RESUMO
Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome-positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Linfócitos T/imunologia , Quinases da Família src/antagonistas & inibidores , Linfócitos B , Dasatinibe/farmacologia , Humanos , Mesilato de Imatinib/farmacologia , Imidazóis/farmacologia , Testes de Liberação de Interferon-gama , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Mutação de Sentido Incorreto , Proteínas de Neoplasias/fisiologia , Fosforilação/efeitos dos fármacos , Mutação Puntual , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/genética , Piridazinas/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Quinases da Família src/fisiologiaRESUMO
Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27(CK-)) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27(T187A)) or nuclear retention (p27(S10A)) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization.
Assuntos
Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Citoplasma/metabolismo , Proteínas de Fusão bcr-abl/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Animais , Células Cultivadas , Genes Supressores de Tumor , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Oncogênicas/metabolismo , Transporte Proteico/genéticaAssuntos
Aurora Quinase A , Leucemia-Linfoma Linfoblástico de Células Precursoras , Aurora Quinase A/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Humanos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação GenéticaRESUMO
Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.
Assuntos
Transformação Celular Neoplásica/metabolismo , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Substituição de Aminoácidos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/sangue , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/tratamento farmacológico , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mutantes/metabolismo , Células Progenitoras Mieloides/metabolismo , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/genética , Receptores de Fator Estimulador de Colônias/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Humanos , Células K562 , Mutação com Perda de Função , Camundongos , MutaçãoRESUMO
Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both 'native' CD4+ CD25+ and 'inducible' CD4+ CD25- peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.
Assuntos
Fatores de Transcrição Forkhead/sangue , Esclerose Múltipla/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Adulto , Idoso , Autoantígenos/imunologia , Autoimunidade/imunologia , Regiões Determinantes de Complementaridade/imunologia , Feminino , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Humanos , Tolerância Imunológica/imunologia , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Proteínas do Tecido Nervoso/imunologia , Linfócitos T Reguladores/imunologia , Vacinação/métodosRESUMO
6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3ß, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association.
Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Tretinoína/farmacologia , ADP-Ribosil Ciclase 1/metabolismo , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Novel-targeted therapies are in rapid development for the treatment of acute lymphoblastic leukemia (ALL) to overcome resistance and decrease toxicity. Survivin, a member of the inhibitor of apoptosis gene family and chromosome passenger complex, is critical in a variety of human cancers, including ALL. A well-established suppressor of survivin has been the small molecule, YM155. Reports are identifying other mechanisms of action for YM155. Therefore, we sought to investigate the mode of action and role of YM155 for therapeutic use in the context of ALL. METHODS: Primary ALL samples and ALL cell lines were interrogated with YM155 to identify drug sensitivity. Ph(+)ALL harboring the BCR-ABL1 oncogene were tested for any interaction with YM155 and the multi-kinase inhibitor dasatinib. Representative ALL cell lines were tested to identify the response to YM155 using standard biochemical assays as well as RNA expression and phosphorylation arrays. RESULTS: ALL samples exhibited significant sensitivity to YM155, and an additive response was observed with dasatinib in the setting of Ph(+)ALL. ALL cells were more sensitive to YM155 during S phase during DNA replication. YM155 activates the DNA damage pathway leading to phosphorylation of Chk2 and H2AX. Interestingly, screening of primary patient samples identified unique and exquisite YM155 sensitivity in some but not all ALL specimens. CONCLUSION: These results are the first to have screened a large number of primary patient leukemic samples to identify individual variations of response to YM155. Our studies further support that YM155 in ALL induces DNA damage leading to S phase arrest. Finally, only subsets of ALL have exquisite sensitivity to YM155 presumably through both suppression of survivin expression and activation of the DNA damage pathway underscoring its potential for therapeutic development.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Imidazóis/farmacologia , Naftoquinonas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Ensaio Cometa , Relação Dose-Resposta a Droga , Humanos , Immunoblotting , Concentração Inibidora 50RESUMO
Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Ensaios Clínicos como Assunto , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinase/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais , Quinase Syk , Regulação para Cima , Quinases da Família src/metabolismoRESUMO
UNLABELLED: Since the advent of tyrosine kinase inhibitors as targeted therapies in cancer, several receptor tyrosine kinases (RTK) have been identified as operationally important for disease progression. Rhabdomyosarcoma (RMS) is a malignancy in need of new treatment options; therefore, better understanding of the heterogeneity of RTKs would advance this goal. Here, alveolar RMS (aRMS) tumor cells derived from a transgenic mouse model expressing two such RTKs, platelet-derived growth factor (PDGFR)α and insulin-like growth factor (IGF)-1R, were investigated by fluorescence-activated cell sorting (FACS). Sorted subpopulations that were positive or negative for PDGFRα and IGF-1R dynamically altered their cell surface RTK expression profiles as early as the first cell division. Interestingly, a difference in total PDGFRα expression and nuclear IGF-1R expression was conserved in populations. Nuclear IGF-1R expression was greater than cytoplasmic IGF-1R in cells with initially high cell surface IGF-1R, and cells with high nuclear IGF-1R established tumors more efficiently in vivo. RNA interference-mediated silencing of IGF-1R in the subpopulation of cells initially harboring higher cell surface and total IGF-1R resulted in significantly reduced anchorage-independent colony formation as compared with cells with initially lower cell surface and total IGF-1R expression. Finally, in accordance with the findings observed in murine aRMS, human aRMS also had robust expression of nuclear IGF-1R. IMPLICATIONS: RTK expression status and subcellular localization dynamics are important considerations for personalized medicine.
Assuntos
Núcleo Celular/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Rabdomiossarcoma Alveolar/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Modelos Animais de Doenças , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Camundongos Transgênicos , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Análise Serial de TecidosRESUMO
The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Apoptose/efeitos dos fármacos , Benzamidas/farmacocinética , Benzamidas/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/farmacocinética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Chronic beryllium disease (CBD) is characterized pathologically by granulomatous inflammation in the lung, composed of a large core of epithelioid cells surrounded by a dense shell of CD4+ T cells. Using beryllium-specific CD4+ T cell lines derived from the bronchoalveolar lavage (BAL) fluid of CBD patients, we show that purified CD4+ T cells produced significant amounts of IFN-gamma and TNF-alpha upon exposure to beryllium in the absence of antigen-presenting cells (APC). However, unlike BAL T cells stimulated by beryllium in the presence of APC, self-presentation by BAL T cells did not induce detectable IL-2 production, and in its absence these activated T cells die from programmed cell death. Resting BAL CD4+ T cells constitutively express high levels of HLA-DP, lymphocyte function-associated antigen 1 (LFA-1) and ICAM-3. When stimulated with beryllium/APC, the adhesion molecule ICAM-1 was up-regulated, as well as several costimulation molecules including CD28, OX-40 (CD134), 4-1-BB (CD137) and B7-1 (CD80). Notably, CD28 was not up-regulated during self-presentation by BAL T cells, and these cells do not express OX-40L, suggesting that lack of appropriate costimulation was responsible for programmed cell death observed upon beryllium self-presentation. Restricting anti-MHC class II mAb completely eliminated beryllium-induced T cell proliferation during self-presentation and significantly reduced IFN-gamma and TNF-alpha production. Our data demonstrate for the first time that self-presentation by BAL T cells in response to beryllium can occur ex vivo, in the absence of professional APC, with a specific dependence on T cell-expressed MHC class II molecules and exogenous IL-2 for survival.
