RESUMO
Kinetics of mature T cell generation in the thymus of normal or major histocompatibility complex (MHC) class I- or II-deficient mice were studied by the bromodeoxyuridine pulse labeling method. As previously described, the early activation and final maturation phases were found to be synchronous for the two T cell lineages, but CD4+8- cells were generated faster than CD4-8+ cells in MHC class I- and II-deficient mice, respectively. CD8 downregulation started on day 2 after cell proliferation even in the absence of MHC class II expression. CD8 downregulation thus appears to be stochastic at its beginning. By contrast, CD4 shut-off was found totally instructive, as the generation of CD4lo8+ cells with a high TCR density was not observed in class I-deficient mice. The analysis of the V beta 14 TCR frequencies in CD4/8 subsets in normal and MHC-deficient mice confirmed that CD4 and CD8 generation pathways are not symmetrical. These findings show that commitment towards the CD4+8- or CD4-8+ phenotype is controlled at the CD8lo step for the former and at the CD4+8+ double-positive stage for the latter.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Animais , Bromodesoxiuridina/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , CamundongosRESUMO
We have fused an H-2- thymoma (BM5R.9) with an H-2+ thymoma (BW5147) and have found that many of the resulting hybrids exhibit an H-2- phenotype. In several hybrids that were analyzed in detail, this phenotype is related to the absence of steady-state H-2 mRNA and shows some instability, possibly related to the loss of chromosomes in segregants. We conclude from our studies that BM5R.9 cells display a trans-acting mechanism that can repress the expression of H-2 antigens, and that the gene(s) causing the repression are not located on chromosome 17. This mechanism is not sufficient to explain the H-2- phenotype of BM5R.9, for which an additional, cis-acting process, must be postulated. We discuss these results in the context of the regulation of expression of the major class I transplantation antigens.
Assuntos
Regulação da Expressão Gênica , Antígenos H-2/genética , Timoma/imunologia , Animais , Fusão Celular , Linhagem Celular , Antígenos H-2/análise , Células Híbridas , Camundongos , Fenótipo , Timoma/genética , Transcrição GênicaRESUMO
We analyzed the phenotype and V beta-T cell receptor (TCR) repertoire, together with interleukin 7 receptor (IL-7R) expression in unfractionated thymocytes stimulated in vitro with IL-7. This culture system results in a specific proliferation of mature thymocytes belonging to the CD3+CD4-, CD4+8-, and CD4-8+ subsets. IL-7 induced a preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4-8- thymocytes. This phenomenon is not observed in beta 2-microglobulin-deficient mice, showing that a fraction of CD4+8- thymocytes, enriched in V beta 8.2+ cells, is selected by class I molecules in normal mice, as are a large proportion of CD4-8- alpha beta TCR+ thymocytes. Our findings also establish that IL-7 plays a major role in the expansion of rare thymocyte subsets, which could exert important functions in inflammatory and immune responses.
Assuntos
Antígenos CD4/imunologia , Antígenos CD8/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interleucina-7/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C3H , Fenótipo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-7 , Timo/citologia , Timo/imunologia , Microglobulina beta-2/imunologiaRESUMO
Intrathymic (IT) transfer of bone marrow (BM) precursor cells in sublethally irradiated hosts has been widely used to study T cell differentiation and maturation. In this report we have used double congenic mice Ly 5.1 Thy 1.1 (host) and Ly 5.2 Thy 1.2 (donor) and detected cycling Ly 5.2+ BM cells by in vivo bromodeoxyuridine incorporation, before induction of the Thy 1.2 antigen. Until Day 9 post-transfer, some donor type cells express a high level of Thy 1.2 together with macrophage and granulocyte markers. A few days later, a Thy 1.2low population transiently B220+ was detected. Thereafter, donor type cells expressed an intermediate Thy 1.2 brightness; this population then persisted and surpassed the other subsets. Our findings permitted to establish a relationship between cell cycle and Thy 1 fluorescence intensity according to the sequence: Thy 1low resting, Thy 1low cycling, Thy 1high cycling, Thy 1high resting. Moreover, we have shown that cells from the myeloïd and B lineages can, in vivo, transiently express the Thy 1 antigen, develop and differentiate within the thymus microenvironment.
