulatory complexity that very likely laid the foundations for the evolutionary success of vertebrates, and opened the trajectory towards Darwinian immunity. To summarize, the lack of a selective scenario specific to vertebrates argues very strongly for a ‘Big Bang’‐type origin of Darwinian immunity, limited by a difficult evolutionary innovation; the abrupt increase in regulatory complexity at the origin of vertebrates was very likely a prerequisite (and possible trigger) to passing this bottleneck; and antigen‐specific dominant immune tolerance is a plausible (but not the sole) candidate for the limiting evolutionary innovation. (5) The chicken and egg problem of Darwinian immunity Beyond the initial bottleneck for Darwinian immunity, an apparent chicken and egg problem arises. Clonal amplification of immune responses with stochastic (somatically diversified) targeting is unsafe without specific (dominant) tolerance; however, specific tolerance might not make much sense without stochastic immune targeting. We argued previously that the emergence of dominant tolerance might have been the key to the evolution of Darwinian immunity in vertebrates – but what drove it in the first place? If specific tolerance evolved against the backdrop of innate or shotgun immunity that did not allow for the clonal amplification of somatically diversified immune responses, what was then the initial selective advantage? There are two ways to resolve this apparent paradox. First, some limited form of clonal amplification, involving immune responses with a limited scope of diversified targeting, might be beneficial even without specific tolerance, if the benefits of improved defence outweigh the costs (including some limited auto‐immunity). Below we shall discuss possible incremental stages in the evolution of randomized immune targeting: it is not impossible that the very first steps could be taken without dominant immune tolerance. In this scenario, a slightly enhanced form of proto‐Darwinian immunity (with restricted somatic diversification, and amplification limited in both space and time) might have preceded the emergence of dominant immune tolerance, and mitigating the low‐level auto‐immunity associated with the former might have provided an immediate evolutionary benefit. If this is true, this level of proto‐Darwinian immunity should eventually be found in extant invertebrates [the near‐monoclonally expressed 185/333 immune‐response genes of sea urchins (Majeske et al., 2014) might constitute a candidate system]. Note that while this scenario somewhat blurs the line between shotgun immunity and Darwinian immunity, a large gap still remains, and bridging that gap very likely required the evolution of specific immune tolerance. Alternatively, Treg‐mediated dominant tolerance might have evolved first to afford specific tolerance to beneficial symbiotic bacteria (Weaver & Hatton, 2009). Innate and shotgun immunity tend to target broad classes of conserved microbial patterns and cannot discriminate and selectively spare potentially beneficial species. By providing this function (downregulating innate mechanisms with narrow targeting), specific tolerance, even in early rudimentary forms, might have provided an immediate benefit even in the absence of somatically diversified immune effector targeting. Remarkably, the gut of extant vertebrates (mice), which holds the largest diversity and biomass of the microbiome, is enriched in Tregs that are reactive to commensal microbes and are essential for the maintenance of immune tolerance against these (Chai, Zhou & Hsieh, 2014; Sefik et al., 2015). Improved microbiome management might afford a huge metabolic benefit (McFall‐Ngai, 2007), and is thought to have been a major driver of immune evolution from the earliest animals (Bosch, 2014). In this scenario, Tregs might even have been the first cell type to evolve somatically diversified targeting, which could then be co‐opted for effector targeting, as the broadening scope of specific tolerance allowed it. Because the generation of regulatory cells depends on an education period when they encounter antigens under non‐inflammatory conditions, the scope of specific tolerance under this scenario could easily be extended to cover self‐antigens. (6) Stepwise evolution of Darwinian immunity after the ‘Big Bang’ After the emergence of an early form of specific immune tolerance, the subsequent evolution of vertebrate Darwinian immunity could proceed in small incremental steps, increasing the potential of somatic receptor diversification and clonal amplification to match and drive further the improving capacity of specific tolerance. We consider in turn how the scope and potential of somatic receptor diversification, clonal amplification and specific immune memory, and specific tolerance might have evolved through a series of gradual improvements. Receptor targeting might have evolved in terms of broadening epitope coverage, shifting from germline‐encoded receptors to increasing somatic diversification, and towards higher specificity. Mechanisms of receptor diversification very likely existed even before the immunological ‘Big Bang’ (Loker et al., 2004), either to generate shotgun immunity [e.g. by somatic hypermutation (Du Pasquier et al., 1998; Lee et al., 2002)] or expressed in the germline to generate variation rapidly across generations. LRR‐ and RAG/Ig‐based systems of gene assembly might also have had their origin at this stage, e.g. the sea urchin homologues of Rag1/2 are expressed in coelomocytes that perform immune functions (Fugmann et al., 2006), and RAG transposition still appears to play a role in generating germline‐encoded receptor diversity across generations in sharks (Lee et al., 2000; Hsu et al., 2006). Such pre‐existing mechanisms of receptor diversity could then be conveniently co‐opted for clonally selected lymphocytes once specific tolerance had appeared. Initially, the germline‐encoded receptors must have targeted safe molecular patterns that were reliably associated with potential pathogens but were absent from the host species (Ohno, 1990), and the scope of somatic diversification in the frame of shotgun immunity must have been optimized (limited) to keep the repertoire within these safe boundaries. Then, after the ‘Big Bang’, the gradual improvements of specific tolerance allowed these safe boundaries to