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Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

BackgroundUlcerative colitis (UC) and Crohn's disease are 2 types of inflammatory bowel disease (IBD), a group of chronic digestive disorders caused by aberrant immune responses to intestinal microbes. Although changes in the composition of immune cell subsets in the context of IBD have been previously described, the interactions and communication among cells are less well understood. Moreover, the precise mechanisms of action underlying many biologic therapies, including the anti-α4β7 integrin antagonist vedolizumab, remain incompletely understood. Our study aimed to explore possible additional mechanisms through which vedolizumab acts.MethodsWe performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on peripheral blood and colon immune cells derived from patients with ulcerative colitis treated with the anti-α4β7 integrin antagonist vedolizumab. We applied a previously published computational approach, NicheNet, to predict immune cell-cell interactions, revealing putative ligand-receptor pairs and key transcriptional changes downstream of these cell-cell communications (CCC).ResultsWe observed decreased proportions of T helper 17 (TH17) cells in UC patients who responded to vedolizumab and therefore focused the study on identifying cell-cell communications and signals of TH17 cells with other immune cells. For example, we observed that colon TH17 cells from vedolizumab nonresponders were predicted to have a greater degree of interactions with classical monocytes compared with responders, whereas colon TH17 cells from vedolizumab responders exhibited more interactions with myeloid dendritic cells compared with nonresponders.ConclusionsOverall, our results indicate that efforts to elucidate cell-cell communications among immune and nonimmune cell types may increase the mechanistic understanding of current and investigational therapies for IBD.

Canonical interleukin-2 (IL-2) signaling via the high-affinity CD25-containing IL-2 receptor-Janus kinase (JAK)1,3-signal transducer and activator of transcription 5 (STAT5) pathway is essential for development and maintenance of CD4+CD25HiFoxp3+ regulatory T cells (Tregs) that support immune homeostasis. Here, we report that IL-2 signaling via an alternative CD25-chemokine receptor pathway promotes the suppressive function of Tregs. Using an antibody against CD25 that biases IL-2 signaling toward this alternative pathway, we establish that this pathway increases the suppressive activity of Tregs and ameliorates murine experimental autoimmune encephalomyelitis (EAE). Furthermore, heparan sulfate, an IL-2-binding element of cell surfaces and extracellular matrix, or an engineered IL-2 immunocytokine can also direct IL-2 signaling toward this alternative pathway. Overall, these data reveal a non-canonical mechanism for IL-2 signaling that promotes suppressive functions of Tregs, further elucidates how IL-2 supports immune homeostasis, and suggests approaches to promote or suppress Treg functions.

Tissue-resident memory CD8+ T (TRM) cells are a subset of memory T cells that play a critical role in limiting early pathogen spread and controlling infection. TRM cells exhibit differences across tissues, but their potential heterogeneity among distinct anatomic compartments within the small intestine and colon has not been well recognized. Here, by analyzing TRM cells from the lamina propria and epithelial compartments of the small intestine and colon, we showed that intestinal TRM cells exhibited distinctive patterns of cytokine and granzyme expression along with substantial transcriptional, epigenetic, and functional heterogeneity. The T-box transcription factor Eomes, which represses TRM cell formation in some tissues, exhibited unexpected context-specific regulatory roles in supporting the maintenance of established TRM cells in the small intestine, but not in the colon. Taken together, these data provide previously unappreciated insights into the heterogeneity and differential requirements for the formation vs. maintenance of intestinal TRM cells.

IntroductionCrohn's disease (CD) is a major subtype of inflammatory bowel disease (IBD), a spectrum of chronic intestinal disorders caused by dysregulated immune responses to gut microbiota. Although transcriptional and functional changes in a number of immune cell types have been implicated in the pathogenesis of IBD, the cellular interactions and signals that drive these changes have been less well-studied.MethodsWe performed Cellular Indexing of Transcriptomes and Epitopes by sequencing on peripheral blood, colon, and ileal immune cells derived from healthy subjects and patients with CD. We applied a previously published computational approach, NicheNet, to predict immune cell types interacting with CD8 + T-cell subsets, revealing putative ligand-receptor pairs and key transcriptional changes downstream of these cell-cell communications.ResultsAs a number of recent studies have revealed a potential role for CD8 + T-cell subsets in the pathogenesis of IBD, we focused our analyses on identifying the interactions of CD8 + T-cell subsets with other immune cells in the intestinal tissue microenvironment. We identified ligands and signaling pathways that have implicated in IBD, such as interleukin-1β, supporting the validity of the approach, along with unexpected ligands, such as granzyme B, which may play previously unappreciated roles in IBD.DiscussionOverall, these findings suggest that future efforts focused on elucidating cell-cell communications among immune and nonimmune cell types may further our understanding of IBD pathogenesis.

