ABSTRACT
BACKGROUND: Thymus hypoplasia due to stromal cell problems has been linked to mutations in several transcription factors, including Forkhead box N1 (FOXN1). FOXN1 supports T-cell development by regulating the formation and expansion of thymic epithelial cells (TECs). While autosomal recessive FOXN1 mutations result in a nude and severe combined immunodeficiency phenotype, the impact of single-allelic or compound heterozygous FOXN1 mutations is less well-defined. OBJECTIVE: With more than 400 FOXN1 mutations reported, their impact on protein function and thymopoiesis remains unclear for most variants. We developed a systematic approach to delineate the functional impact of diverse FOXN1 variants. METHODS: Selected FOXN1 variants were tested with transcriptional reporter assays and imaging studies. Thymopoiesis was assessed in mouse lines genocopying several human FOXN1 variants. Reaggregate thymus organ cultures were used to compare the thymopoietic potential of the FOXN1 variants. RESULTS: FOXN1 variants were categorized into benign, loss- or gain-of-function, and/or dominant-negatives. Dominant negative activities mapped to frameshift variants impacting the transactivation domain. A nuclear localization signal was mapped within the DNA binding domain. Thymopoiesis analyses with mouse models and reaggregate thymus organ cultures revealed distinct consequences of particular Foxn1 variants on T-cell development. CONCLUSIONS: The potential effect of a FOXN1 variant on T-cell output from the thymus may relate to its effects on transcriptional activity, nuclear localization, and/or dominant negative functions. A combination of functional assays and thymopoiesis comparisons enabled a categorization of diverse FOXN1 variants and their potential impact on T-cell output from the thymus.
Subject(s)
T-Lymphocytes , Thymus Gland , Animals , Humans , Mice , Cell Differentiation , Epithelial Cells/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Phenotype , T-Lymphocytes/metabolismABSTRACT
X-linked severe combined immunodeficiency (X-SCID) is a disorder of adaptive immunity caused by mutations in the IL-2 receptor common gamma chain gene resulting in deficiencies of T and natural killer cells, coupled with severe dysfunction in B cells. X-SCID is lethal without allogeneic stem cell transplant or gene therapy due to opportunistic infections. An infant with X-SCID became infected with SARS-CoV-2 while awaiting transplant. The patient developed severe hepatitis without the respiratory symptoms typical of COVID-19. He was treated with convalescent plasma, and thereafter was confirmed to have SARS-CoV-2 specific antibodies, as detected with a microfluidic antigen array. After resolution of the hepatitis, he received a haploidentical CD34 selected stem cell transplant, without conditioning, from his father who had recovered from COVID-19. SARS CoV-2 was detected via RT-PCR on nasopharyngeal swabs until 61 days post transplantation. He successfully engrafted donor T and NK cells, and continues to do well clinically.
Subject(s)
COVID-19/complications , COVID-19/therapy , Hepatitis/virology , Severe Combined Immunodeficiency/complications , Humans , Immunization, Passive/methods , Infant , Male , SARS-CoV-2 , COVID-19 SerotherapyABSTRACT
PURPOSE: The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses. METHODS: An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses. RESULTS: IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (17%) had elevated IgG responses to multiple RNA and DNA viruses (e.g., respiratory syncytial virus, Epstein-Barr virus, adenovirus, Coxsackievirus) and this correlated with increased responses to live attenuated viral vaccines and certain autoantigens. This high group was more likely to be associated with gestational diabetes and an older age. Genetic analyses identified polymorphisms in the IL2RB, TNFSF4, and INS genes in two high responder individuals that were associated with their elevated cytokine levels and clinical history of eczema and asthma. CONCLUSION: Serum IgG profiling of toddlers reveals correlations between the magnitude of the antibody responses towards viruses, live attenuated vaccines, and certain autoantigens. A low responder group had much weaker responses overall, including against vaccines. The serum antibody screen also identifies individuals with IgG responses to less common infections (West Nile virus, parvovirus, tuberculosis). The characterization of the antibody responses in combination with the identification of genetic risk alleles provides an opportunity to identify children with increased risk of clinical disease.
