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Innate immune receptors for cross-presentation: The expanding role of NLRs.
A critical role of pattern recognition receptors (PRRs) is to influence adaptive immune responses by regulating antigen presentation. Engagement of PRRs in dendritic cells (DCs) increases MHC class I antigen presentation and CD8+ T-cell activation by cross-presented peptides but the molecular mechanisms underlying these effects are not completely understood. Studies looking at the role of PRRs in cross-presentation have been largely limited to TLRs but the role of other PRRs such as cytosolic nucleotide-binding oligomerization domain-like (NOD-like) receptors remains particularly enigmatic. Here we discuss recent evidence of the role of PRRs on cross-presentation and consider how cytosolic NLR-associated pathways, such as NOD2, may integrate these signals resulting in effective adaptive CD8+ T cells responses.
HLA-dependent variation in SARS-CoV-2 CD8 + T cell cross-reactivity with human coronaviruses.
The conditions and extent of cross-protective immunity between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and common-cold human coronaviruses (HCoVs) remain open despite several reports of pre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure. Using a pool of functionally evaluated SARS-CoV-2 peptides, we report a map of 126 immunogenic peptides with high similarity to 285 MHC-presented peptides from at least one HCoV. Employing this map of SARS-CoV-2-non-homologous and homologous immunogenic peptides, we observe several immunogenic peptides with high similarity to human proteins, some of which have been reported to have elevated expression in severe COVID-19 patients. After combining our map with SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls, we show that public repertoires for the majority of convalescent patients are dominated by TCRs cognate to non-homologous SARS-CoV-2 peptides. We find that for a subset of patients, >50% of their public SARS-CoV-2-specific repertoires consist of TCRs cognate to homologous SARS-CoV-2-HCoV peptides. Further analysis suggests that this skewed distribution of TCRs cognate to homologous or non-homologous peptides in COVID-19 patients is likely to be HLA-dependent. Finally, we provide 10 SARS-CoV-2 peptides with known cognate TCRs that are conserved across multiple coronaviruses and are predicted to be recognized by a high proportion of the global population. These findings may have important implications for COVID-19 heterogeneity, vaccine-induced immune responses, and robustness of immunity to SARS-CoV-2 and its variants.
Invasive Salmonella exploits divergent immune evasion strategies in infected and bystander dendritic cell subsets.
Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4+ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.
Human Enteric Glia Diversity in Health and Disease: New Avenues for the Treatment of Hirschsprung Disease.
BACKGROUND & AIMS: The enteric nervous system (ENS), which is composed of neurons and glia, regulates intestinal motility. Hirschsprung disease (HSCR) results from defects in ENS formation; however, although neuronal aspects have been studied extensively, enteric glia remain disregarded. This study aimed to explore enteric glia diversity in health and disease. METHODS: Full-thickness intestinal resection material from pediatric controls and patients with HSCR was collected, dissociated, and enriched for the ENS population through fluorescence-activated cell sorting. Single-cell RNA sequencing was performed to uncover the transcriptomic diversity of the ENS in controls and HSCR patients, as well as in wild-type and ret mutant zebrafish. Immunofluorescence and fluorescence in situ hybridization confirmed the presence of distinct subtypes. RESULTS: Two major enteric glial classes emerged in the pediatric intestine: Schwann-like enteric glia, which are reminiscent of Schwann cells, and enteric glia expressing classical glial markers. Comparative analysis with previously published datasets confirmed our classification and revealed that although classical enteric glia are predominant prenatally, Schwann-like enteric glia become more abundant postnatally. In HSCR, ganglionic segments mirrored controls and aganglionic segments featured only Schwann-like enteric glia. Leveraging the regenerative potential of Schwann cells, we explored therapeutic options using a ret mutant zebrafish. Prucalopride, a serotonin-receptor (5-HT) agonist, induced neurogenesis partially rescuing the HSCR phenotype in ret+/- mutants. CONCLUSIONS: Two major enteric glial classes were identified in the pediatric intestine, highlighting the significant postnatal contribution of Schwann-like enteric glia to glial heterogeneity. Crucially, these glial subtypes persist in aganglionic segments of patients with HSCR, offering a new target for their treatment using 5-HT agonists.
Cannabis, Cannabinoids, and the Endocannabinoid System-Is there Therapeutic Potential for Inflammatory Bowel Disease?
Cannabis sativa and its extracts have been used for centuries, both medicinally and recreationally. There is accumulating evidence that exogenous cannabis and related cannabinoids improve symptoms associated with inflammatory bowel disease [IBD], such as pain, loss of appetite, and diarrhoea. In vivo, exocannabinoids have been demonstrated to improve colitis, mainly in chemical models. Exocannabinoids signal through the endocannabinoid system, an increasingly understood network of endogenous lipid ligands and their receptors, together with a number of synthetic and degradative enzymes and the resulting products. Modulating the endocannabinoid system using pharmacological receptor agonists, genetic knockout models, or inhibition of degradative enzymes have largely shown improvements in colitis in vivo. Despite these promising experimental results, this has not translated into meaningful benefits for human IBD in the few clinical trials which have been conducted to date, the largest study being limited by poor medication tolerance due to the Δ9-tetrahydrocannabinol component. This review article synthesises the current literature surrounding the modulation of the endocannabinoid system and administration of exocannabinoids in experimental and human IBD. Findings of clinical surveys and studies of cannabis use in IBD are summarised. Discrepancies in the literature are highlighted together with identifying novel areas of interest.
