RESUMO
A study describing the (1)H, (13)C and (15)N backbone and side chain chemical shift assignments and secondary structure of Skint-1 a prototypic member of a family of mouse genes, of which Skint-1 is involved in the development of the dendritic epidermal T cell (DETC) subset of γδ T cells.
Assuntos
Imunoglobulinas/química , Imunoglobulinas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Subpopulações de Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Animais , Ligantes , Camundongos , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
The potent anti-tumour activities of gammadelta T cells have prompted the development of protocols in which gammadelta-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vgamma9Vdelta2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vgamma9Vdelta2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vgamma9Vdelta2 T cell numbers emerged, as seven patients who failed to sustain Vgamma9Vdelta2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vgamma9Vdelta2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vgamma9Vdelta2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatment-refractory patients with advanced breast cancer.
Assuntos
Neoplasias da Mama/terapia , Difosfonatos/uso terapêutico , Imidazóis/uso terapêutico , Imunoterapia/métodos , Interleucina-2/uso terapêutico , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/citologia , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Idoso , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Proliferação de Células/efeitos dos fármacos , Quimiocinas/sangue , Citocinas/sangue , Difosfonatos/efeitos adversos , Difosfonatos/farmacologia , Progressão da Doença , Esterases/metabolismo , Feminino , Hemiterpenos/farmacologia , Humanos , Imidazóis/efeitos adversos , Imidazóis/farmacologia , Interferon gama/metabolismo , Interleucina-2/efeitos adversos , Interleucina-2/farmacologia , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Lisina/análogos & derivados , Lisina/metabolismo , Pessoa de Meia-Idade , Mucina-1/sangue , Compostos Organofosforados/farmacologia , Indução de Remissão , Terapia de Salvação , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Resultado do Tratamento , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Ácido ZoledrônicoRESUMO
Studies on physiology and pathology as they relate to the immune system draw heavily upon rodent models. With the increasing impetus provided by initiatives in translational medicine, the demand for ever more sophisticated, 'humanized' murine models is greater than ever. However, the design and implementation of studies in such mice is far from trivial. Here we provide a technical perspective on the increasing interest in developing humanized mice. We give examples of primary data starting with the routine procurement of human donor material, through CD34(+) cell purification prior to engraftment to injection into immunocompromised mice. Our goal is to provide practical advice to the many investigators who may be commencing or considering such studies.
Assuntos
Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Antígenos CD34/análise , Separação Celular/métodos , Humanos , Camundongos , Camundongos SCID , Transplante HeterólogoRESUMO
Intraepithelial lymphocytes (IELs) are abundant, evolutionarily conserved T cells, commonly enriched in T cell receptor (TCR) gammadelta expression. However, their primary functional potential and constitutive activation state are incompletely understood. To address this, serial analysis of gene expression (SAGE) was applied to murine TCRgammadelta+ and TCRalphabeta+ intestinal IELs directly ex vivo, identifying 15,574 unique transcripts that collectively portray an "activated yet resting," Th1-skewed, cytolytic, and immunoregulatory phenotype applicable to multiple subsets of gut IELs. Expression of granzymes, Fas ligand, RANTES, prothymosin beta4, junB, RGS1, Btg1, and related molecules is high, whereas expression of conventional cytokines and high-affinity cytokine receptors is low. Differentially expressed genes readily identify heterogeneity among TCRalphabeta+ IELs, whereas differences between resident TCRgammadelta+ IELs and TCRalphabeta+ IELs are less obvious.
Assuntos
Células Epiteliais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/metabolismo , Animais , Diferenciação Celular , Bases de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Biblioteca Gênica , Imunofenotipagem , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The localization of gammadelta T cells within epithelia suggests that these cells may contribute to the down-regulation of epithelial malignancies. We report that mice lacking gammadelta cells are highly susceptible to multiple regimens of cutaneous carcinogenesis. After exposure to carcinogens, skin cells expressed Rae-1 and H60, major histocompatibility complex-related molecules structurally resembling human MICA. Each of these is a ligand for NKG2d, a receptor expressed by cytolytic T cells and natural killer (NK) cells. In vitro, skin-associated NKG2d+ gammadelta cells killed skin carcinoma cells by a mechanism that was sensitive to blocking NKG2d engagement. Thus, local T cells may use evolutionarily conserved proteins to negatively regulate malignancy.
