David R. Withers

Exogenous Stimuli Maintain Intraepithelial Lymphocytes via Aryl Hydrocarbon Receptor Activation

The bodys surfaces form the interface with the external environment, protecting the host. These epithelial barriers are also colonized by a controlled diversity of microorganisms, disturbances of which can give rise to disease. Specialized intraepithelial lymphocytes (IELs), which reside at these sites, are important as a first line of defense as well as in epithelial barrier organization and wound repair. We show here that the aryl hydrocarbon receptor (AhR) is a crucial regulator in maintaining IEL numbers in both the skin and the intestine. In the intestine, AhR deficiency or the lack of AhR ligands compromises the maintenance of IELs and the control of the microbial load and composition, resulting in heightened immune activation and increased vulnerability to epithelial damage. AhR activity can be regulated by dietary components, such as those present in cruciferous vegetables, providing a mechanistic link between dietary compounds, the intestinal immune system, and the microbiota.

Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells.

Innate lymphoid cells keep gut T cells in check Trillions of bacteria inhabit our guts. So do many types of immune cells, including T cells, which might be expected to attack these bacteria. How, then, do our bodies manage to keep the peace? Working in mice, Hepworth et al. report one such mechanism. A population of immune cells, called innate lymphoid cells, directly killed CD4+ T cells that react to commensal gut microbes. Some of the specifics of this process parallel how the immune system keeps developing self-reactive T cells in check in the thymus. Furthermore, this peacekeeping process may be disrupted in children with inflammatory bowel disease. Science, this issue p. 1031 Innate lymphoid cells delete commensal bacteria–specific CD4+ T cells from the intestine in mice. Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria–specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)–intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria–specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria–specific CD4+ T cells in the intestine and suggest that this process is dysregulated in human IBD.

Distinct Roles for CCR4 and CXCR3 in the Recruitment and Positioning of Regulatory T Cells in the Inflamed Human Liver

Regulatory T cells (Tregs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of Treg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)+ Tregs. We isolated CD4+CD25+CD127lowFoxP3+ Tregs from chronically inflamed human liver removed at transplantation; compared with blood-derived Tregs, liver-derived Tregs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, Tregs used CXCR3 and α4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4+ Tregs in the parenchyma and septal areas. Ex vivo, liver-derived Tregs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of Tregs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit Tregs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of Tregs at sites of hepatitis in chronic liver disease.

Roquin differentiates the specialized functions of duplicated T cell costimulatory receptor genes CD28 and ICOS.

During evolutionary adaptation in the immune system, host defense is traded off against autoreactivity. Signals through the costimulatory receptor CD28 enable T cells to respond specifically to pathogens, whereas those through the related costimulatory receptor, ICOS, which arose by gene duplication, are critical for affinity maturation and memory antibody responses. ICOS ligand, unlike the pathogen-inducible CD28 ligands, is widely and constitutively expressed in the immune system. Here, we show that crosstalk between these two pathways provides a mechanism for obviating the normal T cell dependence on CD28. Several CD28-mediated responses-generation of follicular helper T cells, germinal center formation, T helper 1 cell-dependent extrafollicular antibody responses to Salmonella and bacterial clearance, and regulatory T cell homeostasis-became independent of CD28 and dependent on ICOS when the E3 ubiquitin ligase Roquin was mutated. Mechanisms to functionally compartmentalize ICOS and CD28 signals are thus critical for two-signal control of normal immune reactions.

Rank Signaling Links the Development of Invariant γδ T Cell Progenitors and Aire+ Medullary Epithelium

Summary The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.

CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver

Background & Aims IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. Methods The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. Results IL-17+ T cells comprised 1–3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17+ cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. Conclusions CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

CCR7-dependent trafficking of RORγ + ILCs creates a unique microenvironment within mucosal draining lymph nodes

Presentation of peptide:MHCII by RORγ-expressing group 3 innate lymphoid cells (ILC3s), which are enriched within gut tissue, is required for control of CD4 T-cell responses to commensal bacteria. It is not known whether ILC populations migrate from their mucosal and peripheral sites to local draining secondary lymphoid tissues. Here we demonstrate that ILC3s reside within the interfollicular areas of mucosal draining lymph nodes, forming a distinct microenvironment not observed in peripheral lymph nodes. By photoconverting intestinal cells in Kaede mice we reveal constitutive trafficking of ILCs from the intestine to the draining mesenteric lymph nodes, which specifically for the LTi-like ILC3s was CCR7-dependent. Thus, ILC populations traffic to draining lymph nodes using different mechanisms.

Sequential phases in the development of Aire-expressing medullary thymic epithelial cells involve distinct cellular input

Intrathymic deletion of immature thymocytes that express self‐reactive TCR specificities is essential in the generation of self tolerance. Medullary thymic epithelial cells (mTEC) expressing the transcriptional regulator Aire play a key role in this process by regulating expression of tissue‐restricted antigens to ensure tolerance to peripheral tissues. Here, we have analysed the cellular and molecular requirements for the initial appearance of Aire+ mTEC in the embryonic thymus, in addition to their persistence in the adult thymus. Analysis of thymic ontogeny shows that the emergence of embryonic Aire+ mTEC occurs prior to the appearance of mature thymocytes, and depends upon lymphoid tissue inducer cells expressing retinoic acid receptor‐related orphan receptor γ. In the adult thymus, we show that Aire+ mTEC develop in the absence of thymocyte positive and negative selection and CD40 signalling, but are present at reduced frequency. Collectively these data support a model where the initial differentiation of Aire+ mTEC involves receptor activator of NF‐κB (RANK)‐RANKL interactions with lymphoid tissue inducer cells, with subsequent mTEC turnover and/or survival involving CD40‐mediated signalling following interactions with mature CD4+ thymocytes that express CD40L.

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs

Significance Ectopic clusters of immune cells that mimic the structure and function of secondary lymphoid organs are defined as tertiary lymphoid organs (TLOs). They have been observed at sites of chronic inflammation for decades, but their formation and function have remained enigmatic. TLOs are thought to contribute to disease pathogenesis by promoting autoreactive lymphocyte survival and autoantibody production. In this study we identify a novel role for the cytokine IL-22 in TLO development and biology. We provide evidence that IL-22 expression within TLOs is instrumental for the production of the lymphoid chemokines, chemokine (C-X-C motif) ligand 13 and chemokine (C-X-C motif) ligand 12, which in turn orchestrate B-cell clustering, lymphoid aggregation, and autoantibody production. Our data provide a strong rationale for targeting IL-22 in TLO-associated autoimmune diseases. The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22–blocking agents in B-cell–mediated autoimmune conditions.

Transient inhibition of ROR-[gamma]t therapeutically limits intestinal inflammation by reducing TH17 cells and preserving group 3 innate lymphoid cells

RAR-related orphan receptor-γt (ROR-γt) directs differentiation of proinflammatory T helper 17 (TH17) cells and is a potential therapeutic target in chronic autoimmune and inflammatory diseases. However, ROR-γt–dependent group 3 innate lymphoid cells ILC3s provide essential immunity and tissue protection in the intestine, suggesting that targeting ROR-γt could also result in impaired host defense after infection or enhanced tissue damage. Here, we demonstrate that transient chemical inhibition of ROR-γt in mice selectively reduces cytokine production from TH17 but not ILCs in the context of intestinal infection with Citrobacter rodentium, resulting in preserved innate immunity. Temporal deletion of Rorc (encoding ROR-γt) in mature ILCs also did not impair cytokine response in the steady state or during infection. Finally, pharmacologic inhibition of ROR-γt provided therapeutic benefit in mouse models of intestinal inflammation and reduced the frequency of TH17 cells but not ILCs isolated from primary intestinal samples of individuals with inflammatory bowel disease (IBD). Collectively, these results reveal differential requirements for ROR-γt in the maintenance of TH17 cell and ILC3 responses and suggest that transient inhibition of ROR-γt is a safe and effective therapeutic approach during intestinal inflammation.