RESUMEN
BACKGROUND: Biomarkers to objectively measure disease severity and predict therapeutic responses are needed in atopic dermatitis (AD). OBJECTIVE: Primary aim: To identify biomarkers reflecting therapeutic response in patients with AD treated systemically. Secondary aims: (i) To identify a biomarker pattern predicting responsiveness to systemic treatment. (ii) To identify differences in expression of biomarker in filaggrin gene (FLG) mutation carriers vs. non-FLG mutations carriers. METHODS: Thirty-eight severe AD patients treated with methotrexate or azathioprine participated. Serum levels of a proliferation-inducing ligand, B-cell activating factor of the TNF family, thymus and activation-regulated chemokine (chemokine (C-C motif) ligand 17) (TARC (CCl-17)), interleukin-1 receptor antagonist (IL-1RA), interleukin-1 bèta, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-18, IL-31, interferon gamma, tumour necrosis factor alpha, vascular endothelial growth factor (VEGF), monokine induced by interferon gamma (chemokine (C-X-C motif) ligand 9), interferon gamma-induced protein 10 (C-X-C motif chemokine Ligand 10), monocyte chemoattractant protein-1 (chemokine (C-C Motif) ligand 2), macrophage inflammatory protein-1 beta (chemokine (C-C motif) ligand 4), regulated on activation, normal T cell expressed and secreted (chemokine (C-C motif) ligand 5), Cutaneous T-cell-attracting chemokine (chemokine (C-C motif) ligand 27) (CTACK (CCL-27)), thymic stromal lymphopoietin, IL-5, interleukin-1 alpha and granulocyte-colony stimulating factor were analysed by ELISA and Luminex. The primary outcomes were differences in mean absolute change of SCORing Atopic Dermatitis (SCORAD) between groups after 12 weeks compared with baseline. Responders to treatment were defined by a SCORAD reduction in ≥50%. Buccal mucosa swabs were collected to determine FLG genotype status. RESULTS: Thymus and activation-regulated chemokine, CTACK, IL-13 and VEGF showed a significant decrease after treatment with methotrexate or azathioprine. However, no decrease in individual cytokine levels was significantly correlated with a change in any of the outcome parameters. In addition, baseline biomarker levels were not significantly different between responders and non-responders, and FLG and non-FLG mutants showed similar biomarker profiles. CONCLUSION: Thymus and activation-regulated chemokine and CTACK were confirmed as potential biomarkers. VEGF and IL-13 have a potential value as well. Biomarkers could not be used to discriminate at baseline between responders and non-responders, or FLG genotype status.
Asunto(s)
Dermatitis Atópica , Terapia de Inmunosupresión , Adulto , Biomarcadores , Quimiocina CCL17/genética , Quimiocinas , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/genética , Proteínas Filagrina , Humanos , Factor A de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: The in vivo levels of inflammatory mediators in chronic atopic dermatitis (AD) skin are not well-defined due to the lack of a non-invasive or minimally invasive sampling technique. OBJECTIVES: To investigate the cytokine milieu in interstitial fluid (ISF) collected from chronic lesional AD skin as compared to ISF from non-lesional AD skin and/or healthy donor skin. METHODS: ISF was obtained using a minimally invasive technique of creating micropores in the skin by a laser, and harvesting ISF through aspiration. We determined the levels of 33 cytokines by Luminex and ELISA in ISF and plasma from sixteen AD patients and twelve healthy individuals. In seven AD patients, we analysed the IL-13, IL-31, IL-17, IL-22 and IFN-γ production by T cells isolated from lesional skin. AD patients were genotyped for the filaggrin gene (FLG)-null mutations 2282del4, R501X, R2447X and S3247X. RESULTS: Twenty-five of 33 examined mediators were detected in the ISF. The levels of IL-1α, IL-1ß, IL-18, IL-1RA, IL-5, IL-13, IL-6, IL-8, TNF-α, RANTES(CCL-5), MIG(CXCL-9), IP-10(CXCL-10), TARC(CCL-17), VEGF and G-CSF showed significant differences between either lesional, non-lesional and/or healthy skin. IP-10 levels in ISF from lesional and non-lesional AD skin showed significant correlation with IP-10 blood levels. IP-10 also showed a significant correlation with clinical severity (SCORAD), as did IL-13. Levels of both IP-10 and IL-13 were more pronounced in patients with FLG-null mutations. Furthermore, FLG-null mutation carriers had more severe AD. CONCLUSION: The presented minimally invasive technique is a valuable tool to determine the in vivo cytokine profile of AD skin.
