RESUMEN
CD69 is a lymphoid activation antigen whose rapid expression (< or = 2 h postactivation) makes it amenable for the early detection of T-cell activation and for subset activation analyses. In the present study we evaluated the utility of flow cytometric detection of CD69 expression by T cells activated with polyclonal stimuli (anti-CD3 and staphylococcal enterotoxin B [SEB]) and oligoclonal stimuli (tetanus toxoid and allogeneic cells) using flow cytometry. Following activation of T cells with anti-CD3 or SEB, CD69 is detectable at < or = 4 h following activation, with anti-CD3 peaks at 18 to 48 h. Dose titration experiments indicated that CD69 expression largely paralleled that in [3H]thymidine incorporation assays, although the former offered a more sensitive measure of T-cell activation at limiting doses of activator than [3H]thymidine incorporation when cells were activated with either anti-CD3 or SEB. However, activation of T cells with either tetanus toxoid or allogeneic stimulator cells failed to induce detectable CD69 expression at up to 7 days of culture. Subset analyses of anti-CD3- and SEB-activated T cells indicated that populations other than T cells can express CD69 following stimulation with T-cell-specific stimuli, indicating that CD69 can be induced indirectly in non-T cells present in the population. These findings indicate that CD69 is a useful marker for quantifying T-cell and T-cell subset activation in mixed populations but that its utility might be restricted to potent stimuli that are characterized by their ability to activate large numbers of cells with rapid kinetics.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Citometría de Flujo/métodos , Activación de Linfocitos , Adulto , Anticuerpos/farmacología , Complejo CD3/inmunología , Femenino , Humanos , Lectinas Tipo C , Activación de Linfocitos/inmunología , MasculinoRESUMEN
CD30 has been extensively studied as a cell surface marker expressed by Reed-Sternberg cells of Hodgkin's disease and other hematologic malignancies, although little is known about its expression by normal lymphoid cells. We therefore characterized the requirements for the induction of CD30 expression and identified the subsets of T cells that express CD30. CD30 is inducible on approximately 15% of normal PBMC stimulated with any of a variety of nonspecific T cell activators, including PHA, Con A, anti-T11(2) + T11(3), and anti-CD3; ionomycin alone induced lower percentages of CD30+ T cells (3 +/- 2%) compared to other stimuli. Maximal numbers of CD30+ cells were observed at 48 to 72 h of activation and the addition of rIL-2 did not affect these kinetics. However, CD30 expression was enhanced by the addition of exogenous rIL-2 to any of the stimuli tested, although rIL-2 alone did not lead to CD30 expression. The induction of CD30 during anti-CD3 mitogenesis was completely inhibitable by anti-IL-2 antibodies and partially inhibitable by rIL-4, indicating a requirement for both TCR triggering and IL-2 for its expression. Dual immunofluorescence analysis revealed that CD30+ cells were confined to CD3+ T cells that coexpressed higher levels of the p55 IL-2 receptor (CD25) than the CD30- population. Furthermore, CD30 expression was restricted to a subset of cells derived from CD45RO+ T cell precursors. Cell cycle analysis showed that CD30+ expression was not cell cycle dependent. Cross-linking of membrane CD30 induced Ca2+ in TCR+, but not TCR- Jurkat T cells. These results demonstrate that CD30 can serve as a T cell signal-transducing molecule and expressed by a unique subset of activated CD45RO+ T cells.
