Sarah Allan

Vaccines: Explaining alum: immunologists' dirty little secret

widely used adjuvant in human vaccines, but its mechanism of action remains ill-defined. In a new study, Lambrecht and colleagues find that a key factor in the adjuvant activity of alum is the activation of a population of inflammatory dendritic cells (DCs) through the induction of uric acid production, which leads to enhanced humoral and cell-mediated immunity. Lambrecht and colleagues showed that immunization of DO11.10 mice — which have CD4+ T cells specific for an ovalbumin (OVA)-derived peptide — with OVA alone led to transient T-cell proliferation followed by their deletion. By contrast, injection with alum-emulsified OVA (OVA–alum) resulted in an enhanced, persistent and recirculating T-helper-2-cell response. So, alum clearly enhances T-cell responses to an immunized antigen, but what triggers this effect? Emulsification of OVA in alum resulted in significantly increased recruitment of innate immune cells to the peritoneal cavity as early as 6 hours after intraperitoneal injection. CD11c+ myeloid DCs isolated from the peritoneal cavity of OVA–alum-immunized mice were more efficient at antigen uptake and processing, and showed greater signs of functional maturation, compared with DCs from mice injected with OVA alone. In line with these observations, DCs isolated from OVA–alum-immunized mice were more potent stimulators of T-cell proliferation than control DCs. Inflammatory monocytes, identified on the basis of a CD11b+Ly6C+Ly6G–F4/80mid phenotype, were also found to take up significantly more antigen in the presence of alum and to traffic to mediastinal lymph nodes, the primary draining site of the peritoneum, within 24 hours of immunization. When isolated from mediastinal lymph nodes, Ly6C+ inflammatory monocytes were even more potent T-cell stimulators than myeloid DCs from the same site and, most importantly, acquisition of CD11c expression by Ly6C+ monocytes indicated their conversion to a DC phenotype. Depletion of recruited inflammatory monocytes and DCs from the peritoneum abolished both T-cell proliferation and IgG1 production following OVA–alum immunization, indicating that these antigen-presenting cells (APCs) were essential for alum to modulate the adaptive immune response. Exposure to alum in vitro has no effect on the activation state of APCs, so by what mechanism does alum mediate its adjuvant activity in vivo? Examination of the peritoneal lavage fluid of immunized mice revealed that alum strongly induced the production of uric acid, an endogenous danger signal. Uricase, an enzyme that rapidly breaks down uric acid, abolished the recruitment of inflammatory monocytes and antigen-specific T-cell proliferation in OVA–alum-immunized mice, indicating that uric acid is crucial for alum’s mechanism of action. Furthermore, mice lacking the adaptor molecule MyD88 (and therefore deficient in interleukin-1-receptordependent responses to uric acid) had severely impaired recruitment of inflammatory monocytes to the mediastinal lymph nodes compared with wild-type mice. Unlike many mouse models, human vaccines are normally administered subcutaneously or intramuscularly. Importantly, the authors report that intramuscular administration of OVA–alum had similar effects to intraperitoneal immunization in mice. Therefore, these findings should further our understanding of how alum-containing vaccines trigger effective adaptive immune responses in humans. Sarah Allan

