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1.
J Exp Med ; 183(3): 1111-8, 1996 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8642253

RESUMO

The common cytokine receptor gamma chain (gammac), which is a functional subunit of the receptors for interleukins (IL)-2, -4, -7, -9, and -15, plays an important role in lymphoid development. Inactivation of this molecule in mice leads to abnormal T cell lymphopoiesis characterized by thymic hypoplasia and reduced numbers of peripheral T cells. To determine whether T cell development in the absence of gammac is associated with alterations of intrathymic and peripheral T cell selection, we have analyzed gammac-deficient mice made transgenic for the male-specific T cell receptor (TCR) HY (HY/gammac- mice). In HY/gammac- male mice, negative selection of autoreactive thymocytes was not diminished; however, peripheral T cells expressing transgenic TCR-alpha and -beta chains (TCR-alphaT/betaT) were absent, and extrathymic T cell development was completely abrogated. In HY/gammac- female mice, the expression of the transgenic TCR partially reversed the profound thymic hypoplasia observed in nontransgenic gammac- mice, generating increased numbers of thymocytes in all subsets, particularly the TCR-alphaT/betaT CD8+ single-positive thymocytes. Despite efficient positive selection, however, naive CD8+ TCR-alphaT/betaT T cells were severely reduced in the peripheral lymphoid organs of HY/gammac- female mice. These results not only underscore the indispensible role of gammac in thymocyte development, but also demonstrate the critical role of gammac in the maintenance and/or expansion of peripheral T cell pools.


Assuntos
Receptores de Citocinas/química , Receptores de Citocinas/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Linfócitos T CD4-Positivos , Células Cultivadas , Cruzamentos Genéticos , Duodeno/imunologia , Feminino , Citometria de Fluxo , Ativação Linfocitária , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Interleucina-2/genética , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Timo/citologia
2.
J Exp Med ; 185(8): 1395-401, 1997 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-9126920

RESUMO

In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells. For this purpose, we studied common gamma chain (gamma c)-deficient mice, which demonstrate a selective defect in CD3- NK cell development relative to conventional TCR-alphabeta T cells. NK thymocytes differentiate in gamma c- mice as shown by the normal percentage of TCR Vbeta8+ CD4-CD8- cells and the normal quantity of thymic Va14-Jalpha281 mRNA that characterize the NK T repertoire. However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta. Despite these phenotypic abnormalities, gamma c- NK thymocytes could produce normal amounts of IL-4. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4-producing capacity can be clearly dissociated from the acquisition of the NK phenotype. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-alphabeta T cells than to NK cells with respect to cytokine dependency. We also failed to detect peripheral NK T cells in these mice, demonstrating that gamma c-dependent interactions are required for export and/or survival of NK T cells from the thymus. These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.


Assuntos
Antígenos Ly , Antígenos de Superfície/fisiologia , Células Matadoras Naturais/citologia , Lectinas Tipo C , Glicoproteínas de Membrana/fisiologia , Linfócitos T/citologia , Timo/citologia , Animais , Diferenciação Celular , Citometria de Fluxo , Imunofenotipagem , Interleucina-4/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Knockout , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Receptores Semelhantes a Lectina de Células NK , Subpopulações de Linfócitos T/citologia
3.
J Exp Med ; 185(9): 1541-7, 1997 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-9151891

RESUMO

The development of pre-T cells with productive TCR-beta rearrangements can be mediated by each the pre-T cell receptor (pre-TCR), the TCR-alphabeta as well as the TCR-gammadelta, albeit by distinct mechanisms. Although the TCR-gammadelta affects CD4-8- precursor cells irrespective of their rearrangement status by TCR-beta mechanisms not involving TCR-beta selection, both the pre-TCR and the TCR-alphabeta select only cells with productive TCR-beta genes for expansion and maturation. The TCR-alphabeta appears to be much less effective than the pre-TCR because of the paucity of TCR-alpha proteins in TCR-beta-positive precursors since an early expressed transgenic TCR-alphabeta can largely substitute for the pre-TCR. Thus, the TCR-alphabeta can assume a role not only in the rescue from programmed cell death of CD4+8+ but also of CD4-8- thymocytes. In evolution this double function of the TCR-alphabeta may have been responsible for the maturation of alphabeta T cells before the advent of the pre-TCR-alpha chain.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Linfócitos T/citologia , Animais , Diferenciação Celular , Receptores de Hialuronatos/fisiologia , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-2/metabolismo , Subpopulações de Linfócitos T/citologia , Timo/citologia
4.
J Exp Med ; 186(8): 1277-85, 1997 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-9334367