Asunto(s)
Citocinas/metabolismo , Dermatitis Atópica/metabolismo , Líquido Extracelular/metabolismo , Piel/metabolismo , Estudios de Casos y Controles , Quimiocina CXCL10/metabolismo , Enfermedad Crónica , Dermatitis Atópica/genética , Proteínas Filagrina , Genotipo , Humanos , Interleucina-13/metabolismo , Proteínas de Filamentos Intermediarios/genética , Mutación , Índice de Severidad de la Enfermedad , Manejo de Especímenes/instrumentación , Manejo de Especímenes/métodosRESUMEN
As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R(2)≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.
Asunto(s)
Citometría de Flujo/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The case of an infertile couple in which a testicular seminoma and azoospermia were discovered in the husband during infertility treatment is described. A small piece of testicular tissue, obtained by biopsy from the healthy testis [testicular sperm extraction (TESE)], was deep-frozen before oncology therapy was initiated. The patient's lymphocyte karyotype was normal and no Y microdeletions were found. After conclusion of oncology treatment, the tissue was thawed and successfully used in the intracytoplasmic sperm injection (ICSI) procedure. A healthy girl was born. Testicular tumours are known to impair fertility in the majority of patients, and fertility deteriorates further after cytotoxic and surgical oncology treatment. Until recently in Slovenia, for young oncology patients cryopreservation was applied only to high quality ejaculate fulfilling the criteria for intrauterine insemination or in-vitro fertilization after thawing. Failing that, the only remaining options were fertilization by donor spermatozoa or child adoption. New assisted reproductive technologies, of which the ICSI procedure is the most successful, are suitable for the treatment of only the most severe cases of male infertility. It is reasonable to cryopreserve even poor quality ejaculate prior to the oncology therapy, as well as testicular tissue in cases of azoospermia.
Asunto(s)
Criopreservación , Infertilidad Masculina/terapia , Oligospermia/etiología , Seminoma/complicaciones , Neoplasias Testiculares/complicaciones , Testículo/patología , Adulto , Biopsia , Femenino , Humanos , Masculino , Orquiectomía , Embarazo , Resultado del Embarazo , Inducción de Remisión , Seminoma/tratamiento farmacológico , Seminoma/cirugía , Inyecciones de Esperma Intracitoplasmáticas , Neoplasias Testiculares/tratamiento farmacológico , Neoplasias Testiculares/cirugíaRESUMEN
We have characterized dendritic cell precursors (pre-DC) in the human thymus. These CD1a(-)CD3(-)CD4(+)CD8(-) cells express high levels of interleukin-3Ralpha (IL-3Ralpha) on the membrane and are able to develop into mature DC upon culture with IL-3 and CD40 ligation. The DC precursors are predominantly located in the thymic medulla. Interestingly, the pre-DC express pTalpha mRNA, which is also present in CD1a(+)CD3(-)CD4(+)CD8(-) pre-T cells. Yet, the pre-DC lack expression of recombination activating gene-1 mRNA and fail to develop into T cells in appropriate assays. The thymic pre-DC are very similar to the recently characterized pre-DC found in the T cell areas of the tonsil, and it is suggested that these pre-DC populations are of lymphoid origin.
