RESUMO
To investigate the influence of the liver on differentiation of hematopoietic stem cells/pro-T cells, TN-NWP-BMC (athymic nude bone marrow cells that were treated with anti-TCRbeta, anti-CD4, and anti-CD8 Abs plus complement and then passed through a nylon wool column) were cultured on parenchymal liver cells. After culture for 2.5 days, CD3-4-8-TCRbeta+ cells and CD3-CD4+/CD8+TCRbeta- cells were developed from TN-NWP-BMC. TCRVbeta8+ cells comprised 19.9% of CD3-4-8-TCRbeta+ cells, and Vbeta8 mRNA was detected in the CD3-4-8-TCRbeta+ cells by reverse transcriptase-polymerase chain reaction. The CD3-CD4+/CD8+TCRbeta- cells contained not only single-positive cells but also CD4+8+ double-positive cells. The CD8 protein consisted of 88.9% CD8alpha+beta-, 10.1% CD8alpha+beta+, and 1% CD8alpha-beta+ molecules. From these results and the finding of co-expressed antigens, CD3-4-8-TCRbeta+ cells and CD3-CD4+/CD8+TCRbeta- cells appear to be immature cells not committed to a certain cell lineage.
Assuntos
Fígado/citologia , Camundongos Nus/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/citologia , Animais , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciação Celular , Células Cultivadas , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Antígenos Thy-1/metabolismo , Fatores de TempoRESUMO
It has been reported that the intrahepatic lymphocyte (IHL) population is somewhat differently constituted from lymphocytes in other lymphoid tissues. Staphylococcal enterotoxin B (SEB) is a superantigen which can induce T-cell tolerance in mice. The authors investigated the in vitro and in vivo responses of mouse IHL to SEB. An intravenous injection of SEB did not result in the augmentation of the proliferative response of IHL, while mesenteric and splenic lymphocytes (mLNC and SPC, respectively) had augmented responses. Interleukin-2 (IL-2) mRNA was clearly detected in mLNC and SPC by reverse transcriptase-polymerase chain reaction (RT-PCR) shortly after the administration of SEB, but it was scarcely expressed in IHL. The expression of CD25 (IL-2 receptor) was also augmented in mLNC and SPC in the early period, while it was not changed in IHL. These findings suggested that the time required for tolerance induction is different locally and that the loss of augmentation of IL-2 and IL-2 receptor production by IHL may be relevant to the rapid induction of T-cell tolerance in the liver.
Assuntos
Enterotoxinas/farmacologia , Tolerância Imunológica , Fígado/imunologia , Staphylococcus aureus , Linfócitos T/imunologia , Animais , Primers do DNA/química , Feminino , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária , Mesentério , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Interleucina-2/metabolismo , Baço/imunologia , Superantígenos/farmacologiaRESUMO
Amounts of co-precipitating CD3 components by anti-T-cell receptor (TCR)V beta or anti-CD4/8 monoclonal antibodies were compared between non-stimulated and stimulated splenic T cells. The amounts of co-precipitating CD3 delta, epsilon and gamma chains with TCR alpha beta and with CD4/8 were not significantly changed after TCR ligation. The apparent amount of CD3 zeta chain co-precipitated with TCR alpha beta increased up to threefold, while the actual amount of co-precipitating CD3 zeta with TCR alpha beta and the total amount of specifically precipitated CD3 zeta are not changed after cross linking of TCR. The apparent amount of CD3 zeta chain co-precipitated with CD4/8 also increased. Unlike co-precipitation with TCR alpha beta, the actual amount of CD3 zeta co-precipitated with CD4/8 increased significantly. This observation suggests a conformational change as well as the relocation of CD3 zeta molecules within the TCR complex after the signal delivery. After TCR ligation, CD3 zeta chains relocate to the vicinity of either CD4 or CD8 molecules. In addition, when cross linking and binding signals are compared, CD3 chains undergo two distinct phases of conformational change. The responses, while the later conformational change caused by the cross linking of TCR does not induce but enhances the proliferative response.
