ABSTRACT
Medullary thymic epithelial cells (mTECs) are essential for the establishment of T cell central tolerance. The transcription factor Aire plays a key role in this process, but other factors remain understudied. We found that a small population of mTECs expressed the coinhibitory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These CTLA-4+ cells were detectable in perinates, peaked around young adulthood and expanded sixfold in the absence of Aire. Single-cell transcriptomics revealed CTLA-4+ mTECs to express a distinct gene signature encoding molecules associated with antigen presentation and interferon-gamma signaling. Mice conditionally lacking CTLA-4 in thymic epithelial cells had no major immunological deficiencies but displayed a mildly increased inflammatory tone and a partial defect in the generation of Foxp3+CD4+ regulatory T cells. Consequently, these mice developed modest levels of autoantibodies and lymphocytic infiltration of peripheral tissues. Thus, CTLA-4 expression in mTECs complements Aire to establish T cell central tolerance.
Subject(s)
Central Tolerance , Complement System Proteins , Animals , Mice , Central Tolerance/genetics , CTLA-4 Antigen/genetics , Epithelial Cells , Transcription Factors/genetics , T-Lymphocytes/immunologyABSTRACT
A major breakthrough in cancer treatment occurred with the development of strategies that overcome T-cell tolerance toward tumor cells. These approaches enhance antitumor immunity by overcoming mechanisms that are normally in place to prevent autoimmunity but simultaneously prevent rejection of tumor cells. Although tolerance mechanisms that restrict antitumor immunity take place both in the thymus and periphery, only immunotherapies that target peripheral tolerance mechanisms occurring outside of the thymus are currently available. We review here recent gains in our understanding of how thymic tolerance mediated by the autoimmune regulator (Aire) impedes antitumor immunity. It is now clear that transient depletion of Aire-expressing cells in the thymus can be achieved with RANKL blockade. Finally, we discuss key findings that support the repurposing of anti-RANKL as a cancer immunotherapy with a unique mechanism of action.
Subject(s)
Central Tolerance/genetics , Central Tolerance/immunology , Neoplasms/etiology , Neoplasms/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Autoimmune Diseases/etiology , Autoimmune Diseases/metabolism , Autoimmunity/genetics , Clonal Deletion/genetics , Clonal Deletion/immunology , Humans , Immunomodulation , Immunotherapy , Molecular Targeted Therapy , Neoplasms/pathology , Neoplasms/therapy , Organ Specificity/genetics , Organ Specificity/immunology , RANK Ligand/antagonists & inhibitors , RANK Ligand/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , AIRE ProteinABSTRACT
During αßT cell development, the thymus medulla represents an essential microenvironment for T cell tolerance. This functional specialization is attributed to its typical organized topology consisting of a branching structure that contains medullary thymic epithelial cell (mTEC) networks to support negative selection and Foxp3+ T-regulatory cell (T-reg) development. Here, by performing TEC-specific deletion of the thymus medulla regulator lymphotoxin ß receptor (LTßR), we show that thymic tolerance mechanisms operate independently of LTßR-mediated mTEC development and organization. Consistent with this, mTECs continue to express Fezf2 and Aire, regulators of intrathymic self-antigens, and support T-reg development despite loss of LTßR-mediated medulla organogenesis. Moreover, we demonstrate that LTßR controls thymic tolerance by regulating the frequency and makeup of intrathymic dendritic cells (DCs) required for effective thymocyte negative selection. In all, our study demonstrates that thymus medulla specialization for thymic tolerance segregates from medulla organogenesis and instead involves LTßR-mediated regulation of the thymic DC pool.