expand, and the mechanisms of somatic receptor diversity had multiple ways to take advantage of this opportunity and expand accordingly (Fig. 4). First, germline‐encoded receptor genes (or their modular components) might have expanded by gene duplication and divergence to allow the targeting of novel domains in the ‘epitope space’ of possible targeting motifs. Second, the extent of somatic diversification (the possible distance from the germline‐encoded target specificities) might also have increased gradually, e.g. by increasing the rate of hypermutation or by expanding the genomic regions affected. The first mechanism could create new foci of epitope targeting, while the second could increase the action radius of existing foci in epitope space. Both would allow immune targeting to expand gradually into domains of epitope space that used to carry a high risk of autoimmunity, but were becoming safe due to improving specific tolerance. Mechanisms based on gene assembly also offer multiple ‘scalable’ solutions for both aspects of expanding epitope coverage. The number of genomic segments is freely scalable, and the set and probability of possible combinations can also be regulated. For example, Ig genes of cartilaginous fish are still characterized by the (probably) ancestral cluster organization of V(D)J miniloci, which involves very limited numbers of gene segments within each locus, and rearrangements are allowed only within the miniloci (Hsu et al., 2006) (Fig. 5A). This genomic arrangement constrains the possible foci of epitope targeting. By contrast, most Ig genes in tetrapods feature translocon organization, in which multiple gene segments are allowed to recombine, generating much greater combinatorial diversity (Fig. 5B). Remarkably, teleost fish have both cluster and translocon organization in different Ig genes or in different species, underlining the flexibility of gradual evolution towards increasing (or decreasing) combinatorial diversity (Hsu et al., 2006). Furthermore, even with translocon organization, ‘random’ somatic recombination does not necessarily imply that all possible combinations (specificities) are produced with the same probability. The generation of V(D)J recombinants can be skewed (Jackson et al., 2013; Elhanati et al., 2014), and some lymphocyte subsets [e.g. several types of unconventional T cells (Godfrey et al., 2015)] are characterized by a highly focused receptor repertoire with limited gene combinations and diversity. The degrees of freedom in combinatorial diversity might have evolved gradually with improving specific tolerance, and if some parts of ‘receptor space’ were more likely to be useful, regulatory mechanisms could apparently evolve to ensure the skewed production of these predictably useful specificities. There is also no reason why additional mechanisms of somatic receptor diversity could not be fine‐tuned towards generating broader or more constrained diversity. For example, some vertebrates first generate a limited repertoire relying on somatic recombination only, and switch on the expression of terminal deoxynucleotidyl transferase (TdT; responsible for nucleotide addition diversity) only at later stages of ontogeny (Schwager et al., 1991; Bogue et al., 1992). Knock‐out mice lacking TdT display reduced lymphocyte receptor diversity (Gilfillan, Benoist & Mathis, 1995), but are also less prone to autoimmune disease (Conde et al., 1998). The extent of TdT‐mediated junctional diversity could probably be flexibly tuned during evolution to match the evolving capabilities of tolerance mechanisms, and the same is likely to be true for the additional mechanisms of hypermutation and gene conversion. In jawed vertebrates, MHC restriction of adaptive immune responses offers a further scalable solution for the coverage of somatic receptor diversity. Most peptide antigens are able to elicit an immune response only when presented on the surface of a cell bound to an MHC molecule. MHC presentation requires the successive steps of proteasomal cleavage (for class I MHC only), translocation into the lumen of the endoplasmic reticulum (where MHC molecules are loaded), and binding to an MHC molecule. Each of these steps are selective (Hoof et al., 2012), and the degree of selectivity can be fine‐tuned by the substrate specificity of cleavage and translocation, and the number and binding specificity of MHC alleles. The analysis of the highly conserved genome of the elephant shark (Callorhinchus milii) (Venkatesh et al., 2014) suggests that MHC alleles were originally in genetic linkage with the genes of the antigen receptors that could bind to them. Such an arrangement might have facilitated the control of the set of peptides involved in MHC presentation, and might also have allowed somatic diversity to get started without thymic positive selection of lymphocytes (because coupled MHC–TCR pairs could be selected for binding over generations). Tissue‐specific restriction offers a further solution to restricting autoimmune collateral damage when specific tolerance is not (yet) efficient. Of note, unconventional T cells tend to recognize antigens in the context of non‐polymorphic antigen‐presenting molecules, some of which are expressed in a tissue‐specific manner (Godfrey et al., 2015). Clonal amplification and specific memory might also have evolved in incremental steps, contributing to the stepwise co‐evolution of the effector and regulatory arms of Darwinian immunity. Clonal amplification can be safe even without specific tolerance for effector cells bearing germline‐encoded receptors that are selected for safe targeting across generations (Boehm, 2006), and the genetic circuitry for inducible expansion might have evolved prior to the origins of Darwinian immunity for such cell types (as in NK cells). Then clonal amplification might have been co‐opted for cell types using a limited repertoire of somatically diversified receptors [focused on patterns typically associated with pathogens, similar to some classes of unconventional T cells (Godfrey et al., 2015) in extant organisms], and finally also for cells with the broadest diversity of targeting. The evolving genetic circuitry of programmed cell expansion and contraction also incorporated transcription factors that were created in the ancient vertebrate genome duplication event (Rothenberg & Pant, 2004). In addition to