Assuntos
Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Complexo CD3/química , Complexo CD3/isolamento & purificação , Complexo CD3/metabolismo , Antígenos CD4/isolamento & purificação , Antígenos CD4/metabolismo , Antígenos CD8/isolamento & purificação , Antígenos CD8/metabolismo , Reagentes de Ligações Cruzadas , Técnicas In Vitro , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Testes de Precipitina , Conformação Proteica , Receptores de Antígenos de Linfócitos T/isolamento & purificaçãoRESUMO
The feeding of cloned helper T cells by irradiated spleen cells results in the modulation of T-cell antigen receptors (TCRs) as detected by FACS. When two groups of anti-TCR antibodies--anti-idiotypic and anti-V beta--are compared, time-dependent patterns of modulation of TCR are drastically different. Whereas up to 80% of the idiotypic determinants disappear within 2 days after stimulation, almost no change is observed for the determinants recognized by V beta-specific antibodies within the same period. Sequential immunoprecipitations clearly showed that anti-idiotypic and anti-V beta antibodies recognize the same population of TCR. Moreover, surface labelling followed by immunoprecipitation of TCR did not show rapid and drastic changes in the amount of specifically precipitated material. The modulation is not significantly affected by the addition of specific antigen, but is specific for the clone's MHC restriction element. Functionally, the modulation of TCR epitopes affects the ability of anti-TCR antibody but not of the MHC/antigen complex to stimulate T cells. We conclude that the observed modulation of fluorescence intensity in the TCR complex after MHC recognition (1) is epitope specific, (2) is independent of the presence of nominal antigen and (3) does not affect the efficiency of antigen-specific stimulation.
Assuntos
Antígenos CD/imunologia , Antígenos CD4/imunologia , Epitopos/química , Receptores de Antígenos de Linfócitos T/química , Animais , Anticorpos Monoclonais , Antígenos CD11 , Linhagem Celular , Células Clonais/imunologia , Epitopos/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.
Assuntos
Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Linfócitos T/imunologia , Animais , Eletroforese em Gel de Poliacrilamida , Ativação Linfocitária , Camundongos , Testes de PrecipitinaRESUMO
Variant lines expressing high and low surface densities of the accessory molecule CD4 have been developed by repeated preparative flow cytometric cell sortings from the murine Th cell clone D10.G4.1 (D10). The high CD4 variant line (D10H) fully maintained the original I-Ak restricted specificity for conalbumin of wild-type D10 cells. In contrast, the low CD4 variant line (D10L) showed a strong autoreactivity to I-Ak carrying stimulator cells alone which was only slightly augmented by addition of conalbumin. Cell surface molecules other than CD4, including TCR, CD3, CD11a, CD2, CD45, CD44, and MHC class I, remained identical on D10H and D10L sublines as on D10 wild-type cells. The possibility that D10L cells had suffered alterations of their TCR-alpha beta was excluded by demonstrating their reactivity with a panel of eight different anti-clonotypic mAb specific for various epitopes of the D10 TCR. By limiting dilution analysis we show that the majority of responding cells of D10L sublines were autoreactive. Although the reactivity for allogeneic I-A also increased as compared with D10H cells, a clear preference for self-I-Ak was maintained so that a true autoreactive phenotype was evident. The results indicate that the surface concentration of CD4 has a decisive influence on self-non-self discrimination of MHC class II-restricted Th cells.