Subject(s)
Central Tolerance/immunology , Epithelial Cells/immunology , Lymphotoxin beta Receptor/immunology , Thymus Gland/immunology , Animals , Autoantigens/immunology , Central Tolerance/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Epithelial Cells/metabolism , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Lymphotoxin beta Receptor/genetics , Lymphotoxin beta Receptor/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Organogenesis/immunology , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Thymus Gland/embryology , Thymus Gland/metabolism , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , AIRE ProteinABSTRACT
The chemokine receptor CCR7 directs T cell relocation into and within lymphoid organs, including the migration of developing thymocytes into the thymic medulla. However, how three functional CCR7 ligands in mouse, CCL19, CCL21Ser, and CCL21Leu, divide their roles in immune organs is unclear. By producing mice specifically deficient in CCL21Ser, we show that CCL21Ser is essential for the accumulation of positively selected thymocytes in the thymic medulla. CCL21Ser-deficient mice were impaired in the medullary deletion of self-reactive thymocytes and developed autoimmune dacryoadenitis. T cell accumulation in the lymph nodes was also defective. These results indicate a nonredundant role of CCL21Ser in the establishment of self-tolerance in T cells in the thymic medulla, and reveal a functional inequality among CCR7 ligands in vivo.
Subject(s)
Central Tolerance/immunology , Chemokine CCL21/immunology , Self Tolerance/immunology , T-Lymphocytes/immunology , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Central Tolerance/genetics , Chemokine CCL21/genetics , Chemokine CCL21/metabolism , Dacryocystitis/genetics , Dacryocystitis/immunology , Dacryocystitis/metabolism , Flow Cytometry , Gene Expression/immunology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Mice, Transgenic , Microscopy, Confocal , Receptors, CCR7/immunology , Receptors, CCR7/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Self Tolerance/genetics , T-Lymphocytes/metabolism , Thymocytes/immunology , Thymocytes/metabolism , Thymus Gland/immunology , Thymus Gland/metabolismABSTRACT
Autoimmune polyendocrine syndrome type 1 (APS-1, OMIM 2403000) is a rare autosomal recessive disease that is caused by autoimmune regulator (AIRE). The main symptoms of APS-1 are chronic mucocutaneous candidiasis, autoimmune adrenocortical insufficiency (Addison's disease) and hypoparathyroidism. We collected APS-1 cases and analysed them. The AIRE genes of the patient and his family members were sequenced to identify whether the APS-1 patient had an AIRE mutation. We discovered a mutation site (c.206A>C) that had never before been reported in the AIRE gene located in exon 2 of the AIRE gene. This homogyzous mutation caused a substitution of the 69th amino acid of the AIRE protein from glutamine to proline (p.Q69P). A yeast two-hybrid assay, which was used to analyse the homodimerization properties of the mutant AIRE protein, showed that the mutant AIRE protein could not interact with the normal AIRE protein. Flow cytometry and RT-qPCR analyses indicated that the new mutation site could decrease the expression levels of the AIRE, glutamic acid decarboxylase 65 (GAD65) and tryptophan hydroxylase-1 (TPH1) proteins to affect central immune tolerance. In conclusion, our research has shown that the new mutation site (c.206A>C) may influence the homodimerization and expression levels and other aspects of the AIRE protein. It may also impact the expression levels of tissue-restricted antigens (TRAs), leading to a series of autoimmune diseases.
Subject(s)
Central Tolerance/genetics , Point Mutation , Polyendocrinopathies, Autoimmune/genetics , Transcription Factors/genetics , Amino Acid Substitution , Base Sequence , Exons , Gene Expression Regulation , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/immunology , Humans , Male , Pedigree , Polyendocrinopathies, Autoimmune/diagnosis , Polyendocrinopathies, Autoimmune/immunology , Polyendocrinopathies, Autoimmune/pathology , Protein Multimerization , Sequence Analysis, DNA , Transcription Factors/chemistry , Transcription Factors/immunology , Tryptophan Hydroxylase/genetics , Tryptophan Hydroxylase/immunology , Two-Hybrid System Techniques , Young Adult , AIRE ProteinABSTRACT
Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.
