RESUMO
The X-linked CBA/N defect in B cell function precludes an antibody response to phosphorylcholine (PC). Accordingly, (CBA/N X BALB/c)F1 male mice are unresponsive to PC and lack circulating immunoglobulin bearing the T15 idiotype characteristic of BALB/C anti-PC antibody. In contrast, (CBA/N X BALB/c)F1 female mice respond to PC and greater than 80% of the anti-PC antibody is T15+. No T-cell abnormalities are known to be associated with the CBA/N mutation. These experiments compared the ability of helper T cells from either (CBA/N X BALB/c)F1 male (T15-) or F1 female (T15+) mice to help F1 female B cells respond to PC and to influence the level of T15 expression. The results indicate that although F1 male T cells collaborated with F1 female B cells just as efficiently as F1 female T cells for the total anti-PC response, the percentage of T15 expression induced by F1 male T cells fell dramatically. The (CBA/N X BALB/c)F1 male thus appear to lack a helper T-cell subset required for dominant idiotype production. This helper T cell defect could be repaired by adding F1 female T cells primed to a second carrier to F1 male T cells and restimulating the cell mixture with PC coupled to the antigen used to prime the F1 male cells plus free second carrier. This result implies that conventional helper T cells derived from the F1 male donor can collaborate with a distinct helper T-cell subset from the F1 female donor which recognizes both carrier and idiotype to induce an anti-PC antibody response dominated by the T15 clonotype.
Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Idiótipos de Imunoglobulinas , Cooperação Linfocítica , Linfócitos T/imunologia , Animais , Especificidade de Anticorpos , Proteínas de Transporte/imunologia , Epitopos , Feminino , Ligação Genética , Masculino , Camundongos , Camundongos Endogâmicos , Fosforilcolina/imunologia , Coelhos , Cromossomo XRESUMO
Responder and nonresponder mice primed with poly-(L-glutamic acid,L-lysine,L-phenylalanine) (GLPhe), the response to which is under the control of immune response (Ir) genes, were used as a source of both types of helper T cells required for a T15 idiotype dominated T-dependent anti-phosphorylcholine (PC) response. It was found that the activity of one of the helper T cells needed for an anti-PC response was under major histocompatibility complex (MHC)-linked Ir gene control, and only GLPhe-primed responder mice could be used as a source of these cells. These T cells (ThMHC) whose presence is required for in vivo T-B collaboration are found in normal and anti-mu-treated mice, and their activity depends on the hapten being physically linked to the carrier molecule. By contrast, the activity of the second helper T cell (ThId) required for a T15-dominated anti-PC response was present in both GLPhe-primed responder and nonresponder mice. The ThId cell set that is missing or deficient in anti-mu treated mice can be restored by the addition of T cells from normal, carrier-primed donors and restimulating with the priming carrier. When T cells from GLPhe-primed donors are used as a source of ThId cells, both responder and nonresponder donors provide helper cells capable of inducing syngeneic B cells to produce a T15 dominated anti-Pc response. These results are interpreted to suggest that idiotype recognizing helper T cells (ThId) recognize antigen independent of known Ir gene products.
Assuntos
Genes MHC da Classe II , Idiótipos de Imunoglobulinas/genética , Cooperação Linfocítica , Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Proteínas de Transporte/imunologia , Epitopos , Haptenos , Memória Imunológica , Camundongos , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Two synergizing antigen-specific helper T (Th) cell populations are required for an optimal TEPC15 (T15)-dominated antiphosphorylcholine (PC) plaque- forming cell response . In these studies, the two Th cell sets are shown to differ in their requirements for recognition of self-major histocompatibility complex (MHC)-encoded determinants by testing the ability of Th cells from F(1) {arrow} parent bone marrow chimeras to collaborate with PC-specific B cells bearing MHC-encoded determinants of either parental haplotypes. Previous studies have shown that one antigen-specific Th cell population is required for T-dependent anti-PC responses and activates PC-specific B cells only if the hapten, PC, is physically linked to the priming antigen. This Th cell, referred to as ThMHC, induces anti-PC responses that are mainly non-T15 in character, and it appears to be identical to the conventional antigen- specific Th cell. In these experiments, using T cells from (A X B)F(1) {arrow} parent A chimeras, ThMHC cells requiring hapten-carrier association provide help for F(1) and parent A B cells but not for B cells from parent B, thus confirming that the activity of the conventional Th cell is H-2 restricted . The second antigen-specific Th cell population, whose function is measured in the presence of the ThMHC cell set, preferentially activates T15-bearing B cells. This Th cell set (ThId) is missing in mice expressing low levels of T15-bearing antibody and can be restored by the addition of antigen-specific T cells from donors expressing high levels of circulating T15 Id. These studies demonstrate that T cells from F(1) {arrow} parent chimeras that express substantial levels of T15-bearing anti-PC antibody could provide ThId cell activity for the selective activation of T15-bearing B cells of F(1) and both parental H-2 types. These results imply that whereas the activity of conventional, ThMHC, cells is clearly H-2 restricted, ThId cells from the same chimeric donors are not required to recognize antigen in association with self-MHC-encoded determinants for successful T-B collaboration .