Assuntos
Antígenos de Superfície/biossíntese , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Linfócitos T/citologia , Animais , Transplante de Medula Óssea , Ciclo Celular , DNA/biossíntese , Marcadores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/metabolismo , Antígenos Thy-1 , Fatores de TempoRESUMO
OBJECTIVE: To evaluate the role of cell proliferation in peripheral blood lymphocyte (PBL) dynamics during HIV infection and potent antiretroviral therapy including protease inhibitors. DESIGN: Transverse study of 150 patients at different stages of infection. Longitudinal study of 50 patients on triple combination antiretroviral therapy with 9-month follow-up. METHODS: Ex vivo incubation of fresh PBL with the DNA biosynthetic marker bromodeoxyuridine (BrdU). Flow cytometric analysis of cell phenotypes and BrdU incorporation. Parallel determination of plasma virus load and CD4+ cell counts. RESULTS: Percentages of BrdU+ B and T lymphocytes found in patients with asymptomatic HIV infection were not different from the low values found in HIV-seronegative controls, and were not correlated with the CD4+ cell count. DNA synthesis increased significantly only during acute opportunistic infections occurring in patients with high plasma viral load and fewer than 100 x 10(6) CD4+ cells/l. Triple combination therapy induced a decrease of plasma virus load and a rise of CD4+ cell counts, whereas BrdU incorporation remained low or decreased. CONCLUSION: Proliferation of peripheral blood T cells observed at late stages of HIV infection corresponds to a response to opportunistic infections. Apart from these particular cases, proliferation in this compartment does not appear as a critical parameter of CD4+ cell kinetics during chronic HIV infection and potent therapy.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1 , Ativação Linfocitária/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular , DNA/biossíntese , Quimioterapia Combinada , Citometria de Fluxo , Seguimentos , Infecções por HIV/imunologia , Humanos , Estudos Longitudinais , RNA Viral/sangue , Carga ViralRESUMO
Bromodeoxyuridine (BrdUrd) was incorporated in vivo or in vitro into the DNA of proliferating murine thymocytes. Surface antigens Thy1, Lyt2 (CD8), L3T4 (CD4), interleukin-2 receptor (IL2-R), and the V beta 8 chain of the T-cell receptor were detected using specific monoclonal antibodies with the biotin-avidin system, and cells were then treated for DNA denaturation. Simultaneous detection of BrdUrd and surface markers was performed on cell smears and frozen sections by double-color immunofluorescence. The phenotype of cycling cells, determined in fetal thymus and in the thymus of mice from birth to one year of age, showed relative stability after the initial growth period, despite severe involution of the gland. Phenotypic evolution of cycling cells and their progeny was also studied in colchicine-treated animals and was shown to reproduce sequential events of T-cell differentiation. On sections, the highest frequency of cycling cells was observed in the outer cortex in normal thymus, but the first cells to start proliferation during regeneration were mostly located in the deep cortex and corticomedullary junction. These results show the high potential of this method, as compared to autoradiography of radiolabeled cells.
