RESUMEN
The gene transfer efficiency into nonobese diabetic/severe combined immunodeficient (NOD/SCID)-repopulating cells (SRCs) derived from umbilical cord blood (UCB) (n = 11 NOD/SCID mice) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) (n = 64 NOD/SCID mice) was compared using a clinically relevant protocol and a retrovirus vector expressing the enhanced green fluorescent protein (EGFP). At 6-9 weeks after transplantation, the frequency of transduced human cells in the bone marrow (BM) (40.5% +/- 2.4% [mean +/- SE]) and spleen (SPL) (36.4% +/- 3.2%) in recipients of UCB cells was significantly higher (p < 0.001) than that observed in the BM (2.2% +/- 1.8%) and SPL (2.0% +/- 2.6%) in recipients of MPB. In subsequent studies, MPB was cultured for 2-8 days in cytokines prior to transduction to determine if longer prestimulation was required for optimal gene transfer. A significant increase in gene transfer into CD45(+) human cells and clonogenic cells derived from MPB SRCs was observed when cells were prestimulated for 6 days compared to 2 days prior to transduction (p = 0.019). However, even after 6 days of prestimulation, transduction was still significantly less than UCB. A substantial discrepancy exists in the ability to introduce genes effectively via retrovirus vectors into SRCs derived from MPB as compared to UCB.
Asunto(s)
Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Transfusión Sanguínea , Sangre Fetal/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Inmunodeficiencia Combinada Grave/inmunología , Transducción Genética/métodos , Animales , Células Sanguíneas/citología , Células Sanguíneas/trasplante , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Sangre Fetal/citología , Citometría de Flujo , Expresión Génica , Terapia Genética/métodos , Proteínas Fluorescentes Verdes , Humanos , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/inmunología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Reacción en Cadena de la Polimerasa , Retroviridae/genética , Bazo/citología , Bazo/metabolismo , Factores de Tiempo , Transgenes/genética , Inmunología del TrasplanteRESUMEN
Recent studies have detected significant elevations of interleukin (IL)-5 mRNA in the liver parenchyma of patients with both primary biliary cirrhosis and acute rejection after liver transplantation. In both of these disorders, intrahepatic biliary epithelial cells (BECs) are the targets of injury. We hypothesized that BECs may themselves express IL-5 receptors that may modulate key biliary functions. RNAs coding for IL-5alpha and -beta receptors were amplified by RT/PCR from a biliary cell line derived from a human cholangiocarcinoma (Mz-ChA-1) and verified by DNA sequencing. IL-5 receptor distribution was detected immunocytochemically on Mz-ChA-1 cells, immortalized murine BEC, bile duct-ligated rat liver, and isolated cholangiocytes. Patch-clamp studies on Mz-ChA-1 cells showed that IL-5 inhibits 5'-N-ethylcarboxamidoadenosine-stimulated chloride currents. Additional functional studies showed that IL-5 inhibits secretin-induced bile flow. We conclude that BECs express IL-5 receptors and that IL-5 modulates BEC chloride currents and fluid secretion. Since IL-5 has previously been associated with cholestatic liver disease, we speculate that IL-5 may contribute to liver injury through its effects on biliary secretion.
Asunto(s)
Bilis/fisiología , Sistema Biliar/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Interleucina-5/farmacología , Animales , Sistema Biliar/citología , Sistema Biliar/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Interleucina-5/biosíntesis , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Técnicas de Placa-Clamp , Ratas , Ratas Endogámicas F344 , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/virología , Citomegalovirus/inmunología , Herpesvirus Humano 4/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos Virales/inmunología , Línea Celular Transformada , Transformación Celular Viral , Citomegalovirus/genética , Citotoxicidad Inmunológica , Herpesvirus Humano 4/genética , Humanos , Cooperación Linfocítica , Recombinación GenéticaRESUMEN
Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.