Tissue-resident memory T cells (TRM cells) provide protective immunity, but the contributions of specific tissue environments to TRM cell differentiation and homeostasis are not well understood. In the present study, the diversity of gene expression and genome accessibility by mouse CD8+ TRM cells from distinct organs that responded to viral infection revealed both shared and tissue-specific transcriptional and epigenetic signatures. TRM cells in the intestine and salivary glands expressed transforming growth factor (TGF)-β-induced genes and were maintained by ongoing TGF-β signaling, whereas those in the fat, kidney and liver were not. Constructing transcriptional-regulatory networks identified the transcriptional repressor Hic1 as a critical regulator of TRM cell differentiation in the small intestine and showed that Hic1 overexpression enhanced TRM cell differentiation and protection from infection. Provision of a framework for understanding how CD8+ TRM cells adapt to distinct tissue environments, and identification of tissue-specific transcriptional regulators mediating these adaptations, inform strategies to boost protective memory responses at sites most vulnerable to infection.

During an immune response to microbial infection, CD8+ T cells give rise to short-lived effector cells and memory cells that provide sustained protection. Although the transcriptional programs regulating CD8+ T cell differentiation have been extensively characterized, the role of long noncoding RNAs (lncRNAs) in this process remains poorly understood. Using a functional genetic knockdown screen, we identified the lncRNA Malat1 as a regulator of terminal effector cells and the terminal effector memory (t-TEM) circulating memory subset. Evaluation of chromatin-enriched lncRNAs revealed that Malat1 grouped with trans lncRNAs that exhibit increased RNA interactions at gene promoters and gene bodies. Moreover, we observed that Malat1 was associated with increased H3K27me3 deposition at a number of memory cell-associated genes through a direct interaction with Ezh2, thereby promoting terminal effector and t-TEM cell differentiation. Our findings suggest an important functional role of Malat1 in regulating CD8+ T cell differentiation and broaden the knowledge base of lncRNAs in CD8+ T cell biology.

RAR-related orphan receptor-γ (RORγt) is an essential transcription factor for thymic T cell development, secondary lymphoid tissue organogenesis, and peripheral immune cell differentiation. Serine 182 phosphorylation is a major post-translational modification (PTM) on RORγt. However, the in vivo contribution of this PTM in health and disease settings is unclear. We report that this PTM is not involved in thymic T cell development and effector T cell differentiation. Instead, it is a critical regulator of inflammation downstream of IL-1β signaling and extracellular signal regulated kinases (ERKs) activation. ERKs phosphorylation of serine 182 on RORγt serves to simultaneously restrict Th17 hyperactivation and promote anti-inflammatory cytokine IL-10 production in RORγt+ Treg cells. Phospho-null RORγtS182A knockin mice experience exacerbated inflammation in models of colitis and experimental autoimmune encephalomyelitis (EAE). In summary, the IL-1β-ERK-RORγtS182 circuit protects against T cell-mediated inflammation and provides potential therapeutic targets to combat autoimmune diseases.

Immune-modulating medications for inflammatory bowel diseases (IBDs) have been associated with suboptimal vaccine responses. There are conflicting data with SARS-CoV-2 vaccination. We therefore assessed SARS-CoV-2 vaccine immunogenicity at 2 weeks after second mRNA vaccination in 29 patients with IBD compared with 12 normal healthy donors. We observed reduced humoral immunity in patients with IBD on infliximab. However, we observed no difference in humoral and cell-mediated immunity in patients with IBD on infliximab with a thiopurine or vedolizumab compared with normal healthy donors. This is the first study to demonstrate comparable cell-mediated immunity with SARS-CoV-2 vaccination in patients with IBD treated with different immune-modulating medications.