Autoimmunity: Type I interferon target revealed

reduces disease exacerbation in many patients with multiple sclerosis, but how this cytokine inhibits autoimmune inflammation in the central nervous system (CNS) is largely unknown. Now, Prinz, Kalinke and colleagues have revealed that the effect of type I IFNs on myeloid cells is crucial to the protective effect of this cytokine in the relevant animal model of experimental autoimmune encephalomyelitis (EAE). Investigation of the expression of IFNβ in mice with EAE revealed elevated levels of this cytokine in the CNS, but not the blood, of mice with active disease. Mice deficient in type I IFN receptor (IFNAR; Ifnar1–/– mice), developed more severe EAE, characterized by a greater degree of demyelination and increased lethality. IFNAR is expressed by nearly all cell types and tissues, so it has been a challenge to evaluate the specific effects of type I IFNs in the context of EAE. As EAE is known to be a T-cell-mediated disease, the effect of type I IFNs on lymphocytes was investigated. Interestingly, the balance between T helper 1 (TH1)and TH17cell responses was unchanged in Ifnar1–/– mice with EAE, and specific ablation of Ifnar1 in either T cells or B cells had no effect on the course of the disease. Moreover, the lack of IFNAR expression by CNS tissue cells had no impact on EAE severity, indicating that the protective effects of type I IFNs in CNS inflammation are mediated by neither lymphocytes nor CNS-resident cells. However, specific ablation of Ifnar1 in myeloid cells resulted in significant aggravation of the effector phase of EAE, with elevation of pro-inflammatory mediators including tumour-necrosis factor and chemokines. Closer investigation revealed that exposure to IFNβ reduced myelin uptake and impaired the upregulation of MHC class II molecules by macrophages, thereby inhibiting their activation state and antigen-presenting capacity. So, this study has revealed a nonredundant role for myeloid cells in type I IFN-mediated protection of autoimmune inflammation in the CNS. This work provides clues as to how IFNβ therapy might work in patients with multiple sclerosis and could help to develop more cellspecific therapies that reduce the side effects of the treatment. Sarah Allan

Immune tolerance: A T-cell-independent function for AIRE?

regulator (AIRE) gene cause a breakdown in immune tolerance to self antigens, which leads to severe multiorgan autoimmunity and increased levels of circulating autoantibodies. Although this is thought to be mediated by self-reactive T cells that escape negative selection in the thymus, Karlsson and colleagues now provide evidence to suggest that AIRE is also important for controlling T-cell-independent B-cell responses in the periphery. When the authors examined B-cell responses to a T-cellindependent antigen, they observed an increase in immunoglobulin production and activation level, but not in the total number, of B cells in Aire–/– mice compared with wild-type mice. Owing to the similarity between the phenotypes of Aire–/– mice and mice that overexpress the cytokine BAFF (B-cell activating factor), the authors then investigated whether BAFF might be responsible for B-cell hyperactivation in Aire–/– mice. Indeed, both mice and humans with AIRE mutations had increased serum levels of BAFF compared with wild-type controls. Furthermore, in agreement with the fact that marginal-zone B cells are highly responsive to BAFF, the authors found that the levels of immunoglobulin subclasses that are predominantly produced by marginal-zone B cells were increased in Aire–/– mice. So, loss-of-function AIRE mutations cause an increase in circulating levels of BAFF, thereby promoting B-cell hyperactivation and antibody production, but what cells produce this cytokine? The authors used bone-marrow reconstitution experiments to show that the autoreactive T cells in Aire–/– mice do not seem to be involved in increased BAFF production; by contrast, bonemarrow-derived dendritic cells (DCs) from Aire–/– mice produced increased levels of BAFF in response to interferon-γ (IFNγ) compared with wild-type DCs. Although serum IFNγ levels were not increased in Aire–/– mice, the authors provided evidence to suggest that the increase in BAFF production might be caused by a cell-intrinsic dysregulation of DC responses to IFNγ: when Aire–/– DCs were treated with IFNγ in vitro, abnormal expression of a gene induced by signal transducer and activator of transcription 1, a key protein of the IFNγ-mediated signalling pathway, was observed. Together, these results suggest that AIRE is important for immune homeostasis not only by ensuring the generation of a self-tolerant T-cell repertoire, but also through the regulation of peripheral DCs. In addition, this work helps to explain how mutations in AIRE contribute to the hyperactivation of B cells and, in particular, the BAFF-sensitive B-cell populations that are found in marginal zones. Sarah Allan