Assuntos
Antígenos de Superfície/análise , Bromodesoxiuridina/análise , Linfócitos T/análise , Animais , Anticorpos Monoclonais , Ciclo Celular , Demecolcina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Antígenos de Linfócitos T/análise , Receptores Imunológicos/análise , Receptores de Interleucina-2 , Linfócitos T/citologia , Antígenos Thy-1Assuntos
Células-Tronco Hematopoéticas/citologia , Linfócitos T/citologia , Timo/citologia , Animais , Diferenciação Celular , Divisão Celular , Embrião de Galinha , DNA Nucleotidilexotransferase/biossíntese , Células-Tronco Hematopoéticas/enzimologia , Cinética , Camundongos , Modelos Biológicos , Linfócitos T/enzimologia , Timo/enzimologiaAssuntos
DNA Nucleotidilexotransferase/isolamento & purificação , DNA Nucleotidiltransferases/isolamento & purificação , Timo/enzimologia , Animais , Galinhas , Cromatografia de Afinidade , DNA Nucleotidilexotransferase/metabolismo , Imunofluorescência , Soros Imunes , Cinética , Peso Molecular , Especificidade por SubstratoRESUMO
Spontaneously cycling thymocytes have been labeled in vitro and in vivo by bromodeoxyuridine (BUdR), a non-reutilized precursor of DNA that is detectable by a monoclonal antibody. Studies of BUdR-labeled cells have included the determination of their anatomical location, size, and nuclear aspects and of their cell surface phenotype. Dividing blasts were initially located in the cortex (mainly but not exclusively in the subcapsular region) and expressed the double-negative (Lyt-2- L3T4-) and double-positive (Lyt-2+ L3T4+) phenotypes. The fate of these cells have been determined in days after BUdR administration, and we observed an initial double-negative to double-positive transition that was followed by the death of the majority of labeled cells in the cortex. As of day 3, the few surviving cells acquired a mature helper phenotype (Lyt-2- L3T4+) and began migrating into the thymic medulla. The exclusive medullary location of blast cell progeny was observed between days 5 and 10 post-BUdR administration. These results suggest a direct precursor-product relationship between dividing cortical cells and mature medullary thymocytes, and therefore support the single lineage model of intrathymic differentiation.
Assuntos
Linfócitos T/citologia , Timo/citologia , Animais , Antígenos de Diferenciação de Linfócitos T , Antígenos de Superfície/análise , Bromodesoxiuridina , Ciclo Celular , Diferenciação Celular , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
The expression of differentiation antigens and terminal deoxynucleotidyl transferase (TdT) were studied with cultured murine thymocytes using the immunofluorescent technique. Four different culture conditions were used: i) medium alone; ii) addition of Con A; iii) addition of Con A stimulated splenocyte supernatant (CSS); iv) addition of both Con A and CSS. The percentages of viable cells and their absolute numbers were evaluated. TL expression was strongly decreased in all four cases, with the almost total disappearance of TL+ cells in Con A + CSS-stimulated cultures. In contrast, TdT+ cells remained well represented. The distribution of cells expressing or not expressing Lyt antigens varied according to the different culture conditions. The only cell type to exceed its initial number was the TdT+ Lyt- one, in cultures performed in the presence of Con A + CSS. The absolute number of Qa-2+ cells was initially reduced by 50% after 2 h in culture, and was then maintained for 3 days. This number was increased only in the presence of Con A + CSS. Therefore, Qa-2+ cells appear much more resistant than the other thymocyte subpopulations in culture, and even proliferate under stimulating conditions. The proliferating Qa-2+ cells also expressed TdT, as detected by the immunofluorescent technique, but enzymatic detection of the enzyme gave unsignificant results. Proliferating Qa-2+ cells presented either Lyt1 or both Lyt1 and Lyt2 antigens. The expression of H-2K antigen was also strongly increased in stimulated cultures, but Qa-4 and Qa-5 antigens could not be detected. These results suggest that Con A and CSS stimulated both mature cells and precursor cells (as shown by TdT expression). The possible relevance of these findings for the theories of thymocyte ontogeny is discussed.
