RESUMO
In allogeneic bone marrow transplantation (BMT) donor T cells are primarily responsible for antihost activity, resulting in graft-versus-host disease (GVHD), and for antileukemia activity, resulting in the graft-versus-leukemia (GVL) effect. The relative contributions of the Fas ligand (FasL) and perforin cytotoxic pathways in GVHD and GVL activity were studied by using FasL-defective or perforin-deficient donor T cells in murine parent --> F1 models for allogeneic bone marrow transplantation. It was found that FasL-defective B6.gld donor T cells display diminished GVHD activity but have intact GVL activity. In contrast, perforin-deficient B6.pfp(-/-) donor T cells have intact GVHD activity but display diminished GVL activity. Splenic T cells from recipients of B6.gld or B6.pfp(-/-) T cells had identical proliferative and cytokine responses to host antigens; however, splenic T cells from recipients of B6.pfp(-/-) T cells had no cytolytic activity against leukemia cells in a cytotoxicity assay. In experiments with selected CD4(+) or CD8(+) donor T cells, the FasL pathway was important for GVHD activity by both CD4(+) and CD8(+) T cells, whereas the perforin pathway was required for CD8-mediated GVL activity. These data demonstrate in a murine model for allogeneic bone marrow transplantation that donor T cells mediate GVHD activity primarily through the FasL effector pathway and GVL activity through the perforin pathway. This suggests that donor T cells make differential use of cytolytic pathways and that the specific blockade of one cytotoxic pathway may be used to prevent GVHD without interfering with GVL activity.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Leucemia/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Animais , Transplante de Medula Óssea , Citotoxicidade Imunológica , Proteína Ligante Fas , Feminino , Camundongos , Perforina , Proteínas Citotóxicas Formadoras de Poros , Imunologia de Transplantes , Transplante HomólogoRESUMO
Manganese superoxide dismutase 2 (SOD2) is a critical component of the mitochondrial pathway for detoxification of O2(-), and targeted disruption of this locus leads to embryonic or neonatal lethality in mice. To follow the effects of SOD2 deficiency in cells over a longer time course, we created hematopoietic chimeras in which all blood cells are derived from fetal liver stem cells of Sod2 knockout, heterozygous, or wild-type littermates. Stem cells of each genotype efficiently rescued hematopoiesis and allowed long-term survival of lethally irradiated host animals. Peripheral blood analysis of leukocyte populations revealed no differences in reconstitution kinetics of T cells, B cells, or myeloid cells when comparing Sod2(+/+), Sod2(-/-), and Sod2(+/-) fetal liver recipients. However, animals receiving Sod2(-/-) cells were persistently anemic, with findings suggestive of a hemolytic process. Loss of SOD2 in erythroid progenitor cells results in enhanced protein oxidative damage, altered membrane deformation, and reduced survival of red cells. Treatment of anemic animals with Euk-8, a catalytic antioxidant with both SOD and catalase activities, significantly corrected this oxidative stress-induced condition. Such therapy may prove useful in treatment of human disorders such as sideroblastic anemia, which SOD2 deficiency most closely resembles.
Assuntos
Anemia Hemolítica/tratamento farmacológico , Anemia Hemolítica/genética , Antioxidantes/uso terapêutico , Etilenodiaminas/uso terapêutico , Mitocôndrias/enzimologia , Compostos Organometálicos/uso terapêutico , Superóxido Dismutase/deficiência , Anemia Hemolítica/sangue , Animais , Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Transplante de Células , Transplante de Tecido Fetal/fisiologia , Sequestradores de Radicais Livres/uso terapêutico , Genótipo , Heterozigoto , Isoenzimas/deficiência , Isoenzimas/genética , Fígado/citologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Baço/imunologia , Superóxido Dismutase/genética , Linfócitos T/imunologia , Timo/imunologia , Quimeras de TransplanteRESUMO
To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, approximately 5--10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3 zeta are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1--14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3 zeta/epsilon, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.