Subject(s)
Central Tolerance/genetics , Central Tolerance/immunology , Cytidine Deaminase/genetics , Lymphocyte Activation/immunology , Precursor Cells, B-Lymphoid/immunology , Adolescent , Adult , Aged , Animals , Apoptosis/genetics , Apoptosis/immunology , Case-Control Studies , Child , Child, Preschool , DNA-Binding Proteins/genetics , Female , Genes, Immunoglobulin/genetics , Genes, Immunoglobulin/immunology , Humans , Lymphocyte Activation/genetics , Male , Mice , Middle Aged , Nuclear Proteins/genetics , Recombination, Genetic/genetics , Recombination, Genetic/immunology , Somatic Hypermutation, Immunoglobulin/genetics , Somatic Hypermutation, Immunoglobulin/immunology , Young AdultABSTRACT
The establishment of thymic central tolerance is a critical process to prevent the development of autoimmune diseases. Medullary thymic epithelial cells (mTEC) are essential to this process through the expression of the transcription factor Aire, which controls the transcription of many genes encoding tissue-restricted antigens. Mutations in the Aire gene are responsible for a rare autoimmune disorder called APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy). This review summarizes our current knowledge on the mode of action of Aire at the molecular and epigenetic levels in controlling the expression of tissue-restricted antigens. We also discuss recently described additional roles of this transcription factor in the induction of central T-cell tolerance.
Subject(s)
Central Tolerance/genetics , Epigenesis, Genetic , Transcription Factors/genetics , Humans , AIRE ProteinABSTRACT
Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic features.
Subject(s)
B-Lymphocytes/physiology , CD4-Positive T-Lymphocytes/immunology , CD40 Antigens/metabolism , Thymus Gland/immunology , Transcription Factors/metabolism , Animals , Antigen Presentation/genetics , Autoantigens/immunology , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , CD40 Antigens/genetics , Cell Differentiation/genetics , Cells, Cultured , Central Tolerance/genetics , Clonal Selection, Antigen-Mediated/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Immunoglobulin Class Switching/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Signal Transduction , Transcription Factors/genetics , AIRE ProteinABSTRACT
Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.
Subject(s)
Central Tolerance/immunology , Sirtuin 1/immunology , Transcription Factors/immunology , Transcriptional Activation/immunology , Acetylation , Animals , Antigens/immunology , Central Tolerance/genetics , Epithelial Cells/immunology , Epithelial Cells/metabolism , Flow Cytometry , HEK293 Cells , Humans , Immunoblotting , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Organ Specificity/immunology , Protein Binding/immunology , Reverse Transcriptase Polymerase Chain Reaction , Sirtuin 1/genetics , Sirtuin 1/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/immunology , AIRE ProteinABSTRACT
No disponible
Subject(s)
Humans , Male , Child , Breast-Milk Substitutes , Central Tolerance/genetics , Milk Hypersensitivity/classification , Milk Hypersensitivity/complications , Infant Formula/administration & dosage , Asthma/diagnosis , Central Tolerance/physiology , Milk Hypersensitivity/metabolism , Milk Hypersensitivity/prevention & control , Infant Formula , Asthma/complicationsABSTRACT
The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1(-/-) mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.
Subject(s)
Central Tolerance/immunology , DNA-Binding Proteins/immunology , Gene Expression Regulation/immunology , Immune Tolerance , Repressor Proteins/immunology , Transcription Factors/immunology , Animals , Autoantigens/immunology , Central Tolerance/genetics , DNA-Binding Proteins/genetics , Flow Cytometry , HEK293 Cells , Humans , Immunoblotting , Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Protein Binding , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transfection , Two-Hybrid System Techniques , AIRE ProteinABSTRACT
Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.
