Dorothée Bourges

Cutting Edge: Pulmonary Legionella pneumophila Is Controlled by Plasmacytoid Dendritic Cells but Not Type I IFN

Plasmacytoid dendritic cells (pDCs) are well known as the major cell type that secretes type I IFN in response to viral infections. Their role in combating other classes of infectious organisms, including bacteria, and their mechanisms of action are poorly understood. We have found that pDCs play a significant role in the acute response to the intracellular bacterial pathogen Legionella pneumophila. pDCs were rapidly recruited to the lungs of L. pneumophila-infected mice, and depletion of pDCs resulted in increased bacterial load. The ability of pDCs to combat infection did not require type I IFN. This study points to an unappreciated role for pDCs in combating bacterial infections and indicates a novel mechanism of action for this cell type.

Differential use of autophagy by primary dendritic cells specialized in cross-presentation

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed “cross-presentation.” The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.

Helios defines T cells being driven to tolerance in the periphery and thymus.

The expression of the Ikaros transcription factor family member, Helios, has been shown to be associated with T‐cell tolerance in both the thymus and the periphery. To better understand the importance of Helios in tolerance pathways, we have examined the expression of Helios in TCR‐transgenic T cells specific for the gastric H+/K+ ATPase, the autoantigen target in autoimmune gastritis. Analysis of H+/K+ ATPase‐specific T cells in mice with different patterns of H+/K+ ATPase expression revealed that, in addition to the expression of Helios in CD4+Foxp3+ regulatory T (Treg) cells, Helios is expressed by a large proportion of CD4+Foxp3− T cells in both the thymus and the paragastric lymph node (PgLN), which drains the stomach. In the thymus, Helios was expressed by H+/K+ ATPase‐specific thymocytes that were undergoing negative selection. In the periphery, Helios was expressed in H+/K+ ATPase‐specific CD4+ T cells following H+/K+ ATPase presentation and was more highly expressed when T‐cell activation occurred in the absence of inflammation. Analysis of purified H+/K+ ATPase‐specific CD4+Foxp3−Helios+ T cells demonstrated that they were functionally anergic. These results demonstrate that Helios is expressed by thymic and peripheral T cells that are being driven to tolerance in response to a genuine autoantigen.

Glucocorticoid-Induced TNF Receptor Expression by T Cells Is Reciprocally Regulated by NF-κB and NFAT

Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-κB pathway (RelA, cRel, and NF-κB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-κB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-κB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-κB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

Transient Treg-cell depletion in adult mice results in persistent self-reactive CD4(+) T-cell responses.

Depletion of Foxp3+CD4+ regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg‐cell depletion on self‐reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4+ T‐cell responses to endogenous self‐antigens. Short‐term ablation of Treg cells in mice resulted in rapid activation of CD4+ T cells, increased percentage of IFN‐γ+ and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self‐reactive responses, we analyzed the activation of naïve gastric‐specific CD4+ T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric‐specific T cells in the stomach draining LNs of Treg‐cell‐depleted mice, compared with untreated mice, either during Treg‐cell depletion or after Treg‐cell reconstitution. Moreover, the hyperproliferation of gastric‐specific T cells in the Treg‐cell‐ablated mice was predominantly antigen‐dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg‐cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg‐cell depletion results in ongoing antigen‐driven self‐reactive T‐cell responses and emphasize the continual requirement for an intact Treg‐cell population.

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.

Targeting the gut vascular endothelium induces gut effector CD8 T cell responses via cross-presentation by dendritic cells

Systemic delivery of Ag usually induces poor mucosal immunity. To improve the CD8 T cell response at mucosal sites, we targeted the Ag to MAdCAM-1, a mucosal addressin cell adhesion molecule expressed mainly by high endothelial venules (HEV) in mesenteric lymph nodes (MLN) and Peyer’s patches of gut-associated lymphoid tissue. When chemical conjugates of anti-MAdCAM-1 Ab and model Ag OVA were injected i.v., a greatly enhanced proliferative response of Ag-specific OT-I CD8 T cells was detected in MLN. This was preceded by prolonged accumulation, up to 2 wk, of the anti-MAdCAM OVA conjugate on HEV of Peyer’s patches and MLN. In contrast, nontargeted OVA conjugate was very inefficient in inducing OT-I CD8 T cell proliferation in MLN and required at least 20-fold more Ag to induce a comparable response. In addition, MAdCAM targeting elicits an endogenous OVA-specific CD8 T cell response, evident by IFN-γ production and target killing. Induced response offers protection against an OVA-expressing B cell lymphoma. We propose that the augmentation of gut CD8 T cell responses by MAdCAM targeting is due to both accumulation of Ag in the HEV and conversion of a soluble Ag to a cell-associated one, allowing cross-presentation by DCs.

Shaping the T-cell repertoire in the periphery

Selection of T cells does not end with events in the thymus, but continues in extrathymic tissues and for the life of the organism. In this review, we examine how self‐reactive T cells are rendered harmless and the processes that select for T cells that are most efficient at combating pathogens. The implications of peripheral T‐cell selection for the immune response as animals age are discussed as is the critical role of dendritic cells in directing T‐cell differentiation.

Pathogenic T cells persist after reversal of autoimmune disease by immunosuppression with regulatory T cells.

Autoimmune disease can be prevented with immunosuppressive agents; however, the effectiveness of these treatments in advanced stage of disease and the fate of pathogenic T cells following such treatments are not clear. In this study we demonstrate that a single dose of in vitro‐induced Treg cells (iTreg cells) resulted in the functional repair and restitution of stomach tissue that had been severely damaged in advanced autoimmune gastritis. iTreg cells caused depletion or inactivation of autoreactive naïve T cells that were antigen inexperienced, however, autoreactive effector/memory T cells persisted in treated mice, resulting in residual cellular infiltrates within the repaired stomach tissue. The persisting autoreactive T cells were able to rapidly cause autoimmune disease if iTreg cells were removed. Similar data were obtained from mice treated continuously with corticosteroid, in that there was substantial restitution of the gastric mucosa; however, effector T cells persisted and rapidly caused pathology following drug removal. Therefore, iTreg cells or corticosteroid can suppress pathogenic autoreactive cells in advanced autoimmune disease, reversing tissue damage and improving tissue function. However, the persistence of pathogenic T cells represents a disease risk.

Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen

Legionella pneumophila is the causative agent of Legionnaires’ disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.