the breadth of targeting involved in clonal amplification, the extent and durability of the amplification could also evolve in gradual steps. In particular, if an immune reaction is short‐lived and no memory cells survive, then collateral damage is limited to the time span of the primary immune reaction (launched against an invading pathogen), and this one‐time cost might be outweighed by the benefit of efficient defence against the pathogen. That immune effector cells cross‐reactive to self would be induced against potentially dangerous non‐self antigens, but not to self tissues in the first place, could be ensured by the dependence of clonal amplification on danger signals from the very beginning of Darwinian immunity. The use of danger signals was probably easy to evolve: the new effector mechanisms simply needed to be built on top of the original (innate) decision cascades, co‐opting pre‐existing inducers of innate immunity as ‘danger signals’ for evolving Darwinian immunity. This way, self‐reactive cells inflicted only limited collateral damage during acute immune responses, and starting from such a situation, any (initially imperfect) measure of specific tolerance would have been useful and favoured by selection. The scalability of clonal amplification and specific immune memory can still be observed in the immune systems of extant vertebrates. For example, in sharks ‘the memory response is clearly inferior to that of the higher vertebrates’ (Flajnik & Kasahara, 2010, p. 50), and repeated challenge with an antigen cannot boost the antibody response beyond the peak of the initial response (Dooley & Flajnik, 2005). Even mammals have several lymphocyte subsets that display limited receptor diversity, tend to target conserved microbial structures, and are able to launch very rapid responses, but generate limited immune memory (Baumgarth, Tung & Herzenberg, 2005; Godfrey et al., 2015). This combination of characteristics may reconstitute (or preserve) the early stages of the evolution of Darwinian immunity, in that restricted somatic diversity and limited memory allow for safe responses without strict check‐points (that delay the response of highly diverse classes of lymphocytes) and advanced mechanisms of immune tolerance. The efficiency of tolerance mechanisms is also likely to have evolved in a stepwise manner. Genome duplication created surplus copies of regulatory factors, but wiring these into a genetic circuitry for regulatory T cells must have taken considerable evolutionary time, and each improvement in regulatory function could further potentiate the evolution of the effector components of Darwinian immunity. Of note, the deletion of Foxp3 in zebrafish results in only a moderate inflammatory phenotype (in contrast to the fatal autoimmune disease observed in Foxp3‐deficient mice) (Sugimoto et al., 2017), which is compatible with the view that the capacity of both effector and regulatory immune mechanisms has improved gradually during the evolution of vertebrates. The action of Treg cells could then also be complemented by the evolution of negative selection, improving not only the reliability, but also the cost efficiency of immune tolerance, by neutralizing autoreactive cells before they had their first chance to expand. In principle, some simple form of negative selection might even have preceded Treg‐mediated dominant tolerance in the frame of proto‐Darwinian immunity with restricted germline‐encoded receptor diversity. In a possible extant analogy, mammalian NK cells go through a period of ‘education’ early in their development, during which they are able to tune their responsiveness according to the level of inhibitory and stimulatory ligands in their environment (Orr & Lanier, 2010). However, we note that although NK cells are traditionally regarded as components of innate immunity, they are still embedded in the higher regulatory complexity of vertebrates, and it is unclear whether such fine‐tuned regulation had been possible before the ‘Big Bang’ of the WGD event. We cannot rule out that the ‘Big Bang’ of increasing regulatory complexity opened the way simultaneously to both Treg‐mediated dominant tolerance and recessive tolerance by negative selection; remarkably, Foxn1 transcription factor, a marker of the thymopoietic microenvironment, also originated at the WGD event (Singh, Arora & Isambert, 2015). Then, at least in jawed vertebrates, the evolution of the intricate mechanism of promiscuous gene expression in dedicated cells of the thymus (Derbinski et al., 2001) could extend the education of thymocytes (and thereby improve the efficiency of tolerance) to self antigens that are normally restricted to specific tissues. The gene of the transcription factor Aire, the central orchestrator of promiscuous gene expression in the thymus, has been found in the elephant shark (Venkatesh et al., 2014), an ancestral jawed vertebrate, but not yet in lamprey (Smith et al., 2013). Recent studies in mice indicate that promiscuous gene expression promotes the generation of Treg cells involved in dominant tolerance to tissue‐specific antigens (Aschenbrenner et al., 2007; Yang et al., 2015); in a recurring theme of immune evolution, new components of immunity tend to evolve interdependencies with pre‐existing components. As the increasing capacity for somatic receptor variability and clonal amplification allowed for increasing repertoire diversity, targeting could also evolve towards higher specificity. This allowed the targeting of variable (not evolutionarily conserved) patterns of potential pathogens, and facilitated the differential recognition of not only self and non‐self (Borghans, Noest & De Boer, 1999), but also of distinct pathogens that can be controlled by different effector mechanisms (Borghans & De Boer, 2002). The evolutionary scenario (including pre‐‘Big Bang’ pre‐adaptations and selection pressures) for the evolution of Darwinian immunity in vertebrates is shown in Fig. 6. (7) Darwinian immunity as a key driver of vertebrate evolution Finally, we argue that the origin and evolution of Darwinian immunity might have played a crucial role at several stages in the evolution of vertebrates. There are no known vertebrates without Darwinian immunity. Thus, either the innovation was necessary for the subsequent evolution of the vertebrate body plan, or the evolutionary advantage was