Asunto(s)
Técnicas de Transferencia de Gen , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Linfocitos T/metabolismo , Anticuerpos/inmunología , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Células Cultivadas , Citometría de Flujo , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Interleucina-2/metabolismo , Virus de la Leucemia Murina de Moloney/genética , ARN Mensajero/biosíntesis , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Linfocitos T/inmunologíaRESUMEN
Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica/inmunología , Sangre Fetal/inmunología , Herpesvirus Humano 4/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Anticuerpos Monoclonales/inmunología , Antígenos CD/análisis , Línea Celular Transformada , Células Clonales/inmunología , Técnicas de Cocultivo , Citotoxicidad Inmunológica/efectos de los fármacos , Ácido Egtácico/farmacología , Proteína Ligando Fas , Sangre Fetal/citología , Granzimas , Antígenos HLA-DR/inmunología , Herpesvirus Humano 4/fisiología , Humanos , Inmunoterapia Adoptiva , Recién Nacido , Interleucina-2/farmacología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.
Asunto(s)
Fibronectinas/metabolismo , Receptores Virales/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Retroviridae/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Adulto , Transformación Celular Viral , Humanos , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
A transduction strategy has been developed, using fibronectin (FN)-assisted retroviral-mediated gene transfer, based on the observation that hematopoietic stem and progenitor cells bind to specific adhesion domains of fibronectin, via the integrins, very late antigen-4 (VLA-4)alpha 4 beta 1 and very late antigen-5 (VLA-5)alpha 5 beta 1. Retrovirus-mediated transduction on a recombinant FN fragment, FN CH-296, containing binding sites for VLA-4 and VLA-5, separated by type III repeats 12 to 14, makes it possible to efficiently target hematopoietic stem and progenitor cells and T-lymphocytes due to colocalization of target cells and retrovirus particles. These gene therapy strategies are applicable to the potential treatment of a variety of acquired and inherited immune disorders.
Asunto(s)
Fibronectinas/metabolismo , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/metabolismo , Retroviridae/genética , Linfocitos T/metabolismo , Animales , Antígenos CD34/metabolismo , Sitios de Unión/genética , Fibronectinas/genética , Vectores Genéticos , Células Madre Hematopoyéticas/inmunología , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Primates , Receptores de Fibronectina/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfocitos T/inmunología , Transducción GenéticaRESUMEN
Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.
Asunto(s)
Adenosina Desaminasa/deficiencia , Antígeno B7-1/genética , Fibronectinas/genética , Técnicas de Transferencia de Gen , Activación de Linfocitos , Linfocitos T/fisiología , Adenosina Desaminasa/genética , Células Cultivadas , Fibronectinas/administración & dosificación , Vectores Genéticos , Humanos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , RetroviridaeRESUMEN
Posttransplant Epstein-Barr virus-related lymphoproliferative disease (PT-LPD) is a common and often fatal complication following solid organ and hematopoietic stem cell transplantation. PT-LPD following solid organ transplantation generally occurs in B cells of recipient origin in contrast to PT-LPD in marrow transplant recipients, which is exclusively of donor origin. The efficacy of adoptive immunotherapy using donor leukocytes to treat PT-LPD in bone marrow transplant recipients has recently been reported. Because PT-LPD in solid organ transplant recipients is generally of recipient origin, the potential application of adoptive immunotherapy of PT-LPD in solid organ recipients obligates the use of either autologous or allogeneic HLA identical leukocytes, with the attendant risk of organ rejection if cells mismatched with the transplanted organ are used. Nonirradiated allogeneic mononuclear cells from an Epstein-Barr virus (EBV)-seropositive, HLA-identical normal sibling were used to treat a monoclonal EBV lymphoma of recipient origin in the central nervous system of a child who had undergone an HLA-mismatched cadaveric lung transplant. The patient received three separate mononuclear cell infusions over a 9-month period, each containing 1 x 10(6) CD3+ mononuclear cells per kilogram. Complete clinical, radiological, and pathological remission was achieved with this treatment regimen. The response correlated with in vivo reconstitution of normal EBV-specific cytotoxic activity and cytotoxic T lymphocyte precursor frequency. Use of allogeneic HLA-compatible mononuclear cells may thus offer an additional mode of therapy for EBV-related lymphoproliferative disease in selected solid organ transplant recipients refractory to conventional therapies.