Assuntos
Antígenos H-2/genética , Idiótipos de Imunoglobulinas/genética , Linfócitos T/imunologia , Animais , Células da Medula Óssea , Quimera , Feminino , Complexo Principal de Histocompatibilidade , Masculino , CamundongosRESUMO
An adoptive secondary antibody response to phosphorylcholine (PC) can be generated by the transfer of keyhole limpet hemocyanin (KLH)-primed T cells, PC-bovine gamma globulin-primed B cells, and PC-KLH into irradiated syngeneic BALB/c mice. If the KLH-primed T-cell donors were pretreated with anti-idiotype antibodies directed against the BALB/c PC-binding myeloma TEPC 15, their T cells were unable to collaborate effectively with PC-primed B cells; moreover, they could suppress the helper activity of T cells from normal mice for the PC-KLH response. The Ly phenotype of these T cells was found to be Ly 1-, 2+. The specificity of the suppressor T-cell population induced by anti-T15 treatment appears to be both for idiotype (hapten) and carrier, since the suppressor T cells fail to interfere with the antibody response to PC on a heterologous carrier, nor do they suppress the response to trinitrophenol-KLH.
Assuntos
Colina/análogos & derivados , Idiótipos de Imunoglobulinas , Memória Imunológica , Cooperação Linfocítica , Fosforilcolina/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Anti-Idiotípicos , Antígenos de Superfície/análise , Linfócitos B/imunologia , Hemocianinas/imunologia , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos BALB C , FenótipoRESUMO
We have investigated in vitro the induction of antibody responses to phosphorylcholine (PC) by cloned T helper (Th) cell lines. The cloned Th cells are antigen specific, in this case ovalbumin (OVA), self-Ia recognizing, and induce antibody secretion only if the hapten, PC, is physically linked to the carrier (OVA) molecule. The plaque-forming cell (PFC) response generated in the presence of cloned Th cells is idiotypically diverse with 5-40% of the secreting B cells bearing the TEPC-15 (T15) idiotype. The interaction of the cloned Th cells and unprimed B cells requires recognition of B cell surface Ia glycoproteins for all B cells activated to secrete anti-PC antibody, whether they be T15-bearing or not. More importantly, however, effective interaction between a cloned Th cell and a B cell is determined by the quantity of B cell surface Ia glycoproteins. Our results indicate that quantitative differences in B cell surface Ia antigens are directly related to B cell activation by the cloned Th cell. The high Ia density B cells are most easily activated by cloned Th cells, and these appear to be mainly non-T15-bearing. These data suggest that the failure of cloned Th cells to effectively activate T15-bearing B cells in vitro may be due to the lower relative Ia density of these B cells and therefore to their inability to interact effectively with cloned Ia-recognizing Th cells. These results imply that monoclonal T cells may distinguish between T15-bearing and non-T15-bearing B cells based on their Ia density.