so large that all other forms without it were outcompeted and went extinct without descendants. The latter possibility becomes highly unlikely once considerable adaptive radiation has occurred, so the emergence of the fundamental framework of vertebrate Darwinian immunity must have happened either shortly after the adaptive radiation of early vertebrates, or even before it, possibly contributing to the evolutionary success of vertebrates. The situation is somewhat analogous to the origin of eukaryotes and mitochondria. All extant eukaryotes either possess mitochondria or are derived from ancestors that had them. While it is unclear whether it was the acquisition of mitochondria that triggered the burst of evolutionary innovations that led to the last common eukaryotic ancestor (Poole & Gribaldo, 2014), the symbiogenetic event conferred sufficient selective advantage to drive all other protoeukaryotic lineages to extinction. Darwinian immunity evolved along with a whole package of evolutionary innovations triggered by the WGD event. While the exact contribution of Darwinian immunity to the evolutionary success of vertebrates cannot be directly estimated, the apparent extinction of several intermediate stages of its evolutionary trajectory argues that it must have been a major driver of vertebrate evolution. As argued in previous sections, the selective advantage provided by Darwinian immunity might have included improved cost efficiency of defence against pathogens and/or improved microbiome management. In addition, the pattern of two alternative implementations of Darwinian immunity in jawless and jawed vertebrates is far from straightforward to explain, and may have further implications for the evolution of vertebrates. Assuming that the evolution of Darwinian immunity was indeed initiated by the establishment of a framework for specific immune tolerance in the common ancestor of all vertebrates, two alternative scenarios can explain the extant pattern of two unrelated implementations of receptor diversity. In the first scenario, one of the two systems (VLR in jawless fish; TCR/BCR in jawed vertebrates) evolved first in the common ancestor of both lineages, but was then replaced by the other system in one of the lineages. It has been speculated that VLR might have evolved first, because all the required genes seem to have been present in the last common vertebrate ancestor, while the horizontal gene transfer that inserted RAG genes into an ancestral TCR/BCR‐like gene locus occurred after the split, in the jawed vertebrate lineage (Kato et al., 2012; Kasahara & Sutoh, 2014). Alternatively, the two systems might have arisen independently, each in the common ancestor of one of the lineages, over the background of some form of shotgun and/or proto‐Darwinian immunity. The first scenario (replacement) would imply that the more recent of the two systems had, already in its early rudimentary form, a selective advantage over the more ancient system, which at that time had already undergone some period of adaptive evolution. If VLR is indeed more ancient (Kato et al., 2012; Kasahara & Sutoh, 2014), then the BCR/TCR system must be more efficient, and it is tempting to speculate that it might have contributed to the much greater evolutionary success of jawed versus jawless vertebrates. Under the replacement scenario (irrespective of which system appeared first), the evolution of the second, more powerful system might have been helped by the presence of the tolerance mechanisms that co‐evolved with the first system of somatic diversity. In turn, the alternative scenario of independent origins of both systems from shotgun immunity would imply that two vertebrate lineages that acquired Darwinian immunity remained successful to this day, while all ancestral lineages without it have (apparently) been lost. Finally, we note that while at the moment, Darwinian immunity is practically synonymous with vertebrate adaptive immunity, independently evolved systems of Darwinian immunity might yet be found in invertebrates. The lessons from vertebrates suggest that higher developmental complexity and, in particular, extensive genome duplications might be prerequisites for the emergence of Darwinian immunity, while filter‐feeding and/or reliance on symbiotic microorganisms might give rise to particularly strong selection pressure for improved immunity: invertebrate groups displaying combinations of these traits should be investigated with particular scrutiny. If the last decade has taught us anything, it was that the diversity and ingenuity of invertebrate immune systems is far greater than previously thought: we are certain that the explosive growth of comparative immunology will not fail to deliver further surprises. We list some of the outstanding questions below. (8) Outstanding questions of Darwinian immunity What conditions (life‐history traits) favour Darwinian immunity over other types of adaptive and innate immunity? Has Darwinian immunity evolved in any invertebrate taxa? Is the monoallelic expression of variable immune receptors in sea urchins associated with clonal selection? Is there clonal selection (based on clonally stable receptor identity) in NK cells? Has Darwinian immunity been lost completely in any vertebrate? How exactly did the genomic duplication(s) at the origin of vertebrates facilitate the emergence of specific immune tolerance? What drove the exceptional increase in regulatory complexity, in contrast to other genome duplication events? How do species that appear to have no homologues of Foxp3 [some birds (Andersen et al., 2012); possibly sea lamprey] operate dominant immune tolerance? Do they have a divergent form of the gene (Denyer et al., 2016), or an alternative mechanism has taken over its function? Is the LRR‐based somatic receptor diversity of jawless vertebrates the ancestral vertebrate condition, or did both LRR‐based and Ig/RAG‐based somatic diversity evolve after the split of jawless and jawed vertebrates? Is MHC restriction a fortuitous ‘complication’ in jawed vertebrates, or is this function necessary (inevitable) beyond some level of complexity or potency of Darwinian immunity? In the latter case, are jawless fish below this level, or do they have an analogous system to perform this function?
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Mysterious ILC2 tissue adaptation