Asunto(s)
Neoplasias del Sistema Nervioso Central/terapia , Inmunoterapia Adoptiva , Trasplante de Pulmón/efectos adversos , Trastornos Linfoproliferativos/terapia , Suero Antilinfocítico/uso terapéutico , Niño , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Inmunosupresores/uso terapéutico , Transfusión de Leucocitos , Trasplante de Pulmón/inmunología , Linfoma/terapia , Linfoma/virología , Masculino , Linfocitos T Citotóxicos/virología , Trasplante HomólogoRESUMEN
Epstein Barr virus induced lymphoproliferative disease (EBV-LPD) is a heterogeneous disorder, ranging from polyclonal lymphoproliferations to malignant lymphoma, typically seen in individuals with inadequate cellular immunity to EBV. The diagnosis of EBV-LPD following transplant requires a high index of suspicion in those patients at risk. Unlike organ transplant patients, in whom lymphomas are generally of host origin and respond to decreases in immunosuppression, bone marrow transplant recipients have donor origin tumors that are not as responsive to conservative treatment modalities. For the latter group, adoptive immunotherapy with donor lymphocytes is the treatment of choice and generally results in complete eradication of these tumors. Whether adoptive immunotherapy can be used for EBV-LPD in patients following organ transplantation is currently being investigated.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/patogenicidad , Trastornos Linfoproliferativos/microbiología , Inmunología del Trasplante , Trasplante de Médula Ósea/inmunología , Humanos , Inmunidad Celular , Trastornos Linfoproliferativos/terapia , Trasplante de ÓrganosRESUMEN
The expression of the murine T cell Ag 4-1BB, a member of the TNF-R family, is induced by T cell activation. Previously, we and others had shown that signaling through 4-1BB enhanced proliferative T cell responses. To investigate a potential role for the interaction of 4-1BB with its ligand (4-1BBL) in T cell activation, we studied the ability of a soluble chimera of 4-1BB (4-1BBFc) to interfere with proliferative responses and cytokine production in models of activation dependent in intercellular interactions. The potential blocking effect of 4-1BBFc was compared with that of the chimeric molecule CTLA-4Ig, a reagent known to interfere with the interaction of CD28 (and/or CTLA-4) with B7 costimulatory receptors. In this study, we report that 4-1BBFc partially blocked both the activation of unfractionated splenocytes triggered by soluble anti-CD3 (anti-CD3s), and the more physiologically relevant responses to alloantigen. In addition, we show that both chimeric molecules partially blocked proliferative responses and IL-2 secretion by highly purified resting T cells activated with anti-CD3s in the presence of fixed accessory cells that express B7 receptors and 4-1BBL. Furthermore, in this model system, the blocking capacity of 4-1BBFc and CTLA-4Ig appears to correlate with the relative expression of their respective cognate receptors (4-1BBL and B7) on the accessory cell. Simultaneous addition of both blocking reagents produced an additive effect in the model systems studied.
Asunto(s)
Antígenos CD28/inmunología , Activación de Linfocitos , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD , Antígenos CD28/metabolismo , Complejo CD3/inmunología , División Celular , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B cells with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells.
Asunto(s)
Antígeno B7-1/fisiología , AMP Cíclico/farmacología , Inmunoconjugados , Activación de Linfocitos , Receptores de Factor de Crecimiento Nervioso/fisiología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Ligando 4-1BB , Abatacept , Fosfatasa Alcalina/fisiología , Animales , Presentación de Antígeno , Antígenos CD , Antígenos de Diferenciación/fisiología , Antígeno CTLA-4 , Línea Celular , Femenino , Ligandos , Linfoma de Células B/inmunología , Ratones , Ratones Endogámicos BALB C , Regulación hacia ArribaRESUMEN
The human homologue of 4-1BB (H4-1BB) cDNA was isolated from PMA plus ionomycin-treated human peripheral T-cell cDNA libraries. The amino acid sequence deduced from the nucleotide sequence showed that the protein is composed of 255 amino acids with 2 potential N-linked glycosylation sites. The molecular weight of its protein backbone is calculated to be 27 kDa. The H4-1BB contains features such as signal sequence and transmembrane domain, indicating that it is a receptor protein. This protein showed 60% identity of amino acid sequence to mouse 4-1BB. In the cytoplasmic domain there are 5 regions of amino acid sequences conserved from mouse to human, indicating that these residues might be important in the 4-1BB function. H4-1BB mRNA was detected in unstimulated peripheral blood T cells and was inducible in T-cell lines such as Jurkat and CEM. H4-1BB-AP, a fusion protein between the H4-1BB extracellular domain and alkaline phosphatase, was used to identify the ligand for the H4-1BB. Although the H4-1BB ligand was detected in both T and B cells of human peripheral blood, the ligand was preferentially expressed in primary B cells and B-cell lines. Daudi, a B-cell lymphoma, was one of the B-cell lines that carried a higher number of ligands. Scatchard analysis showed that the Kd = 1.4 x 10(9) M and the number of ligands in Daudi cell was 4.2 x 10(3).