RESUMO

We have investigated the role of common gamma chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma/delta T cells. TCR-gamma/delta-bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma/delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma/delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR V gamma 1 or TCR V gamma 1/V delta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma/delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma/delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma/delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma/delta lineage.


Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Receptores de Citocinas/fisiologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T/genética , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T/genética , Linfopenia/genética , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/análise , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Citocinas/deficiência , Receptores de Citocinas/genética , Subpopulações de Linfócitos T/fisiologia
5.
J Exp Med ; 171(5): 1697-704, 1990 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-2139699

RESUMO

We have characterized a child with a severe combined immunodeficiency disease syndrome with increased numbers, but a normal distribution, of CD3+ T cells. This patient's immunological defect appears to be attributable to a selective deficiency in T cell production of IL-2, which may reflect a subtle abnormality in the IL-2 gene locus or a defect in a regulatory factor necessary for IL-2 transcription. The increased numbers of phenotypically normal T cells in this patient suggest that alternative pathways of T cell development exist in man or that IL-2 production intra- and extrathymically is controlled via distinct regulatory mechanisms.


Assuntos
Síndromes de Imunodeficiência/imunologia , Interleucina-2/deficiência , Linfócitos T/imunologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3 , Células Cultivadas , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Recém-Nascido , Interleucina-2/biossíntese , Interleucina-2/genética , Ionomicina/farmacologia , Ativação Linfocitária , Masculino , Receptores de Antígenos de Linfócitos T/análise , Linfócitos T/efeitos dos fármacos
6.
Curr Biol ; 7(7): R424-6, 1997 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-9210372

RESUMO

That the signal transduction pathways used by the cytokines IL-2 and IL-15 are identical would suggest that these cytokines have redundant roles in lymphoid development; instead, IL-2 is the guardian of thymus-derived T-cell homeostasis, while interleukin-15 promotes extrathymic development of T and NK cells.


Assuntos
Interleucina-15/metabolismo , Interleucina-2/metabolismo , Células Matadoras Naturais/citologia , Proteínas do Leite , Linfócitos T/citologia , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Janus Quinase 1 , Janus Quinase 3 , Células Matadoras Naturais/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Interleucina-2/metabolismo , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Linfócitos T/metabolismo , Transativadores/metabolismo
7.
J Immunol Methods ; 141(1): 123-31, 1991 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-1830894

RESUMO

The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8 alpha chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8 alpha/beta heterodimer. In order to generate antibodies specific for CD8 beta, murine fibroblast transfectants were constructed which express the human CD8 beta chain in combination with either the human CD8 alpha chain or the murine CD8 alpha homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8 alpha/beta heterodimers induced moderate anti-CD8 alpha titers, but were weakly effective in generating anti-CD8 beta titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8 alpha and human CD8 beta) induced high anti-CD8 beta titers in immunized mice. Following fusion of splenocytes from mice immunized with mixed-species CD8 transfectants, the mAb 5F2 was isolated which specifically recognizes the human CD8 beta chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8 beta chain irrespective of its pairing partner, and can immunoprecipitate the CD8 beta protein from cells transfected with the CD8 beta gene in the absence of the human or mouse CD8 alpha gene product. Anti-CD8 beta antibodies should help elucidate the extent of biochemical heterogeneity of the CD8 beta protein, and will also be useful in defining the role of the CD8 beta protein in thymocyte and lymphocyte development, recognition and activation.


Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Transfecção , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/genética , Complexo CD3 , Antígenos CD8 , Feminino , Imunofluorescência , Humanos , Células L , Camundongos , Camundongos Endogâmicos C3H , Receptores de Antígenos de Linfócitos T/análise
8.
Immunol Lett ; 57(1-3): 5-8, 1997 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9232417

RESUMO

The development of pre-T-cells with productive T-cell receptor beta (TCR beta) rearrangements can be furthered by each of the pre-T-cell receptors (pre-TCR), the alpha beta TCR as well as the gamma delta TCR, albeit by distinct mechanisms. While the gamma delta TCR affects CD4-8- precursor cells irrespective of their TCR beta rearrangement status both the pre-TCR and the alpha beta TCR select only cells with productive TCR beta genes for expansion and maturation. The alpha beta TCR is much less effective than the pre-TCR because of the paucity of TCR alpha proteins in TCR beta positive precursors.


Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Animais , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética
9.
J Reprod Immunol ; 35(2): 111-33, 1997 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-9421796

RESUMO

Maternal lymphocytes having a large and granulated morphology accumulate at healthy implantation sites in normal mice. Insight into the functions of these cells has come from a previous study of two independent lines of mice deficient in natural killer (NK) cells. In pregnant Tg epsilon 26 mice, vascular pathology was found that led to the major complications of either fetal death or intrauterine growth retardation. In pregnant p56lck null x IL-2R beta null mice, extensive distension of the decidua was observed that separated the placenta from the myometrium and appeared to be interstitial edema. To strengthen assignment of uterine large granulated lymphocytes to the NK cell lineage and to understand which aspects of NK cell biology may be important for a uterine-based, pregnancy-associated subset, mid-gestation implantation sites from a new series of mice having gene deletions which alter NK cells (IL-2R gamma null, Stat4 null, IL-12 p40 null, beta 7 integrin null and Muc-1 null) have been examined histologically. The findings support the assignment of pregnancy-associated large granulated cells of mice to the NK cell lineage and suggest that the primary functions of these tissue-based NK cells are to support normal development of the decidua and/or its vasculature using pathways that involve IL-12 mediated signal transduction.


Assuntos
Cadeias beta de Integrinas , Células Matadoras Naturais/metabolismo , Útero/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Deleção de Genes , Integrinas/genética , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Mutantes , Placenta/metabolismo , Placenta/patologia , Gravidez , Receptores de Interleucina-2/genética , Fator de Transcrição STAT4 , Transdução de Sinais , Transativadores/metabolismo , Útero/citologia
10.
Hybridoma ; 7(4): 397-405, 1988 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3169807

RESUMO

We have analyzed the expression of cyclophosphamide (CY) resistance in somatic cell hybrids of mouse LPC-1/CY-R plasmacytoma and P3-X63-Ag8.653 (Ag8) myeloma cells. LPC-1/CY-R tumor is resistant to curative doses (60-250 mg/kg body weight) of CY. The median survival time (MST) of drug treated LPC-1/CY-R tumor bearing mice is 25 days, similar to that of untreated mice. LPC-1/CY-R tumor cells secrete an IgG 2a kappa M component and do not survive in tissue culture. Ag8 tumor cells are CY sensitive, are selected out in hypoxanthine-aminopterin-thymidine (HAT) media and do not secrete any immunoglobulin. Hybrids formed between these two cell lines survived in HAT and secreted IgG 2a kappa. Hybrid cells exhibited greater ploidy than that of their parents and contained the metacentric marker chromosome of the Ag8 cell line. Hybrid cells exhibited the same growth characteristics in BALB/c mice as that of their LPC-1/CY-R parent and were resistant to curative doses of CY. These studies demonstrate that, CY resistance is a somatic cell dominant trait which can be transferred to daughter cells via somatic cell hybridization. Availability of somatic cell hybrids produced between CY sensitive and resistant tumor cells may provide useful tools to study the biochemical nature and the somatic cell genetics of this drug resistant trait.