Assuntos
Antígenos de Superfície/análise , Antígenos de Histocompatibilidade Classe I , Timo/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T , Antígenos Ly/análise , Diferenciação Celular , Células Cultivadas , DNA Nucleotidilexotransferase/metabolismo , Imunofluorescência , Camundongos , Camundongos Endogâmicos , Timo/citologiaRESUMO
T cell antigen receptor expression by cycling and post-cycling thymocytes has been analysed by flow cytometry. Normal mice were pulsed with 5-bromo-2'-deoxyuridine (BrdUrd), a thymidine analogue detectable with a monoclonal antibody. Thymocytes were surface-stained with antibodies against several V beta gene products and against whole alpha beta receptors and detection of BrdUrd in the nuclei was performed after enzymatic generation of single-stranded DNA. A significant (10%) percentage of thymocytes expressing high levels of alpha beta TCR were found in the cycle: these cells were immature, as shown by the CD4+8+ phenotype and by high HSA expression. After division, most alpha beta high BrdUrd+ cells entered a resting state and their number remained constant for 3 days, decreasing in two steps thereafter. This post-mitotic evolution was not modified by injection of an anti-mitotic drug. After day 4, a majority of the studied subset acquired a single positive phenotype. Location of BrdUrd+ V beta 8.2 high cells studied on frozen sections was found cortical at early times and medullary after day 3. V beta 6 expression by cycling and post-cycling thymocytes was analysed in various mouse strains, and early high expression by cycling thymocytes was found to be restricted to MIs 1b strains. These results suggest that high alpha beta TCR expression by cycling immature thymocytes corresponds to positive selection, which must therefore be considered as an early event in intrathymic differentiation.
Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Divisão Celular , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
After sublethal (6 Gy) whole-body irradiation, the C57BL/Ba (Thy-1.1) murine thymus regenerated in two waves, on days 3-10 and 25-32, separated by a severe relapse. The second phase of depletion-reconstitution reproduced the first one, in a less synchronous manner. The depletion affected all cell subsets, but CD4+ CD8- cells decreased later than immature cells. Cell proliferation, measured by BrdUrd incorporation, started on day 3 after irradiation and concerned CD4- CD8-, CD4- CD8+, and CD4+ CD8+ cells, sequentially. CD4+ CD8- cells never represented a significant percentage of cycling cells. When irradiation was immediately followed by an intrathymic injection of 10(5) C57BL/Ka (Thy-1.2) bone marrow cells, the relapse in thymus reconstitution was no longer observed. Detected with anti-Thy-1.2 antibodies, donor cells started cycling on day 14 and showed only one wave of proliferation. In these chimeras, recipient thymocytes behave exactly like thymocytes of solely irradiated mice. Intrathymically transferred CD4- CD8- thymocytes (10(5] showed the same proliferation kinetics as endogenous cells, with a peak in number on day 10 but completely disappeared from the thymus on days 14-21. These data reflect maturational differences between intrathymic and bone marrow precursor cells and suggest different radiosensitivities not linked to proliferative status. The resting state of the thymus immigrants was shown by the absence of Thy-1 acquisition by bone marrow cells continuously labeled for 10 days with BrdUrd in vivo before intrathymic transfer. When such labeled bone marrow cells were injected in the thymus, only the minor BrdUrd- subset gave rise to Thy-1+ cells.
Assuntos
Ativação Linfocitária , Linfócitos T/imunologia , Timo/efeitos da radiação , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Bromodesoxiuridina , Antígenos CD8 , Replicação do DNA/efeitos da radiação , Cinética , Ativação Linfocitária/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mitose/efeitos da radiação , Regeneração/efeitos da radiação , Especificidade da Espécie , Linfócitos T/classificação , Linfócitos T/efeitos da radiação , Timo/citologia , Timo/imunologia , Irradiação Corporal TotalRESUMO
The proliferative status of thymocyte cell subsets in vivo was assessed by observing the effect of two antimitotic drugs, hydroxyurea (HU) and demecolcine. Both drugs had the greatest effect on the double-positive (DP) subset followed by the L3T4+ single-positive (SP) subset. However, the decrease in the latter type was delayed by several days, showing that their precursors rather than the cells themselves were killed by HU. Double-negative (DN) cells were less affected, indicating that they contain a resting subset and that they are renewed by emigration rather than by autonomous in situ proliferation. After drug treatment all cycling cells were eliminated from the thymus but, as shown by in vivo and in vitro bromodeoxyuridine incorporation, new cells rapidly reentered in cycle, starting from DN cells and Lyt-2+ SP cells and followed by DP cells. Lyt-2+ SP cycling cells represent an intermediary stage between DN and DP cells, and they are very transient. Injection of HU 24 h after in vivo BrdUrd labeling eliminated most labeled DN cells, but did not prevent the emergence of L3T4+ SP-labeled cells on day 3 as observed in control thymuses. These results suggest that these L3T4+ SP cells are generated from DP cells in the absence of proliferation. Cycling cells in the regenerating thymus were first located at the corticomedullary junction and then in the subcapsular region, suggesting a reverse migration process to that observed after cessation of proliferation. A model is proposed to summarize these sequential events.