Assuntos
Autoimunidade , Antígenos CD4/fisiologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Separação Celular , Células Clonais , Genes MHC da Classe II , Haplótipos , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Camundongos , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Repertoire sizes of T cells and B cells were estimated by frequency studies using mouse helper T cell clone D10G4.1 (D10 for short) and monoclonal antibodies raised against the antigen recognition structures on D10 cells. The soluble form of these antibodies was capable of activating D10 cells to proliferate in the presence of exogenous IL-1. Moreover, among the heterogeneous peripheral T cells, there is a population of cells which respond to those antibodies. Normal spleen B cells were non-clonally activated by LPS in limiting dilution cultures and the supernatant was tested for its activation capacity on D10 cells. Precursor frequencies of B cells which produce D10 activating antibodies were in the range of one in millions. The presence of D10 cells in the limiting dilution cultures increased this frequency by a factor of more than ten. For the estimation of frequencies of a given T cell clone which is activated by anti-D10 antibodies, normal spleen T cells were activated either by beads coupled monoclonal antibodies or by Con A followed by specific soluble antibodies in the limiting dilution cultures, and thereafter specific proliferation was assayed. A group of monoclonal anti-receptor antibodies gave rise to a single hit curve with relatively low frequency of one in 10(4) to 10(5). Another group of antibodies revealed in addition to the low frequency population, a higher frequency population of one in thousands. The data taken together suggest the frequencies of mutually interactive B and T cells are in the range of one in 10(4) to 10(5), while among T cells, there can be seen another population of cells of ten to a hundred fold higher frequency. Consequently, direct idiotype-antiidiotypic interactions between lymphocytes should occur only once in 10(8) to 10(10) cells in the normal unstimulated immune system. Thus, it is more likely that interactions will take place between cells of more degenerated specificities. The implications of these findings in favour of multiple levels of connection among T cells which reflect the multiple levels of affinity of the recognition complex of T cells are discussed.
Assuntos
Linfócitos B/imunologia , Linfócitos T/imunologia , Animais , Formação de Anticorpos , Soro Antilinfocitário/imunologia , Células Clonais , Contagem de Leucócitos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Eicosanoids play a prominent role in trauma. Such mediators of inflammation negatively influence cell-mediated immunity (CMI). There is, however, no information available on the effect of eicosanoids on a critical event in CMI, i.e., antigen-presenting (AP) cell function of macrophages (M luminal diameter), a cellular process responsible for the activation of T and B lymphocytes. The aim of this study, therefore, was to examine the effect of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) on AP cell function of the peritoneal M luminal diameter. To study this, a T-helper-cell clone, D10.G4.1 was employed. This cell clone proliferates in the presence of Iak (Class II glycoprotein, MAC product) bearing M luminal diameter and specific antigen (conalbumin A) thus directly reflecting the AP capability of the M luminal diameter. Peritoneal M luminal diameter were harvested from B10.BR mice (H2k) and their AP was tested in vitro by incubating varying numbers of M luminal diameter with 2 X 10(4) D10.G4.1 cells/well and conalbumin (400 micrograms/ml) in the presence and absence of different concentrations of PGE2 or TXB2. Cultures were incubated for 72 hr, pulsed with [3H]-thymidine, and harvested. At concentrations of 10, 30, and 100 nM of PGE2, D10.G4.1 proliferations were 38, 35, and 20% of control, respectively (P less than 0.05 compared to control). TXB2 added at the above-mentioned concentrations did not suppress the proliferative response of D10. Thus, PGE2 but not TXB2 has a potent immunosuppressive effect on AP of peritoneal M luminal diameter.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Células Apresentadoras de Antígenos/fisiologia , Macrófagos/imunologia , Peritônio/citologia , Prostaglandinas E/farmacologia , Animais , Dinoprostona , Imunidade Celular/efeitos dos fármacos , Tromboxano B2/farmacologiaRESUMO