Subject(s)
Exosomes/chemistry , MicroRNAs/genetics , Proteome/genetics , T-Lymphocytes, Regulatory/cytology , Thymocytes/cytology , Thymus Gland/cytology , Biomarkers/metabolism , Cell Communication , Cell Differentiation , Central Tolerance/genetics , Exosomes/immunology , Gene Expression , Gene Expression Profiling , Humans , Infant , Infant, Newborn , MicroRNAs/immunology , Molecular Sequence Annotation , Organ Specificity , Proteome/immunology , T-Lymphocytes, Regulatory/immunology , Thymocytes/immunology , Thymus Gland/immunologyABSTRACT
The thymus is a specialized organ that provides an inductive environment for the development of T cells from multipotent hematopoietic progenitors. Self-nonself discrimination plays a key role in inducing a productive immunity and in preventing autoimmune reactions. Tolerance represents a state of immunologic nonresponsiveness in the presence of a particular antigen. The immune system becomes tolerant to self-antigens through the two main processes, central and peripheral tolerance. Central tolerance takes place within the thymus and represents the mechanism by which T cells binding with high avidity self-antigens, which are potentially autoreactive, are eliminated through so-called negative selection. This process is mostly mediated by medullary thymic epithelia cells (mTECs) and medullary dendritic cells (DCs). A remarkable event in the process is the expression of tissue-specific antigens (TSA) by mTECs driven by the transcription factor autoimmune regulator (AIRE). Mutations in this gene result in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a rare autosomal recessive disease (OMIM 240300). Thus far, this syndrome is the paradigm of a genetically determined failure of central tolerance and autoimmunty. Patients with APECED have a variable pattern of autoimmune reactions, involving different endocrine and nonendocrine organs. However, although APECED is a monogenic disorder, it is characterized by a wide variability of the clinical expression, thus implying a further role for disease-modifying genes and environmental factors in the pathogenesis. Studies on this polyreactive autoimmune syndrome contributed enormously to unraveling several issues of the molecular basis of autoimmunity. This review focuses on the developmental, functional, and molecular events governing central tolerance and on the clinical implication of its failure.
Subject(s)
Autoantigens/genetics , Central Tolerance/genetics , Mutation , Polyendocrinopathies, Autoimmune/genetics , T-Lymphocytes/immunology , Transcription Factors/genetics , Autoantigens/immunology , Autoimmunity/genetics , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dendritic Cells/pathology , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Peripheral Tolerance/genetics , Polyendocrinopathies, Autoimmune/metabolism , Polyendocrinopathies, Autoimmune/pathology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Thymus Gland/immunology , Thymus Gland/metabolism , Thymus Gland/pathology , Transcription Factors/immunology , AIRE ProteinABSTRACT
BACKGROUND: Escape mutations potentially allow viruses to avoid detection and clearance by the host immune system and may represent a mechanism through which infections may persist in some patients. The association of the mutations in the HBcAg gene with Hepatitis B asymptomatic carriers (ASC) has not been studied adequately. The current study was aimed to investigate HBcAg18-27 CTL epitope mutations in ASC patients in the South-Eastern region of Iran. METHODS: 100 ASC patients were selected for this study and screened for HLA-A2 using flow cytometry. HBV-DNA was extracted from the HLA-A2 positive patients and the HBc gene was amplified using PCR. Direct double sequencing was performed to analyse mutations in the HBc gene of HBV isolates from patients with ASC. RESULTS: Overall, 25 (25%) of individuals were HLA-A2 positive. Direct double sequencing indicated no mutations in the HBcAg18-27 epitope. However, four mutations within the T helper and three mutations within the B cell epitopes of ASC patients were identified. CONCLUSIONS: The lack of mutations within the HBcAg18-27 epitope suggests that the antigenicity of this region is not altered in HBV isolates of our patients and therefore antigen presentation would occur normally to the patient's immune system through HLA-A2. However, in the course of this study we revealed some novel mutations within the T helper and B cell epitopes that may affect the efficiencies of immune response of ASC patients against these novel HBV epitopes.
Subject(s)
Asymptomatic Infections , B-Lymphocytes/immunology , Central Tolerance/immunology , Hepatitis B Core Antigens/genetics , Hepatitis B/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adult , Amino Acid Sequence , Antibodies, Viral , B-Lymphocytes/cytology , Base Sequence , Central Tolerance/genetics , DNA, Viral/analysis , Female , Hepatitis B Core Antigens/chemistry , Host-Pathogen Interactions , Humans , Male , Molecular Sequence Data , Mutation , T-Lymphocytes, Helper-Inducer/cytologyABSTRACT
Autoimmune regulator (AIRE) directs the expression of otherwise tissue-restricted antigens (TRAs) in medullary thymic epithelial cells, allowing their presentation to developing T cells, which leads to central tolerance. We addressed the conundrum of how AIRE is recruited to these otherwise silent genes in cells. Our studies confirmed that interactions between AIRE and the unmodified histone H3K4 (H3K4me0) are important for targeting AIRE to the mouse insulin promoter in chromatin. By replacing its H3K4me0-binding module with one that binds to the methylated H3K4me3, we redirected the mutant AIRE.ING protein to an actively transcribed gene. Nevertheless, the mutant AIRE D297A protein, which could not bind to H3K4me0, still activated the human insulin promoter on an episomal plasmid target. This targeting was due to DNA-dependent protein kinase (DNA-PK). Thus, in cells that lacked the catalytic subunit of DNA-PK (DNA-PKcs), the assembly and activity of AIRE on DNA, whether in chromatin or on episomal plasmids, was abrogated. However, by the heterologous tethering of AIRE to DNA, we could restore its activity on a plasmid target in DNA-PKcs-negative cells. Importantly, mutations in the putative DNA-binding residues in its SAND domain had no effect on the transcriptional effects of AIRE. Thus, AIRE is recruited to TRA genes in chromatin via cooperative interactions with H3K4me0 and DNA-PK.