Transcription factors (TFs) are crucial for regulating cell differentiation during the development of the immune system. However, the key TFs for orchestrating the specification of distinct immune cells are not fully understood. Here, we integrated the transcriptomic and epigenomic measurements in 73 mouse and 61 human primary cell types, respectively, that span the immune cell differentiation pathways. We constructed the cell-type-specific transcriptional regulatory network and assessed the global importance of TFs based on the Taiji framework, which is a method we have previously developed that can infer the global impact of TFs using integrated transcriptomic and epigenetic data. Integrative analysis across cell types revealed putative driver TFs in cell lineage-specific differentiation in both mouse and human systems. We have also identified TF combinations that play important roles in specific developmental stages. Furthermore, we validated the functions of predicted novel TFs in murine CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Thus, we have developed a bioinformatic approach that provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions.

Background & aimsHyperbaric oxygen therapy (HBOT) is a promising treatment for moderate-to-severe ulcerative colitis. However, our current understanding of the host and microbial response to HBOT remains unclear. This study examined the molecular mechanisms underpinning HBOT using a multi-omic strategy.MethodsPre- and post-intervention mucosal biopsies, tissue, and fecal samples were collected from HBOT phase 2 clinical trials. Biopsies and fecal samples were subjected to shotgun metaproteomics, metabolomics, 16s rRNA sequencing, and metagenomics. Tissue was subjected to bulk RNA sequencing and digital spatial profiling (DSP) for single-cell RNA and protein analysis, and immunohistochemistry was performed. Fecal samples were also used for colonization experiments in IL10-/- germ-free UC mouse models.ResultsProteomics identified negative associations between HBOT response and neutrophil azurophilic granule abundance. DSP identified an HBOT-specific reduction of neutrophil STAT3, which was confirmed by immunohistochemistry. HBOT decreased microbial diversity with a proportional increase in Firmicutes and a secondary bile acid lithocholic acid. A major source of the reduction in diversity was the loss of mucus-adherent taxa, resulting in increased MUC2 levels post-HBOT. Targeted database searching revealed strain-level associations between Akkermansia muciniphila and HBOT response status. Colonization of IL10-/- with stool obtained from HBOT responders resulted in lower colitis activity compared with non-responders, with no differences in STAT3 expression, suggesting complementary but independent host and microbial responses.ConclusionsHBOT reduces host neutrophil STAT3 and azurophilic granule activity in UC patients and changes in microbial composition and metabolism in ways that improve colitis activity. Intestinal microbiota, especially strain level variations in A muciniphila, may contribute to HBOT non-response.

Inflammatory disorders of the skin are frequently associated with inflammatory bowel diseases (IBDs). To explore mechanisms by which these organs communicate, we performed single-cell RNA-Seq analysis on fibroblasts from humans and mice with IBD. This analysis revealed that intestinal inflammation promoted differentiation of a subset of intestinal stromal fibroblasts into preadipocytes with innate antimicrobial host defense activity. Furthermore, this process of reactive adipogenesis was exacerbated if mouse skin was inflamed as a result of skin wounding or infection. Since hyaluronan (HA) catabolism is activated during skin injury and fibroblast-to-adipocyte differentiation is dependent on HA, we tested the hypothesis that HA fragments could alter colon fibroblast function by targeted expression of human hyaluronidase-1 in basal keratinocytes from mouse skin. Hyaluronidase expression in the skin activated intestinal stromal fibroblasts, altered the fecal microbiome, and promoted excessive reactive adipogenesis and increased inflammation in the colon after challenge with dextran sodium sulfate. The response to digested HA was dependent on expression of TLR4 by preadipocytes. Collectively, these results suggest that the association between skin inflammation and IBD may be due to recognition by mesenchymal fibroblasts in the colon of HA released during inflammation of the skin.