Asunto(s)
Células Dendríticas/citología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/biosíntesis , Células Madre/citología , Timo/citología , Animales , Antígenos CD/análisis , Ligando de CD40 , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Preescolar , Técnicas de Cocultivo , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Lactante , Recién Nacido , Interleucina-3/farmacología , Tejido Linfoide/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Noqueados , Microscopía Confocal , Especificidad de Órganos , Tonsila Palatina/citología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T alfa-beta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Proteínas de Neoplasias , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos T/metabolismo , Factores de Transcripción/genética , Antígenos CD/inmunología , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Reordenamiento Génico de Linfocito T/genética , Secuencias Hélice-Asa-Hélice , Humanos , Proteínas Inhibidoras de la Diferenciación , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo , Transducción GenéticaRESUMEN
The thymus is populated by hematopoietic cells that have the capacity to develop into at least three different hematopoietic lineages, T, NK and dendritic cells. While developing into T cells these cells pass a series of developmental stages that can be discriminated on the basis of expression of a number of antigens. The availability of a myriad of monoclonal anti- bodies against human differentiation antigens has permitted a detailed analysis of the various cellular stages in the human thymus. This analysis not only comprised investigation of molecular but also of functional features of purified thymocyte subsets, since more recently assays were set up that allowed investigation of the hematopoietic precursor activities of human thymic progenitor cells. Here we review the current status of knowledge with regard to early developmental stages in the human thymus. In addition, we discuss recent data on later developmental stages, in particular concerning positive selection and maturation of T cells.
Asunto(s)
Antígenos de Diferenciación/inmunología , Linaje de la Célula/inmunología , Células Dendríticas/citología , Células Asesinas Naturales/citología , Linfocitos T/citología , Timo , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Humanos , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Timo/citología , Timo/embriología , Timo/inmunologíaRESUMEN
T-cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow to migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T-cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD1a. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T-cell commitment, which correlates with appearance of CD1a and involves the loss of capacity to develop into NK cells and DC and the initiation of T-cell receptor (TCR) gene rearrangements. Basic helix-loop-helix transcription factors play a role in induction of T-cell commitment. CD1a+CD34+ cells develop into CD4+CD8 alpha+ beta+ cells by upregulating first CD4, followed by CD8 alpha and then CD8 beta. Selection for productive TCR beta gene rearrangements (beta selection) likely occurs in the CD4+CD8 alpha+ beta- and CD4+CD8 alpha+ beta+ populations. Although the T and NK-cell lineages are closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK-cell development.
Asunto(s)
Células Dendríticas/citología , Células Asesinas Naturales/citología , Linfocitos T/citología , Timo/citología , Animales , Diferenciación Celular , Movimiento Celular , Células Madre Hematopoyéticas/citología , HumanosRESUMEN
The thymus is seeded at week 7-8 of gestation with hematopoietic progenitor cells derived from the liver. By week 15-16 of gestation a fully differentiated thymus with a cortical/medullary junction and Hassal's corpuscles has been formed. The thymus is continuously populated by progenitor cells first from the liver and then from bone marrow. This process continues in childhood after which the thymus starts to involute. Recent information indicates that the cells that populate the thymus are not committed to the T cell lineage. When developing to T cells these progenitor cells traverse a series of cellular stages that can be discriminated on the basis of cell surface and cytoplasmic markers, status of TCR gene rearrangements and precursor cell activities. The early stages of T cell development in the mouse thymus have been described in detail. The recent development of assays to measure the T cell precursor activity of human thymic and extrathymic progenitor cell subsets has led to a rapid accumulation of data on early events in human thymic development as well. The information available now permits a comparison of early cellular stages of T cell development in mice and man. Some of the extrinsic and intrinsic factors that govern T cell differentiation will be discussed. Data on the role of the cytokine, interleukin-7, in human and mouse T cell development will be summarized. Furthermore, recent data on the involvement of transcription factors in early T cell development are reviewed.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Linfocitos T/inmunología , Animales , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Linfocitos T/citologíaRESUMEN
In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with anti-CD34 and Thy-1 antibodies revealed that the double-labeled cells were located in the pericortical area. In addition, it was found that the CD34+Thy-1+ cells lacked CD45 and CD50, indicating that these cells are not of hematopoietic origin; this was confirmed by the finding that these cells could be cultured as adherent cells in a medium with cholera toxin and dexamethasone, but failed to grow in mixtures of hematopoietic growth factors. Further analysis indicated that most cultured CD34+Thy-1+ cells expressed cytokeratin (CK) 14 but lacked CK 13, suggesting that these cells are immature epithelial cells. Cultured CD34+Thy-1+ cells were able to induce differentiation of CD1-CD34+CD3-CD4-CD8- thymic precursors into CD4+CD8+ cells in a reaggregate culture in the absence of exogenous cytokines. The CD4+CD8+ cells that developed in these cultures did not express CD3, indicating that CD34+Thy-1+ thymic stromal cells are not capable of completing full T cell differentiation of thymic hematopoietic progenitor cells.
