Ever since it was discovered that central tolerance to self is imposed on developing T cells in the thymus through their interaction with self-peptide major histocompatibility complexes on thymic antigen-presenting cells, immunologists have speculated about the nature of these peptides, particularly in humans. Here, to shed light on the so-far unknown human thymic peptide repertoire, we analyse peptides eluted from isolated thymic dendritic cells, dendritic cell-depleted antigen-presenting cells and whole thymus. Bioinformatic analysis of the 842 identified natural major histocompatibility complex I and II ligands reveals significant crosstalk between major histocompatibility complex-class I and II pathways and differences in source protein representation between individuals as well as different antigen-presenting cells. Furthermore, several autoimmune-and tumour-related peptides, from enolase and vimentin for example, are presented in the healthy thymus. 302 peptides are directly derived from negatively selecting dendritic cells, thus providing the first global view of the peptide matrix in the human thymus that imposes self-tolerance in vivo.
Nude severe combined immunodeficiency is a rare inherited disease caused by autosomal recessive loss-of-function mutations in FOXN1. This gene encodes a transcription factor essential for the development of the thymus, the primary lymphoid organ that supports T-cell development and selection. To date nine cases have been reported presenting with the clinical triad of absent thymus resulting in severe T-cell immunodeficiency, congenital alopecia universalis and nail dystrophy. Diagnosis relies on testing for FOXN1 mutations, which allows genetic counselling and guides therapeutic management. Options for treating the underlying immune deficiency include HLA-matched genoidentical haematopoietic cell transplantation containing mature donor T-cells or thymus tissue transplantation. Experience from other severe combined immune deficiency syndromes suggests that early diagnosis, supportive care and definitive management result in better patient outcomes. Without these the prognosis is poor due to early-onset life threatening infections.
The conduits of life; the animal oviducts and human fallopian tubes are of paramount importance for reproduction in amniotes. They connect the ovary with the uterus and are essential for fertility. They provide the appropriate environment for gamete maintenance, fertilization and preimplantation embryonic development. However, serious pathologies, such as ectopic pregnancy, malignancy and severe infections, occur in the oviducts. They can have drastic effects on fertility, and some are life-threatening. Despite the crucial importance of the oviducts in life, relatively little is known about the molecular drivers underpinning the embryonic development of their precursor structures, the Müllerian ducts, and their successive differentiation and maturation. The Müllerian ducts are simple rudimentary tubes comprised of an epithelial lumen surrounded by a mesenchymal layer. They differentiate into most of the adult female reproductive tract (FRT). The earliest sign of Müllerian duct formation is the thickening of the anterior mesonephric coelomic epithelium to form a placode of two distinct progenitor cells. It is proposed that one subset of progenitor cells undergoes partial epithelial-mesenchymal transition (pEMT), differentiating into immature Müllerian luminal cells, and another subset undergoes complete EMT to become Müllerian mesenchymal cells. These cells invaginate and proliferate forming the Müllerian ducts. Subsequently, pEMT would be reversed to generate differentiated epithelial cells lining the fully formed Müllerian lumen. The anterior Müllerian epithelial cells further specialize into the oviduct epithelial subtypes. This review highlights the key established molecular and genetic determinants of the processes involved in Müllerian duct development and the differentiation of its upper segment into oviducts. Furthermore, an extensive genome-wide survey of mouse knockout lines displaying Müllerian or oviduct phenotypes was undertaken. In addition to widely established genetic determinants of Müllerian duct development, our search has identified surprising associations between loss-of-function of several genes and high-penetrance abnormalities in the Müllerian duct and/or oviducts. Remarkably, these associations have not been investigated in any detail. Finally, we discuss future directions for research on Müllerian duct development and oviducts.
In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.
Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5′-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.
Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.
The thymic stroma is composed of epithelial and nonepithelial cells providing separate microenvironments controlling homing, differentiation, and selection of hematopoietic precursor cells to functional T cells. Here, we explore at single-cell resolution the complex composition and dynamic changes of the nonepithelial stromal compartment across different developmental stages in the human and mouse thymus, and in an experimental model of the DiGeorge syndrome, the most common form of human thymic hypoplasia. The detected gene expression signatures identify previously unknown stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions, revealing an unprecedented heterogeneity of different cell types that emerge at discrete developmental stages and vary in their expression of key regulatory signaling circuits and extracellular matrix components. Together, these findings highlight the dynamic complexity of the nonepithelial thymus stroma and link this to separate instructive roles essential for normal thymus organogenesis and tissue maintenance.
BACKGROUND The lymphocyte-depleting antibody alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis (RRMS); however, 50% of patients develop novel autoimmunity after treatment. Most at risk are individuals who reconstitute their T cell pool by proliferating residual cells, rather than producing new T cells in the thymus, raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in nonhuman primates. METHODS Following a dose tolerability substudy, individuals with RRMS (duration ≤10 years; expanded disability status scale ≤5.0, with ≥2 relapses in the previous 2 years) were randomized to placebo or 180 μg/kg/d palifermin, given for 3 days immediately before and after each cycle of alemtuzumab, with repeat doses at month 1 (M1) and M3. The interim primary endpoint was naive CD4 + T cell count at M6. Exploratory endpoints included number of recent thymic emigrants (RTEs) and signal joint T cell receptor excision circles/ml (sjTRECs/ml) of blood. The trial’s primary endpoint was incidence of autoimmunity at M30. RESULTS At M6, individuals receiving palifermin had fewer naive CD4 + T cells (2.229 × 10 7 /l vs. 7.733 × 10 7 /l; P = 0.007), RTEs (16% vs. 34%), and sjTRECs/ml (1100 vs. 3396), leading to protocol-defined termination of recruitment. No difference was observed in the rate of autoimmunity between the 2 groups. CONCLUSION In contrast with animal studies, palifermin reduced thymopoiesis in our patients. These results offer a note of caution to those using palifermin to promote thymopoiesis in other settings, particularly in the oncology/hematology setting, where alemtuzumab is often used as part of the conditioning regime. TRIAL REGISTRATION ClinicalTrials.gov NCT01712945. FUNDING MRC and Moulton Charitable Trust.
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