Assuntos
Complexo CD3/fisiologia , Regulação para Baixo/imunologia , Ativação Linfocitária , Microdomínios da Membrana/enzimologia , Proteínas Nucleares , Proteínas Tirosina Fosfatases/fisiologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Domínios de Homologia de src/imunologia , Complexo CD3/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Marcação de Genes , Humanos , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Fatores de Transcrição NFATC , Octoxinol/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Solubilidade , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Tirosina/antagonistas & inibidores , Tirosina/metabolismo , Domínios de Homologia de src/genéticaRESUMO
Rapamycin is a fungal macrolide that inhibits the proliferation of T cells. Studies in both animals and humans have found that rapamycin significantly reduces graft rejection. However, though CD8(+) T cells are involved in graft infiltration and rejection, little is known regarding the effects of rapamycin on CD8(+) human T cell responses. In this study, we examined the mechanism of rapamycin-induced inhibition of Ag-driven activation of CD8(+) T cells. Surprisingly, a heterogeneous proliferative response in the presence of rapamycin was observed among different Ag-specific CD8(+) T cell clones; this was also observed in CD8(+) peripheral blood T cells activated with TCR cross-linking ex vivo. Inhibition of T cell proliferation by rapamycin was controlled by both the strength of signal delivered through the Ag receptor as well as the specific costimulatory signals received by the T cell. Rapamycin-resistant proliferation occurred despite inhibition of p70(s6) kinase activity. Moreover, rapamycin-resistant proliferation of the CD8(+) T cell clones was blocked by anti-IL-2 Abs, suggesting that while some of the parallel pathways triggered by IL-2R signaling are sensitive to the effects of rapamycin, others account for the Ag-driven rapamycin resistance. These data provide a new framework for examining the specific mechanism of action of rapamycin in human disease.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirolimo/farmacologia , Anticorpos Monoclonais/metabolismo , Antígenos CD2/imunologia , Antígenos CD2/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Transformada , Células Clonais , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Resistência a Medicamentos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/fisiologia , Humanos , Interleucina-2/fisiologia , Complexo Principal de Histocompatibilidade/imunologia , Modelos Imunológicos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologiaRESUMO
CD43, one of the most abundant glycoproteins on the T cell surface, has been implicated in selection and maturation of thymocytes and migration, adhesion, and activation of mature T cells. The adapter molecule Cbl has been shown to be a negative regulator of Ras. Furthermore, it may also regulate intracellular signaling through the formation of several multi-molecular complexes. Here we investigated the role of Cbl in the CD43-mediated signaling pathway in human T cells. Unlike T cell receptor signaling, the interaction of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase, resulting from CD43-specific signals, is independent of Cbl tyrosine phosphorylation, suggesting an alternative mechanism of interaction. CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the tau-isoform of 14-3-3. protein. Protein kinase C-mediated Cbl serine phosphorylation was required for this interaction, because the PKC inhibitor RO-31-8220 prevented it, as well as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the CD43-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and AP-1 transcriptional activity, confirming nevertheless a negative role for Cbl in T cell signaling. However, under normal conditions, PKC activation resulting from CD43 engagement was required to activate the MAPK pathway, suggesting that phosphorylation of Cbl on serine residues by PKC and its association with 14-3-3 molecules may play a role in preventing the Cbl inhibitory effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate the molecular associations and functions of the Cbl adapter protein.