Assuntos
Linfócitos B/classificação , Antígenos de Histocompatibilidade Classe II/análise , Idiótipos de Imunoglobulinas/análise , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Monoclonais/imunologia , Células Produtoras de Anticorpos/classificação , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Células Clonais/imunologia , Epitopos , Técnica de Placa Hemolítica , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Ovalbumina/imunologia , Fosforilcolina/imunologiaRESUMO
Cell lines expressing specific variants of murine CD45 (T200) were established by infection with retroviral constructs of four cDNAs encoding different forms of CD45. These lines were then used to determine which sequences encoded by alternatively spliced exons of CD45 were required to generate antigenic determinants recognized by anti-CD45 mAbs. The binding of two B220 antibodies (14.8 and RA32C2) to CD45 was dependent on the expression of the first alternatively spliced exon (exon A). A third B220 antibody, RA3-6B2, did not bind to any of the forms of CD45 expressed on fibroblasts. A newly defined anti-CD45R antibody, C363.16A, reacts with an antigenic site dependent upon the expression of the second alternatively spliced exon, exon B.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação/genética , Éxons , Antígenos de Histocompatibilidade/genética , Animais , Antígenos de Diferenciação/imunologia , Linfócitos B/imunologia , Linhagem Celular , DNA/genética , Epitopos/genética , Epitopos/imunologia , Regulação da Expressão Gênica , Variação Genética , Antígenos de Histocompatibilidade/imunologia , Antígenos Comuns de Leucócito , Glicoproteínas de Membrana , Camundongos , Processamento de Proteína Pós-Traducional , Splicing de RNA , Proteínas Recombinantes/genética , Linfócitos T Reguladores/imunologia , TransfecçãoRESUMO
Naive CD4+ T cells can differentiate into cells predominantly involved in humoral immunity, known as T helper type 2 cells (Th2), or cells involved in cell-mediated immunity, known as Th1 cells. In this report, we show that priming of CD4+ T cells bearing a transgene-encoded T cell receptor can lead to differentiation into Th1-like cells producing abundant interferon gamma when the cells are exposed to high antigen doses, while low doses of the same peptide induce cells with the same T cell receptor to differentiate into Th2-like cells producing abundant interleukin 4. Thus antigen dose is one factor that can control the differentiation fate of a naive CD4+ T cell.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Grupo dos Citocromos c/imunologia , Relação Dose-Resposta Imunológica , Feminino , Interferon gama/biossíntese , Interleucina-4/biossíntese , Masculino , Camundongos , Camundongos Transgênicos , Mariposas , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/química , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologiaRESUMO
Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased mucus secretion is an important clinical symptom and contributes to airway obstruction in asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4-deficient mice were not recruited to the lung and did not induce mucus production. When this defect in homing was overcome by administration of TNF-alpha, IL-4 -/- Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.
Assuntos
Brônquios/metabolismo , Muco/metabolismo , Células Th2/imunologia , Transferência Adotiva , Animais , Brônquios/patologia , Movimento Celular/imunologia , Epitopos/imunologia , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-4/biossíntese , Interleucina-4/fisiologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Ativação Linfocitária , Transfusão de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Muco/química , Muco/efeitos dos fármacos , Ovalbumina/imunologia , Coloração e Rotulagem , Células Th2/metabolismo , Células Th2/transplanteRESUMO
CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor-transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-gamma? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-gamma? In IFN-gamma receptor(-/-) recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-gamma receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-gamma. The blockade of eosinophilia and mucus production by IFN-gamma likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-gamma signaling in tissue of recipient mice.
Assuntos