Goblet cell differentiation subgroups in colorectal cancer.
The poor prognosis of relatively undifferentiated cancers has long been recognized, suggesting that selection against differentiation and in favor of uncontrolled growth is one of the most powerful drivers of cancer progression. Goblet cells provide the mucous surface of the gut, and when present in colorectal cancers (CRC), the cancers are called mucinous. We have used the presence of MUC2, the main mucous product of goblet cells, and an associated gene product, TFF3, to classify a large panel of nearly 80 CRC-derived cell lines into five categories based on their levels of MUC2 and TFF3 expression. We have then shown that these five patterns of expression can be easily identified in the direct analysis of tumor specimens allowing a much finer characterization of CRCs with respect to the presence of goblet cell differentiation. In particular, about 30% of all CRCs fall into the category of expressing TFF3 but not MUC2, which has not previously been acknowledged. Using the cell line data, we suggest that there are up to 12 genes (MUC2, TFF3, ATOH1, SPDEF, CDX1, CDX2, GATA6, HES1, ETS2, OLFM4, TOX3, and LGR5) that may be involved in selection against goblet cell differentiation in CRC by changes in methylation rather than mutations. Of these, LGR5, which is particularly associated with lack of goblet cell features, may function in the control of differentiation rather than direct control of cell growth, as has so far mostly been assumed. These results emphasize the importance of methylation changes in driving cancer progression.
Bone Morphogenetic Protein Pathway Antagonism by Grem1 Regulates Epithelial Cell Fate in Intestinal Regeneration.
BACKGROUND & AIMS: In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. METHODS: A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS: In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS: BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.
Large-scale sequencing identifies multiple genes and rare variants associated with Crohn's disease susceptibility.
Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation.
Single-Cell and Time-Resolved Profiling of Intracellular Salmonella Metabolism in Primary Human Cells.
The intracellular pathogen Salmonella enterica has evolved an array of traits for propagation and invasion of the intestinal layers. It remains largely elusive how Salmonella adjusts its metabolic states to survive inside immune host cells. In this study, single-cell Raman biotechnology combined with deuterium isotope probing (Raman-DIP) have been applied to reveal metabolic changes of the typhoidal Salmonella Typhi Ty2, the nontyphoidal Salmonella Typhimurium LT2, and a clinical isolate Typhimurium D23580. By initially labeling the Salmonella strains with deuterium, we employed reverse labeling to track their metabolic changes in the time-course infection of THP-1 cell line, human monocyte-derived dendritic cells (MoDCs) and macrophages (Mf). We found that, in comparison with a noninvasive serovar, the invasive Salmonella strains Ty2 and D23580 have downregulated metabolic activity inside human macrophages and dendritic cells and used lipids as alternative carbon source, perhaps a strategy to escape from the host immune response. Proteomic analysis using high sensitivity mass spectrometry validated the findings of Raman-DIP analysis.
Lymphatic endothelia stakeout cryptic stem cells.
A trio of studies in this issue of Cell Stem Cell catalogs the anatomical and functional relationship of intestinal lymphatics with epithelial stem cells, defining an important niche role for the lymphatic endothelium.
Spatiotemporal analysis of human intestinal development at single-cell resolution.
Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.
Publisher Correction: Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.
In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.
Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.
Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.
Remission of Inflammatory Bowel Disease in Glucose-6-Phosphatase 3 Deficiency by Allogeneic Haematopoietic Stem Cell Transplantation.
Mendelian disorders in glucose-6-phosphate metabolism can present with inflammatory bowel disease [IBD]. Using whole genome sequencing we identified a homozygous variant in the glucose-6-phosphatase G6PC3 gene [c.911dupC; p.Q305fs*82] in an adult patient with congenital neutropenia, lymphopenia and childhood-onset, therapy-refractory Crohn's disease. Because G6PC3 is expressed in several haematopoietic and non-haematopoietic cells it was unclear whether allogeneic stem cell transplantation [HSCT] would benefit this patient with intestinal inflammation. We show that HSCT resolves G6PC3-associated immunodeficiency and the Crohn's disease phenotype. It illustrates how even in adulthood, next-generation sequencing can have a significant impact on clinical practice and healthcare utilization in patients with immunodeficiency and monogenic IBD.
Association of Genetic Variants in NUDT15 With Thiopurine-Induced Myelosuppression in Patients With Inflammatory Bowel Disease.