Mysterious ILC2 tissue adaptation | Immunology | Scoop.it
Group 2 innate lymphoid cells (ILC2s) that produce the cytokine IL-5 are found in lung, gut, fat and skin tissues. New findings indicate that ILC2s in different tissues selectively express distinct functional cytokine receptors for cell activation in response to the local environment.
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Tissue signals imprint ILC2 identity with anticipatory function

Tissue signals imprint ILC2 identity with anticipatory function | Immunology | Scoop.it
Locksley et al. show tissue-specific imprinting dictates the activating receptors ILC2s express, even in germ-free mice. Skin ILC2s are preferentially activated by IL-18, and IL-18 contributes to inflammation in a mouse model of atopic dermatitis.
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Human thymopoiesis is influenced by a common genetic variant within the TCRA-TCRD locus

Human thymopoiesis is influenced by a common genetic variant within the TCRA-TCRD locus | Immunology | Scoop.it
Personalized medicine partly depends on understanding what causes variance even outside the context of overt disease. The Milieu Intérieur Consortium enrolled 1000 healthy adults to study how genetics and the environment influence the immune system. Clave et al . leveraged samples from this cohort to see how thymic output, known to decrease over time, is affected by other factors. In addition to seeing sex-dependent differences, a genome-wide association study revealed variants that were associated with thymic output, which was confirmed in an independent cohort and mouse models. The authors have also developed a Web application for other investigators to examine the Milieu Intérieur data.