Asunto(s)
Antígenos Thy-1/análisis , Timo/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Humanos , Microscopía Confocal , Técnicas de Cultivo de Órganos , Células del Estroma/inmunología , Timo/citología , Timo/embriologíaRESUMEN
To investigate the role of Yersinia persistence in chronic undifferentiated arthritis, two patients who had chronic undifferentiated polyarthritis and circulating IgA and IgG antibodies to Yersinia outer proteins were studied. Immunofluorescence using antibodies directed against Yersinia adhesin A was performed on colonic and synovial tissue. Synovial tissue T cells were cloned aspecifically and screened for their proliferative responses to Yersinia enterocolitica. Furthermore, a Yersinia-specific polymerase chain reaction (PCR) was performed on synovial tissue. Both patients were found to have Yersinia antigens in colonic and synovial tissue. Y. enterocolitica-positive T-cell clones were grown from the synovial tissue: 4 CD4+ clones of 37 clones from patient 1 and 6 CD4+ clones of 53 clones from patient 2. Yersinia-specific PCR products were not detected in the synovial tissue specimens. The results support the hypothesis that an immune-mediated response to Yersinia antigens may play an important role in the pathogenesis of chronic undifferentiated arthritis.
Asunto(s)
Antígenos Bacterianos/análisis , Artritis/inmunología , Artritis/microbiología , Yersiniosis/complicaciones , Yersinia enterocolitica/inmunología , Adhesinas Bacterianas/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Enfermedad Crónica , Colon/microbiología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Activación de Linfocitos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Líquido Sinovial/microbiología , Yersiniosis/inmunologíaRESUMEN
Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.
Asunto(s)
Inhibidores de Crecimiento/fisiología , Secuencias Hélice-Asa-Hélice/inmunología , Células Asesinas Naturales/citología , Proteínas de Neoplasias , Subgrupos de Linfocitos T/citología , Factores de Transcripción/fisiología , Animales , Antígenos CD1/análisis , Antígenos CD34/análisis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Niño , Feto , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/efectos de los fármacos , Secuencias Hélice-Asa-Hélice/fisiología , Humanos , Proteínas Inhibidoras de la Diferenciación , Interleucina-7/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Factor de Células Madre/farmacología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Timo , Factores de Transcripción/biosíntesisRESUMEN
The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Células Asesinas Naturales/citología , Hígado/citología , Linfocitos T/citología , Animales , Antígenos CD34/inmunología , Diferenciación Celular/inmunología , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Hígado/inmunología , Ratones , Embarazo , Linfocitos T/inmunologíaRESUMEN
Progenitor cells that seed the fetal thymus are derived from the fetal liver and the bone marrow. These cells migrate through the fetal blood to the thymus. In this work, we address which peripheral progenitor cells have the potential to become T cells and whether these progenitor cells are already committed to the T cell lineage. All CD34+CD38- precursor cells, regardless of their origin, are able to develop into T cells in a hybrid human/mouse fetal thymic organ culture. Previously, we found that the more differentiated CD34+CD38+ progenitor cells from fetal liver cannot develop into T cells. In this work, we show that CD34+CD38+ cells from fetal bone marrow and cord blood are capable of T cell development. In spite of the T cell-developing potential, we did not detect rearrangements of TCR-delta or TCR-beta loci in any of the CD34+ peripheral precursors. CD34+ fetal bone marrow cell subpopulations express pre-TCR-alpha. However, we could not detect expression of pT alpha or of recombination-activating gene 1 in CD34+ cord blood cells. Since cord blood CD34+ cells should contain the direct progenitors of the CD34+ thymocytes, our data do not support the notion that in humans commitment to the T cell lineage occurs before the cells migrate into the thymus.