Subject(s)
Antibodies, Viral/blood , Autoantigens/immunology , Bacteria/immunology , DNA Viruses/immunology , Immunoglobulin G/blood , RNA Viruses/immunology , Vaccines/immunology , Child, Preschool , Cytokines/blood , Female , Genotype , Humans , Immunoglobulin M/blood , Infant , Male , Microfluidic Analytical TechniquesABSTRACT
The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.
Subject(s)
Autoimmunity/physiology , CD3 Complex/physiology , Receptors, Antigen, T-Cell/physiology , Animals , CD3 Complex/genetics , CD3 Complex/immunology , Mice , Receptors, Antigen, T-Cell/metabolismABSTRACT
The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tissues in the body. Stress, in the form of infections, radiation exposure, and steroids, impairs thymic epithelial cell (TEC) functions and induces the programmed cell death of immature thymocytes. MicroRNAs are small noncoding RNAs involved in tissue repair and homeostasis, with several supporting T cell development. We report that miR-205, an epithelial-specific miR, maintains thymopoiesis following inflammatory perturbations. Thus, the activation of diverse pattern recognition receptors in mice causes a more severe thymic hypoplasia and delayed T cell recovery when miR-205 is conditionally ablated in TECs. Gene expression comparisons in the TECs with/without miR-205 revealed a significant differential regulation of chemokine/chemokine receptor pathways, antigen processing components, and changes in the Wnt signaling system. This was partly a consequence of reduced expression of the transcriptional regulator of epithelial cell function, Forkhead Box N1 (Foxn1), and its two regulated targets, stem cell factor and ccl25, following stress. miR-205 mimics supplemented into miR-205-deficient fetal thymic organ cultures restored Foxn1 expression along with ccl25 and stem cell factor A number of putative targets of miR-205 were up-regulated in TECs lacking miR-205, consistent with an important role for this miR in supporting T cell development in response to stress.
Subject(s)
Cell Differentiation , Chemokines, CC/metabolism , Forkhead Transcription Factors/genetics , MicroRNAs/metabolism , Stem Cell Factor/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Animals , Cells, Cultured , Chemokines, CC/genetics , Female , Forkhead Transcription Factors/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Stem Cell Factor/genetics , Thymocytes/cytology , Thymocytes/metabolism , Thymus Gland/cytology , Thymus Gland/growth & development , Thymus Gland/metabolismABSTRACT
Type I interferon (IFN-α/ß) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon-sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN-α/ß-mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN-α/ß signaling suppressed IL5 and IL13 mRNA expression during recall responses to T-cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN-α/ß-mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-α/ß signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.
Subject(s)
Gene Expression Regulation , Immunologic Memory , Interleukin-5/genetics , STAT4 Transcription Factor/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Transcription, Genetic , Biomarkers , Cytokines/metabolism , Gene Expression Regulation/drug effects , Humans , Inflammation Mediators/metabolism , Interferon-alpha/metabolism , Interferon-alpha/pharmacology , Interferon-beta/metabolism , Interferon-beta/pharmacology , Interleukin-13/genetics , Interleukin-13/metabolism , Interleukin-5/metabolism , Promoter Regions, Genetic , Signal TransductionABSTRACT
miR-185 is a microRNA (miR) that targets Bruton's tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90-95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor ß (TCRß) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.
