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Splenic dendritic cells (DCs) present blood-borne antigens to lymphocytes to promote T cell and antibody responses. The cues involved in positioning DCs in areas of antigen exposure in the spleen are undefined. Here we show that CD4+ DCs highly express EBI2 and migrate to its oxysterol ligand, 7α,25-OHC. In mice lacking EBI2 or the enzymes needed for generating normal distributions of 7α,25-OHC, CD4+ DCs are reduced in frequency and the remaining cells fail to situate in marginal zone bridging channels. The CD4+ DC deficiency can be rescued by LTβR agonism. EBI2-mediated positioning in bridging channels promotes DC encounter with blood-borne particulate antigen. Upon exposure to antigen, CD4+ DCs move rapidly to the T-B zone interface and promote induction of helper T cell and antibody responses. These findings establish an essential role for EBI2 in CD4+ DC positioning and homeostasis and in facilitating capture and presentation of blood-borne particulate antigens. One of the main roles of the spleen is to make the antibodies that protect the body against viruses, bacteria and other microorganisms. Antibodies are made by B cells, which are a type of white blood cell, after they have been exposed to antigens. For most antibody responses, it is also necessary for the B cells to get help from other white blood cells called T cells that have been exposed to antigens. Specialized cells called dendritic cells have a central role in bringing the antigens—which are usually fragments of the infectious agents that have invaded the body—to the T cells. One subset of dendritic cells, called CD4+ dendritic cells, are found in large numbers in a part of the spleen called the bridging channel, but the process by which these cells become localized in this channel has not been fully understood. Now, Yi and Cyster show that a receptor called EBI2, which is found on the surface of CD4+ dendritic cells, binds to a type of organic molecule called an oxysterol that is produced in the bridging channel. In mice that had been genetically engineered to lack EBI2 or the enzymes needed to make this particular oxysterol—which is known as 7α,25-dihydroxycholesterol, or 7α,25-OHC for short—the CD4+ dendritic cells were no longer clustered in the bridging channel and their number was markedly decreased. This showed that the interaction between EBI2 and the oxysterol was essential for ensuring that the CD4+ dendritic cells were in the right place. The correct positioning of the CD4+ dendritic cells was, in turn, necessary for maintaining cell numbers. Moreover, these mice had a weakened immune response because of the very low number of antigens that were being presented to the T cells. A number of autoimmune diseases, such as lupus, are caused by the body developing an immune response to its own cells and tissues. One implication of the work of Yi and Cyster is that if small molecule inhibitors of EBI2 could be designed, they might be able to suppress the onset of such autoimmune responses.

NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O 2 •− ), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation. NADPH oxidase NOX2 produces superoxide, a reactive oxygen species essential in innate immunity. Here, the authors reveal the structure of the NOX2 core, rationalize disease-causing mutations, and suggest avenues for selective NOX2 pharmacological modulation.

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering. β-tryptases are responsible for most of the proteolytic activity during mast cell activation. Here, the authors develop β-tryptase-inhibiting antibodies and provide structural and biochemical evidence that the bivalency of the antibodies is a prerequisite for their inhibitory activity.

EBI2 receptors revealed as oxysterols The EBI2 receptor (Epstein–Barr virus-induced gene 2, also known as GPR183) was recently shown to be linked to autoimmune disease, and is a critical regulator of the humoral immune response. It is a G-protein-coupled receptor, and its natural ligand has been unknown. Two groups now bring an end to the 'orphan' status of this receptor with identification of specific oxysterols as its natural ligands. The most potent ligand and activator is 7a,25-dihydroxycholesterol, and the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response. Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases 1 , 2 , 3 . The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo . A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H) 4 . Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples.