Assuntos
Bromodesoxiuridina/metabolismo , DNA/metabolismo , Mitose/efeitos dos fármacos , Regeneração , Linfócitos T/citologia , Timo/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , DNA/biossíntese , Feminino , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T/classificação , Linfócitos T/metabolismo , Timo/metabolismo , Timo/fisiologiaRESUMO
The relationships between cell proliferation and cell differentiation during thymus ontogeny were studied by labeling DNA-synthesizing thymocytes with bromodeoxyuridine and staining with antibodies against CD4, CD8, J11d, phagocytic glycoprotein 1, TCR V beta 8 chain, Thy-1, and IL-2R surface proteins. The development of the thymus was discontinuous, with two well defined growth periods from 13 days to 18 days of fetal life and from 3 days to 6 days after birth, and more progressive growth from day 8 to 2 wk. Cell proliferation started on fetal day 12, 1 day after the arrival of hemopoietic stem cells in the third branchial pouch. These cells were phagocytic glycoprotein 1-positive but IL-2R and Thy-1 negative. Thus, cell proliferation preceded IL-2R expression. Until day 15, CD4-8- thymocytes expanded without differentiation. Then CD4-8+ and CD4+8+ cells appeared; this induction was proliferation dependent and occurred on cells which had already lost IL-2R, but just after maximum expression of this receptor. During several days, the thymus remained of constant size (around 10(7) cells) and behaved like the steady state thymus. On day 3 after birth, expansion started again and was correlated with an increase in CD4-8- proliferation index and IL-2R expression. At the same time, the thymic subset capable of expansion without differentiation was again, transiently, detectable. These results suggest that the inflow of precursor cells into the thymus is permanent but transiently increased at several times during ontogeny. Moreover, the behavior of fetal CD4-8- cells does not appear radically different from that of adult precursors, but the actual difference resides in the variation of the relative proportion of CD4-8- cells at different maturation stages, as revealed by striking variations of IL-2R expression by cycling cells.
Assuntos
Animais Recém-Nascidos/imunologia , Desenvolvimento Embrionário e Fetal , Ativação Linfocitária , Timo/fisiologia , Animais , Animais Recém-Nascidos/fisiologia , Antígenos de Diferenciação de Linfócitos T , Ciclo Celular , Diferenciação Celular , Sobrevivência Celular , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T/classificação , Linfócitos T/fisiologia , Timo/crescimento & desenvolvimento , Timo/imunologiaRESUMO
Deoxyguanosine (dGuo) inhibits thymic blast DNA synthesis and then induces thymocyte cell death. The dGuo inhibitory action, measured with four different assays (spontaneous thymidine incorporation, immunofluorescent detection of 5-bromodeoxyuridine incorporation, dye exclusion, tetrazolium salt cleavage), was suppressed in the presence of supernatants from cultures containing phagocytic cells. In particular, we studied the anti-dGuo activity in supernatants from thymic reticulum cultures (TRS) and in those from phagocytic cells isolated from TR cultures (P-TR). The anti-dGuo substance was identified as deoxycytidine (dCyd) by high performance liquid chromatography and other physicochemical studies. Secretion of dCyd by P-TR is accompanied by thymidine but not by purine nucleoside secretion. A dual mechanism of thymocyte rescue by dCyd was demonstrated by a study of the dose-dependencies of dCyd-mediated prevention and reversal, respectively, of the dGuo inhibition. In addition to this exogenously added anti-dGuo action, dCyd and dCyd-containing TRS induced significant stimulation of spontaneous thymic blast proliferation, and the kinetics of both effects were identical. These findings might suggest that a major role of thymic phagocytic cells is the supply of pyrimidine nucleosides to thymocytes resulting in the maintenance of proliferation and protection of at least some thymic blasts from the toxic effects of dGTP accumulation. The role of this system in the intrathymic selection process is discussed.