Subject(s)
Calcium Signaling , Cytokines/biosynthesis , MicroRNAs/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Thymocytes/metabolism , Adaptor Proteins, Signal Transducing , Animals , Calcium/immunology , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 4/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 4/immunology , Calcium-Calmodulin-Dependent Protein Kinase Type 4/metabolism , Cytokines/genetics , Cytokines/immunology , Humans , Mice , Mice, Transgenic , MicroRNAs/genetics , MicroRNAs/immunology , NFATC Transcription Factors/genetics , NFATC Transcription Factors/immunology , NFATC Transcription Factors/metabolism , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Messenger/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Thymocytes/cytology , Thymocytes/immunology , Transgenes/genetics , Transgenes/immunologyABSTRACT
Patients with 22q11.2 deletion syndrome have heterogeneous clinical presentations including immunodeficiency, cardiac anomalies, and hypocalcemia. The syndrome arises from hemizygous deletions of up to 3Mb on chromosome 22q11.2, a region that contains 60 genes and 4 microRNAs. MicroRNAs are important post-transcriptional regulators of gene expression, with mutations in several microRNAs causal to specific human diseases. We characterized the microRNA expression patterns in the peripheral blood of patients with 22q11.2 deletion syndrome (n=31) compared to normal controls (n=22). Eighteen microRNAs had a statistically significant differential expression (p<0.05), with miR-185 expressed at 0.4× normal levels. The 22q11.2 deletion syndrome cohort exhibited microRNA expression hyper-variability and group dysregulation. Selected microRNAs distinguished patients with cardiac anomalies, hypocalcemia, and/or low circulating T cell counts. In summary, microRNA profiling of chromosome 22q11.2 deletion syndrome/DiGeorge patients revealed a signature microRNA expression pattern distinct from normal controls with clinical relevance.
Subject(s)
DiGeorge Syndrome/genetics , Gene Expression Profiling , MicroRNAs/genetics , Oligonucleotide Array Sequence Analysis/methods , Adolescent , Child , Child, Preschool , Chromosome Deletion , Chromosomes, Human, Pair 22/genetics , Cohort Studies , Female , Heart Defects, Congenital/genetics , Humans , Hypocalcemia/genetics , Infant , Lymphocyte Count , Male , T-Lymphocytes/metabolismABSTRACT
T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.
Subject(s)
CD3 Complex/metabolism , Immunological Synapses , Phosphatidylinositols/metabolism , Receptor-CD3 Complex, Antigen, T-Cell/metabolism , Amino Acids, Basic , Animals , Binding Sites/immunology , CD3 Complex/chemistry , CD3 Complex/immunology , Cell Line , Humans , Lymphocyte Activation/immunology , Mice , Phosphatidylinositols/immunology , Protein Binding/immunology , Signal Transduction/immunology , TransfectionABSTRACT
The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.
Subject(s)
Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , CD3 Complex/metabolism , Proline/metabolism , Amino Acid Motifs/immunology , Animals , CD3 Complex/genetics , CD3 Complex/immunology , Cell Line, Tumor , Epitopes, T-Lymphocyte/physiology , Hybridomas , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/metabolism , Proline/immunology , Receptor-CD3 Complex, Antigen, T-Cell/genetics , Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptor-CD3 Complex, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an "antibiotic resistance arrow of time."
Subject(s)
ATP-Binding Cassette Transporters/genetics , Antitubercular Agents/pharmacology , Azithromycin/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Fungal Proteins/genetics , Mycobacterium avium/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Area Under Curve , Conserved Sequence , Drug Resistance, Multiple, Bacterial/drug effects , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal/drug effects , Macrophages/drug effects , Macrophages/microbiology , Microbial Sensitivity Tests , Molecular Sequence Data , Mycobacterium avium/drug effects , Mycobacterium avium/metabolism , Polymerase Chain Reaction , Protein Structure, Secondary , Sequence Alignment , Time FactorsABSTRACT
Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZα promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.