The thymus is the primary lymphoid organ where naïve T cells are generated; however, with the exception of age, the parameters that govern its function in healthy humans remain unknown. We characterized the variability of thymic function among 1000 age- and sex-stratified healthy adults of the Milieu Intérieur cohort, using quantification of T cell receptor excision circles (TRECs) in peripheral blood T cells as a surrogate marker of thymopoiesis. Age and sex were the only nonheritable factors identified that affect thymic function. TREC amounts decreased with age and were higher in women compared to men. In addition, a genome-wide association study revealed a common variant (rs2204985) within the T cell receptor TCRA-TCRD locus, between the DD2 and DD3 gene segments, which associated with TREC amounts. Strikingly, transplantation of human hematopoietic stem cells with the rs2204985 GG genotype into immunodeficient mice led to thymopoiesis with higher TRECs, increased thymocyte counts, and a higher TCR repertoire diversity. Our population immunology approach revealed a genetic locus that influences thymopoiesis in healthy adults, with potentially broad implications in precision medicine.
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CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway | Immunology | Scoop.it
Human innate lymphoid cells (ILCs) develop from tissue-resident ILC precursors (ILCPs)
in secondary lymphoid tissues. Here, Chen et al. describe a model of human ILC development in tonsils in which NK cells and group 3 ILCs derive from a CD56+ subset of ILCPs that cannot differentiate into group 2...
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Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria

Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria | Immunology | Scoop.it
Cytotoxic granule proteins secreted by CD8+ T cells contribute to host immunity to intracellular bacterial pathogens by assisting in the killing of both infected cells and intracellular bacteria. Balin et al . studied the ability of subsets of human CD8+ T cells expressing different combinations of granule proteins to kill macrophages infected with Mycobacterium leprae . The CD8+ T cell subset with the highest efficiency of mycobacterial killing simultaneously expressed three granule proteins: granzyme B, perforin, and granulysin. Transcriptional profiling of CD8+ T cell subsets identified the NK-activating receptor NKG2C as a valuable surface marker for identification and enrichment of these potent antimicrobial CD8+ T cells. The results of this study define a specific lymphocyte subset needed for effective immune defense against human leprosy.

Human CD8+ cytotoxic T lymphocytes (CTLs) contribute to antimicrobial defense against intracellular pathogens through secretion of cytotoxic granule proteins granzyme B, perforin, and granulysin. However, CTLs are heterogeneous in the expression of these proteins, and the subset(s) responsible for antimicrobial activity is unclear. Studying human leprosy, we found that the subset of CTLs coexpressing all three cytotoxic molecules is increased in the resistant form of the disease, can be expanded by interleukin-15 (IL-15), and is differentiated from naïve CD8+ T cells by Langerhans cells. RNA sequencing analysis identified that these CTLs express a gene signature that includes an array of surface receptors typically expressed by natural killer (NK) cells. We determined that CD8+ CTLs expressing granzyme B, perforin, and granulysin, as well as the activating NK receptor NKG2C, represent a population of “antimicrobial CTLs” (amCTLs) capable of T cell receptor (TCR)–dependent and TCR-independent release of cytotoxic granule proteins that mediate antimicrobial activity.

Via Krishan Maggon
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Development of Damaging Immune Cells in Tuberculosis Infection

Development of Damaging Immune Cells in Tuberculosis Infection | Immunology | Scoop.it
Development of damaging white blood cells occurs during Tuberculosis infection leading to a maladaptive immune response.
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The Role of Autoimmune Regulator (AIRE) in Peripheral Tolerance

The Role of Autoimmune Regulator (AIRE) in Peripheral Tolerance | Immunology | Scoop.it
Journal of Immunology Research is a peer-reviewed, Open Access journal that provides a platform for scientists and clinicians working in different areas of immunology and therapy. The journal publishes research articles, review articles, as well as clinical studies related to classical immunology,...
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Induction of innate immune memory: the role of cellular metabolism - ScienceDirect

Induction of innate immune memory: the role of cellular metabolism - ScienceDirect | Immunology | Scoop.it
The paradigm that only adaptive immunity can develop immunological memory has recently been challenged by studies showing that cells from the innate i…
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Roles of the RANKL–RANK axis in antitumour immunity — implications for therapy