Assuntos
Desoxicitidina/farmacologia , Desoxiguanosina/antagonistas & inibidores , Timo/citologia , Animais , Células Apresentadoras de Antígenos/fisiologia , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , Replicação do DNA/efeitos dos fármacos , Desoxicitidina/fisiologia , Desoxiguanosina/farmacologia , Relação Dose-Resposta Imunológica , Imunofluorescência , Linfocinas/biossíntese , Linfocinas/farmacologia , Camundongos , Timidina/metabolismo , Timo/efeitos dos fármacosRESUMO
We have recently shown that thymocyte activation is associated with a strong increase of Qa-2 antigen expression. Contrary to what could be predicted from these results, the initial depletion of Qa-2+ cells from PNA- thymocytes increases the Con-A-induced proliferation. Qa-2+ cells not only reappeared after activation, but were enhanced in Qa-2-depleted PNA- cells compared to the control. The newly formed Qa-2+ population contained a higher percentage of Lyt 1+2- cells and thus seems to be different from the Qa-2+ pool initially present.
Assuntos
Antígenos de Superfície/imunologia , Concanavalina A/farmacologia , Antígenos de Histocompatibilidade Classe I , Linfócitos T/imunologia , Timo/efeitos dos fármacos , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos Ly/análise , Antígenos Ly/imunologia , Antígenos de Superfície/isolamento & purificação , Células Cultivadas , Concanavalina A/imunologia , Feminino , Imunofluorescência , Lectinas/farmacologia , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos , Aglutinina de Amendoim , Fenótipo , Timo/citologia , Timo/imunologiaRESUMO
The in vivo cell cycle of normal murine thymocytes was studied by bivariate analysis of bromodeoxyuridine and total DNA content in the 24 h following a single injection of the thymidine analogue. Bromodeoxyuridine incorporation was strictly limited to cells in S phase and 98% of S phase cells were labeled, demonstrating high efficiency and specificity. Cell-cycle parameters were determined by measuring the DNA content of bromodeoxyuridine-labeled cells, related to their distribution in the different phases. The changes of this distribution as a function of time reflected the progression of the cells along the cell cycle. The duration of total cycle, S phase, and both G2/M and G1 was 10 h, 6.5 h and 1.5 h, respectively. All thymocytes labeled in S phase entered G2/M, divided and returned to the G0/G1. Seventy percent of them remained in the resting state, and the other 30% re-entered a second S phase. Cell-cycle parameters of isolated CD4-CD8- cells were also determined. No evidence of cell loss during S or G2/M phase was found, suggesting that intrathymic cell death is not directly linked to the proliferative phases of differentiation.