Asunto(s)
Antígenos CD/análisis , Antígenos de Neoplasias/análisis , Antígenos Comunes de Leucocito/análisis , Activación de Linfocitos , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Antígenos CD/biosíntesis , Antígenos de Neoplasias/biosíntesis , Complejo CD3/fisiología , Linfocitos T CD4-Positivos/inmunología , Calcio/metabolismo , Ciclo Celular , Humanos , Interleucina-2/farmacología , Antígeno Ki-1 , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Carcinoma de Células Renales/sangre , Células Madre Hematopoyéticas/fisiología , Interleucina-2/uso terapéutico , Neoplasias Renales/sangre , Células Asesinas Activadas por Linfocinas/fisiología , Melanoma/sangre , Antígeno CD56 , Carcinoma de Células Renales/tratamiento farmacológico , Separación Celular , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Neoplasias Renales/tratamiento farmacológico , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/fisiología , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/fisiología , Melanoma/tratamiento farmacológico , Fenotipo , Receptores de IgG/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
By traditional definitions, NK cells can be activated by cytokines to exhibit two functionally distinct levels of cytotoxicity. Whereas IL-2-mediated activation of NK cells leads to the development of lymphokine-activated killer (LAK) cytotoxicity, characterized by the acquisition of cytolytic activity against NK-resistant targets, IFN-treated NK cells become activated without the acquisition of novel cytolytic specificities. In this study we show that NK cells activated by 18 to 24 h of stimulation with either IFN-alpha or IFN-gamma do acquire LAK cytolytic activity, demonstrated by the ability of IFN-treated PBMC to lyse NK-resistant COLO 205 cells as well as fresh tumor targets. The level of IFN-alpha-induced LAK activity was significantly greater than that induced by IFN-gamma, although IL-2-induced LAK activity was considerably greater than IFN-alpha-induced LAK cytotoxicity. Maximal IFN-induced LAK cytotoxicity occurred after 24 h of culture, and occurred with the use of IFN-alpha at 500 U/ml and IFN-gamma at 1000 U/ml. Whereas neutralizing antibody experiments demonstrated that IFN-alpha-induced LAK activation did not involve the participation of endogenously produced IL-2, the partial inhibition (63%) of IFN-gamma-induced LAK cytotoxicity by anti-IL-2 and of IL-2-induced LAK by anti-IFN-gamma (33.3%) indicates that the induction of LAK cytotoxicity by either of these individual cytokines involves the endogenous production and participation of the other cytokine. Similar to IL-2-induced LAK cells, phenotypic analysis revealed that IFN-alpha/gamma LAK cells were Leu-19+, although the Leu 19"dim"+ subset exhibited greater IFN-induced LAK activity than the Leu-19"bright"+ subset. The results of this study clearly demonstrate that IFN-alpha and IFN-gamma induce classic LAK activity and IFN-gamma plays a participatory role in the optimal induction of LAK cells by IL-2.
Asunto(s)
Citotoxicidad Inmunológica , Interferón Tipo I/farmacología , Interferón gamma/farmacología , Células Asesinas Activadas por Linfocinas/inmunología , Activación de Linfocitos , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Humanos , Interleucina-2/fisiología , Cinética , Activación de Linfocitos/efectos de los fármacos , Fenotipo , Células Tumorales CultivadasRESUMEN
Cord blood caffeine concentrations were measured by high-pressure liquid chromatography in 79 preterm infants. Eleven infants (14%) had detectable caffeine concentrations ranging from 1.1 to 3.7 micrograms/mL (means +/- SD = 2.5 +/- 0.8), and 68 infants had no measurable caffeine. Seven infants with detectable caffeine (group 1) had impedance pneumograms recorded before 2 weeks of age. Each infant in group 1 was matched with two infants without detectable caffeine by birthweight, gestational age, and chronologic age at pneumogram recording to yield a control group (group 2) of 14 infants. Comparison of the groups using quantitative measures of apnea, bradycardia, and periodic breathing obtained from pneumogram analysis and the incidence of monitor alarms on bedside nursing records showed no significant differences. Thus, caffeine was present infrequently and at low concentrations at birth in 79 preterm infants. The amount of apnea, bradycardia, and periodic breathing experienced before 2 weeks of age in 7 preterm infants with detectable cord blood caffeine was not different from that in 14 similar infants without caffeine. Future studies are planned to examine the relationship between postnatal changes in transplacentally acquired methylxanthine concentrations and quantitative measures of apnea, bradycardia, and periodic breathing in a larger number of preterm infants without cardiorespiratory disease.