Asunto(s)
Glicoproteínas de Membrana/química , Receptores de Factor de Crecimiento Nervioso/química , Receptores del Factor de Necrosis Tumoral/química , Factor de Necrosis Tumoral alfa/química , Células 3T3 , Ligando 4-1BB , Secuencia de Aminoácidos , Animales , Antígenos CD , Linfocitos B/metabolismo , Secuencia de Bases , ADN Complementario/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Activación de Linfocitos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Estructura Terciaria de Proteína , ARN Mensajero/análisis , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/aislamiento & purificación , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35-45% 4-1BB+ by flow cytometric analysis. 4-1BB was expressed on activated CD8+, CD4+, CD28+ and CD45RB+ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 x 10(6)/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB+. In contrast, at lower cell densities (4 x 10(5), 2 x 10(5) and 1 x 10(5)), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1 alpha (IL-1 alpha) or interferon-gamma (IFN-gamma) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1 alpha, IFN-gamma or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.
Asunto(s)
Comunicación Celular , Interleucina-2/farmacología , Interleucina-4/farmacología , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores del Factor de Necrosis Tumoral/análisis , Linfocitos T/química , Animales , Antígenos CD , Antígenos CD28/fisiología , Complejo CD3/inmunología , Femenino , Interferón gamma/farmacología , Interleucina-1/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Linfocitos T/fisiología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The 4-1BB antigen has been recently demonstrated to be a 30 kDa inducible T-cell antigen and is expressed on the cell surface of activated splenic T cells and thymocytes. This novel T-cell antigen also associates physically with the T cell-specific protein tyrosine kinase, p56lck. We show here that the inducible T-cell antigen 4-1BB is expressed on activated intestinal intra-epithelial T lymphocytes (IEL). After activation, or in the presence of IL-2, the activated IELs showed higher levels of 4-1BB. Functional studies revealed that the activated IELs triggered with anti-4-1BB monoclonal antibody could enhance the level of IEL cytotoxicity against anti-CD3-secreting hybridoma cells. Cross-linking of anti-4-1BB antibody also enhanced the proliferation of IELs. These results suggest that the inducible antigen 4-1BB has broad biological functions which not only play a role in activated splenic T cells and thymocytes but also in the mucosal immune system.
Asunto(s)
Antígenos de Superficie/inmunología , Mucosa Intestinal/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/metabolismo , Complejo CD3/inmunología , Células Cultivadas , Femenino , Citometría de Flujo , Immunoblotting , Interleucina-2/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMEN
4-1BB is expressed on activated murine T cells and may function as an accessory signaling molecule during T-cell activation. To identify putative 4-1BB ligands, a fusion protein consisting of the extracellular domain of 4-1BB fused to human placental alkaline phosphatase (4-1BB-AP) was constructed. Alkaline phosphatase activity could then be used as an indicator of the relative amount of bound 4-1BB. These studies indicated that 4-1BB-AP specifically bound to the surface of various mature B and macrophage cell lines. 4-1BB-AP bound at low levels to T cell lines (non-activated and anti-CD3-activated), pre-B-cell lines, and an immature macrophage cell line. 4-1BB-AP did not bind to a glial tumor cell line, HeLa cells, or COS cells. In addition, 4-1BB-AP bound at higher levels to F(ab')2 anti-mu-activated primary B cells compared to anti-CD3-activated primary T cells. Scatchard analysis indicated that the A20 B cell lymphoma expressed 3680 binding sites per cell with a Kd of 1.86 nM. Affinity cross-linking studies demonstrated that a major cell surface species of 120 kDa bound to 4-1BB-AP; 4-1BB-AP also bound to a minor species of approximately 60 kDa. The addition of paraformaldehyde-fixed SF21 cells expressing recombinant 4-1BB synergized with F(ab')2 anti-mu in inducing splenic B cell proliferation suggesting that 4-1BB may function as a regulator of B cell growth.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos B/inmunología , Activación de Linfocitos , Macrófagos/inmunología , Receptores del Factor de Necrosis Tumoral , Linfocitos T/inmunología , Animales , Antígenos CD , Linfocitos B/metabolismo , Comunicación Celular , Femenino , Ligandos , Cooperación Linfocítica , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Recombinantes de Fusión , Bazo/citología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