Subject(s)
Gene Expression , Genetic Vectors , Genetics, Microbial/methods , Macrophages/microbiology , Molecular Biology/methods , Mycobacterium/genetics , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli/genetics , Fluorescence , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Immune Evasion , Immune Tolerance , Mycobacterium/pathogenicity , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Virulence Factors/biosynthesis , Virulence Factors/geneticsABSTRACT
Invariant NKT (iNKT) cells regulate early immune responses to infections, in part because of their rapid release of IFN-gamma and IL-4. iNKT cells are proposed to reduce the severity of Lyme disease following Borrelia burgdorferi infection. Unlike conventional T cells, iNKT cells express an invariant alphabeta TCR that recognizes lipids bound to the MHC class I-like molecule, CD1d. Furthermore, these cells are positively selected following TCR interactions with glycolipid/CD1d complexes expressed on CD4+CD8+ thymocytes. Whereas conventional T cell development can proceed with as few as 4/10 CD3 immunoreceptor tyrosine-based activation motifs (ITAMs), little is known about the ITAM requirements for iNKT cell selection and expansion. We analyzed iNKT cell development in CD3 zeta transgenic lines with various tyrosine-to-phenylalanine substitutions (YF) that eliminated the functions of the first (YF1,2), third (YF5,6), or all three (YF1-6) CD3 zeta ITAMs. iNKT cell numbers were significantly reduced in the thymus, spleen, and liver of all YF mice compared with wild type mice. The reduced numbers of iNKT cells resulted from significant reductions in the expression of the early growth response 2 and promyelocytic leukemia zinc finger transcription factors. In the mice with few to no iNKT cells, there was no difference in the severity of Lyme arthritis compared with wild type controls, following infections with the spirochete B. burgdorferi. These findings indicate that a full complement of functional CD3 zeta ITAMs is required for effective iNKT cell development.
Subject(s)
CD3 Complex/immunology , Natural Killer T-Cells/immunology , Receptors, Antigen, T-Cell/immunology , Amino Acid Motifs , Animals , Cell Differentiation/immunology , Cell Separation , Flow Cytometry , Interleukin-4/immunology , Interleukin-4/metabolism , Lyme Disease/immunology , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , Natural Killer T-Cells/cytology , Receptors, Antigen, T-Cell/metabolism , Reverse Transcriptase Polymerase Chain ReactionSubject(s)
Chromosomes, Human, Pair 9/chemistry , Guanine Nucleotide Exchange Factors/genetics , Immunologic Deficiency Syndromes/genetics , Mutagenesis, Insertional , Adult , Base Sequence , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Child , Consanguinity , Exons , Female , Guanine Nucleotide Exchange Factors/immunology , High-Throughput Nucleotide Sequencing , Humans , Immunologic Deficiency Syndromes/diagnosis , Immunologic Deficiency Syndromes/immunology , Immunologic Deficiency Syndromes/pathology , Lymphocyte Count , Male , Pedigree , Primary Immunodeficiency Diseases , Siblings , Exome SequencingABSTRACT
The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from the 3rd pharyngeal pouch during embryogenesis, this organ functions throughout life. Yet, thymopoiesis can be transiently or permanently damaged contingent on the types of systemic stresses encountered. The thymus also undergoes a functional decline during aging, resulting in a progressive reduction in naïve T cell output. This atrophy is evidenced by a deteriorating thymic microenvironment, including, but not limited, epithelial-to-mesenchymal transitions, fibrosis and adipogenesis. An exploration of cellular changes in the thymus at various stages of life, including mouse models of in-born errors of immunity and with single cell RNA sequencing, is revealing an expanding number of distinct cell types influencing thymus functions. The thymus microenvironment, established through interactions between immature and mature thymocytes with thymus epithelial cells (TEC), is well known. Less well appreciated are the contributions of neural crest cell-derived mesenchymal cells, endothelial cells, diverse hematopoietic cell populations, adipocytes, and fibroblasts in the thymic microenvironment. In the current review, we will explore the contributions of the many stromal cell types participating in the formation, expansion, and contraction of the thymus under normal and pathophysiological processes. Such information will better inform approaches for restoring thymus functionality, including thymus organoid technologies, beneficial when an individuals' own tissue is congenitally, clinically, or accidentally rendered non-functional.
Subject(s)
Endothelial Cells , Thymocytes , Adipogenesis , Animals , Epithelial Cells/metabolism , Mice , Stromal Cells , Thymocytes/metabolism , Thymus GlandABSTRACT
The CD3 epsilon subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 epsilon, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 epsilon to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P(3), and PI(4,5)P(2). Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 epsilon localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 epsilon lipid-binding domain in T cell biology.