Asunto(s)
Células Madre Hematopoyéticas/citología , Linfocitos T/citología , Timo/citología , Animales , Antígenos CD34 , Diferenciación Celular , Linaje de la Célula , Femenino , Humanos , Ratones , Técnicas de Cultivo de Órganos , Embarazo , Timo/embriologíaRESUMEN
Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4+CD3+ alpha beta + mature T cells. These cells, which represent 0.1-0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J). Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm. Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas de Homeodominio , Receptores de Antígenos de Linfocitos T alfa-beta/aislamiento & purificación , Linfocitos T/fisiología , Adulto , Animales , Antígenos CD , Antígenos de Diferenciación , Circulación Sanguínea , Diferenciación Celular , Citometría de Flujo , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Humanos , Masculino , Ratones , Técnicas de Cultivo de Órganos , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T , Timo/citología , Timo/inmunología , Transcripción GenéticaRESUMEN
We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a- cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR alpha beta+CD4+ SP cells, indicating that mouse MHC class II can positively select TCR alpha beta +CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.
Asunto(s)
Antígenos CD1/biosíntesis , Antígenos CD/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Linfocitos T/inmunología , Timo/inmunología , Envejecimiento/inmunología , Animales , Antígenos CD4/biosíntesis , Diferenciación Celular , Niño , Preescolar , Cartilla de ADN , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Lactante , Activación de Linfocitos , Ratones , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Timo/crecimiento & desarrolloRESUMEN
Recently we reported that the human thymus contains a minute population of CD34+CD38dim cells that do not express the T-cell lineage markers CD2 and CD5. The phenotype of this population resembled that of CD34+CD38dim cells present in fetal liver, umbilical cord blood, and bone marrow known to be highly enriched for pluripotent hematopoietic stem cells. In this report we tested the hypothesis that the CD34+CD38dim thymocytes constitute the most primitive hematopoietic cells in the thymus using a combination of phenotypic and functional analyses. It was found that in contrast to CD34+CD38dim cells from fetal liver and bone marrow, CD34+CD38dim cells from the thymus express high levels of CD45RA and are negative for Thy-1. These data indicate that the CD34+CD38dim thymocytes are distinct from pluripotent stem cells. CD34+CD38dim thymocytes differentiate into T cells when cocultured with mouse fetal thymic organs. In addition, individual cells in this population can differentiate either to natural killer cells in the presence of stem cell factor (SCF), interleukin-7 (IL-7), and IL-2 or to dendritic cells in the presence of SCF, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha(TNFalpha), indicating that CD34+CD38dim thymocytes contain multi-potential hematopoietic progenitors. To establish which CD34+ fetal liver subpopulation contains the cells that migrate to the thymus, we investigated the T-cell-developing potential of CD34+CD38dim and CD34+CD38+ fetal liver cells and found that the capacity of CD34+ fetal liver cells to differentiate into T cells is restricted to those cells that are CD38dim. Collectively, these findings indicate that cells from the CD34+CD38dim fetal liver cell population migrate to the thymus before upregulating CD38 and committing to the T-cell lineage.