Roles of the RANKL–RANK axis in antitumour immunity — implications for therapy | Immunology | Scoop.it
Intriguing evidence suggests that expression of RANK or RANKL by various cells of the tumour microenvironment modulates the anticancer immune response. Herein, the authors review this evidence, discuss the current preclinical and clinical data supporting a potential of RANKL inhibition to improve...
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Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation | Scientific Reports

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Full text from first author. Congratulations

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Plant Immunity: From Signaling to Epigenetic Control of Defense

Plant Immunity: From Signaling to Epigenetic Control of Defense | Immunology | Scoop.it
Pathogen recognition by plants results in the activation of signaling pathways that
induce defense reactions. There is growing evidence indicating that epigenetic mechanisms
directly participate in plant immune memory.
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Zika virus strips immune cells of their identity: New lab technique for separating infected and uninfected cells helps reveal how Zika virus manipulates the human immune system

Zika virus strips immune cells of their identity: New lab technique for separating infected and uninfected cells helps reveal how Zika virus manipulates the human immune system | Immunology | Scoop.it
Macrophages are immune cells that are supposed to protect the body from infection by viruses and bacteria. Yet Zika virus preferentially infects these cells. Researchers have now unraveled how the virus shuts down the genes that make macrophages function as immune cells.
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Natural killer cells and other innate lymphoid cells in cancer

Natural killer cells and other innate lymphoid cells in cancer | Immunology | Scoop.it
Eric Vivier and colleagues discuss the emerging roles for natural killer (NK) cells and other innate lymphoid cell populations in cancer immunity. The authors highlight the current immunotherapy trials that are targeting NK cells to treat patients with cancer.
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Endogenous Regulators of the Immune System (sCD100, Malonic Dialdehyde, and Arginase). - PubMed - NCBI

Endogenous Regulators of the Immune System (sCD100, Malonic Dialdehyde, and Arginase). - PubMed - NCBI | Immunology | Scoop.it
Bull Exp Biol Med. 2018 Mar;164(5):693-700. doi: 10.1007/s10517-018-4061-6. Epub 2018 Mar 26. Review
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Distinguishing Th1 and Th2 Cells

Distinguishing Th1 and Th2 Cells | Immunology | Scoop.it
Reagents That Distinguish Th1 and Th2 cells Courtesy of R&D SystemsSchematic representation of cytokines influencing the development of antigen-activated naive CD4+ T cells into Th1 and Th2 cells.
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Recording the wild lives of immune cells

Recording the wild lives of immune cells | Immunology | Scoop.it
Intravital microscopic imaging can uncover fundamental aspects of immune cell behavior in real time in both healthy and pathological states. Here, we discuss approaches for single-cell imaging of adaptive and innate immune cells to explore how they migrate, communicate, and mediate regulatory or effector functions in various tissues throughout the body. We further review how intravital single-cell imaging can be used to study drug effects on immune cells.
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Research uncovers how the immune system detects DNA damage

Research uncovers how the immune system detects DNA damage | Immunology | Scoop.it
Our immune system is working every day to protect us from bacteria, viruses, and parasites, but it can also detect when our own cells are damaged.
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The human lymph node microenvironment unilaterally regulates T-cell activation and differentiation

The human lymph node microenvironment unilaterally regulates T-cell activation and differentiation | Immunology | Scoop.it
Author summary The lymph node microenvironment contains an abundance of immune cells that interact with and within an intricate structural framework created by fibroblastic reticular cells. In mice, fibroblastic reticular cells are known to regulate T-cell activation, proliferation, and function,...
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Runx-dependent and silencer-independent repression of a maturation enhancer in the Cd4 gene

Runx-dependent and silencer-independent repression of a maturation enhancer in the Cd4 gene | Immunology | Scoop.it
The commitment of helper and cytotoxic lineages for CD4 and CD8 T cells, respectively, is associated with the regulation of Cd4 gene expression. Here the authors show that an intronic silencer, S4, has differential effects and synergy with the RUNX complex to act on two enhancer elements of the...
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