Assuntos
Antígenos CD , Proteínas de Ciclo Celular , Proteínas Oncogênicas de Retroviridae/metabolismo , Sialoglicoproteínas/metabolismo , Linfócitos T/metabolismo , Proteínas 14-3-3 , Anticorpos Monoclonais , Ativação Enzimática , Genes Reporter , Humanos , Células Jurkat , Leucossialina , Ativação Linfocitária , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica v-cbl , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteína Quinase C/metabolismo , Subunidades Proteicas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Agregação de Receptores , Proteínas Oncogênicas de Retroviridae/imunologia , Serina/genética , Serina/metabolismo , Sialoglicoproteínas/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Transfecção , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Biblioteca de Peptídeos , Peptídeos/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Inibidores Enzimáticos/síntese química , Genes Reporter , Humanos , Interleucina-2/genética , Isoenzimas/metabolismo , Células Jurkat , Cinética , Ativação Linfocitária , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Fosfolipase C gama , Linfócitos T/enzimologia , Linfócitos T/imunologia , Transfecção , Fosfolipases Tipo C/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
Phosphatidylinositol 3'-kinase (PI3K) is a key component of multiple signaling pathways, where it typically promotes survival, proliferation, and/or adhesion. Here, we show that in TCR signaling, the scaffolding adapter Gab2 delivers an inhibitory signal via PI3K. Overexpression of Gab2 in T cell lines inhibits TCR-evoked activation of the IL-2 promoter, blocking NF-AT- and NF-kappaB-directed transcription. Inhibition is abrogated by mutating the Gab2 p85-binding sites, by treatment with PI3K inhibitors or by cotransfection of phosphatase homolog of tensin. Our findings provide the first evidence of a negative function for a scaffolding adapter in T cells and identify Gab2/PI3K-containing complexes as novel regulators of TCR signaling.
Assuntos
Inibidores Enzimáticos/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/fisiologia , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Regulação da Expressão Gênica/imunologia , Hemaglutininas/genética , Humanos , Interleucina-2/antagonistas & inibidores , Interleucina-2/genética , Células Jurkat , Ativação Linfocitária/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Plasmídeos/imunologia , Regiões Promotoras Genéticas/imunologia , Transdução de Sinais/genética , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
Lymphokine gene transcription involves numerous signal transduction molecules and second messengers. The serine/threonine phosphatase calcineurin has been demonstrated to play a central role in the immediate, early activation of numerous lymphokines (such as interleukin [IL]-2) and in the regulation of cell surface receptors such as CD40L, CD95, and recently CD25 alpha (the alpha chain of the IL-2 receptor). In addition to lymphocyte activation, calcineurin functions include control of neuronal signaling, muscle contraction, muscle hypertrophy and cellular death. Therefore, calcineurin not only plays a vital role in the regulation of T lymphocyte function, but also functions in cellular environments outside the immune system.
Assuntos
Calcineurina/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Humanos , Linfocinas/imunologiaAssuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Ligases/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Oncogênicas de Retroviridae/genética , Animais , Regulação da Expressão Gênica , Modelos Moleculares , Proteína Oncogênica v-cbl , Fosforilação , Conformação Proteica , Proteínas Proto-Oncogênicas c-cbl , Transdução de Sinais , Ubiquitina-Proteína Ligases , Domínios de Homologia de srcRESUMO
BACKGROUND: The Fas/Fas ligand (FasL) pathway plays an important role in a number of apoptotic processes that could be important for the development of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), such as cytolysis of target cells by cytotoxic T cells, regulation of inflammatory responses, peripheral deletion of autoimmune cells, costimulation of T cells, and activation-induced cell death. METHODS: To study the role of the Fas/FasL pathway in the complex pathophysiology of graft versus host disease (GVHD), we used FasL-deficient B6.gld mice as recipients in a Major Histocompatibility Antigen Complex-matched minor Histocompatibility Antigen-mismatched murine model for GVHD after allogeneic BMT (C3H.SW-->B6). RESULTS: We found a significantly higher morbidity and mortality from GVHD compared to control B6 recipients. Histopathological analysis of the GVHD target organs demonstrated that B6.gld recipients developed significantly more thymic and intestinal GVHD. B6gld recipients with GVHD demonstrated an increased expansion of donor T cells and monocytes/ macrophages compared to control B6 recipients, whereas serum TNF-alpha levels were equivalent in B6.gld recipients and control B6 recipients. CONCLUSION: This study demonstrates that the expression of FasL in the BMT recipient is important for the host's ability to control GVHD.