RESUMO
Pathogenic variants in the transcription factor TP63 are associated with clinically overlapping syndromes including ectrodactyly-ectodermal dysplasia clefting (EEC) and ankyloblepharon-ectodermal defects-cleft lip/palate (AEC). T cell lymphopenia has rarely been described in individuals with TP63 variants and the cause of the T cell defect is unclear. Here, we present a case of a female infant born with TP63-related syndrome and profound T cell lymphopenia, first uncovered through newborn screening. Flow cytometry analysis revealed low CD4+ naïve T cells and nearly absent CD8+ T cells with intact B and NK cell compartments. A de novo heterozygous pathogenic variant c.1040 G>A (C347Y) in exon 8 of TP63 was identified. An artificial thymic organoid system, to assess the intrinsic ability of the patient's hematopoietic cells to develop into T cells, was performed twice using separate peripheral blood samples. Ex vivo T cell differentiation was evident with the artificial organoid system, suggesting that a thymic stromal cell defect may be the cause of the T cell lymphopenia. Consistent with this, interrogation of publicly available data indicated that TP63 expression in the human thymus is restricted to thymic epithelial cells. Based on these data, congenital athymia was suspected and the patient received an allogenic cultured thymus tissue implant (CTTI). This is the first report of suspected congenital athymia and attempted treatment with CTTI associated with TP63 variant. At 9 months post-implant, peripheral lymphocyte analysis revealed measurable T cell receptor excision circles and presence of CD4+ recent thymic emigrants suggestive of early thymopoiesis. She will continue regular monitoring to ensure restoration of T cell immunity.
Assuntos
Linfopenia , Organoides , Timo , Fatores de Transcrição , Proteínas Supressoras de Tumor , Humanos , Linfopenia/genética , Linfopenia/imunologia , Feminino , Timo/imunologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Recém-Nascido , Linfócitos T/imunologia , Diferenciação Celular , MutaçãoRESUMO
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.
Assuntos
Linfócitos T , Timo , Animais , Humanos , Camundongos , Diferenciação Celular , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fenótipo , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Células Endoteliais , Timócitos , Adipogenia , Animais , Células Epiteliais/metabolismo , Camundongos , Células Estromais , Timócitos/metabolismo , TimoRESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Autoantígenos/imunologia , Bactérias/imunologia , Vírus de DNA/imunologia , Imunoglobulina G/sangue , Vírus de RNA/imunologia , Vacinas/imunologia , Pré-Escolar , Citocinas/sangue , Feminino , Genótipo , Humanos , Imunoglobulina M/sangue , Lactente , Masculino , Técnicas Analíticas MicrofluídicasRESUMO
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.
Assuntos
COVID-19/complicações , COVID-19/terapia , Hepatite/virologia , Imunodeficiência Combinada Severa/complicações , Humanos , Imunização Passiva/métodos , Lactente , Masculino , SARS-CoV-2 , Soroterapia para COVID-19RESUMO
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.
Assuntos
Síndrome Brânquio-Otorrenal/complicações , Síndrome CHARGE/complicações , Síndrome de DiGeorge/complicações , Síndromes de Imunodeficiência/etiologia , Imunodeficiência Combinada Severa/complicações , Timo/anormalidades , Animais , Síndrome Brânquio-Otorrenal/genética , Síndrome Brânquio-Otorrenal/imunologia , Síndrome CHARGE/genética , Síndrome CHARGE/imunologia , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/imunologia , Modelos Animais de Doenças , Humanos , Síndromes de Imunodeficiência/imunologia , Camundongos , Mutação , Ratos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Timo/embriologia , Timo/imunologia , Peixe-ZebraRESUMO
MicroRNAs (miRNAs) are processed from primary miRNA transcripts (pri-miRNAs), many of which are annotated as long noncoding RNAs (lncRNAs). We assessed whether MIR205HG, the host gene for miR-205, has independent functions as an lncRNA. Comparing mice with targeted deletions of MIR205HG and miR-205 revealed a functional role for the lncRNA in the anterior pituitary. Mice lacking MIR205HG had a temporal reduction in Pit1, growth hormone, and prolactin. This was mediated, in part, through the ability of this lncRNA to bind and regulate the transcriptional activity of Pit1 in conjunction with Zbtb20. Knockdown of MIR205HG in lactotropes decreased the expression of Pit1, Zbtb20, prolactin, and growth hormone, while its overexpression enhanced the levels of these transcripts. The effects of MIR205HG on the pituitary were independent of miR-205. The data support a role for MIR205HG as an lncRNA that regulates growth hormone and prolactin production in the anterior pituitary.