4-1BB is a 30-kDa inducible T cell Ag, and is expressed predominantly as a 55-kDa dimer on both CD4+ and CD8+ T lymphocytes. The cytoplasmic tail of 4-1BB contains the sequence Cys-Arg-Cys-Pro, which is similar to the sequence Cys-X-Cys-Pro, which mediates the binding of the CD4 and CD8 molecules to the protein tyrosine kinase p56lck. An anti-4-1BB mAb, 53A2, was used to determine whether 4-1BB may associate with p56lck. The 53A2 mAb specifically recognized 4-1BB on a CD8+ T cell line, CTLL-2, and coimmunoprecipitated a 56-kDa protein along with 4-1BB. Peptide mapping indicated that the 56-kDa phosphoprotein was identical to p56lck. The coimmunoprecipitation of p56lck with 4-1BB also occurred in nonlymphoid cells such as insect (Sf-21) and HeLa cells when the two recombinant proteins were coexpressed. Analysis of mutant p56lck recombinant proteins showed that two cysteine residues critical for p56lck-CD4 (or -CD8) complex formation are also required for the p56lck-4-1BB interaction. These studies establish that 4-1BB physically associates with p56lck.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Activación de Linfocitos , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Cisteína/química , Células HeLa , Humanos , Técnicas In Vitro , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Unión Proteica , Proteínas Recombinantes/metabolismo , Linfocitos T/ultraestructuraRESUMEN
Evidence is presented to demonstrate that murine B lymphocytes receive growth stimulatory signals from their surface class II molecules. Monoclonal anti-Ia antibodies enhanced anti-mu-induced B cell proliferative response. The signals through surface immunoglobulin (Ig) and Ia appeared to act sequentially since preexposure to anti-mu was required to observe anti-Ia-induced increase in B cell proliferation. Anti-Ia antibodies did not increase the number of B cells entering the G1 phase of cell cycle but always enhanced transition from G1 into the S phase in response to stimulation with anti-mu. Analysis of early gene expression revealed that signaling through class II molecules led to an increase in anti-mu-induced expression of c-myc, a proto-oncogene, and of ornithine decarboxylase, a key enzyme in polyamine biosynthesis that has been shown to be intimately related to increased cell proliferation.
Asunto(s)
Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/fisiología , Activación de Linfocitos , Animales , Ciclo Celular , Expresión Génica , Genes myc , Ratones , Ratones Endogámicos , Ornitina Descarboxilasa/genética , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de Transferrina/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
4-1BB is an inducible receptor-like protein expressed in both cytolytic and Th cells. Optimal induction of 4-1BB mRNA in T cells required both PMA and ionomycin stimulation, indicating that protein kinase C activation and increases in intracellular Ca2+ were required for its expression. 4-1BB was categorized as an early activation gene since the protein synthesis inhibitor, cycloheximide, blocked the induction of 4-1BB mRNA. A rat mAb, 53A2, was generated against recombinant soluble 4-1BB and was used to characterize this molecule. 4-1BB is a 30-kDa glycoprotein and appears to exist as both a monomer and a 55-kDa dimer on the cell surface of a T cell clone. The 4-1BB protein may be post-translationally modified since its predicted backbone is 25 kDa. FACS analysis indicated that 4-1BB was inducible and expressed on the cell surface of activated splenic T cells and thymocytes. Cross-linking of 4-1BB on anti-CD3-stimulated T cells with 53A2 resulted in a dramatic enhancement of T cell proliferation. This suggests that 4-1BB may function as an accessory signaling molecule during T cell activation.
Asunto(s)
Receptores de Antígenos de Linfocitos T/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Secuencia de Bases , Antígenos CD40 , Femenino , Sueros Inmunes/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunologíaRESUMEN
In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.