Subject(s)
CD3 Complex/metabolism , Phospholipids/metabolism , T-Lymphocytes/immunology , Amino Acid Motifs , Amino Acids, Basic , Animals , Binding Sites , CD3 Complex/genetics , CD3 Complex/physiology , Cell Line , Cytoplasm/chemistry , DNA, Complementary , Humans , Mice , Mutation , Receptors, Antigen, T-Cell , Thymus Gland/cytologyABSTRACT
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells, and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50 and 350 nts in length and smaller RNAs (sncRNA) < 50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists, and locked nucleic acid sequence-specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs are summarized.
ABSTRACT
INTRODUCTION: We describe a previously unreported 437 TâG missense mutation producing a V146G substitution in the first coiled-coil (CC1) domain of nuclear factor-κB essential modulator (NEMO) in a 9-month-old boy with ectodermal dysplasia with immunodeficiency who presented with methicillin-resistant Staphylococcus aureus subdural empyema. We performed in vitro experiments to determine if this novel mutation resulted in impaired NF-κB signaling. METHODS: IκBα phosphorylation experiments were performed using a Jurkat T cell line lacking endogenous NEMO expression that was transfected with vectors containing either the wild type or the patient's V146G mutation. The cells were stimulated with TNF-α to activate the NF-κB pathway. Phosphorylated IκBα was detected by immunoblotting with anti-phospho-IκBα antibodies. Peripheral blood mononuclear cells from the patient were stimulated with TNF-α or anti-CD3 and anti-CD28. Impaired IκBα degradation was detected using antibodies against the IκBα protein. RESULTS: While TNF-α stimulation resulted in IκBα phosphorylation in NEMO-deficient Jurkat cells reconstituted with wild-type NEMO, cell transfected with the V146G mutant exhibited a 75% reduction in phospho-IκBα. Peripheral blood mononuclear cells from the patient showed impaired degradation of IκBα after stimulation when compared with normal controls. CONCLUSIONS: The patient's V146G mutation results in impaired NF-κB activation in vitro. The mutation extends the known N-terminal boundary within the CC1 domain that produces an ectodermal dysplasia phenotype, and defines an infectious susceptibility previously unappreciated in ectodermal dysplasia with immunodeficiency (methicillin-resistant S. aureus subdural empyema), broadening the clinical spectrum associated with the disease.
Subject(s)
Drug Resistance, Bacterial , Empyema, Subdural/genetics , I-kappa B Kinase/metabolism , Staphylococcus aureus/immunology , Ectodermal Dysplasia , Empyema, Subdural/drug therapy , Empyema, Subdural/metabolism , Empyema, Subdural/physiopathology , Humans , I-kappa B Kinase/genetics , Infant , Jurkat Cells , Male , Methicillin/therapeutic use , Mutant Proteins/genetics , Mutation, Missense/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Signal Transduction , Staphylococcus aureus/drug effects , Staphylococcus aureus/pathogenicity , Transcriptional Activation/genetics , Transgenes/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
22q11.2 deletion syndrome (DiGeorge), CHARGE syndrome, Nude/SCID and otofaciocervical syndrome type 2 (OTFCS2) are distinct clinical conditions in humans that can result in hypoplasia and occasionally, aplasia of the thymus. Thymic hypoplasia/aplasia is first suggested by absence or significantly reduced numbers of recent thymic emigrants, revealed in standard-of-care newborn screens for T cell receptor excision circles (TRECs). Subsequent clinical assessments will often indicate whether genetic mutations are causal to the low T cell output from the thymus. However, the molecular mechanisms leading to the thymic hypoplasia/aplasia in diverse human syndromes are not fully understood, partly because the problems of the thymus originate during embryogenesis. Rodent and Zebrafish models of these clinical syndromes have been used to better define the underlying basis of the clinical presentations. Results from these animal models are uncovering contributions of different cell types in the specification, differentiation, and expansion of the thymus. Cell populations such as epithelial cells, mesenchymal cells, endothelial cells, and thymocytes are variably affected depending on the human syndrome responsible for the thymic hypoplasia. In the current review, findings from the diverse animal models will be described in relation to the clinical phenotypes. Importantly, these results are suggesting new strategies for regenerating thymic tissue in patients with distinct congenital disorders.