Assuntos
Hormônio do Crescimento/biossíntese , MicroRNAs/metabolismo , Adeno-Hipófise/metabolismo , Prolactina/biossíntese , RNA Longo não Codificante/metabolismo , Animais , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Prolactina/genética , Prolactina/metabolismo , RNA Longo não Codificante/genética , Ratos , Fator de Transcrição Pit-1/genética , Fator de Transcrição Pit-1/metabolismo , TranscriptomaRESUMO
Chromosome 22q11.2 deletion syndrome (22q11.2del) is a complex, multi-organ disorder noted for its varying severity and penetrance among those affected. The clinical problems comprise congenital malformations; cardiac problems including outflow tract defects, hypoplasia of the thymus, hypoparathyroidism, and/or dysmorphic facial features. Additional clinical issues that can appear over time are autoimmunity, renal insufficiency, developmental delay, malignancy and neurological manifestations such as schizophrenia. The majority of individuals with 22q11.2del have a 3 Mb deletion of DNA on chromosome 22, leading to a haploinsufficiency of ~106 genes, which comprise coding RNAs, noncoding RNAs, and pseudogenes. The consequent haploinsufficiency of many of the coding genes are well described, including the key roles of T-box Transcription Factor 1 (TBX1) and DiGeorge Critical Region 8 (DGCR8) in the clinical phenotypes. However, the haploinsufficiency of these genes alone cannot account for the tremendous variation in the severity and penetrance of the clinical complications among those affected. Recent RNA and DNA sequencing approaches are uncovering novel genetic and epigenetic differences among 22q11.2del patients that can influence disease severity. In this review, the role of coding and non-coding genes, including microRNAs (miRNA) and long noncoding RNAs (lncRNAs), will be discussed in relation to their bearing on 22q11.2del with an emphasis on TBX1.
RESUMO
Germline coding mutations in different telomere-related genes have been linked to autosomal-dominant familial pulmonary fibrosis. Individuals with these inherited mutations demonstrate incomplete penetrance of clinical phenotypes affecting the lung, blood, liver, skin, and other organs. Here, we describe the somatic acquisition of promoter mutations in telomerase reverse transcriptase (TERT) in blood leukocytes of approximately 5% of individuals with inherited loss-of-function coding mutations in TERT or poly(A)-specific ribonuclease (PARN), another gene linked to telomerase function. While these promoter mutations were initially identified as oncogenic drivers of cancer, individuals expressing the mutations have no history of cancer. Neither promoter mutation was found in population-based cohorts of similar or advanced age. The TERT promoter mutations were found more frequently in cis with the WT allele than the TERT coding sequence mutation. EBV-transformed lymphoblastoid B cell lines (LCLs) derived from subjects with TERT promoter mutations showed increased telomerase expression and activity compared with cell lines from family members with identical coding mutations. TERT promoter mutations resulted in an increased proliferation of LCLs and demonstrated positive selection over time. The persistence and recurrence of noncoding gain-of-function mutations in these cases suggests that telomerase activation is not only safely tolerated but also advantageous for clonal expansion.
Assuntos
Alelos , Linfócitos B/metabolismo , Seleção Clonal Mediada por Antígeno/genética , Mutação , Regiões Promotoras Genéticas , Telomerase , Linhagem Celular Transformada , Proliferação de Células/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Humanos , Masculino , Telomerase/genética , Telomerase/metabolismoRESUMO
Physiological stress resulting from infections, trauma, surgery, alcoholism, malnutrition, and/or pregnancy results in a substantial depletion of immature CD4(+)CD8(+) thymocytes. We previously identified 18 distinct stress-responsive microRNAs (miRs) in the thymus upon systemic stress induced by lipopolysaccharide (LPS) or the synthetic glucocorticoid, dexamethasone (Dex). MiRs are short, non-coding RNAs that play critical roles in the immune system by targeting diverse mRNAs, suggesting that their modulation in the thymus in response to stress could impact thymopoiesis. MiR-181d is one such stress-responsive miR, exhibiting a 15-fold down-regulation in expression. We utilized both transgenic and gene-targeting approaches to study the impact of miR-181d on thymopoiesis under normal and stress conditions. The over-expression of miR-181d in developing thymocytes reduced the total number of immature CD4(+)CD8(+) thymocytes. LPS or Dex injections caused a 4-fold greater loss of these cells when compared with the wild type controls. A knockout mouse was developed to selectively eliminate miR-181d, leaving the closely spaced and contiguous family member miR-181c intact. The targeted elimination of just miR-181d resulted in a thymus stress-responsiveness similar to wild-type mice. These experiments suggest that one or more of three other miR-181 family members have overlapping or compensatory functions. Gene expression comparisons of thymocytes from the wild type versus transgenic mice indicated that miR-181d targets a number of stress, metabolic, and signaling pathways. These findings demonstrate that selected miRs enhance stress-mediated thymic involution in vivo.