Asunto(s)
Antígenos CD , Células Dendríticas/citología , Células Madre Hematopoyéticas/citología , Células Asesinas Naturales/citología , Subgrupos de Linfocitos T/citología , Timo/citología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD34/análisis , Antígenos de Diferenciación/análisis , Médula Ósea/embriología , Células de la Médula Ósea , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Células Cultivadas , Preescolar , Técnicas de Cocultivo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/clasificación , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Separación Inmunomagnética , Inmunofenotipificación , Lactante , Hígado/citología , Hígado/embriología , Glicoproteínas de Membrana , Ratones , N-Glicosil Hidrolasas/análisis , Subgrupos de Linfocitos T/efectos de los fármacosRESUMEN
Retroviral vectors have been used in most human gene therapy trials that have been undertaken. Many of these therapies have focused on the introduction of genes into hematopoietic stem cells with the goal of obtaining expression in the mature T lymphocytic progeny. It has proven difficult to achieve expression in the lymphoid lineage, although several groups have demonstrated low expression of transduced genes in the myeloid lineage. In this study we used an in vitro thymic organ culture in which stem/progenitor cells can develop into T cells and all intermediate stages can be studied and manipulated to investigate the fate of a retrovirally introduced Escherichia coli LacZ gene in this system. Here we show that certain conditions can transduce Jurkat T cells, three different antigen-specific T cell clones and CD34+CD3-CD4-CD8- thymocytes (progenitor T cells) with high (> 80%) efficiency. Moreover, retroviral transduction with the LacZ gene does not inhibit T and NK cell differentiation of progenitor cells in fetal thymic organ cultures (FTOC). The LacZ gene also is functionally expressed at all stages of development, although the expression decreases somewhat during differentiation. This experimental system, combining FTOC and retroviral transduction, provides a genetic tool for the study of human T cell development.
Asunto(s)
Vectores Genéticos/inmunología , Retroviridae/genética , Células Madre/virología , Subgrupos de Linfocitos T/virología , Secuencia de Bases , Diferenciación Celular/inmunología , Preescolar , Marcadores Genéticos , Humanos , Lactante , Células Asesinas Naturales/inmunología , Operón Lac/inmunología , Datos de Secuencia Molecular , Retroviridae/inmunología , Células Madre/enzimología , Células Madre/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Timo/virología , Transfección/inmunología , beta-Galactosidasa/genéticaRESUMEN
The histopathological features of rheumatoid joint-inflammation suggest that an antigen-driven activation of T cells plays a central role in the onset and/or perpetuation of the inflammatory process. However, the disease-associated antigens responsible for the activation of T cells in the joint are unknown. In this project we study the response of IL-2 expanded T-cell lines from the synovial fluid (SF) of rheumatoid arthritis (RA) patients against autologous SF in a proliferation assay. Sixteen out of 32 RA patients were found to have CD4+ T cells that proliferate in response to autologous SF. The presence of T cells able to respond to SF antigens in inflamed joints suggests that these T cells play an active role in the pathogenesis of RA. T cell clones reactive to autologous SF were isolated from SF-derived T-cell lines of two RA patients. All clones were of the CD4+, CD8-, alpha/beta+ phenotype. SF-reactivity of T-cell clones from the DR4/DR12-positive RA patient was restricted via the Dw4 subtype of DR4. SF reactivity of T cells of the DR12/DR15 patient was DP-restricted. Some of the T-cell clones responded specifically to autologous and not to allogeneic SF, whereas others revealed responsiveness against a limited number of allogeneic SF samples. The (restricted) specificity of T cells towards autologous SF antigens is indicative for heterogeneity of the epitopes recognized and argues against ubiquitous nonpolymorphic joint constituents as the relevant antigens recognized by the SF-autoreactive T cells.