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Doença Enxerto-Hospedeiro/etiologia , Glicoproteínas de Membrana/fisiologia , Animais , Suscetibilidade a Doenças , Proteína Ligante Fas , Feminino , Macrófagos/fisiologia , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Linfócitos T/imunologia , Transplante Homólogo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Although numerous reports document a role for CD43 in T cell signaling, the direct participation of this molecule in cell activation has been questioned. In this study we show that CD43 ligation on human normal peripheral T cells was sufficient to induce interleukin-2, CD69, and CD40-L gene expression, without requiring signals provided by additional receptor molecules. This response was partially inhibited by cyclosporin A and staurosporine, suggesting the participation of both the Ca(2+) and the protein kinase C pathways in CD43 signaling. Consistent with the transient CD43-dependent intracellular Ca(2+) peaks reported by others, signals generated through the CD43 molecule resulted in the induction of NF-AT DNA binding activity. CD43-dependent signals resulted also in AP-1 and NFkappaB activation, probably as a result of protein kinase C involvement. AP-1 complexes bound to the AP-1 sequence contained c-Jun, and those bound to the NF-AT-AP-1 composite site contained c-Jun and Fos. NFkappaB complexes containing p65 could be found as early as 1 h after CD43 cross-linking, suggesting that CD43 participates in early events of T cell activation. The induction of the interleukin-2, CD69, and CD-40L genes and the participation of AP-1, NF-AT, and NFkappaB in the CD43-mediated signaling cascade implicate an important role for this molecule in the regulation of gene expression and cell function.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares , Sialoglicoproteínas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Ligante de CD40/metabolismo , Inibidores de Calcineurina , Cálcio/metabolismo , Núcleo Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Ciclosporina/farmacologia , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Interleucina-2/metabolismo , Íons , Células Jurkat , Lectinas Tipo C , Leucossialina , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , NF-kappa B/genética , Fatores de Transcrição NFATC , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Estaurosporina/farmacologia , Sinaptotagmina I , Sinaptotagminas , Linfócitos T/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genéticaRESUMO
Cyclin dependent kinase 4 (cdk4) activity is controlled by the binding of regulatory subunits and inhibitory factors, as well as tyrosine and serine/threonine phosphorylation. More recently the influence of calcium levels have been demonstrated. Using transient transfections in Jurkat cells, we observed specific binding between cdk4 and the calcium and calmodulin activated serine/threonine phosphatase, calcineurin. Furthermore, we demonstrated that the inhibition of the phosphatase activity of calcineurin with FK506 and cyclosporin A resulted in an overall increase in cdk4 kinase activity, suggesting that the phosphatase activity of calcineurin was inhibitory to the kinase activity of cdk4. In contrast, we were not able to observe a similar effect on the kinase activity of either cdk6 or cdk2, indicating that the phosphatase activity of calcineurin was specific for cdk4. In addition, using an in vitro phosphatase assay for calcineurin, we observed that the exogenous addition of calcineurin resulted in the dephosphorylation of cdk4, an event that downregulated the kinase activity of cdk4. Calcineurin could, therefore, play an opposing role to the action of the cyclin activating kinase complex, an enzyme that upregulates the kinase activity of cdk4, an important G0/G1 checkpoint element in mammalian cells. Oncogene (2000) 19, 2820 - 2827
Assuntos
Calcineurina/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Proteínas Proto-Oncogênicas , Quinase 4 Dependente de Ciclina , Humanos , Células Jurkat , Mitose , Monoéster Fosfórico Hidrolases/fisiologia , FosforilaçãoRESUMO