Hipersensibilidade/imunologia , Inflamação/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Receptores de Interferon/genética , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Asma/imunologia , Modelos Animais de Doenças , Eosinofilia/imunologia , Citometria de Fluxo , Interferon gama/farmacologia , Interleucinas/metabolismo , Camundongos , Camundongos Knockout , Muco/imunologia , Ovalbumina/imunologia , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Analysis of activation of phosphorylcholine (PC)-specific B cells by a large number of different cloned, self Ia-specific helper T cell (Th) clones has permitted the classification of such T cells into four distinct functional types. Types 1 and 2 induce B cells to secrete anti-PC antibody in an antigen-specific, Ia-restricted fashion. Type 3 cells induce antigen-specific, Ia-restricted B cell proliferation, but do not lead to specific antibody formation, and have been shown previously to have suppressor functions. Type 4 cells are autoreactive, and induce antigen-independent B cell activation and antibody secretion. The distinction between type 1 and type 2 Th clones was analyzed in detail. In bulk cultures, type 1 cloned lines generate an idiotypically heterogeneous anti-PC antibody response, whereas type 2 cloned lines induce a larger response that is dominated by the T15 idiotype. In limiting-dilution analyses, type 2 cells induce fourfold more T15+, PC-specific precursor B cells than do type 1 cells, and in addition, induce larger burst sizes for T15+, PC-specific B cells. Type 4 clones can also be subdivided into cells that are type 1-like, and cells that are type 2-like. These differences in functional phenotype are seen over a broad range of antigen and cell doses. Detailed analysis of the behavior of these distinct functional types of Th should allow a better understanding of the functional properties of mixed populations of antigen-primed, Ia-restricted Th cells.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos B/imunologia , Células Clonais/imunologia , Haptenos/imunologia , Imunização , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Fosforilcolina/imunologiaRESUMO
The inflammatory response in the lungs of mice infected with an influenza A virus consists largely of macrophages and CD3+ T cells. Most T lymphocytes recovered before day 7 after infection express mRNA for the T cell receptor alpha/beta (TCR-alpha/beta), while TCR-gamma/delta mRNA+ cells are found at much higher frequency over the next 7 d. The predominant surface phenotype for the TCR-gamma/delta mRNA+ population is CD3+4-8-TCR-alpha/beta-. Some lymphocytes expressing all the known V gamma genes are found in the inflammatory exudate, but V gamma 2+/V gamma 1+ and V gamma 4+ T cells are present at highest frequency. The response is staged, with maximal numbers of V gamma 4+ cells occurring on day 10 after infection, while the predominant phenotype on day 13 is V gamma 2/V gamma 1+. The emerging peak in numbers of V gamma 4+ lymphocytes is paralleled by increasing numbers of macrophages expressing hsp mRNA. The later maxima found for the V gamma 2+/V gamma 1+ T cells is consistent with the possibility that at least some of these lymphocytes are responding to the hsp+ cells and are functioning to resolve the inflammatory process.
Assuntos
Vírus da Influenza A , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T/análise , Linfócitos T/imunologia , Animais , Feminino , Proteínas de Choque Térmico/genética , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Pneumonia/imunologia , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T gama-deltaRESUMO
To better understand the requirement for interleukin 2 (IL-2) in specific immune responses, we have established the use of cell ablation to selectively eliminate T cells that produce IL-2. To accomplish this we have generated transgenic mice that express the herpes simplex virus 1-thymidine kinase (HSV-TK) gene under the transcriptional control of the murine IL-2 promoter that renders IL-2-producing cells sensitive to the cytotoxic effects of the antiviral drug ganciclovir (GANC). HSV-TK activity was specifically expressed in activated T cells from transgenic mice. When CD4 T cells from transgenic mice were stimulated with the superantigen staphylococcal enterotoxin A (SEA) in the presence of GANC, proliferation and IL-2 production were almost completely inhibited and the activated CD4+V beta 3+ T cell population, eliminated. Proliferation was not restored by adding IL-2, showing that most proliferating cells are not bystander cells. In contrast, the proliferative response to concanavalin A (Con A) was only partially inhibited by treatment of CD4 T cells with GANC, although the efficiency of eliminating IL-2-producing cells was shown to be comparable with that achieved using SEA. This suggests that a portion of the proliferative response to Con A occurs via an alternative pathway not requiring IL-2 synthesis and release.
Assuntos
Interleucina-2/fisiologia , Linfócitos T/citologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Morte Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Separação Celular , Concanavalina A/farmacologia , Enterotoxinas/farmacologia , Ganciclovir/farmacologia , Interleucina-2/biossíntese , Camundongos , Camundongos Transgênicos , Simplexvirus/enzimologia , Baço/citologia , Baço/enzimologia , Staphylococcus aureus/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , TitulometriaRESUMO
The present results demonstrate that CD4+ T cells activated in the primary in vivo response to antigen produce distinct patterns of cytokines depending upon the MHC class II haplotype of the responding mice. I-As mice were found to selectively activate IL-2/IFN-gamma-producing CD4+ T cells, whereas I-Ab mice exhibited selective activation of IL-4-producing CD4+ T cells in response to collagen IV. The effector response phenotype was found to correlate with the cytokine phenotype of CD4+ T cells activated in vivo; IL-2/IFN-gamma-producing cells giving rise to proliferative (cell-mediated) responses, IL-4-producing cells leading to secondary IgG (humoral) responses. Together the data support the notion that the outcome of a given immune response (e.g., protection vs. onset, tolerance vs. autoimmunity) may be determined in part by the type of CD4+ T cells initially activated by antigen. Moreover, the present experiments demonstrate for the first time that polymorphism in class II MHC can determine such selective activation of different cytokine-producing CD4+ T cell phenotypes.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Genes MHC da Classe II , Ativação Linfocitária , Animais , Fatores Biológicos/biossíntese , Colágeno/imunologia , Citocinas , Feminino , Imunoglobulina G/biossíntese , Camundongos , Trinitrobenzenos/imunologiaRESUMO
To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR alpha chain of the D10.G4.1 (D10) TCR and bred them to D10 beta chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by approximately 100-fold. A mutation in the reference peptide CA134-146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex-bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Imunidade Celular , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Células Th1/imunologia , Células Th2/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Interferon gama/biossíntese , Interleucina-4/biossíntese , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutação , Peptídeos/imunologia , Células Th1/citologia , Células Th2/citologiaRESUMO
Antigen priming of naive CD4 T cells can generate effector CD4 T cells that produce interleukin 4 (T helper [Th]2-like) or interferon-gamma (Th1-like). Using a system in which priming leads to responses dominated by one or the other of these cell types, we show that varying either the antigenic peptide or the major histocompatibility complex class II molecule can determine whether Th1-like or Th2-like responses are obtained. Our results show that peptide/major histocompatibility complex class II complexes that interact strongly with the T cell receptor favor generation of Th1-like cells, while those that bind weakly favor priming of Th2-like T cells. Thus, signals from the T cell receptor can influence the differentiation of CD4 T cells into specific types of effector cells.
Assuntos
Apresentação de Antígeno , Colágeno/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th1/citologia , Células Th2/citologia , Animais , Sequência de Bases , Diferenciação Celular , Colágeno/genética , Humanos , Interferon gama/biossíntese , Interleucina-4/biossíntese , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
The CD45 tyrosine phosphatase plays an important role in regulating T lymphocyte activation, but the function of the different isoforms of CD45 is not known. T cell transfectants have been prepared that express individual CD45 isoforms in cells with a well-defined T cell receptor (TCR) from the D10 T helper 2 clone. We find that cells bearing low molecular weight CD45 isoforms are far more efficient in responding to stimulation with peptide and antigen-presenting cells compared with cells bearing high molecular weight CD45 isoforms. One hypothesis for the preferential activation of cells that express low molecular weight CD45 isoforms is that they interact with other cell surface antigens important in TCR signaling, altering their phosphorylation status and affecting the character of the signal transduction pathway. In this report, using cells expressing single isoforms, we demonstrate that low molecular weight isoforms of CD45 preferentially associate with CD4 and the TCR complex compared with high molecular weight isoforms. The molecular basis for this interaction was further examined using a glycosyl phosphatidyl inositol (GPI)-linked form of CD45Null (lacking tyrosine phosphatase domains), which preferentially associated with CD4 compared with GPI-linked CD45ABC, and cytoplasmic tail mutants of CD4, which retained the ability to coassociate. Using this panel of transfectants, it is clear that the interaction between CD4 and CD45 does not require the cytoplasmic domains of CD45, but is dependent on the specific external domain of the various isoforms: low molecular weight species were more likely to associate with the CD4-TCR complex than the higher molecular weight isoforms, and their ability to coassociate correlated with the magnitude of the response to specific antigen.
