High-content multimodal analysis supports the IL-7/IL-7 receptor axis as a relevant therapeutic target in primary Sjögren's syndrome.

OBJECTIVE: While the involvement of IL-7/IL-7R axis in pSS has been described in relation to T cells, little is known about the contribution of this pathway in relationship with other immune cells, and its implication in autoimmunity. Using high-content multiomics data, we aimed at characterizing IL-7R expressing cells and the involvement of IL-7/IL-7R pathway in pSS pathophysiology. METHODS: An IL-7 signature established using RNA-sequencing of human PBMCs incubated with IL-7 was applied to 304 pSS patients, and on RNA-Seq datasets from tissue biopsies. High-content immunophenotyping using flow and imaging mass cytometry was developed to characterize peripheral and in situ IL-7R expression. RESULTS: We identified a blood 4-gene IL-7 module (IKZF4, KIAA0040, PGAP1 and SOS1) associated with anti-SSA/Ro positiveness in patients as well as disease activity, and a tissue 5-gene IL-7 module (IL7R, PCED1B, TNFSF8, ADAM19, MYBL1) associated with infiltration severity. We confirmed expression of IL-7R on T cells subsets, and further observed upregulation of IL-7R on double-negative (DN) B cells, and especially DN2 B cells. IL-7R expression was increased in pSS compared to sicca patients with variations seen according to the degree of infiltration. When expressed, IL-7R was mainly found on epithelial cells, CD4+ and CD8+ T cells, switched memory B cells, DN B cells and M1 macrophages. CONCLUSION: This exhaustive characterization of the IL-7/IL-7R pathway in pSS pathophysiology established that two IL-7 gene modules discriminate pSS patients with a high IL-7 axis involvement. Their use could guide the implementation of an anti-IL-7R targeted therapy in a precision medicine approach.

ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates.

ANISEED (https://www.aniseed.cnrs.fr) is the main model organism database for the worldwide community of scientists working on tunicates, the vertebrate sister-group. Information provided for each species includes functionally-annotated gene and transcript models with orthology relationships within tunicates, and with echinoderms, cephalochordates and vertebrates. Beyond genes the system describes other genetic elements, including repeated elements and cis-regulatory modules. Gene expression profiles for several thousand genes are formalized in both wild-type and experimentally-manipulated conditions, using formal anatomical ontologies. These data can be explored through three complementary types of browsers, each offering a different view-point. A developmental browser summarizes the information in a gene- or territory-centric manner. Advanced genomic browsers integrate the genetic features surrounding genes or gene sets within a species. A Genomicus synteny browser explores the conservation of local gene order across deuterostome. This new release covers an extended taxonomic range of 14 species, including for the first time a non-ascidian species, the appendicularian Oikopleura dioica. Functional annotations, provided for each species, were enhanced through a combination of manual curation of gene models and the development of an improved orthology detection pipeline. Finally, gene expression profiles and anatomical territories can be explored in 4D online through the newly developed Morphonet morphogenetic browser.

Evolution of embryonic cis-regulatory landscapes between divergent Phallusia and Ciona ascidians.

Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages. Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns. We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of "shadow" enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge.

Linking genetic variation with epigenetic profiles in Sjögren's syndrome.

DNA methylation represents an important regulatory event governing gene expression that is dysregulated in Sjögren's syndrome (SjS) and a number of autoimmune/inflammatory diseases. As disease-associated single-nucleotide polymorphisms (SNPs) have relevance in controlling DNA methylation, 94 non-HLA SjS-SNPs were investigated, among them 57 (60.6%) with widespread effects on 197 individual DNA methylation quantitative trait loci (meQTL) were selected. Typically, these SNPs are intronic, possess an active promoter histone mark, and control cis-meQTLs located around transcription start sites. Interplay is independent of the physical distance between SNPs and meQTLs. Using epigenome-wide association study datasets, SjS-meQTLs were characterized (41 genes and 13 DNA methylation CpG motifs) and for the most part map to a pro-inflammatory cytokine pathway, which is important for the control of DNA methylation in autoimmune diseases. In conclusion, exploring meQTLs represents a valuable tool to predict and investigate downstream effects of genetic factors in complex diseases such as SjS.