BACKGROUND: We studied whether T-cell clones, which appear in the periphery as a result of the failure of thymic negative selection during graft-versus-host disease (GVHD), have any in vivo anti-host reactivity and can cause GVHD in an adoptive transfer model. METHODS: We performed our studies in a murine model (B10.BR into CBA/J) for allogeneic bone marrow transplantation with major histocompatibility complex-matched and minor histocompatibility antigen-mismatched unrelated donors and unique Vbeta T-cell deletion patterns in donors and recipients. RESULTS: GVHD resulted in the appearance of Vbeta6+ T cells as a result of a loss of negative selection. We found that Vbeta6+ T cells from normal donors proliferated in vitro and in vivo. Depletion of Vbeta6+ T cells from the donor T-cell inoculum resulted in less GVHD morbidity and a decrease in the loss of thymic cellularity. To test the anti-host reactivity of de novo generated Vbeta6+ T cells in animals with GVHD, we developed an adoptive transfer model of splenic T cells from CBA/J host animals with GVHD into sublethally irradiated CBA/J recipients Depletion of Vbeta6+ T cells from the splenic T cells before adoptive transfer could significantly decrease the transient GVHD morbidity in the sublethally irradiated hosts. CONCLUSIONS: Our data indicate that GVHD-associated thymic damage results in a loss of thymic negative selection, which leads to the appearance of T-cell clones with anti-host reactivity in vitro and in vivo.
Assuntos
Transplante de Medula Óssea , Doença Enxerto-Hospedeiro/imunologia , Subpopulações de Linfócitos T/imunologia , Imunologia de Transplantes , Animais , Apresentação de Antígeno , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Camundongos , Timo/imunologia , Transplante HomólogoRESUMO
STAT5, a member of the signal transducers and activators of transcription (STATs), is important in modulating T cell functions through interleukin-2 (IL-2) receptors. Like other STAT proteins, STAT5 undergoes a rapid activation and inactivation cycle upon cytokine stimulation. Tyrosine phosphorylation and dephosphorylation are critical in regulating STAT5 activity. A number of protein tyrosine kinases have been shown to phosphorylate STAT5; however, the phosphatases responsible for STAT5 dephosphorylation remain unidentified. Using CTLL-20 as a model system, we provide evidence that tyrosine dephosphorylation of STAT5 subsequent to IL-2-induced phosphorylation occurs in the absence of STAT5 nuclear translocation and new protein synthesis. Nevertheless, down-regulation of the upstream Janus kinase activity during the deactivation cycle of IL-2-induced signaling does involve new protein synthesis. These findings point to the constitutive presence of STAT5 tyrosine phosphatase activity in the cytosolic compartment. We further demonstrate that SHP-2, but not SHP-1, directly dephosphorylates STAT5 in an in vitro tyrosine phosphatase assay with purified proteins. Furthermore, tyrosine-phosphorylated STAT5 associates with the substrate-trapping mutant (Cys --> Ser) of SHP-2 but not SHP-1. These results suggest a potential role for cytoplasmic protein-tyrosine phosphatases in directly dephosphorylating STAT proteins and in maintaining a basal steady state level of STAT activity.
Assuntos
Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transativadores/metabolismo , Transporte Biológico , Linhagem Celular , Núcleo Celular/metabolismo , Regulação para Baixo , Interleucina-2/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Linfoma de Células T , Mutação , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT5 , Células Tumorais CultivadasRESUMO
The Fas/Fas ligand (FasL) pathway is involved in a variety of regulatory mechanisms that could be important for the development of graft-vs-host disease (GVHD) after bone marrow transplantation (BMT), such as cytolysis of target cells by cytotoxic T cells, regulation of inflammatory responses, peripheral deletion of autoimmune cells, costimulation of T cells, and activation-induced cell death. To further evaluate the role of Fas/FasL in the complex pathophysiology of GVHD, we used Fas-deficient B6.lpr mice as recipients in a MHC-matched minor histocompatibility Ag-mismatched murine model for GVHD after allogeneic BMT (C3H.SW-->B6). We found a significantly higher morbidity and mortality from GVHD compared with control B6 recipients. In contrast, B6.lpr recipients had very little hepatic GVHD, although all other specific GVHD target organs (skin, intestines, and thymus) were more severely affected than in the control B6 recipients. B6.lpr recipients with GVHD demonstrated intact donor lymphoid engraftment and an increase in expansion of donor T cells and monocytes/macrophages compared with control B6 recipients. Serum levels of IFN-gamma and TNF-alpha were higher in B6.lpr recipients than in control B6 recipients, and monocytes/macrophages in B6.lpr recipients appeared more sensitized. B6.lpr recipients had more residual peritoneal macrophages after BMT, and peritoneal macrophages from B6.lpr mice could induce a greater proliferative response from C3H.SW splenocytes. This study demonstrates that the expression of Fas in the recipient is required for GVHD of the liver, but shows unexpected consequences when host tissues lack the expression of Fas for the development of GVHD in other organs and systemic GVHD.
