Within the human gut reside diverse microbes coexisting with the host in a mutually advantageous relationship. Evidence has revealed the pivotal role of the gut microbiota in shaping the immune ...system. To date, only a few of these microbes have been shown to modulate specific immune parameters. Herein, we broadly identify the immunomodulatory effects of phylogenetically diverse human gut microbes. We monocolonized mice with each of 53 individual bacterial species and systematically analyzed host immunologic adaptation to colonization. Most microbes exerted several specialized, complementary, and redundant transcriptional and immunomodulatory effects. Surprisingly, these were independent of microbial phylogeny. Microbial diversity in the gut ensures robustness of the microbiota’s ability to generate a consistent immunomodulatory impact, serving as a highly important epigenetic system. This study provides a foundation for investigation of gut microbiota-host mutualism, highlighting key players that could identify important therapeutics.
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•Human gut microbiota comprises a treasure trove of immunomodulatory bacteria•Diverse and redundant immune and transcriptional responses follow monocolonization•Immunologic and transcriptional changes are not related to microbial phylogeny•Following monocolonization, immune recalibration varies to strains within a species
Each of 53 human-resident bacterial species studied in monoculture in mice modulates the host immune system, providing a baseline for investigating how consortia of gut microbes interact with their host.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration ...and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L‐cells overexpressing WNT3a or control un‐transduced L‐cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B‐GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate ...the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Motivation: Typical analysis of microarray data has focusedon spot by spot comparisons within a single organism. Less analysis has been done on the comparison of the entire distribution of spot ...intensities between experiments and between organisms. Results: Here we show that mRNA transcription data from a wide range of organisms and measured with a range of experimental platforms show close agreement with Benford’s law (Benford, Proc. Am. Phil. Soc. , 78, 551–572, 1938) and Zipf’s law (Zipf, The Psycho-biology of Language: an Introduction to Dynamic Philology , 1936 and Human Behaviour and the Principle of Least Effort , 1949). The distribution of the bulk of microarray spot intensities is well approximated by a log-normal with the tail of the distribution being closer to power law. The variance, σ2, of log spot intensity shows a positive correlation with genome size (in terms of number of genes) and is therefore relatively fixed within some range for a given organism. The measured value of σ2 can be significantly smaller than the expected value if the mRNA is extracted from a sample of mixed cell types. Our research demonstrates that useful biological findings may result from analyzing microarray data at the level of entire intensity distributions. Contact: david.c.hoyle@man.ac.uk * To whom correspondence should be addressed.
The metabolic pathways encoded by the human gut microbiome constantly interact with host gene products through numerous bioactive molecules
. Primary bile acids (BAs) are synthesized within ...hepatocytes and released into the duodenum to facilitate absorption of lipids or fat-soluble vitamins
. Some BAs (approximately 5%) escape into the colon, where gut commensal bacteria convert them into various intestinal BAs
that are important hormones that regulate host cholesterol metabolism and energy balance via several nuclear receptors and/or G-protein-coupled receptors
. These receptors have pivotal roles in shaping host innate immune responses
. However, the effect of this host-microorganism biliary network on the adaptive immune system remains poorly characterized. Here we report that both dietary and microbial factors influence the composition of the gut BA pool and modulate an important population of colonic FOXP3
regulatory T (T
) cells expressing the transcription factor RORγ. Genetic abolition of BA metabolic pathways in individual gut symbionts significantly decreases this T
cell population. Restoration of the intestinal BA pool increases colonic RORγ
T
cell counts and ameliorates host susceptibility to inflammatory colitis via BA nuclear receptors. Thus, a pan-genomic biliary network interaction between hosts and their bacterial symbionts can control host immunological homeostasis via the resulting metabolites.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
T regulatory cells that express the transcription factor Foxp3 (Foxp3+ Tregs) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a ...distinct Treg population in the mouse colon, which constrains immuno-inflammatory responses. This induction–which we find to map to a broad, but specific, array of individual bacterial species–requires the transcription factor Rorγ, paradoxically, in that Rorγ is thought to antagonize FoxP3 and to promote T helper 17 (TH17) cell differentiation. Rorγ's transcriptional footprint differs in colonic Tregs and TH17 cells and controls important effector molecules. Rorγ, and the Tregs that express it, contribute substantially to regulating colonic TH1/TH17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell types.