Assuntos
Apresentação de Antígeno , Antígenos CD4/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Linhagem Celular , Citometria de Fluxo , Capeamento Imunológico , Antígenos Comuns de Leucócito/genética , Dados de Sequência Molecular , Fenótipo , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases , Ligação Proteica , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
The role of STAT (signal transducer and activator of transcription) proteins in T cell receptor (TCR) signaling was analyzed. STAT5 became immediately and transiently phosphorylated on tyrosine 694 in response to TCR stimulation. Expression of the protein tyrosine kinase Lck, a key signaling protein in the TCR complex, activated DNA binding of transfected STAT5A and STAT5B to specific STAT inducible elements. The role of Lck in STAT5 activation was confirmed in a Lck-deficient T cell line in which the activation of STAT5 by TCR stimulation was abolished. Expression of Lck induced specific interaction of STAT5 with the subunits of the TCR, indicating that STAT5 may be directly involved in TCR signaling. Stimulation of T cell clones and primary T cell lines also induced the association of STAT5 with the TCR complex. Inhibition of STAT5 function by expression of a dominant negative mutant STAT5 reduced antigen-stimulated proliferation of T cells. Thus, TCR stimulation appears to directly activate STAT5, which may participate in the regulation of gene transcription and T cell proliferation during immunological responses.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Ativação Linfocitária , Proteínas do Leite , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Transativadores/metabolismo , Animais , Anticorpos , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Divisão Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Interferon gama/farmacologia , Interleucina-2/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT5 , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Transativadores/genética , TransfecçãoRESUMO
Atopic individuals are predisposed to mounting vigorous Th2-type immune responses to environmental allergens. To determine the factors responsible, animal models that closely mimic natural modes of allergen exposure should prove most informative. Therefore, we investigated the role of IL-4, a known Th2-promoting cytokine, in generation of Th2 responses after exposure of either the skin or airway to soluble protein. Compared with wild-type (WT) mice, IL-4-deficient (IL-4(-/-)) mice showed markedly impaired Th2 activation after primary exposure to inhaled ovalbumin (OVA), with decreased OVA-specific IgG1 and IgE, and significantly fewer eosinophils in bronchoalveolar lavage (BAL) fluid after airway challenge. In contrast, IL-4(-/-) mice initially exposed to epicutaneous (e.c.) OVA mounted Th2 responses equivalent to responses in WT mice, with high numbers of eosinophils in BAL fluid. Because Th2 responses were not induced by e.c. OVA exposure in Stat6(-/-) mice (mice lacking signal transducer and activator of transcription 6), the role of IL-13 was tested. In vivo depletion of IL-13 prevented Th2 responses induced by e.c. OVA exposure in IL-4(-/-) mice. These data demonstrate a marked difference in the IL-4 dependence of Th2 responses generated at two anatomic sites of natural allergen encounter and identify the skin as a particularly potent site for Th2 sensitization.
Assuntos
Hipersensibilidade Imediata/imunologia , Interleucina-4/fisiologia , Ovalbumina/administração & dosagem , Células Th2/imunologia , Administração Cutânea , Administração por Inalação , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Eosinófilos/imunologia , Feminino , Imunização , Interleucina-13/antagonistas & inibidores , Interleucina-13/fisiologia , Interleucina-4/deficiência , Interleucina-4/genética , Pulmão/metabolismo , Pulmão/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologiaRESUMO
By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO(+), but not CD45ABC(+), T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO(+) expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO(+) T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC(+) T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.
Assuntos
Proteínas de Ligação a DNA , Antígenos Comuns de Leucócito/metabolismo , Receptor Cross-Talk , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Ativação Linfocitária , Modelos Biológicos , Isoformas de Proteínas , Proteínas Proto-Oncogênicas , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio etsRESUMO
Interleukin-1 (IL-1), an immune monokine secreted by activated macrophages and monocytes, appears to be intimately involved in endocrine phenomena. It is well established that IL-1 mediates a number of immune and inflammatory responses. In addition, the production of IL-1 appears to be modulated by the gonadal estradiol and progesterone, leading to this study of IL-1 secretion by cultured peripheral monocytes isolated at varying times during the menstrual cycle and pregnancy. Circulating peripheral monocytes were isolated from eight women undergoing human menopausal gonadotropin-stimulated ovulation induction during the late follicular phase just before hCG administration and from eight similarly stimulated women on the 12th day after hCG administration. Peripheral monocytes were also isolated from six women during both second and third trimester pregnancy. After 48 h in culture, conditioned media were assayed for IL-1 bioactivity using the D1O lymphocyte proliferation assay. Significantly more IL-1 bioactivity was secreted by monocytes isolated in the luteal phase of the cycle (52.4 +/- 17.5 IU/mL) compared to that in late follicular phase (5.2 +/- 0.9 IU/mL; P = 0.001) or third trimester of pregnancy (7.0 +/- 1.5 IU/mL; P = 0.006). Thus, peripheral monocyte IL-1 secretion appears to be increased by luteal levels of progesterone, although further elevation of progesterone during pregnancy returns IL-1 levels to the preovulatory baseline. In addition, basal body temperatures obtained in four women during third trimester pregnancy were 98 F or less. Thus, IL-1 secretion from cultured monocytes appears to increase with luteal concentrations of progesterone and decrease to preovulatory levels at higher concentrations of the steroid during pregnancy, which may account for the dissociation of high progesterone levels and elevated basal body temperature during late pregnancy.