Background IL-22 is induced by aryl hydrocarbon receptor (AhR) signaling and plays a critical role in gastrointestinal barrier function through effects on antimicrobial protein production, mucus secretion, and epithelial cell differentiation and proliferation, giving it the potential to modulate the microbiome through these direct and indirect effects. Furthermore, the microbiome can in turn influence IL-22 production through the synthesis of L-tryptophan (L-Trp)-derived AhR ligands, creating the prospect of a host-microbiome feedback loop. We evaluated the impact IL-22 may have on the gut microbiome and its ability to activate host AhR signaling by observing changes in gut microbiome composition, function, and AhR ligand production following exogenous IL-22 treatment in both mice and humans. Results Microbiome alterations were observed across the gastrointestinal tract of IL-22-treated mice, accompanied by an increased microbial functional capacity for L-Trp metabolism. Bacterially derived indole derivatives were increased in stool from IL-22-treated mice and correlated with increased fecal AhR activity. In humans, reduced fecal concentrations of indole derivatives in ulcerative colitis (UC) patients compared to healthy volunteers were accompanied by a trend towards reduced fecal AhR activity. Following exogenous IL-22 treatment in UC patients, both fecal AhR activity and concentrations of indole derivatives increased over time compared to placebo-treated UC patients. Conclusions Overall, our findings indicate IL-22 shapes gut microbiome composition and function, which leads to increased AhR signaling and suggests exogenous IL-22 modulation of the microbiome may have functional significance in a disease setting. Cw21cUiGabCHLYfc9EL5iq Video Abstract

IntroductionTargeting integrins that mediate adhesion and migration of lymphocytes to the gastrointestinal (GI) tract is an effective therapy in inflammatory bowel disease (IBD). α4β7 and α4β1 are expressed on circulating lymphocytes that may mediate inflammation, while αEβ7 integrin is expressed primarily on a subset of T cells within the mucosa. Etrolizumab is a humanized monoclonal antibody that selectively binds the β7 subunit of the α4β7 and αEβ7 integrin heterodimers. The relative role of individual integrin heterodimers in lymphocyte migration and retention in the GI tract remains to be characterized.MethodspSMAD3, MAdCAM, VCAM and ICAM levels were measured in colonic and ileal biopsies. α4β7+ and α4β7- human T cells were induced to express αE integrin by TGF-β1 stimulation followed by qPCR array gene expression analysis. A murine photo-convertible reporter system was used to determine the effect of blockade of α4β7 and/or αEβ7 integrins on lymphocyte migration and retention. T cell-epithelial cell interactions were evaluated using intravital two-photon microscopy.ResultspSMAD3 was observed in the epithelium and lamina propria in IBD biopsies, suggesting active TGF-β signalling. Adhesion molecule expression was increased in inflamed biopsies. TGF-β1 stimulation induced αE integrin expression on both α4β7+ and α4β7- circulating T cells. αEβ7+ cells derived from α4β7+ and α4β7- progenitors had similar cytokine, chemokine, transcription factors and effector molecule gene expression. In a mouse model of T cell migration, combined blockade of both α4β7 and αEβ7 with anti-β7 (etrolizumab surrogate) led to a greater reduction of T cell accumulation in the intestinal mucosa and epithelium compared to single blockade of either α4β7 or αEβ7. Further intravital two-photon microscopy and photo-specific labelling experiments revealed that blockade of αEβ7 reduces T cell:epithelial cell interactions, increases the migratory speed of activated T cells in the intestinal mucosa, and facilitates effector T cell egress from the intestinal mucosa through lymphatic vessels.ConclusionsαEβ7 is induced by TGF-β1 on both α4β7+ or α4β7- T cells. Co-blockade of α4β7 and αEβ7 together leads to greater inhibition of T cell accumulation in gastrointestinal tissues through a stepwise inhibition of T cell migration and subsequent tissue retention.

Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.