Dendritic cells: Assessing cross-presentation in vivo

(DCs) to cross-present exogenous antigens on MHC class I molecules is well established, although the importance of cross-presentation for different types of immune response in vivo is not clear. In a recent report published in Science, Murphy and colleagues show that deficiency in the transcription factor BATF3 (basic leucine zipper transcription factor, ATF-like 3) leads to impaired immune responses in vivo owing to the absence of a DC subset that is particularly important for cross-presentation. The authors used global gene expression analysis to search for genes that might be involved in the development of different DC subsets and found that BATF3 was highly expressed by conventional DCs (cDCs). Deletion of Batf3 in mice caused a selective loss of the CD8α+ subset of cDCs in the spleen without affecting the development of other haematopoietic cell types. CD8α+ cDCs are characterized by their efficient ability to cross-present antigen and their responsiveness to Toll-like receptor 3 (TLR3) ligands. Indeed, when the function of cDCs from Batf3–/– and wild-type mice was compared, those from the mutant mice showed marked impairments in TLR3-mediated cytokine production and antigen cross-presentation in vitro. Having established that the transcription factor BATF3 is required for CD8α+ cDC development in mice, the authors next investigated how a deficiency in this DC subset influenced immune responses in which cross-presentation is important in vivo. First, using a model of West Nile virus (WNV) infection, the authors found that Batf3–/– mice had a markedly impaired CD8+ T-cell response, but had normal B-cell and CD4+ T-cell responses to WNV. Second, the authors investigated the importance of CD8α+ cDCs in tumour immunity. Wild-type mice could reject syngeneic fibrosarcomas, whereas these tumours grew rapidly in Batf3–/– mice. The failure to reject the tumours was associated with reduced infiltration of CD8+ but not CD4+ T cells into the tumours and an impaired development of tumour-specific CD8+ T cells. To rule out the possibility that the impaired antiviral and antitumour responses in Batf3–/– mice were due to intrinsic T-cell defects, the authors carried out several assays of T-cell differentiation and function. Defects in Batf3–/– T cells could not be detected, which supported the authors’ conclusion that impaired antiviral and antitumour responses in Batf3–/– mice were due to the lack of CD8α+ cDCs. However, a closer examination of antitumour responses in Batf3–/– mice indicated that immune-cell populations other than CD8α+ cDCs were sufficient to promote an antitumour response in mice that carried a low tumour burden. Therefore, although cross-presentation that is mediated by CD8α+ cDCs is important for optimal priming of CD8+ T-cell responses, other subsets of antigenpresenting cells can also carry out this function in vivo. This work shows that BATF3 is required for the development of CD8α+ cDCs, a subset with crosspresenting ability that is required for optimal immune responses to viral infections and tumours in mice. Sarah Allan

Immune regulation: Tripping a tissue-specific switch

tissue-specific mechanisms in place to regulate the threshold of immune responses and resolve inflammation. A recent study in Nature Immunology has now shown that a homeostatic loop involving CD200 and its receptor (CD200R) is an important regulator of steady-state immune homeostasis and inflammation in the airways. Ligation of CD200R, which is expressed almost exclusively by myeloid cells, triggers negative regulatory pathways in the cells that express it. In this study, Snelgrove et al. first showed that alveolar macrophages that were isolated from normal mice have a higher basal expression level of CD200R than macrophages that were isolated from other tissues. The exposure of nonmucosal splenic macro phages to the immuno suppressive cytokines interleukin-10 (IL-10) or transforming growth factor-β was found to upregulate the expression of CD200R, thereby linking CD200R-mediated inhibition with other negative regulatory pathways. When cultured in vitro with CD200+ epithelial cells, alveolar macrophages isolated from CD200R-deficient mice were found to produce increased amounts of the pro-inflammatory cytokines IL-6 and tumour-necrosis factor (TNF) compared with wild-type macrophages, in which the CD200–CD200R axis was intact. In addition, the lungs of mice that lacked CD200 expression were found to contain increased numbers of CD11c+ macrophages that were hyperresponsive to activation ex vivo, which demonstrates the importance of CD200–CD200R interactions for maintaining the steady-state homeostasis of macrophages in the airways in vivo. Although many different cell types express CD200, including haemato poietic cells, the authors showed that the epithelial cells of the airway lumen are most likely to modulate the responses of CD200R+ alveolar macrophages. The same mechanisms that maintain immune homeostasis are commonly involved in the resolution of inflammation. Indeed, disabling CD200–CD200R interactions led to more severe immuno pathology and increased mortality following influenza virus infection. Interestingly, heightened immunopathology in CD200-deficient mice corresponded with a lower viral load compared with that of wild-type mice, which indicates that the hyperresponsiveness of macrophages in CD200-deficient mice led to more efficient viral clearance but also to more severe ‘collateral damage’. The authors went on to demonstrate that the treatment of wild-type mice with a CD200R agonist decreased influenza virus-induced immunopathology, which indicates that this regulatory pathway could be a relevant therapeutic target for inhibiting inflammation in the lung. These findings provide evidence of a non-redundant role for CD200 in modulating the function of alveolar macrophages and illustrate a new tissue-specific means of immune modulation in the airways. Whether this mechanism of myeloid-cell restraint also operates in other mucosal tissues remains an open question. Sarah Allan