Assuntos
MicroRNAs/genética , Timócitos/metabolismo , Timo/metabolismo , Animais , Sequência de Bases , Antígenos CD4/genética , Antígenos CD4/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Dexametasona/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/imunologia , Dados de Sequência Molecular , Cultura Primária de Células , Estresse Fisiológico , Timócitos/efeitos dos fármacos , Timócitos/imunologia , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
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."
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antituberculosos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas Fúngicas/genética , Mycobacterium avium/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Área Sob a Curva , Sequência Conservada , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium avium/efeitos dos fármacos , Mycobacterium avium/metabolismo , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Alinhamento de Sequência , Fatores de TempoRESUMO
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.
Assuntos
Expressão Gênica , Vetores Genéticos , Genética Microbiana/métodos , Macrófagos/microbiologia , Biologia Molecular/métodos , Mycobacterium/genética , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Evasão da Resposta Imune , Tolerância Imunológica , Mycobacterium/patogenicidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Physiological stress evokes rapid changes in both the innate and adaptive immune response. Immature αß T cells developing in the thymus are particularly sensitive to stress, with infections and/or exposure to lipopolysaccharide or glucocorticoids eliciting a rapid apoptotic program. MicroRNAs are a class of small, non-coding RNAs that regulate global gene expression by targeting diverse mRNAs for degradation. We hypothesized that a subset of thymically encoded microRNAs would be stress responsive and modulate thymopoiesis. We performed microRNA profiling of thymic microRNAs isolated from control or stressed thymic tissue obtained from mice. We identified 18 microRNAs that are dysregulated >1.5-fold in response to lipopolysaccharide or the synthetic corticosteroid dexamethasone. These included the miR-17-90 cluster, which have anti-apoptotic functions, and the miR-181 family, which contribute to T cell tolerance. The stress-induced changes in the thymic microRNAs are dynamically and distinctly regulated in the CD4(-)CD8(-), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) thymocyte subsets. Several of the differentially regulated murine thymic miRs are also stress responsive in the heart, kidney, liver, brain, and/or spleen. The most dramatic thymic microRNA down modulated is miR-181d, exhibiting a 15-fold reduction following stress. This miR has both similar and distinct gene targets as miR-181a, another member of miR-181 family. Many of the differentially regulated microRNAs have known functions in thymopoiesis, indicating that their dysregulation will alter T cell repertoire selection and the formation of naïve T cells. This data has implications for clinical treatments involving anti-inflammatory steroids, ablation therapies, and provides mechanistic insights into the consequences of infections.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Estresse Fisiológico/genética , Timo/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/metabolismo , Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Genes Reporter/genética , Humanos , Fator Inibidor de Leucemia/genética , Lipopolissacarídeos/farmacologia , Luciferases/genética , Masculino , Camundongos , Especificidade de Órgãos , Estresse Fisiológico/efeitos dos fármacos , Timócitos/citologia , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Timo/citologia , Timo/efeitos dos fármacos , Timo/fisiologia , Fatores de Tempo , Transcriptoma/efeitos dos fármacosRESUMO
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.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Complexo CD3/metabolismo , Prolina/metabolismo , Motivos de Aminoácidos/imunologia , Animais , Complexo CD3/genética , Complexo CD3/imunologia , Linhagem Celular Tumoral , Epitopos de Linfócito T/fisiologia , Hibridomas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Prolina/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Farmacorresistência Bacteriana , Empiema Subdural/genética , Quinase I-kappa B/metabolismo , Staphylococcus aureus/imunologia , Displasia Ectodérmica , Empiema Subdural/tratamento farmacológico , Empiema Subdural/metabolismo , Empiema Subdural/fisiopatologia , Humanos , Quinase I-kappa B/genética , Lactente , Células Jurkat , Masculino , Meticilina/uso terapêutico , Proteínas Mutantes/genética , Mutação de Sentido Incorreto/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Ativação Transcricional/genética , Transgenes/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Complexo CD3/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Motivos de Aminoácidos , Animais , Diferenciação Celular/imunologia , Separação Celular , Citometria de Fluxo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Doença de Lyme/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Células T Matadoras Naturais/citologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Haemophilus ducreyi/imunologia , Macrófagos/enzimologia , Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Cátions Bivalentes/farmacologia , Chlorocebus aethiops , Ativadores de Enzimas/farmacologia , Células HeLa , Humanos , Lectinas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.
Assuntos
Complexo CD3/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/fisiologia , Quinases de Receptores Adrenérgicos beta/metabolismo , Animais , Complexo CD3/genética , Quinase 2 de Receptor Acoplado a Proteína G , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Quinases de Receptores Adrenérgicos beta/genéticaRESUMO
Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.