IMPORTANCE: Use of thiopurines may be limited by myelosuppression. TPMT pharmacogenetic testing identifies only 25% of at-risk patients of European ancestry. Among patients of East Asian ancestry, NUDT15 variants are associated with thiopurine-induced myelosuppression (TIM). OBJECTIVE: To identify genetic variants associated with TIM among patients of European ancestry with inflammatory bowel disease (IBD). DESIGN, SETTING, AND PARTICIPANTS: Case-control study of 491 patients affected by TIM and 679 thiopurine-tolerant unaffected patients who were recruited from 89 international sites between March 2012 and November 2015. Genome-wide association studies (GWAS) and exome-wide association studies (EWAS) were conducted in patients of European ancestry. The replication cohort comprised 73 patients affected by TIM and 840 thiopurine-tolerant unaffected patients. EXPOSURES: Genetic variants associated with TIM. MAIN OUTCOMES AND MEASURES: Thiopurine-induced myelosuppression, defined as a decline in absolute white blood cell count to 2.5 × 109/L or less or a decline in absolute neutrophil cell count to 1.0 × 109/L or less leading to a dose reduction or drug withdrawal. RESULTS: Among 1077 patients (398 affected and 679 unaffected; median age at IBD diagnosis, 31.0 years [interquartile range, 21.2 to 44.1 years]; 540 [50%] women; 602 [56%] diagnosed as having Crohn disease), 919 (311 affected and 608 unaffected) were included in the GWAS analysis and 961 (328 affected and 633 unaffected) in the EWAS analysis. The GWAS analysis confirmed association of TPMT (chromosome 6, rs11969064) with TIM (30.5% [95/311] affected vs 16.4% [100/608] unaffected patients; odds ratio [OR], 2.3 [95% CI, 1.7 to 3.1], P = 5.2 × 10-9). The EWAS analysis demonstrated an association with an in-frame deletion in NUDT15 (chromosome 13, rs746071566) and TIM (5.8% [19/328] affected vs 0.2% [1/633] unaffected patients; OR, 38.2 [95% CI, 5.1 to 286.1], P = 1.3 × 10-8), which was replicated in a different cohort (2.7% [2/73] affected vs 0.2% [2/840] unaffected patients; OR, 11.8 [95% CI, 1.6 to 85.0], P = .03). Carriage of any of 3 coding NUDT15 variants was associated with an increased risk (OR, 27.3 [95% CI, 9.3 to 116.7], P = 1.1 × 10-7) of TIM, independent of TPMT genotype and thiopurine dose. CONCLUSIONS AND RELEVANCE: Among patients of European ancestry with IBD, variants in NUDT15 were associated with increased risk of TIM. These findings suggest that NUDT15 genotyping may be considered prior to initiation of thiopurine therapy; however, further study including additional validation in independent cohorts is required.
Immunotherapy-related hepatitis: real-world experience from a tertiary centre.
OBJECTIVE: Immune checkpoint inhibitors like anti-programmed cell death protein 1 (PD-1) drugs Nivolumab and Pembrolizumab and anti-cytotoxic T-lymphocyte associated (CTLA-4) drug Ipilimumab have become standard of care in many metastatic cancers. Immunotherapy-related hepatitis and cholangitis present a diagnostic and management challenge, being rare and incompletely characterised. We aim to report the incidence, features and treatments used for this in a real-world setting and to identify useful biomarkers, which can be used to predict effective use of steroids. DESIGN: Retrospective review of 453 patients started on immunotherapy over 7 years. SETTING: Tertiary hepatology and oncology centre. PATIENTS: 21 patients identified with immunotherapy-related hepatotoxicity. RESULTS: Hepatitis was most common in those receiving dual therapy (incidence 20%), with 75% of Grade 4 hepatitis cases occurring with ipilimumab-containing regimens. Corticosteroid monotherapy is first line treatment, but doses above 60 mg OD prednisolone do not demonstrate any additional benefit in time to hepatitis resolution. The alanine transaminase (ALT) reduction in steroid-responsive hepatitis is typically rapid (with a halving of ALT within 11 days). The commencement of additional immunosuppression (typically mycophenolate) appears safe and prompts a more rapid fall in ALT than corticosteroid use alone. Infliximab was safely used twice as hepatitis treatment. We also describe one patient with rare immunotherapy-induced biliary disease. CONCLUSIONS: Vigilance is required for detection of immunotherapy-associated liver disease as, other than dual immunotherapy, we can identify no predictive factors for its development. Our data suggest that corticosteroid response is not dependent on the higher dosing regimens. Early escalation of immunosuppression may be of benefit in the absence of a rapid response to corticosteroids.
Predicting Cross-Reactivity and Antigen Specificity of T Cell Receptors.
Adaptive immune recognition is mediated by specific interactions between heterodimeric T cell receptors (TCRs) and their cognate peptide-MHC (pMHC) ligands, and the methods to accurately predict TCR:pMHC interaction would have profound clinical, therapeutic and pharmaceutical applications. Herein, we review recent developments in predicting cross-reactivity and antigen specificity of TCR recognition. We discuss current experimental and computational approaches to investigate cross-reactivity and antigen-specificity of TCRs and highlight how integrating kinetic, biophysical and structural features may offer valuable insights in modeling immunogenicity. We further underscore the close inter-relationship of these two interconnected notions and the need to investigate each in the light of the other for a better understanding of T cell responsiveness for the effective clinical applications.