Immunological memory: Basophils boost B-cell memory

locytes that is thought to have an important function in allergic reactions and in host defence against parasite infections. Now, Mack and colleagues have revealed that basophils also have an important role in the generation of optimal B-cell memory responses. Through the expression of the high-affinity Fc receptor for IgE, basophils can bind antigen-specific IgE that is produced for long periods of time after immunization with an antigen. Following re-exposure, antigen-reactive basophils bind soluble antigen through this bound antigen-specific IgE and produce interleukin-4 (IL-4) and IL-6, which promotes a T helper 2 (TH2)-cell response. In this study, the authors first confirmed the capacity of basophils to bind small amounts of soluble antigen following re-exposure of immunized mice to the antigen, and showed that these antigen-reactive basophils were the primary producers of IL-4 and IL-6 in the spleen and the bone marrow. If basophils were depleted after immunization with a model antigen, a marked reduction in the magnitude of the B-cell memory response was observed following antigen re-exposure, which was indicated by a significant reduction in the number of antigenspecific plasma cells and by lower concentrations of antigen-specific IgG1 and IgG2a in the serum. Furthermore, the adoptive transfer of basophils from immunized mice enhanced antigen-specific B-cell memory responses in recipient mice following exposure to the same antigen. The capacity of basophils to boost B-cell memory was also shown to be important in the context of pathogenic infection. Mice depleted of basophils before secondary immunization with pneumococcal surface protein A, which is known to provide protection from Streptococcus pneumoniae-induced sepsis, were more susceptible to disease induced by subsequent exposure to the live pathogen. Clearly, basophils are important in the generation of B-cell memory, but how is this effect mediated? Using in vitro assays, the authors showed that this effect relied on cell−cell contact, and that basophilderived IL-6 was also essential. Furthermore, the presence of activated T cells — which were found to differentiate into TH2 cells in the presence of antigen-reactive, activated basophils —were also required for basophil-dependent enhancement of B-cell memory. So, basophils have an important role in the generation of optimal B-cell memory responses by promoting TH2-cell-dependent B-cell help, which could have important implications not only for understanding the role of this granulocyte subset in host defence against invading pathogens, but also for vaccine design in which the generation of B-cell memory is an important aim.