Assuntos
Predisposição Genética para Doença , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Receptor fas/genética , Animais , Transplante de Medula Óssea/imunologia , Divisão Celular/imunologia , Feminino , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/patologia , Interferon gama/sangue , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Fígado/imunologia , Fígado/patologia , Ativação Linfocitária/genética , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Monócitos/citologia , Pele/imunologia , Pele/patologia , Linfócitos T/citologia , Linfócitos T/transplante , Timo/imunologia , Timo/patologia , Transplante Homólogo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pretransplant exposure to allogeneic lymphocytes can result in donor-specific unresponsiveness and prolonged allograft survival. Intracellular signaling events have been described in anergic T cell clones, but the biochemical events underlying in vivo induced unresponsiveness have not been studied in detail. We employed a TCR transgenic mouse, bearing the 2C TCR, providing adequate numbers of homogenous peripheral T cells to study biochemical aspects of T cell unresponsiveness in vivo. 2C mice exposed to semiallogeneic lymphocytes (H-2b x H-2d) experienced prolonged H-2d cardiac allograft survival, and cells from these mice did not proliferate or make IL-2 in response to alloantigen (H-2d). Importantly, there were marked differences in TCR-associated tyrosine phosphorylation activation patterns. The targets for the unresponsive state appear to be diminished Lck activation and absent ZAP-70 and LAT (linker for activation of T cells) phosphorylation. Our study demonstrates that Ag-induced tolerance in vivo is accompanied by altered early TCR-mediated signaling events.
Assuntos
Tolerância Imunológica , Isoantígenos/imunologia , Ativação Linfocitária , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/genética , Injeções Intravenosas , Isoantígenos/administração & dosagem , Linfonodos/citologia , Linfonodos/imunologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/genética , Baço/citologia , Baço/imunologia , Baço/transplante , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Proteínas ras/fisiologiaRESUMO
The tyrosine phosphatase SHP-1 functions as a negative regulator in hematopoietic cell development, proliferation, and receptor-mediated cellular activation. In Jurkat T cells, a major 68-kDa band and a minor 70-kDa band were immunoprecipitated by a monoclonal antibody against the SHP-1 protein-tyrosine phosphatase domain, while an antibody against the SHP-1 C-terminal 19 amino acids recognized only the 68-kDa SHP-1. The SDS-gel-purified 70-kDa protein was subjected to tryptic mapping and microsequencing, which was followed by molecular cloning. It revealed that the 70-kDa protein, termed SHP-1L, is a C-terminal alternatively spliced form of SHP-1. SHP-1L is 29 amino acids longer than SHP-1, and its 66 C-terminal amino acids are different from SHP-1. The C terminus of SHP-1L contains a proline-rich motif PVPGPPVLSP, a potential Src homology 3 domain-binding site. In contrast to SHP-1, tyrosine phosphorylation of SHP-1L is not detected upon stimulation in Jurkat T cells. This is apparently due to the lack of a single in vivo tyrosine phosphorylation site, which only exists in the C terminus of SHP-1 (Y564). COS cell-expressed glutathione S-transferase-SHP-1L can dephosphorylate tyrosine-phosphorylated ZAP70. At pH 7.4, SHP-1L was shown to be more active than SHP-1 in the dephosphorylation of ZAP70. At pH 5.4, SHP-1L and SHP-1 exhibited similar catalytic activity. It is likely that these two isoforms play different roles in the regulation of hematopoietic cell signal transduction.