Modeling human diseases as networks simplify complex multi-cellular processes, helps understand patterns in noisy data that humans cannot find, and thereby improves precision in prediction. Using Inflammatory Bowel Disease (IBD) as an example, here we outline an unbiased AI-assisted approach for target identification and validation. A network was built in which clusters of genes are connected by directed edges that highlight asymmetric Boolean relationships. Using machine-learning, a path of continuum states was pinpointed, which most effectively predicted disease outcome. This path was enriched in gene-clusters that maintain the integrity of the gut epithelial barrier. We exploit this insight to prioritize one target, choose appropriate pre-clinical murine models for target validation and design patient-derived organoid models. Potential for treatment efficacy is confirmed in patient-derived organoids using multivariate analyses. This AI-assisted approach identifies a first-in-class gut barrier-protective agent in IBD and predicted Phase-III success of candidate agents.

BackgroundMany studies have investigated the role of the microbiome in inflammatory bowel disease (IBD), but few have focused on surgery specifically or its consequences on the metabolome that may differ by surgery type and require longitudinal sampling. Our objective was to characterize and contrast microbiome and metabolome changes after different surgeries for IBD, including ileocolonic resection and colectomy.MethodsThe UC San Diego IBD Biobank was used to prospectively collect 332 stool samples from 129 subjects (50 ulcerative colitis; 79 Crohn's disease). Of these, 21 with Crohn's disease had ileocolonic resections, and 17 had colectomies. We used shotgun metagenomics and untargeted liquid chromatography followed by tandem mass spectrometry metabolomics to characterize the microbiomes and metabolomes of these patients up to 24 months after the initial sampling.ResultsThe species diversity and metabolite diversity both differed significantly among groups (species diversity: Mann-Whitney U test P value = 7.8e-17; metabolomics, P-value = 0.0043). Escherichia coli in particular expanded dramatically in relative abundance in subjects undergoing surgery. The species profile was better able to classify subjects according to surgery status than the metabolite profile (average precision 0.80 vs 0.68).ConclusionsIntestinal surgeries seem to reduce the diversity of the gut microbiome and metabolome in IBD patients, and these changes may persist. Surgery also further destabilizes the microbiome (but not the metabolome) over time, even relative to the previously established instability in the microbiome of IBD patients. These long-term effects and their consequences for health outcomes need to be studied in prospective longitudinal trials linked to microbiome-involved phenotypes.

Background & aimsWe created and validated a clinical decision support tool (CDST) to predict outcomes of vedolizumab therapy for ulcerative colitis (UC).MethodsWe performed logistic regression analyses of data from the GEMINI 1 trial, from 620 patients with UC who received vedolizumab induction and maintenance therapy (derivation cohort), to identify factors associated with corticosteroid-free remission (full Mayo score of 2 or less, no subscore above 1). We used these factors to develop a model to predict outcomes of treatment, which we called the vedolizumab CDST. We evaluated the correlation between exposure and efficacy. We validated the CDST in using data from 199 patients treated with vedolizumab in routine practice in the United States from May 2014 through December 2017.ResultsAbsence of exposure to a tumor necrosis factor (TNF) antagonist (+3 points), disease duration of 2 y or more (+3 points), baseline endoscopic activity (moderate vs severe) (+2 points), and baseline albumin concentration (+0.65 points per 1 g/L) were independently associated with corticosteroid-free remission during vedolizumab therapy. Patients in the derivation and validation cohorts were assigned to groups of low (CDST score, 26 points or less), intermediate (CDST score, 27-32 points), or high (CDST score, 33 points or more) probability of vedolizumab response. We observed a statistically significant linear relationship between probability group and efficacy (area under the receiver operating characteristic curve, 0.65), as well as drug exposure (P < .001) in the derivation cohort. In the validation cohort, a cutoff value of 26 points identified patients who did not respond to vedolizumab with high sensitivity (93%); only the low and intermediate probability groups benefited from reducing intervals of vedolizumab administration due to lack of response (P = .02). The vedolizumab CDST did not identify patients with corticosteroid-free remission during TNF antagonist therapy.ConclusionsWe used data from a trial of patients with UC to develop a scoring system, called the CDST, which identified patients most likely to enter corticosteroid-free remission during vedolizumab therapy, but not anti-TNF therapy. We validated the vedolizumab CDST in a separate cohort of patients in clinical practice. The CDST identified patients most likely to benefited from reducing intervals of vedolizumab administration due to lack of initial response. ClinicalTrials.gov no: NCT00783718.