A genome database for a Japanese population of the larvacean Oikopleura dioica.

The larvacean Oikopleura dioica is a planktonic chordate and is a tunicate that belongs to the closest relatives to vertebrates. Its simple and transparent body, invariant embryonic cell lineages, and short life cycle of 5 days make it a promising model organism for the study of developmental biology. The genome browser OikoBase was established in 2013 using Norwegian O. dioica. However, genome information for other populations is not available, even though many researchers have studied local populations. In the present study, we sequenced using Illumina and PacBio RSII technologies the genome of O. dioica from a southwestern Japanese population that was cultured in our laboratory for 3 years. The genome of Japanese O. dioica was assembled into 576 scaffold sequences with a total length and N50 length of 56.6 and 1.5 Mb, respectively. A total of 18,743 gene models (transcript models) were predicted in the genome assembly, named OSKA2016. In addition, 19,277 non-redundant transcripts were assembled using RNA-seq data. The OSKA2016 has global sequence similarity of only 86.5% when compared with the OikoBase, highlighting the sequence difference between the two far distant O. dioica populations on the globe. The genome assembly, transcript assembly, and transcript models were incorporated into ANISEED (https://www.aniseed.cnrs.fr/) for genome browsing and BLAST searches. Mapping of reads obtained from male- or female-specific genome libraries yielded male-specific scaffolds in the OSKA2016 and revealed that over 2.6 Mb of sequence were included in the male-specific Y-region. The genome and transcriptome resources from two distinct populations will be useful datasets for developmental biology, evolutionary biology, and molecular ecology using this model organism.

ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets.

ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.

Epigenetics in Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (SjS) is a chronic and systemic autoimmune epithelitis with predominant female incidence, which is characterized by exocrine gland dysfunction. Incompletely understood, the etiology of SjS is multi-factorial and evidence is growing to consider that epigenetic factors are playing a crucial role in its development. Independent from DNA sequence mutations, epigenetics is described as inheritable and reversible processes that modify gene expression. Epigenetic modifications reported in minor salivary gland and lymphocytes from SjS patients are related to (i) an abnormal DNA methylation process inducing in turn defective control of normally repressed genes involving such matters as autoantigens, retrotransposons, and the X chromosome in women; (ii) altered nucleosome positioning associated with autoantibody production; and (iii) altered control of microRNA. Results from epigenome-wide association studies have further revealed the importance of the interferon pathway in disease progression, the calcium signaling pathway for controlling fluid secretions, and a cell-specific cross talk with risk factors associated with SjS. Importantly, epigenetic modifications are reversible thus opening opportunities for therapeutic procedures in this currently incurable disease.

Convergent Acquisition of Nonembryonic Development in Styelid Ascidians.

Asexual propagation and whole body regeneration are forms of nonembryonic development (NED) widespread across animal phyla and central in life history and evolutionary diversification of metazoans. Whereas it is challenging to reconstruct the gains or losses of NED at large phylogenetic scale, comparative studies could benefit from being conducted at more restricted taxonomic scale, in groups for which phylogenetic relationships are well established. The ascidian family of Styelidae encompasses strictly sexually reproducing solitary forms as well as colonial species that combine sexual reproduction with different forms of NED. To date, the phylogenetic relationships between colonial and solitary styelids remain controversial and so is the pattern of NED evolution. In this study, we built an original pipeline to combine eight genomes with 18 de novo assembled transcriptomes and constructed data sets of unambiguously orthologous genes. Using a phylogenomic super-matrix of 4,908 genes from these 26 tunicates we provided a robust phylogeny of this family of chordates, which supports two convergent acquisitions of NED. This result prompted us to further describe the budding process in the species Polyandrocarpa zorritensis, leading to the discovery of a novel mechanism of asexual development. Whereas the pipeline and the data sets produced can be used for further phylogenetic reconstructions in tunicates, the phylogeny provided here sets an evolutionary framework for future experimental studies on the emergence and disappearance of complex characters such as asexual propagation and whole body regeneration.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition.

Control of nerve cord formation by Engrailed and Gooseberry-Neuro: A multi-step, coordinated process.