Assuntos
Ciclofosfamida/farmacologia , Hibridomas/efeitos dos fármacos , Plasmocitoma/tratamento farmacológico , Animais , Resistência a Medicamentos/genética , Feminino , Imunoglobulinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Plasmocitoma/genética
12.
EMBO J ; 7(11): 3465-70, 1988 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3145196

RESUMO

We have previously identified a monoclonal antibody, T8/2T8-5H7, which clustered serologically with CD8 monoclonal antibodies, but lacked reactivity with L cell transfectants expressing the human CD8 molecule (Lyt-2 homologue). Based on these observations, we postulated that T8/2T8-5H7 might recognize the human Lyt-3 gene product. To test this hypothesis, we have isolated a full-length cDNA encoding the human Lyt-3 molecule and have characterized its product in additional transfection experiments. The results of these studies indicate that the human Lyt-3 cDNA encodes a product recognized by the antibody T8/2T8-5H7. Interestingly, the human Lyt-3 molecule cannot be expressed alone, but requires the human Lyt-2 homologue for efficient cell surface expression. A heterodimer composed of the human Lyt-2 and Lyt-3 molecules may have importance in T cell-target cell interactions.


Assuntos
Antígenos de Diferenciação de Linfócitos T/genética , Antígenos Ly/genética , DNA/genética , Regulação da Expressão Gênica , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos Ly/imunologia , Antígenos de Superfície/genética , Sequência de Bases , Antígenos CD8 , Linhagem Celular , Membrana Celular/imunologia , Imunofluorescência , Humanos , Células L , Camundongos , Dados de Sequência Molecular
13.
Immunogenetics ; 30(6): 494-501, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2512251

RESUMO

The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4+ or CD8+ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4+ or CD8+ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4+ and CD8+ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4+ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8+ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD4/metabolismo , Células L/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos Ly/análise , Antígenos CD4/análise , Antígenos CD8 , Regulação para Baixo , Humanos , Ativação Linfocitária , Camundongos , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Transfecção
14.
Eur J Immunol ; 26(9): 2093-2100, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8814252

RESUMO

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are though to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56lck SH2 domain with zeta-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/zeta complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8 alpha alpha and CD8 alpha beta isoforms to the TCR. We demonstrate in vivo in association of CD8 alpha alpha/p56lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56lck specifically impedes the association of ZAP-70 with CD8 alpha alpha/p56lck without affecting the zeta/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8 alpha alpha or CD8 alpha beta isoform with the TCR/CD3/zeta complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated TCR/CD3/zeta complex.


Assuntos
Antígenos CD8/fisiologia , Ativação Linfocitária , Proteínas de Membrana/fisiologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Domínios de Homologia de src/fisiologia , Quinases da Família src/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Proteínas Tirosina Quinases/fisiologia , Coelhos , Proteína-Tirosina Quinase ZAP-70
15.
DNA ; 7(10): 735-41, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3234179

RESUMO

A general strategy has been developed for expression and rescue of cDNAs in mammalian cells. cDNA libraries are constructed in a new phage vector, lambda PMV, which contains simian virus 40 (SV40) early region promoter sequences for transcription of cDNA inserts, as well as a dominant-acting selectable marker neo. Efficient transfer of the cDNA library to mammalian cells can be achieved by phage particle-mediated transfection. Following selection in the antibiotic G418, cells expressing the phenotype of interest are identified and isolated. Rapid recovery of the transfected cDNA is achieved through cell fusion of the transduced cells with COS cells. Replication at the SV40 origin promotes excision of the integrated cDNA as small circular DNA, which after isolation in this form is used to transform bacteria to ampicillin resistance. To test this strategy, a cDNA encoding the human lymphocyte differentiation antigen CD8 was inserted into lambda PMV. CD8 expression on the surface of mouse L cells and the efficient recovery of full-length CD8 cDNA inserts confirm the feasibility of this system. It is anticipated that the single-step screening of libraries constructed in lambda PMV will allow for the isolation of rare cDNAs and will prove less laborious than methods currently available.


Assuntos
Bacteriófagos , DNA/fisiologia , Vetores Genéticos , Células Eucarióticas , Citometria de Fluxo , Métodos , Transfecção
16.
Eur J Immunol ; 27(4): 990-8, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9130655