Assuntos
Linfócitos T/citologia , Timo/citologia , Animais , Anticorpos Monoclonais , Bromodesoxiuridina/metabolismo , Ciclo Celular/imunologia , Diferenciação Celular/imunologia , Divisão Celular , Células Cultivadas , DNA/análise , Citometria de Fluxo/métodos , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Fenótipo , Linfócitos T/metabolismoRESUMO
We present a comparison of two different methods for simultaneous detection of bromodeoxyuridine and cell surface markers. Both methods use enzymatic generation of single-strand DNA with nuclease. The biological system used is the murine thymus, in which in vivo DNA synthetizing cells were labeled by injection of BrdUrd and analyzed at different time points after the nucleoside pulse. The surface proteins detected were CD4 and CD8 differentiation markers and the T-cell receptor. Extraction of DNA-associated proteins with 0.1N HCl and detergent is necessary for the action of EcoR1 and Exonuclease III, but this treatment destroys phycocyanins and induces cell aggregation, as shown using the doublet-discrimination module. For DNAse I action, cells could be treated with paraformaldehyde and a low concentration of Tween 20, and this treatment was adequate for surface staining preservation (even with phycocyanins) and BrdUrd detection. Both methods were adequate for cell cycle studies, but only 7-amino-actinomycin D could be used as total DNA dye after DNAse action, and good results needed long (48-72 h) incubation in the fixative-detergent mixture. The DNAse I method now allows three-color staining (two surface markers and Brd-Urd), analyzed in a one laser-cytometer for the study of the phenotype of cycling cells, and of their progeny, in vivo and in cell cultures. It also allows the quantitative analysis of cell surface receptor densities in conditions similar to fresh cells.
Assuntos
Bromodesoxiuridina/análise , DNA/análise , Proteínas de Membrana/análise , Subpopulações de Linfócitos T/química , Animais , Antígenos CD4/análise , Antígenos CD8/análise , Diferenciação Celular , Replicação do DNA , Dactinomicina/análogos & derivados , Desoxirribonuclease I , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Antígenos de Linfócitos T/análiseRESUMO
Antibodies specific for chicken terminal deoxynucleotidyl transferase (TdT) were used to develop immunoperoxidase and immunofluorescence assays. The cellular distribution and localisation of TdT during the development of chicken thymus were studied. TdT began to appear in the embryonic thymus in the cytoplasm of large cells, between 11 and 12 days of incubation. Thereafter, the proportion of TdT-positive cells increased and TdT was detected in both nucleus and cytoplasm. The first appearance of TdT positive cells, their increasing proportion and the intracellular localisation of TdT will be discussed in correlation with the developmental stages of the thymus.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidiltransferases/metabolismo , Timo/enzimologia , Animais , Núcleo Celular/enzimologia , Embrião de Galinha , Citoplasma/enzimologia , Imunofluorescência , Técnicas Imunoenzimáticas , Timo/embriologia , Fatores de TempoRESUMO
A small population of DNA-synthesizing mature thymocytes could be defined by analyzing cell surface markers and 5-bromo-2'-deoxyuridine (BrdUrd) labeling by four-color cytofluorometry. These cells have a completely mature phenotype (CD4- CD8+ or CD4+ CD8- TCR(high), HSA-, Qa-2(high)) and expand only weakly after BrdUrd incorporation. They recovered immediately in total number and in DNA synthesis rate after treatment with the antimitotic drug demecolcin, thus much faster than immature CD4+ CD8+ thymocytes. These data demonstrate the existence of a late intrathymic expansion phase, independent of that of developing CD4+ CD8+ immature cells, and involving phenotypically mature cells renewed each day. In mixed chimeras prepared by transfer of bone marrow and lymph node cells into RAG-2(-/-) mice, all cycling mature thymocytes were bone marrow derived. They are thus produced in situ and do not correspond to peripheral T cells reentering the thymus. Double FITC/BrdUrd detection showed that a high proportion (10-20%) of recent thymic emigrants were BrdUrd+ just postcycling cells and that around 50% of cycling mature thymocytes are just ready to emigrate to the periphery in the few hours after DNA synthesis. The late intrathymic expansion phase demonstrated here increases the daily thymic cell export by at least 30%. It could play a role in the adjustment of the T cell repertoire before emigration and in the regulation of the thymic cell output into the peripheral T cell pool.