One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. We previously showed that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. In this report, we identified genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed us to define 175 potential targets of EN-GsbN regulation. We chose a subset of these genes to examine ventral nerve cord (VNC) defects and found that half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (i) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (ii) temporal control of the translation of some mRNAs, (iii) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.

A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation.

Genomic imprinting is an epigenetic mechanism that restrains the expression of ∼ 100 eutherian genes in a parent-of-origin-specific manner. The reason for this selective targeting of genes with seemingly disparate molecular functions is unclear. In the present work, we show that imprinted genes are coexpressed in a network that is regulated at the transition from proliferation to quiescence and differentiation during fibroblast cell cycle withdrawal, adipogenesis in vitro, and muscle regeneration in vivo. Imprinted gene regulation is not linked to alteration of DNA methylation or to perturbation of monoallelic, parent-of-origin-dependent expression. Overexpression and knockdown of imprinted gene expression alters the sensitivity of preadipocytes to contact inhibition and adipogenic differentiation. In silico and in cellulo experiments showed that the imprinted gene network includes biallelically expressed, nonimprinted genes. These control the extracellular matrix composition, cell adhesion, cell junction, and extracellular matrix-activated and growth factor-activated signaling. These observations show that imprinted genes share a common biological process that may account for their seemingly diverse roles in embryonic development, obesity, diabetes, muscle physiology, and neoplasm.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Genome-wide survey of miRNAs and their evolutionary history in the ascidian, Halocynthia roretzi.

BACKGROUND: miRNAs play essential roles in the modulation of cellular functions via degradation and/or translation attenuation of target mRNAs. They have been surveyed in a single ascidian genus, Ciona. Recently, an annotated draft genome sequence for a distantly related ascidian, Halocynthia roretzi, has become available, but miRNAs in H. roretzi have not been previously studied. RESULTS: We report the prediction of 319 candidate H. roretzi miRNAs, obtained through three complementary methods. Experimental validation suggests that more than half of these candidate miRNAs are expressed during embryogenesis. The majority of predicted H. roretzi miRNAs appear specific to ascidians or tunicates, and only 32 candidates, belonging to 25 families, are widely conserved across metazoans. CONCLUSION: Our study presents a comprehensive identification of candidate H. roretzi miRNAs. This resource will facilitate the study of the mechanisms for miRNA-controlled gene regulatory networks during ascidian development. Further, our analysis suggests that the majority of Halocynthia miRNAs are specific to ascidian or tunicates, with only a small number of widely conserved miRNAs. This result is consistent with the general notion that animal miRNAs are less conserved between taxa than plant ones.

Cell-specific epigenome-wide DNA methylation profile in long-term cultured minor salivary gland epithelial cells from patients with Sjögren's syndrome.

OBJECTIVES: The aetiology of primary Sjögren's syndrome (pSS), also referred to as autoimmune epithelitis, is incompletely understood but includes an epigenetic contribution. Accordingly, the aim of this study was to investigate DNA methylation in salivary gland epithelial cells (SGEC), and to compare results with those publicly available from pSS B and T cells. METHODS: Long-term cultured SGEC were selected to conduct an epigenome-wide association study (EWAS) in patients with pSS with comparison to controls using the HumanMethylation 450 K array from Illumina. RESULTS: The analysis of differentially methylated CpG (DMC) uncovered 4662 positions corresponding to 2560 genes, and 575 genes with two or more DMC sites (DMCs), in SGEC as compared with controls. Further analysis highlighted an important proportion of interferon-regulated genes (61%), the calcium pathway (hypomethylated) and the Wnt pathway (hypermethylated). When comparing SGEC with pSS T and/or B cell results, an important overlap was observed with respect to differentially methylated genes (38.8%) and pSS risk factors (71.4%), although such assertion was not true when comparing DMCs. CONCLUSIONS: This study conducted in SGEC emphasises the role of DNA methylation in pSS pathogenesis and supports the necessity to conduct pure cell analysis for future EWAS studies when analysing salivary glands from patients with pSS.

Guidelines for the nomenclature of genetic elements in tunicate genomes.

Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.

Essential requirement for ß-arrestin2 in mouse intestinal tumors with elevated Wnt signaling.

ß-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including Wnt/ß-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in Wnt signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the Wnt pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active ß-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations.

Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein.

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cell-autonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting non-cell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize.

Maf expression in B cells restricts reactive plasmablast and germinal center B cell expansion.

Precise regulation of B cell differentiation is essential for an effective adaptive immune response. Here, we show that B cell development in mice with B cell-specific Maf deletion is unaffected, but marginal zone B cells, germinal centre B cells, and plasmablasts are significantly more frequent in the spleen of naive Maf-deficient mice compared to wild type controls. In the context of a T cell-dependent immunization, Maf deletion causes increased proliferation of germinal centre B cells and extrafollicular plasmablasts. This is accompanied by higher production of antigen-specific IgG1 antibodies with minimal modification of early memory B cells, but a reduction in plasma cell numbers. Single-cell RNA sequencing shows upregulation of genes associated with DNA replication and cell cycle progression, confirming the role of Maf in cell proliferation. Subsequent pathway analysis reveals that Maf influences cellular metabolism, transporter activity, and mitochondrial proteins, which have been implicated in controlling the germinal centre reaction. In summary, our findings demonstrate that Maf acts intrinsically in B cells as a negative regulator of late B cell differentiation, plasmablast proliferation and germinal centre B cell formation.

Epigenetic dysregulation in salivary glands from patients with primary Sjögren's syndrome may be ascribed to infiltrating B cells.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy characterized by an epithelium injury with dense lymphocytic infiltrates, mainly composed of activated T and B cells. Present at the interface of genetic and environmental risk factors, DNA methylation is suspected to play a key role in SS. To clarify this point, global DNA methylation was tested within salivary gland epithelial cells (SGEC), peripheral T cells and B cells from SS patients. Global DNA methylation was reduced in SGEC from SS patients, while no difference was observed in T and B cells. SGEC demethylation in SS patients was associated with a 7-fold decrease in DNA methyl transferase (DNMT) 1 and a 2-fold increase in Gadd45-alpha expression. The other DNA methylation/demethylation partners, tested by real time PCR (DNMT3a/b, PCNA, UHRF1, MBD2, and MBD4), were not different. Interestingly, SGEC demethylation may be attributed in part to the infiltrating B cells as suspected in patients treated with anti-CD20 antibodies to deplete B cells. Such hypothesis was confirmed using co-culture experiments with human salivary gland cells and B cells. Furthermore, B cell-mediated DNA demethylation could be ascribed to an alteration of the PKC delta/ERK/DNMT1 pathway. As a consequence, part of the SGEC dysfunction in SS may be linked to epigenetic modifications, thus opening new therapeutic perspectives in SS.

Deciphering the maturation of tertiary lymphoid structures in cancer and inflammatory diseases of the digestive tract using imaging mass cytometry.

Persistent inflammation can promote the development of tertiary lymphoid structures (TLS) within tissues resembling secondary lymphoid organs (SLO) such as lymph nodes (LN). The composition of TLS across different organs and diseases could be of pathophysiological and medical interest. In this work, we compared TLS to SLO in cancers of the digestive tract and in inflammatory bowel diseases. Colorectal and gastric tissues with different inflammatory diseases and cancers from the department of pathology of CHU Brest were analyzed based on 39 markers using imaging mass cytometry (IMC). Unsupervised and supervised clustering analyses of IMC images were used to compare SLO and TLS. Unsupervised analyses tended to group TLS per patient but not per disease. Supervised analyses of IMC images revealed that LN had a more organized structure than TLS and non-encapsulated SLO Peyer's patches. TLS followed a maturation spectrum with close correlations between germinal center (GC) markers' evolution. The correlations between organizational and functional markers made relevant the previously proposed TLS division into three stages: lymphoid-aggregates (LA) (CD20+CD21-CD23-) had neither organization nor GC functionality, non-GC TLS (CD20+CD21+CD23-) were organized but lacked GC's functionality and GC-like TLS (CD20+CD21+CD23+) had GC's organization and functionality. This architectural and functional maturation grading of TLS pointed to differences across diseases. TLS architectural and functional maturation grading is accessible with few markers allowing future diagnostic, prognostic, and predictive studies on the value of TLS grading, quantification and location within pathological tissues in cancers and inflammatory diseases.