RESUMO

Deficiency of the cytokine receptor common gamma chain (gamma c) results in abnormal lymphoid development and a severe immunodeficiency disease due to the combined loss of the receptors for interleukins (IL)-2, -4, -7, -9, and -15. We have observed the development of secondary hematopoiesis with circulating hematopoietic progenitor cells in adult mice harboring a null mutation in gamma c. These extramedullary changes were not secondary to bone marrow failure or to an inability to maintain circulating blood counts. These results suggested that gamma c-dependent cytokine signaling pathways modulate hematopoietic development. An intrinsic defect in gamma c- hematopoietic stem cell commitment appeared unlikely, as fetal liver hematopoiesis was unaltered in gamma c- embryos. Furthermore, the absence of natural killer cells in gamma c- mice was not responsible for the observed hematopoietic changes. Peripheral TCR alpha beta T cells from gamma c- mice were characterized by an activated phenotype (CD62Llo, CD44hi, CD69hi) and showed increased levels of transcripts for hematopoietic stimulating cytokines, including IL-3 and granulocyte/macrophage-colony-stimulating factor. A predominance of these cells was detected in the bone marrow, suggesting a role for residual T cells in the enhanced hematopoiesis. Strikingly, the elimination of residual T cells from gamma c- mice reduced splenic and circulating hematopoietic precursor frequencies to normal levels. These results clearly implicate a deregulated TCR alpha beta T cell population in the observed hematopoietic changes in gamma c- mice, and emphasize the importance of gamma c-dependent cytokine interactions in modulating mature T cell responses.


Assuntos
Hematopoese Extramedular/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Citocinas/deficiência , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Feto , Hematopoese Extramedular/genética , Células-Tronco Hematopoéticas/imunologia , Células Matadoras Naturais/fisiologia , Camundongos , Camundongos Nus , Receptores de Citocinas/genética , Baço/metabolismo , Baço/fisiologia , Subpopulações de Linfócitos T/citologia
17.
Eur J Immunol ; 27(7): 1762-8, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9247589

RESUMO

The common gamma chain (gamma c) forms a critical component of the receptors for interleukins (IL)-2, IL-4, IL-7, IL-9, and IL-15. We analyzed gamma c-deficient mice to define a role for gamma c signaling in the development and function of the macrophage lineage. No major differences in absolute cell numbers, cell surface phenotype, or in vitro function of gamma c- compared to gamma c+ macrophages were observed. We therefore conclude that signaling through the gamma c chain is not essential for the differentiation of mouse macrophages. Although B and T cells require gamma c for IL-4 responses, IL-4 up-regulated major histocompatibility class II molecules and inhibited nitric oxide production from gamma c- macrophages following stimulation with lipopolysaccharide and interferon-gamma. gamma c- macrophages could also respond to IL-13, consistent with the model of a type II IL-4 receptor alpha/IL-13R which can function in the absence of gamma c. Both IL-4 and IL-13 responses could be completely inhibited with the mouse IL-4 antagonist OY, suggesting that all of the observed IL-13 responses pass through the type II receptor, making it the primary signaling receptor complex for IL-13 in mouse macrophages.


Assuntos
Interleucina-13/metabolismo , Interleucina-4/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Transdução de Sinais/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Imunofenotipagem , Interleucina-13/antagonistas & inibidores , Interleucina-13/fisiologia , Interleucina-4/antagonistas & inibidores , Interleucina-4/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Receptores de Interleucina/fisiologia , Transdução de Sinais/genética
18.
Immunity ; 6(3): 265-72, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9075927

RESUMO

Growth factors have been implicated in thymocyte development, but mutants lacking cytokines, or their receptors, have failed to reveal essential roles for growth/differentiation factors in the thymus. Mutations in the receptor tyrosine kinase c-kit and the common cytokine receptor gamma chain (gamma c) reduce cellularity, but are permissive for thymocyte development. We now report that thymocyte development is completely abrogated in mice lacking both c-kit and gamma c (c-kit-gamma c-). Thymic hypocellularity is so severe that the T cell receptor repertoire fails to form except for monoclonal or oligoclonal beta chain DJ rearrangements. B lymphopoiesis is only mildly reduced in c-kit-gamma c- as compared with c-kit+gamma c- mice, and hematological values are identical comparing c-kit-deficient and c-kit-gamma c- mice. These experiments reveal essential, overlapping, and synergistic functions for two distinct signaling pathways, one utilizing c-kit and the other cytokine receptor gamma c complexes coupling to Janus kinases and signal transducers and activators of transcription.


Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Receptores de Citocinas/fisiologia , Linfócitos T/citologia , Animais , Linfócitos B/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Clonais , Eritropoese/imunologia , Rearranjo Gênico do Linfócito T , Hematopoese/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Proteínas Proto-Oncogênicas c-kit/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Citocinas/genética , Linfócitos T/metabolismo , Timo/citologia , Timo/metabolismo
19.
Eur J Immunol ; 24(9): 1951-5, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8088315

RESUMO

Interleukin-2 (IL-2) and IL-2 receptors (IL-2R) critically regulate the magnitude and duration of T cell expansion required in an immune response. Modulation occurs at the level of receptor number and affinity. IL-2R is a multisubunit receptor which contains at least three chains, IL-2R alpha (p55), IL-2R beta (p70) and IL-2R gamma (p64). Some components of high-affinity receptors (alpha beta gamma) are continuously internalized in the absence as well as in the presence of IL-2. From studies on other receptors, it is known that endocytosis of ligand-receptor complexes is due to an intrinsic property of the receptor. However, the specific chains responsible for endocytosis of high-affinity IL-2 receptors have not been fully elucidated. IL-2R gamma has been reported to be necessary for IL-2 internalization, based on the fact that fibroblasts transfected with IL-2R alpha and -beta do not internalize IL-2. However, IL-2 dissociates too rapidly from IL-2R alpha beta receptors to allow for its internalization. From the reported results on IL-2 internalization in transfected fibroblasts, it cannot be concluded as to the respective roles of IL-2R beta and/or IL-2R gamma in endocytosis. As modulation of receptor number is important for biological activity, we have attempted to define the chains responsible for receptor internalization. In this work, we have studied the endocytic properties of IL-2R beta. We demonstrate that IL-2R beta is constitutively endocytosed in a B cell line, derived from a X-linked severe combined immunodeficiency patient, which lacks expression of IL-2R gamma. IL-2R beta was also constitutively internalized in T and natural killer cell lines independently of IL-2R gamma. These results suggest that IL-2R beta is endowed with endocytic capacity and carries internalization signals.


Assuntos
Endocitose/fisiologia , Interleucina-2/fisiologia , Linfócitos/metabolismo , Receptores de Interleucina-2/metabolismo , Anticorpos Monoclonais , Linhagem Celular Transformada , Meia-Vida , Humanos , Receptores de Interleucina-2/química , Receptores de Interleucina-2/deficiência , Receptores de Interleucina-2/imunologia , Imunodeficiência Combinada Severa/imunologia , Células Tumorais Cultivadas
20.
Eur J Immunol ; 24(2): 475-9, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8299698

RESUMO

Interactions of interleukin-2 (IL-2) with its high-affinity, heterotrimeric receptor (IL-2R alpha beta gamma) play a pivotal role in the autocrine pathway of T lymphocyte expansion required in an immune response. Mutations in the IL-2R gamma chain-encoding gene have been found in SCIDX1, a primary immunodeficiency characterized by the absence of T cell and NK cell development. We have investigated six unrelated SCIDX1 patients for molecular abnormalities of the IL-2R gamma gene. A variety of defects were identified, including the absence of transcripts, frame-shift deletions and point mutations within canonical cytokine receptor motifs (conserved cysteines and the "WS" box). The ability of these mutated IL-2R gamma chains to participate in the function of a high-affinity IL-2R complex was examined by radiolabeled IL-2 binding studies using Epstein-Barr virus-transformed B lymphoblastoid cell lines (B-LCL) derived from SCIDX1 patients. Although normal control B-LCL express high-affinity IL-2 binding sites (Kd = 60 pM, 150 sites/cell), B-LCL derived from SCIDX1 patients failed to bind IL-2 under high-affinity conditions. These SCIDX1 mutations confirm the critical role of the IL-2R gamma chain in T cell and NK cell development. In addition, these data provide insight into the structure/function relationship of the IL-2R gamma chain by identifying residues required for the formation of a high-affinity IL-2R complex.


Assuntos
Receptores de Interleucina-2/genética , Imunodeficiência Combinada Severa/imunologia , Sequência de Bases , Primers do DNA/química , Humanos , Dados de Sequência Molecular , Mutação Puntual , RNA Mensageiro/genética , Receptores de Interleucina-2/metabolismo , Imunodeficiência Combinada Severa/genética , Cromossomo X
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