Subject(s)
Central Tolerance/genetics , Chromatin/metabolism , DNA-Activated Protein Kinase/metabolism , Histones/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Animals , Chromatin/genetics , DNA-Activated Protein Kinase/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Histones/genetics , Humans , Insulin/genetics , Insulin/metabolism , Methylation , Mice , Mutant Proteins/genetics , Mutant Proteins/metabolism , Plasmids/genetics , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , T-Lymphocytes/metabolism , Transcription Factors/genetics , AIRE ProteinABSTRACT
Thymic epithelial cells (TECs) provide a highly specialized microenvironment for the generation of a functional and self-tolerant T cell repertoire. Much of our current view of TEC biology is derived from gain- or loss-of-function approaches, which have significantly contributed to our understanding of gene function in TEC development and T cell repertoire selection. Here, we will review transgenic and viral strategies that have been used to manipulate gene expression in TECs, highlight some of the shortcomings of particular currently available tools and provide a brief outline of our own attempts to more rapidly and/or more specifically assess gene function in TECs.
Subject(s)
Central Tolerance/genetics , Epithelial Cells/metabolism , Gene Expression/immunology , Molecular Biology/methods , T-Lymphocytes/metabolism , Thymus Gland/metabolism , Adenoviridae/genetics , Animals , Cell Differentiation , Cell Lineage , Cellular Microenvironment/genetics , Cellular Microenvironment/immunology , Central Tolerance/immunology , Epithelial Cells/cytology , Epithelial Cells/immunology , Gene Expression Profiling , Gene Knockdown Techniques , Genetic Vectors , Humans , Lentivirus/genetics , Mice , Mice, Knockout , MicroRNAs/genetics , MicroRNAs/immunology , RNA, Small Interfering/genetics , RNA, Small Interfering/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Tissue Culture TechniquesABSTRACT
To analyze B lymphocyte central tolerance in a polyclonal immune system, mice were engineered to express a superantigen reactive to IgM of allotype b (IgM(b)). IgM(b/b) mice carrying superantigen were severely B cell lymphopenic, but small numbers of B cells matured. Their sera contained low levels of IgG and occasionally high levels of IgA. In bone marrow, immature B cells were normal in number, but internalized IgM and had a unique gene expression profile, compared with those expressing high levels of surface IgM, including elevated recombinase activator gene expression. A comparable B cell population was defined in wild-type bone marrows, with an abundance suggesting that at steady state â¼20% of normal developing B cells are constantly encountering autoantigens in situ. In superantigen-expressing mice, as well as in mice carrying the 3H9 anti-DNA IgH transgene, or 3H9 H along with mutation in the murine κ-deleting element RS, IgM internalization was correlated with CD19 downmodulation. CD19(low) bone marrow cells from 3H9;RS(-/-) mice were enriched in L chains that promote DNA binding. Our results suggest that central tolerance and attendant L chain receptor editing affect a large fraction of normal developing B cells. IgH(a/b) mice carrying the superantigen had a â¼50% loss in follicular B cell numbers, suggesting that escape from central tolerance by receptor editing from one IgH allele to another was not a major mechanism. IgM(b) superantigen hosts reconstituted with experimental bone marrow were demonstrated to be useful in revealing pathways involved in central tolerance.