The differentiation of T follicular helper cells requires the G-protein-coupled receptor Ebi2 as well as the interaction with CD25-producing dendritic cells that quench T-cell-derived interleukin-2. EBI2 required for Tfh cell maturation The T-cell subset known as T follicular helper (Tfh) cells are crucial for mounting antibody responses, but the cues that guide their positioning and development are incompletely understood. Here Jianhua Li et al . demonstrate that the differentiation of Tfh cells requires the G-protein-coupled receptor EBI2 (also called GPR183), as well as the interaction with CD25-producing dendritic cells that quench T-cell-derived interleukin-2. T follicular helper (Tfh) cells are a subset of T cells carrying the CD4 antigen; they are important in supporting plasma cell and germinal centre responses 1 , 2 . The initial induction of Tfh cell properties occurs within the first few days after activation by antigen recognition on dendritic cells, although how dendritic cells promote this cell-fate decision is not fully understood 1 , 2 . Moreover, although Tfh cells are uniquely defined by expression of the follicle-homing receptor CXCR5 (refs 1 , 2 ), the guidance receptor promoting the earlier localization of activated T cells at the interface of the B-cell follicle and T zone has been unclear 3 , 4 , 5 . Here we show that the G-protein-coupled receptor EBI2 (GPR183) and its ligand 7α,25-dihydroxycholesterol mediate positioning of activated CD4 T cells at the interface of the follicle and T zone. In this location they interact with activated dendritic cells and are exposed to Tfh-cell-promoting inducible co-stimulator (ICOS) ligand. Interleukin-2 (IL-2) is a cytokine that has multiple influences on T-cell fate, including negative regulation of Tfh cell differentiation 6 , 7 , 8 , 9 , 10 . We demonstrate that activated dendritic cells in the outer T zone further augment Tfh cell differentiation by producing membrane and soluble forms of CD25, the IL-2 receptor α-chain, and quenching T-cell-derived IL-2. Mice lacking EBI2 in T cells or CD25 in dendritic cells have reduced Tfh cells and mount defective T-cell-dependent plasma cell and germinal centre responses. These findings demonstrate that distinct niches within the lymphoid organ T zone support distinct cell fate decisions, and they establish a function for dendritic-cell-derived CD25 in controlling IL-2 availability and T-cell differentiation.

Key Points The oxysterols 25-hydroxycholesterol (25-HC) and 7α,25-dihydroxycholesterol (7α,25-HC) are made by a range of cell types in lymphoid and non-lymphoid tissues, and their production is upregulated by inflammatory stimuli. The ability of 25-HC to regulate the synthesis and compartmentalization of lipids enables it to antagonize the replication of a wide diversity of viruses. 25-HC functions downstream of interferon (IFN) signalling to suppress interleukin-1β (IL-1β) expression and inflammasome activity. This mechanism of IFN-mediated feedback regulation of inflammation occurs at least in part through 25-HC-mediated suppression of sterol response element-binding protein (SREBP) processing. 25-HC augments the expression of some inflammatory cytokines, including IL-6. The mechanisms by which the anti-inflammatory and pro-inflammatory actions of 25-HC might be coordinated are discussed. 7α,25-HC, the most potent ligand of the G protein-coupled receptor EBI2 (also known as GPR183), guides the migration of B cells and dendritic cells to interfollicular regions of lymphoid tissues. EBI2 and 7α,25-HC deficiency both cause defective antibody responses. A model is discussed in which 25-HC and 7α,25-HC produced in overlapping zones might function in a coordinated manner to recruit EBI2 + cells, promote their resistance to viral infection and control their cytokine production. The closely related oxysterols, 25-hydroxycholesterol and 7α,25-dihydroxycholesterol, have important functions in innate and adaptive immune responses, ranging from antiviral and inflammation-regulatory effects to a role as a guidance cue for B cells and dendritic cells. Cholesterol and components of the cholesterol biosynthetic pathway have fundamental roles in all mammalian cells. Hydroxylated forms of cholesterol are now emerging as important regulators of immune function. This involves effects on the cholesterol biosynthetic pathway and cell membrane properties, which can have antiviral and anti-inflammatory influences. In addition, a dihydroxylated form of cholesterol functions as an immune cell guidance cue by engaging the G protein-coupled receptor EBI2, and it is required for mounting adaptive immune responses. In this Review, we summarize the current understanding of the closely related oxysterols 25-hydroxycholesterol and 7α,25-dihydroxycholesterol, and the growing evidence that they have wide-ranging influences on innate and adaptive immunity.