Although major tissue trauma produces profound depression of cell-mediated immunity, it is not known whether surgical trauma (i.e., midline laparotomy) has any adverse effect on the antigen presentation function and membrane interleukin-1 (IL-1) activity of peritoneal macrophages. To study this, C3H/HEJ (endotoxin-tolerant) mice were anesthetized. An approximately 1-inch midline abdominal incision was made, followed by abdominal closure. On days 1, 3, 5, and 7, peritoneal macrophages were harvested by means of peritoneal lavage, and antigen presentation capability was tested by incubating various numbers of peritoneal macrophages with 2 X 10(4) D10.G4.1 cells per well in the presence of conalbumin (400 micrograms/ml). The T helper cell clone (D.10.G4.1) proliferates on recognition of conalbumin in the context of Iak and also proliferates in the presence of membrane-bound IL-1 plus concanavalin A. To measure membrane IL-1 expression in peritoneal macrophages, Concanavalin A (10 micrograms/ml) was substituted for conalbumin. Cultures were incubated for 72 hours, pulsed with tritiated thymidine, and harvested. Peritoneal macrophages from laparotomized mice induced significantly less T helper cell proliferation on days 1 and 3 in the antigen presentation assay (37% and 30%, respectively; p less than 0.05) and in the membrane IL-1 assay (14% and 10%, respectively; p less than 0.05) as compared with the control. This difference was not detectable on day 5. More effective antigen presentation capability (167% of control; p less than 0.05) was seen on day 7. Thus laparotomy by itself produces marked depression of both antigen presentation function and membrane IL-1 activity of peritoneal macrophages, which may enhance susceptibility to intra-abdominal sepsis.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Interleucina-1/imunologia , Laparotomia , Macrófagos/imunologia , Animais , Conalbumina/farmacologia , Concanavalina A/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Peritônio/citologia , Linfócitos T Auxiliares-IndutoresRESUMO
We describe the quantitative measurement of antigen-specific clusters formed by antigen-pulsed macrophages and immunized T cells in mice. We have found the peripheral blood T cells show very little non-specific adhesion to macrophages in mice. By using this population of lymphocytes in the peripheral blood as the source of immunized T cells, we could quantitate antigen-specific cluster formation. On OVA-pulsed monolayers of peritoneal exudate macrophages from normal BALB/c mice, syngeneic peripheral blood T cells from donors immunized with the same antigen develop 20-40 clusters per 1,000 macrophages, whereas the same T cells on non-pulsed monolayers develop only 0-5 cluster-like accumulations of cells. On antigen-pulsed monolayers of macrophages from allogeneic (C57BL/6 or A/J) mice, clusters are developed only in the negative range (0-5/1,000 macrophages). Considering the observation by Braendstrup et al, these data seem to suggest that histocompatibility between macrophages and T cells is required to develop antigen-specific T cell clusters on antigen-pulsed macrophage monolayers, and that the genetic restriction of immune responsiveness may be directly expressed in this initial form of cellular interaction between antigen-bearing macrophages and specific T cells.
Assuntos
Antígenos/imunologia , Macrófagos/imunologia , Linfócitos T/imunologia , Animais , Adesão Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
The intravenous administration of syngeneic spleen cells (SPCs) briefly pulsed with antigen in vitro, results in a profound state of IgE antibody unresponsiveness. In Balb/c mice, the primary response of anti-DNP, anti-beef insulin and anti-ovalbumin IgE antibody is completely suppressed by the administration of antigen-pulsed spleen cells, 1 X 10(7), 5 X 10(7) and 1 X 10(8), respectively. This suppression is antigen specific and effects both primary and secondary immune responses. Furthermore, the immune response to dinitrophenylated Keyhole limpet hemocyanin (DNP-KLH) is most extensively suppressed by DNP-KLH pulsed SPCs, intermediately suppressed by KLH-pulsed SPCs and minimally suppressed by dinitrophenylated mouse gamma globulin or dinitrophenylated mouse serum albumin pulsed SPCs. Suppressing directly cells specific for hapten and carrier, hapten carrier protein pulsed SPCs would caused the additive suppressive effect. The suppression is induced strongly by the intravenous administration of antigen pulsed spleen cells, slightly by the subcutaneous administration and is not induced by the intravenous administration of antigen solution in phosphate buffer saline. This suppression may be mediated by either of two different mechanisms: one of them is responsible for the immediate tolerance which is induced without any suppressor cells 1 day after the administration of antigen pulsed SPCs, and the other is responsible for the suppression transferred by suppressor cells or factors to normal mice 7 days after the administration of antigen pulsed SPCs. This method in which IgE antibody response is suppressed by the administration of cells briefly pulsed in vitro with antigen, provides a powerful tool to analyze the first step of antigen specific suppression developed in vivo by conventional antigens.