Lymphocyte responses: A new role for IL-21 revealed

important for the differentiation of activated B cells into memory B cells and plasma cells in germinal centres. Although recent work has increased what is known about the phenotype and function of these cells, much about what makes them distinct from other CD4+ TH-cell subsets is still unclear. Now, two new studies in Immunity provide further evidence that defines TFH cells as a distinct T-cell subset and shows that interleukin-21 (IL-21) has a pivotal role in their differentiation. In a study by Nurieva et al., the comparison of gene-expression profiles demonstrated that TFH cells have a transcriptome that is distinct from that of TH1, TH2 and TH17 cells. TFH cells were unique in their increased co-expression of genes encoding CXC-chemokine receptor 5 (CXCR5), B-cell lymphoma 6 (BCL-6), programmed cell death-1 (PD1) and IL-21. When the authors investigated whether the generation of TFH cells was dependent on other subsets of TH cells, they found that functional TFH cells were present in mice that were deficient in factors essential for the differentiation of TH1, TH2 or TH17 cells, indicating that TFH cells can differentiate independently of these other T-cell subsets. IL-21 is known to be important for B-cell responses to T-cell-dependent antigens, but the exact role of this cytokine in germinal centres was unknown. Here, both groups observed defective germinal-centre formation and decreased IgG1 production in IL-21-deficient mice, and Vogelzang et al. went on to show that these defects were T-cell, rather than B-cell, intrinsic. It has been established that TFH cells express high levels of IL-21, and that ICOS (inducible co-stimulator) is required for their generation. In these two studies, the authors linked these ideas with the finding that ICOS ligand-expressing B cells are important for regulating the differentiation of TFH cells, and that ICOS–ICOS ligand interactions are necessary for CD4+ T-cell IL-21 production. Vogelzang et al. then showed that IL-21 supports TFH-cell development by promoting the phosphorylation of VAV1, which is involved in the signalling pathway downstream of the T-cell receptor, thereby providing an important costimulatory signal for differentiating TFH cells. Similar to TFH cells, TH17 cells express large amounts of IL-21. In addition, Nurieva et al. showed that both TFH cells and TH17 cells require IL-6 and STAT3 (signal transducer and activator of transcription 3) for their generation. However, unlike TH17 cells, the generation of TFH cells was independent of transforming growth factor-β (TGFβ) and of the transcription factors RORγt (retinoic-acid-receptor-related orphan receptor-γt) and RORα, both of which are necessary for TH17-cell development. These data confirm the identity of TFH cells as a separate THcell subset and open up the possibility that TFH and TH17 cells differentiate in a reciprocal manner. Together, these two studies significantly advance our understanding of the biology of TFH cells and assign an important new role to IL-21 in germinal-centre reactions. Sarah Allan

B-cell responses: Revising autoantibody responses

activating gene (RAG) proteins is required for the rearrangement of immunoglobulin genes and receptor editing in immature B cells in the bone marrow. However, the expression of RAG proteins in peripheral B cells is a topic of some controversy. Recent results published in the Journal of Clinical Investigation now show that the re-expression of RAG proteins enables ‘receptor revision’ in peripheral B cells that are activated by self antigen, thereby preventing autoimmune humoral responses. The authors of this study made use of an established mouse model in which a lupus-like disease is induced following immunization with a mimetope of double-stranded DNA. Mice immunized with a control peptide that does not result in the generation of autoantibodies served as controls. With this system, the authors clearly demonstrate that RAG expression was induced in autoreactive B cells, but not in B cells that were specific for the control peptide, following their activation in vivo. Autoreactive RAG+ B cells were contained within the B-cell fraction that expresses low levels of B220, which contains a large proportion of CD138+ plasma cells, in contrast to the B220hi cell fraction, which is less differentiated. Phenotypic and functional analyses showed that autoreactive B220lowRAG+ B cells that were derived from germinalcentre responses, rather than extrafollicular responses, had undergone isotype switching and extensive somatic hypermutation, and had features of memory or pre-plasma B cells. Importantly, further experiments indicated that, although RAGexpressing B cells had differentiated in germinal centres, RAG expression and subsequent receptor revision were induced following activation by soluble antigen outside of the germinal-centre environment. If the soluble antigen was eliminated from the circulation — which in this model was achieved by the administration of DNase to remove free self DNA — receptor revision did not occur. The authors next showed that, similar to the expression of RAG proteins in immature B cells, their re-expression in peripheral B cells required the expression of interleukin-7 receptor (IL-7R). Blocking of IL-7R inhibited the upregulation of RAG expression by autoreactive B220low B cells, thereby preventing receptor revision following antigen recognition. The ability to prevent the induction of RAG expression allowed the authors to investigate its importance in limiting autoimmune responses. Indeed, blocking of IL-7R led to a significant increase in the level of serum self-DNA-specific autoantibodies, which indicates that receptor revision is an important mechanism of B-cell tolerance. Together, these data establish that the expression of RAG proteins following soluble antigen recognition and IL-7R signalling enables receptor revision in autoreactive B cells, which is important for diminishing autoimmune humoral responses. Sarah Allan