Assuntos
Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Catálise , Clonagem Molecular , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Testes de Precipitina , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/metabolismo , Vanadatos/farmacologia , Proteína-Tirosina Quinase ZAP-70RESUMO
Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins. Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent manner. We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells. In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We have evidence that SLAP forms homodimers through its C-terminal region. Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity. The in vivo association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.
Assuntos
Proteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Células COS , Dimerização , Precursores Enzimáticos/metabolismo , Escherichia coli , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Fosfoproteínas/metabolismo , Mutação Puntual , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae , Quinase Syk , Proteína-Tirosina Quinase ZAP-70RESUMO
Engagement of the TCR determines the fate of T cells to activate their functional programs, proliferate, or undergo apoptosis. The intracellular signal transduction pathways that dictate the specific outcome of receptor engagement have only been partially elucidated. The adapter protein, Shc, is involved in cytokine production, mitogenesis, transformation, and apoptosis in different cell systems. We found that Shc becomes phosphorylated on tyrosine residues upon stimulation of the TCR in DO11.10 hybridoma T cells; therefore, we investigated the role of Shc in activation-induced cell death in these cells by creating a series of stably transfected cell lines. Expression of Shc-SH2 (the SH2 domain of Shc) or Shc-Y239/240F (full-length Shc in which tyrosines 239 and 240 have been mutated to phenylalanine) resulted in the inhibition of activation-induced cell death and Fas ligand up-regulation after TCR cross-linking. Expression of wild-type Shc or Shc-Y317F had no significant effect. In addition, we found that Shc-SH2 and Shc-Y239/240F, but not Shc-Y317F, inhibited phosphorylation of extracellular signal-regulated protein kinase and production of IL-2 after TCR cross-linking. These results indicate an important role for Shc in the early signaling events that lead to activation-induced cell death and IL-2 production after TCR activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Ativação Linfocitária/imunologia , Proteínas/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Domínios de Homologia de src/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Proteína Ligante Fas , Proteína Adaptadora GRB2 , Hibridomas , Interleucina-2/biossíntese , Ligantes , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Camundongos , Mutação , Fosforilação , Mutação Puntual , Biossíntese de Proteínas , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Linfócitos T/metabolismo , Transfecção , Receptor fas/metabolismoRESUMO
T cells can undergo activation-induced cell death (AICD) upon stimulation of the T cell receptor-CD3 complex. We found that the extracellular signal-regulated kinase (ERK) pathway is activated during AICD. Transient transfection of a dominant interfering mutant of mitogen-activated/extracellular signal-regulated receptor protein kinase kinase (MEK1) demonstrated that down-regulation of the ERK pathway inhibited FasL expression during AICD, whereas activation of the ERK pathway with a constitutively active MEK1 resulted in increased expression of FasL. We also found that pretreatment with the specific MEK1 inhibitor PD98059 prevented the induction of FasL expression during AICD and inhibited AICD. However, PD98059 had no effect on other apoptotic stimuli. We found only very weak ERK activity during Fas-mediated apoptosis (induced by Fas cross-linking). Furthermore, preincubation with the MEK1 inhibitor did not inhibit Fas-mediated apoptosis. Finally, we also demonstrated that pretreatment with the MEK1 inhibitor could delay and decrease the expression of the orphan nuclear steroid receptor Nur77, which has been shown to be essential for AICD. In conclusion, this study demonstrates that the ERK pathway is required for AICD of T cells and appears to regulate the induction of Nur77 and FasL expression during AICD.