Assuntos
Epiderme/imunologia , Vigilância Imunológica , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores Imunológicos/imunologia , Neoplasias Cutâneas/imunologia , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Carcinógenos , Linhagem Celular , Citotoxicidade Imunológica , Dimerização , Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Dados de Sequência Molecular , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Conformação Proteica , Dobramento de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores Imunológicos/metabolismo , Receptores de Células Matadoras Naturais , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/induzido quimicamenteRESUMO
c-Myc is associated with cell growth and cycling in many tissues and its deregulated expression is causally implicated in cancer, particularly lymphomagenesis. However, the contribution of c-Myc to lymphocyte development is unresolved. We show here that the formation of normal lymphocytes by c-Myc-/- cells is selectively defective. c-Myc-/- cells are inefficient, in an age-dependent manner, at populating the thymus, and subsequent thymocyte maturation is ineffective: they fail to grow and proliferate normally at the late double-negative (DN) CD4-CD8- stage. Because N-Myc expression in thymocytes usually declines at the late DN stage, these results confirm that the nonredundant contributions of Myc family members to development are related to their distinct patterns of developmental gene expression.
Assuntos
Proteínas Proto-Oncogênicas c-myc/imunologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Divisão Celular , Quimera/genética , Quimera/imunologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes RAG-1 , Genes myc , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/genética , Linfócitos T/citologia , Timo/citologia , Timo/imunologiaAssuntos
Gastroenteropatias/imunologia , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular , Doença de Crohn/imunologia , Desenvolvimento Embrionário e Fetal/imunologia , Gastroenteropatias/microbiologia , Humanos , Tolerância Imunológica , Doenças Inflamatórias Intestinais/imunologia , Tecido Linfoide/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4(-)8(-) thymocytes and is down-regulated across the CD4(-)8(-) to CD4(+)8(+) transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4(-)8(-) cells, a failure to down-regulate CD25 in post-CD4(-)8(-) subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3.
Assuntos
Proteínas I-kappa B , Leucemia de Células T/metabolismo , Linfoma de Células T/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Apoptose/fisiologia , Sequência de Bases , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Sobrevivência Celular , Primers do DNA , Proteínas de Ligação a DNA/fisiologia , Leucemia de Células T/patologia , Linfoma de Células T/patologia , Camundongos , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Receptor Notch3 , Receptor Notch4 , Receptores de Superfície Celular/genética , Receptores Notch , Timo/patologiaRESUMO
The analysis of T cell receptor alpha (TCR alpha) chains in mice transgenic for a TCR beta chain has allowed us to demonstrate a central role for self-peptides in the positive intrathymic selection of major histocompatibility complex (MHC) class II-restricted T cells. Analysis of specific V alpha-J alpha joins in mature CD4+ TCRhigh thymocytes and in peripheral CD4+ T cells revealed a limitation in amino-acid sequences. By analysis of immature thymocytes, we could show that this limited repertoire was selected from a more diverse repertoire. By analysis of the same beta chain-transgenic mice bred to H-2Ma-deficient mice that express one or a very limited number of peptides, we could demonstrate that the V alpha-J alpha join repertoire was now altered and much more limited. Together, these data provide molecular and genetic evidence that the intrathymic positive selection of the TCR repertoire is critically affected by self-peptides presented by MHC class II molecules, most likely on thymic cortical epithelial cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Aminoácidos , Animais , Autoantígenos/imunologia , Sequência de Bases , Conalbumina/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
To explore the mechanisms by which alpha beta T cells and gamma delta T cells regulate systemic autoimmunity, lupus-prone mice were rendered deficient in CD40 ligand and/or alpha beta T cells by intercrossing CD40L -/- and TCR-alpha -/- knockouts, generating CD40L-intact or -deficient (CD40L+ or CD40L-), alpha beta T cell-intact or -deficient (alpha beta+ or alpha beta-) MRL-lpr/lpr animals. As expected, CD40L+ alpha beta+ mice developed high titer autoantibodies along with severe renal and cutaneous disease. CD40L+ alpha beta- animals developed lower levels of autoantibodies, accompanied by less severe or delayed renal and cutaneous disease. CD40L- alpha beta+ mice developed even lower titers of autoantibodies and less severe renal disease yet developed cutaneous lesions indistinguishable from those of CD40L+ alpha beta+ disease. Most surprisingly, CD40L- alpha beta- animals developed higher levels of some autoantibodies than did CD40L- alpha beta+ mice and developed renal disease similar in severity to CD40L+ alpha beta- counterparts; however, they failed to develop skin disease. Thus, disruption of CD40L and alpha beta T cells provides a novel dissection of the physiology and pathology of murine lupus; while these data confirm previous findings demonstrating a role for CD40L-dependent, alpha beta T cell-dependent mechanisms in autoantibody production and renal disease in murine lupus, they also: 1) establish that alpha beta T cells may drive autoimmune skin disease by a CD40L-independent mechanism; 2) identify a role for CD40L in non-alpha beta T cell-dependent autoantibody production and autoimmune skin disease; and 3) suggest a role for alpha beta T cells in the down-regulation of autoimmunity driven by other T cells. Thus, both alpha beta and non-alpha beta T cells, such as gamma delta T cells, regulate systemic autoimmunity by CD40L-dependent and -independent mechanisms.