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Th17 cells accrue in the intestine in response to particular microbes. In rodents, segmented filamentous bacteria (SFB) induce intestinal Th17 cells, but analogously functioning microbes in humans ...remain undefined. Here, we identified human symbiont bacterial species, in particular Bifidobacterium adolescentis, that could, alone, induce Th17 cells in the murine intestine. Similar to SFB, B. adolescentis was closely associated with the gut epithelium and engendered cognate Th17 cells without attendant inflammation. However, B. adolescentis elicited a transcriptional program clearly distinct from that of SFB, suggesting an alternative mechanism of promoting Th17 cell accumulation. Inoculation of mice with B. adolescentis exacerbated autoimmune arthritis in the K/BxN mouse model. Several off-the-shelf probiotic preparations that include Bifidobacterium strains also drove intestinal Th17 cell accumulation.
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Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors ...are unknown. Here, we report that layilin, a C-type lectin domain-containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLβ2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1-dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or "dysfunctional" CD8+ T cells enhance cellular adhesiveness to maintain their cytotoxic potential.
Abstract The presence of tertiary lymphoid structures (TLS) and associated high endothelial venules (HEV) in the tumor microenvironment strongly correlates with improved prognosis and treatment ...outcomes across a wide range of solid tumors. These clinical observations, now replicated repeatedly, highlight the therapeutic potential of induction of TLS and/or boosting TLS functions, which include acting as a point of entry and education for immune effector cells locally in the tumor. Lymphotoxin beta receptor (LTBR) signalling is essential for lymphoid structure development and the ectopic expression of its ligands, lymphotoxin αβ and LIGHT, is sufficient for TLS formation in vivo but challenging for therapeutic development. Here we present human bispecific antibodies (bsAbs) which conditionally agonize LTBR on co-engagement of fibroblast activation protein (FAP), a tumor microenvironment specific marker expressed by cancer associated fibroblasts. In vitro characterisation of these therapeutic bsAbs is presented, along with in vivo preclinical evaluation of surrogate bsAbs binding to mouse LTBR and mouse FAP in murine tumor models. Results - Therapeutic bsAbs were identified following an extensive screening campaign combining LTBR and FAP binding arms on an engineered human Fc with minimal FcyR binding. In vitro assays using primary cancer associated fibroblasts and LTBR positive cells demonstrated conditionally active therapeutic bsAbs potently activated LTBR in the presence of FAP expressing cells, whereas in the absence of FAP expressing cells no activity was observed. Surrogate bsAbs demonstrated monotherapy activity and led to tumor regressions in combination with anti-PDL1 therapy in an EMT6 mouse model, a syngeneic orthotopic model of breast cancer. Flow cytometry of endpoint tumors showed robust formation of HEVs and increased infiltration of T cells and B cells. In addition, we demonstrate that LTBR agonism, but not PD-L1 inhibition, leads to the formation of TLS structures containing organized lymphocyte aggregates with the appearance of germinal centres and accumulations of T and B cells in a mouse model of lung cancer. In conclusion, conditionally active FAP-LTBR bispecifics activate LTBR in the tumor microenvironment and induce HEV and TLS formation, leading to potent monotherapy activity in vivo, and to tumor regression in combination with PD-L1. These data support the development of FAP-LTBR bispecifics for the treatment of solid tumors as monotherapy and in combination with standard of care. Citation Format: Marta Lewandowska, Fevzi Demircioglu, Lucy Penfold, Rebecca B. Riddle, Sameer Sirohi, Floriane Laurent, Richard Brown, Sonia Bains, Dara Bevan, Emma Stanley, Jeanine Pignatelli, James W. Legg, Ray Jupp. Conditionally active, therapeutic lymphotoxin beta receptor (LTBR) agonist bispecific antibodies for induction of tertiary lymphoid structures in the treatment of solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB123.
Our guts harbor trillions of microbial inhabitants, some of which regulate the types of immune cells that are present in the gut. For instance, Clostridium species of bacteria induce a type of T cell ...that promotes tolerance between the host and its microbial contents. Ohnmacht et al. and Sefik et al. characterized a population of gut regulatory T cells in mice, which required gut microbiota to survive. Multiple bacterial species of the microbiota could induce transcription factor-expressing regulatory T cells that helped maintain immune homeostasis. Mice engineered to lack these transcription factors exhibited enhanced susceptibility to colonic inflammation and had elevated amounts of proinflammatory molecules associated with allergies (see the Perspective by Hegazy and Powrie). Science, this issue pp. 989 and 993 T regulatory cells that express the transcription factor Foxp3 (Foxp3+ Tregs) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct Treg population in the mouse colon, which constrains immuno-inflammatory responses. This induction--which we find to map to a broad, but specific, array of individual bacterial species--requires the transcription factor Rorγ, paradoxically, in that Rorγ is thought to antagonize FoxP3 and to promote T helper 17 (TH17) cell differentiation. Rorγ's transcriptional footprint differs in colonic Tregs and TH17 cells and controls important effector molecules. Rorγ, and the Tregs that express it, contribute substantially to regulating colonic TH1/TH17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell types.
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