ABSTRACT
Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes, but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a method to profile Th cell identity using Cas9-targeted single-molecule nanopore sequencing. Targeting as few as 10 selected genomic loci, we were able to distinguish major in vitro polarized murine T cell subtypes, as well as intermediate phenotypes, by their native DNA 5mCpG patterns. Moreover, by using off-target sequences, we were able to infer transcription factor activities relevant to each cell subtype. Detection of 5mCpG and 5hmCpG was validated on intestinal Th17 cells escaping transforming growth factor ß control, using single-molecule adaptive sampling. A total of 21 differentially methylated regions mapping to the 10-gene panel were identified in pathogenic Th17 cells relative to their nonpathogenic counterpart. Hence, our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological transition states of Th cells.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Animals , Mice , Cytosine , DNA/metabolism , Th17 Cells/metabolismABSTRACT
Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development1-4. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota5-8, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells have not been identified. Here we describe the identification of a class of RORγt+ antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pTreg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pTreg generation, including the TGF-ß-activating integrin αvß8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pTreg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pTreg generation, further implicating TC IV as the tolerogenic RORγt+ antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.
Subject(s)
Antigen-Presenting Cells , Dendritic Cells , Epithelial Cells , Gastrointestinal Microbiome , Immune Tolerance , T-Lymphocytes, Regulatory , Thymus Gland , Cell Differentiation , Dendritic Cells/immunology , Dendritic Cells/metabolism , Epithelial Cells/immunology , Epithelial Cells/metabolism , Gastrointestinal Microbiome/immunology , Immunity, Innate , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , Transforming Growth Factor beta/immunology , Antigen-Presenting Cells/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Lymph Nodes/immunologyABSTRACT
TITLE: Le dialogue entre les cellules souches intestinales et les lymphocytes T CD4+ module l'homéostasie des cellules souches - Module d'immunologie virologie et cancer du Master de cancérologie de Lyon. ABSTRACT: Dans le cadre d'un partenariat avec médecine/sciences, et pour la troisième année, des étudiants du module d'immunologie virologie et cancer du Master de cancérologie de Lyon présentent une analyse d'articles scientifiques récents faisant état d'observations innovantes et importantes. Ce travail a été encadré par des chercheurs confirmés du département d'immunologie, virologie et inflammation du CRCL. Le master de cancérologie de Lyon (Lyon1-VetAgroSup) accueille chaque année 30 à 40 étudiants en M1 et en M2. Ce master dit « d'excellence ¼ assure aux étudiants de M1 une formation à la cancérologie reposant sur un socle de base commun (biologie cellulaire, moléculaire, immunologie, bio-statistique...). En M2, les étudiants peuvent choisir l'une des trois spécialités suivantes : le Master recherche « Recherche en cancérologie ¼, le Master recherche et professionnel « Technologie haut débit en cancérologie ¼ et enfin le Master recherche et professionnel « Innovations thérapeutiques en cancérologie ¼. Le Master de cancérologie de Lyon repose sur une forte implication des chercheurs et enseignants-chercheurs du laboratoire d'excellence en développement et cancérologie (LabEx DEVweCAN), ainsi que sur un partenariat solide avec plusieurs instituts dont le MIT (Massachusetts Institute of Technology, Cambridge, États-Unis), l'université d'Harvard (Boston, États-Unis), l'université Johns Hopkins (Baltimore, États-Unis), l'Imperial College of London (Royaume-Uni), les universités de Jiao Tong (République Populaire de Chine) et de Tokyo (Japon), entre autres. Pour plus d'information : http://devwecan.universite-lyon.fr/formation/.