Assuntos
Doenças Autoimunes/imunologia , Antígenos CD40/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Autoanticorpos/biossíntese , Doenças Autoimunes/metabolismo , Antígenos CD40/genética , Ligante de CD40 , Doenças do Complexo Imune/imunologia , Doenças do Complexo Imune/metabolismo , Nefropatias/genética , Nefropatias/imunologia , Ligantes , Linfopenia/genética , Linfopenia/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Mutantes , Dermatopatias/genética , Dermatopatias/imunologiaRESUMO
Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using alpha beta T cell-deficient (TCR alpha -/-) mice, are presented here. The results clearly show that TCR alpha -/- mice cannot mount CS, implicating alpha beta T cells as the critical CS-effector cells. However, TCR alpha -/- mice can, after high dose tolerance, downregulate alpha +/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized alpha -/- mice are shown to include gamma delta TCR+ cells that also can downregulate interferon-gamma production by the targeted CS-effector cells in vitro. Downregulation by gamma delta cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that gamma delta T cells cannot fulfill CS-effector functions performed by alpha beta T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by gamma delta T cells. Comparisons are likewise considered with downregulation by gamma delta T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.
Assuntos
Dermatite de Contato , Interferon gama/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/imunologia , Células Cultivadas , Regulação para Baixo , Tolerância Imunológica , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Baço/imunologiaRESUMO
T cell development is regulated by extracellular signals that mediate cellular proliferation and differentiation via specific signal transduction pathways. To determine the importance of the mitogen-activated protein kinase (MAP kinase) pathway in thymocyte development, we analyzed transgenic mice expressing dominant negative Raf (DN Raf) and a constitutively active v-Raf under the control of the p56lck proximal promoter. DN Raf had a profound effect on T cell receptor (TCR)-mediated signaling events as assessed by the inhibition of mitogen-induced proliferation of thymocytes in vitro. Overall thymocyte numbers were decreased by at most twofold from nontransgenic littermates. Positive selection was inhibited in DN Raf transgenic mice, as evidenced by both reduced numbers of mature thymocytes and a decrease in CD8+ thymocytes in female mice doubly transgenic for DN-Raf and a class I-restricted H-Y TCR. In contrast, the differentiation of double-positive thymocytes to single-positive thymocytes was enhanced in H-YTCR transgenic mice expressing constitutively active Raf (v-Raf). Thus, Raf regulates positive selection in the thymus.
Assuntos
Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Subpopulações de Linfócitos T/citologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Diferenciação Celular , Antígeno H-Y/imunologia , Lectinas Tipo C , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas v-raf , Proteínas Proto-Oncogênicas c-raf , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Oncogênicas de Retroviridae , Transdução de Sinais , Timo/citologia , Timo/crescimento & desenvolvimentoRESUMO
Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.
Assuntos
Precursores Enzimáticos/genética , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Animais , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/metabolismo , Células Epiteliais , Epitélio/metabolismo , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intracelular , Linfócitos/enzimologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Quinase SykRESUMO
Fas (CD95) and its ligand are central regulatory molecules in hematopoietic cells. Previous studies have suggested a role for Fas in the regulation of tumor progression, but Fas has not yet been conclusively identified as a tumor suppressor. Fas-deficient individuals lack malignant tumors, perhaps because of regulation by T cells. To investigate such a possibility, mice deficient in both T cells and Fas were generated, and they were found to develop severe B cell dysregulation characterized by malignant, lethal B cell lymphoma. Lymphoma arose from a monoclonal B220+CD19-CD5-CD23- B cell secreting immunoglobulin M, kappa rheumatoid factor. In contrast, animals containing alpha beta T cells, gamma delta T cells, and/or functional Fas suppressed the development of lymphoma. These data indicate that Fas functions as a tumor suppressor, and identifies roles for both alpha beta T cells and gamma delta T cells in Fas-independent tumor regulation.