Assuntos
Apresentação de Antígeno/imunologia , Beriliose/imunologia , Berílio/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Of potential importance to multiple sclerosis (MS), oligodendroglial alpha B-crystallin is expressed and associated with the myelin sheath at the earliest stage of MS lesion development. We selected T-cell lines specific for human alpha B-crystallin from peripheral blood mononuclear cells (PBMC) of HLA-DR2 homozygous MS patients and found that the alpha B-crystallin-specific T-cells were CD4+ and restricted by DRB1*1501, and expressed Th1 cytokines. The CD4 T-cell epitopes of human alpha B-crystallin were determined by proliferation of alpha B-crystallin-specific T-cell lines to 17 20-mer synthetic overlapping peptides spanning the entire molecule of human alpha B-crystallin. It was found that the HLA-DR2 donor-derived alpha B-crystallin-specific T-cell lines proliferated to alpha B-crystallin peptides 21-40, 41-60, and to a lesser extent, 131-150. These T-cell proliferation responses were associated with intracellular expression of interleukin-2 (IL-2) and secretion of interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). The amino acid sequences of these peptides were compatible with predicted HLA-DR2-restricted binding motifs. PBMC of an early active MS patient proliferated to the epitope-containing peptides significantly better than did those of later stage MS patients or healthy controls. Taken together, these findings suggest that autoreactive alpha B-crystallin-specific Th1 cells may have the potential to contribute to MS pathogenesis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Esclerose Múltipla/metabolismo , Cadeia B de alfa-Cristalina/imunologia , Cadeia B de alfa-Cristalina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/imunologia , Linfócitos T CD4-Positivos/citologia , Divisão Celular/imunologia , Linhagem Celular , Epitopos de Linfócito T/genética , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Análise de Sequência de Proteína/métodos , Cadeia B de alfa-Cristalina/genéticaRESUMO
The goal of this study was to establish an unlimited and standardized source of humanized myelin peptide-specific T cells for in vitro testing of biological function. Thus, we perpetuated myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide-specific T cells obtained from immunized HLA-DRB1*1501-transgenic (Tg) mice by somatic fusions with BW5147 thymoma cells or BW5147 T-cell receptor (TCR) alpha(-)beta(-) variant (BW5147 variant) cells. The resulting T-cell hybridomas responded strongly to both mouse MOG-35-55 (42S) and human MOG-35-55 peptide (42P), regardless of which peptide was used for initial immunization, and were DRB1*1501 restricted. The MOG-35-55-reactive T-cell hybridomas were CD3(+)CD4(+)CD8(-) and expressed intracellular Th1 cytokines upon concanavalin A stimulation. Clones from either human MOG-35-55- or mouse MOG-35-55-selected hybridomas uniquely expressed the TCR BV8 gene in combination with AV17 and AV11 genes. V gene analyses confirmed the expression of TCR AV1, AV11, AV16, BV1, and BV5 gene segments in the widely used fusion partner BW5147 and demonstrated deletion of TCR AV1, AV11, and BV1 in the BW5147 variant. T-cell hybridomas were positively stained with anti-TCR beta-chain antibody on the cell surface, whereas neither BW5147 nor its variant had positive TCR surface expression. For functional application, we found that a monomeric form of the human HLA-DR2-derived recombinant T-cell receptor ligand (RTL) covalently linked to human MOG-35-55 peptide specifically inhibited proliferation of a hybridoma clone selected with human MOG-35-55 but not a different hybridoma clone selected with myelin basic protein. The RTL-induced inhibition in vitro of the human MOG-35-55 peptide-specific hybridoma reflected the ability of the RTL to inhibit experimental autoimmune encephalomyelitis induced by human MOG-35-55 peptide in HLA-DR2 transgenic mice. Thus, the MOG-35-55 peptide-specific T-cell hybridoma from DR2-Tg mice represents a novel humanized T-cell reagent useful for standardized biological screening of both DR2-restricted stimulation and RTL-dependent inhibition of response to human MOG-35-55 peptide.
Assuntos
Glicoproteínas/imunologia , Antígenos HLA-DR/metabolismo , Hibridomas/metabolismo , Fragmentos de Peptídeos/imunologia , Linfócitos T/metabolismo , Animais , Formação de Anticorpos/fisiologia , Southern Blotting/métodos , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Relação Dose-Resposta Imunológica , Citometria de Fluxo/métodos , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade do Receptor de Antígeno de Linfócitos T/fisiologia , Fatores de TempoRESUMO
CD4+CD25+ regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4+CD25+ T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4+CD25- indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25- indicator cells by both CD4+CD25(+high) and CD4+CD25(+low) T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO- (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA-4 and GITR than CD4+CD25- T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.
Assuntos
Envelhecimento/imunologia , Bioensaio/métodos , Linfócitos T CD4-Positivos/citologia , Tolerância Imunológica/imunologia , Receptores de Interleucina-2/imunologia , Linfócitos T/imunologia , Adulto , Anticorpos , Antígenos CD , Antígenos de Diferenciação/imunologia , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4 , Regulação para Baixo/imunologia , Feminino , Citometria de Fluxo/métodos , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Tolerância Imunológica/efeitos dos fármacos , Separação Imunomagnética , Interleucina-2/farmacologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Fatores Sexuais , Linfócitos T/efeitos dos fármacosRESUMO
Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.