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated that circulating memory cells can be further divided into effector memory (Tem) and central memory (Tcm) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was terminally fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally differentiated effector memory CD8 T cell population (terminal-Tem) was conflated within the conventional Tem population, prompting redefinition of the classical characteristics of Tem cells. Murine terminal-Tem were transcriptionally, functionally, and developmentally unique compared to Tem cells. Through mass cytometry and single-cell RNA sequencing (RNA-seq) analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-Tem population of CD8 T cells that was transcriptionally distinct from Tem and Tcm Key findings from this study show that parsing of terminal-Tem from conventionally defined Tem challenge the reported characteristics of Tem biology, including enhanced presence in lymphoid tissues, robust IL-2 production, and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-Tem and clarification of Tem biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a β integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4β1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.

A subsection of the radiating spiral arm, placed in parallel, induces a bandstop response at a notch frequency proportional to the resonant length of the strip. Detailed parametric study on the effect of variation of design parameters for an Archimedean spiral antenna and the resonant parallel strip (RPS) is presented. Empirical analysis on phase velocity on the radiating spiral arms allows characterization of RPS in terms of its resonant length. Identified systematic relation between design parameters and filter response is applied to design an antenna for the 3.1-10.5 GHz operating band with the notch response over the IEEE 802.11a band, 5.15-5.95 GHz. Successful implementation is demonstrated through performance comparison between simulated and experimental results.

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy.

The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

BackgroundThe ability to measure the expression of proinflammatory cytokines from intestinal biopsies in patients with Crohn's disease in an accurate and reproducible way is critical for proof-of-concept and mechanism-of-action trials; however, the number of biopsies from a segment of the ileum or colon required to yield reproducible results has not been rigorously evaluated. We examined intestinal biopsies from patients with Crohn's disease to validate methods for detecting changes in inflammatory gene expression.MethodsTo evaluate the reproducibility of gene expression measurements, intestinal biopsies were obtained from designated segments from 6 healthy controls, 6 patients with active Crohn's disease, and 6 patients with inactive Crohn's disease. Disease activity was based on the simple endoscopic score for Crohn's disease. Expression of 7 proinflammatory genes was measured from each biopsy using quantitative polymerase chain reaction. Using a linear mixed effects model, the power to detect transcriptional changes corresponding to active and inactive Crohn's disease was calculated.ResultsTotal simple endoscopic score for Crohn's disease score corresponds with expression of most inflammatory biomarkers. For most genes, 2 to 5 biopsies are needed to reduce sampling error to

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

BackgroundWhile falls and urinary incontinence are prevalent among older patients, who sometimes rely on proxies to provide their health information, the validity of proxy reports of concern about falls and urinary incontinence remains unknown.MethodsTelephone interviews with 43 consecutive patients with falls or fear of falling and/or bothersome urinary incontinence and their proxies chosen by patients as most knowledgeable about their health. The questionnaire included items derived from the Medical Outcomes Study Short Form 12 (SF-12), a scale assessing concerns about urinary incontinence (UI), and a measure of fear of falling, the Falls Efficacy Scale (FES). Scores were estimated using items asking the proxy perspective (6 items from the SF-12, 10 items from a UI scale, and all 10 FES items). Proxy and patient scores were compared using intraclass correlation coefficients (ICC, one-way model). Variables associated with absolute agreement between patients and proxies were explored.ResultsPatients had a mean age of 81 years (range 75-93) and 67% were female while proxies had a mean age of 70 (range 42-87) and 49% were female. ICCs were 0.63 for the SF-12, 0.52 for the UI scale, and 0.29 for the FES. Proxies tended to understate patients' general health and incontinence concern, but overstate patients' concern about falling. Proxies who lived with patients and those who more often see patients more closely reflected patient FES scores compared to those who lived apart or those who saw patients less often. Internal consistency reliability of proxy responses was 0.62 for the SF-12, 0.86 for the I-QOL, and 0.93 for the FES. In addition, construct validity of the proxy FES scale was supported by greater proxy-perceived fear of falling for patients who received medical care after a fall during the past 12 months (p < .05).ConclusionCaution should be exercised when using proxies as a source of information about older patients' health perceptions. Questions asking about proxies' views yield suboptimal agreement with patient responses. However, proxy scales of UI and fall concern are internally consistent and may provide valid independent information.