Assuntos
Linfoma/patologia , Linfócitos T/fisiologia , Receptor fas/fisiologia , Animais , Feminino , Rim/patologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
SUMMARY: Disruption of the cyclin-dependent kinase-inhibitory domain of p27 enhances growth of mice. Growth is attributed to an increase in cell number, due to increased cell proliferation, most obviously in tissues that ordinarily express p27 at the highest levels. Disruption of p27 function leads to nodular hyperplasia in the intermediate lobe of the pituitary. However, increased growth occurs without an increase in the amounts of either growth hormone or IGF-I. In addition, female mice were infertile. Luteal cell differentiation is impaired, and a disordered estrus cycle is detected. These results reflect a disturbance of the hypothalamic-pituitary-ovarian axis. The phenotypes of these mice suggest that loss of p27 causes an alteration in cell proliferation that can lead to specific endocrine dysfunction.
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Supressoras de Tumor , Animais , Ciclo Celular/genética , Divisão Celular/genética , Inibidor de Quinase Dependente de Ciclina p27 , Feminino , Marcação de Genes , Transtornos do Crescimento/fisiopatologia , Hiperplasia , Sistema Hipotálamo-Hipofisário/patologia , Sistema Hipotálamo-Hipofisário/fisiopatologia , Infertilidade Feminina/genética , Masculino , Camundongos , Camundongos Knockout , Ovário/patologia , Ovário/fisiopatologia , FenótipoRESUMO
To investigate the possibility that non-alpha beta T cell-dependent mechanisms can induce systemic autoimmune disease, and to address the roles of alpha beta T cells in murine lupus, we analyzed lupus-prone MRL mice congenitally deficient in alpha beta T cells. Surprisingly, TCR-alpha-/- MRL mice developed several characteristics of human systemic lupus erythematosus, including hypergammaglobulinemia, autoantibodies against DNA and small nuclear ribonucleoproteins, and immune deposits in kidneys. These results, which contrast with past studies concluding that MRL autoimmunity requires CD4+ alpha beta T cells, demonstrate that non-alpha beta T cell-dependent mechanisms are capable of inducing lupus phenomena, and further suggest that MRL disease may consist of both alpha beta T cell-independent and alpha beta T cell-dependent mechanisms.
Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/patologia , Animais , Anticorpos Antinucleares/análise , Especificidade de Anticorpos/genética , Modelos Animais de Doenças , Humanos , Hipergamaglobulinemia/genética , Hipergamaglobulinemia/imunologia , Lúpus Eritematoso Sistêmico/genética , Nefrite Lúpica/genética , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Camundongos , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Productive gene rearrangement at the T-cell receptor (TCR) beta-chain locus facilitates formation of the "pre-TCR," a molecular complex that is important for the subsequent development of alpha beta T cells. The transition of thymocytes from a population of cells undergoing TCRbeta chain genes to a population enriched in cells with productively rearranged TCRbeta chain genes is known as "beta selection." This is the first point in alpha beta T-cell development at which the products of an activated TCR locus define cell phenotype. Toward an understanding of these events, this study has focused on a set of thymocytes defined by cell surface phenotype as HSA+ CD44low CD25+, in which the bulk of TCRbeta gene rearrangement occurs. The analysis of this set, presented here, allows its novel subdivision into two subsets that are respectively strong candidates for cells immediately prior to and immediately following TCRbeta selection. Cells that have passed beta selection differ from the preceding cells by several criteria, including hyperphosphorylation of Rb, increased expression of cyclins A and B, down-regulation of p27, increased CDK2 activity, an induction of cdc2 activity, and progression through DNA synthesis. Consistent with these changes being attributable to productive TCRbeta chain gene rearrangement, the identified "beta-selected" subset is not detected in mutant mice that cannot assemble a pre-TCR. Interestingly, there is a coincident selective and transient down-regulation of the protein RAG2, on which TCR gene rearrangement obligatorily depends. Together, these findings demonstrate that productive TCR gene rearrangement is associated with events that can ensure thymocyte expansion and monoclonality.
Assuntos
Ciclo Celular , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Proteínas de Homeodomínio , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/citologia , Timo/citologia , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Separação Celular , Células Clonais , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Hialuronatos/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/metabolismo , RNA Mensageiro/genética , Receptores de Interleucina-2/análise , Proteína do Retinoblastoma/metabolismo , Linfócitos T/enzimologiaRESUMO
BACKGROUND: The subdivision of T cells into alpha beta and gamma delta subtypes is conserved throughout vertebrate development. The respective alpha beta and gamma delta T-cell receptors (TCRs) are encoded by somatically rearranged genes. There has been broad speculation as to whether an individual thymocyte can become either a gamma delta T cell or an alpha beta T cell as a result of stochastic gene rearrangement processes, or whether the two types of T cell are derived from separate lineages. Many of the experimental findings are apparently conflicting, however, and the issue--a basic one in immunology and development--remains unresolved. RESULTS: To address this issue, we have used the recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, which allows us to examine quantitatively the status of TCR gamma and delta genes in postnatal alpha beta T cells and their progenitors. Interestingly, such cells are depleted of productively rearranged delta and gamma genes, which can encode delta and gamma TCR polypeptide chains. However, in mice that can rearrange TCR delta gene segments, but in which the TCR delta gene is non-functional in other respects, no such depletion of productive rearrangements is seen. CONCLUSION: The quantitative data that we have obtained fulfill the predictions of the stochastic hypothesis: that is, a progenitor T cell first attempts to become a gamma delta T cell and, if unsuccessful, then attempts to become an alpha beta T cell. Thus, alpha beta and gamma delta T cells can derive from a common precursor thymocyte. In the simplest case, therefore, lineage-determining factors are the successful rearrangement of both gamma and delta genes before TCR alpha gene rearrangements occur, which lead to deletion of the TCR delta locus and thereby preclude further gamma delta T-cell differentiation. In contrast, successful rearrangement of the TCR beta locus remains compatible with cells becoming either gamma delta or alpha beta T cells.
Assuntos
Rearranjo Gênico do Linfócito T , Hematopoese , Células-Tronco Hematopoéticas/citologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/citologia , Animais , Sequência de Bases , Diferenciação Celular , DNA Nucleotidiltransferases/metabolismo , Células Dendríticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Processos Estocásticos , VDJ RecombinasesRESUMO
Retinoids play an important role as differentiating agents in a variety of normal and neoplastic cells and have been reported to induce ICAM-1 levels in melanomas, a phenomenon that we confirm in this paper. The effects of retinoids on gene expression usually involve the binding of specific retinoic acid receptor trans-acting factors (RARs) with their ligands, which then interact with specific target sites, the retinoic acid responsive elements (RAREs) present in the promoters of responsive genes. In the case of ICAM-1, we have cloned and analyzed the proximal regulatory region of the human gene. We show that the ICAM-1 promoter is RA-inducible, that it contains a putative consensus RARE (GGGTCATCGCCCTGCC), which binds in vitro RAR alpha complemented with RXRs, and that mutation of the RARE abrogates promoter responsiveness to RA. These studies allow ICAM-1 to be added to the list of genes transcriptionally activated by RA acting through an RARE element.
Assuntos
Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Regiões Promotoras Genéticas , Tretinoína/farmacologia , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Humanos , Melanoma , Dados de Sequência Molecular , Mutagênese , Metástase Neoplásica , Receptores do Ácido Retinoico/metabolismo , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
gamma delta T-cell hybrid clones were obtained from intestinal intraepithelial lymphocytes (i-IEL) by fusion with the BW5147 thymoma line. Four clones which expressed different V gamma/V delta genes were selected for further study. All of the gamma delta clones secreted interleukin-2 (IL-2) in the presence of the BALB/c-derived B-lymphoma line, A20. No alpha beta T-cell hybrid clones derived from spleen or i-IEL responded to A20. We obtained several pieces of evidence which strongly suggest that these responses are mediated by the gamma delta T-cell receptor (TcR). Class II major histocompatibility complex (MHC), FcR and surface Ig expressed on A20 are not involved in the response. Native i-IEL derived from BALB/c selectively survive in culture in the presence of A20 cells. The ligand may be a superantigen-like molecule because all our gamma delta T-cell clones responded to A20 in spite of their different combinations of V gamma/V delta gene segments.