Asunto(s)
Cicloparafinas/uso terapéutico , Janus Quinasa 3/antagonistas & inhibidores , Cetonas/uso terapéutico , Linfoma de Células T/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Administración Oral , Cicloparafinas/farmacología , Células HEK293 , Células Hep G2 , Humanos , Cetonas/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
We have investigated the potential toxicity of hlL-12 DNA plasmid formulated with 5% polyvinylpyrrolidone (PVP) administered twice weekly via subcutaneous injections to Cynomolgus monkeys for four weeks, and have evaluated recovery from any effects of the test article over a four-week treatment-free period. Doses of the formulated hIL-12 plasmid were selected based on anti-tumour efficacy studies previously conducted in mice. The duration of the study and the frequency of dosing were designed to support clinical trials. No clinical signs indicative of an adverse effect of administration of formulated hIL-12 plasmid were observed. There were no apparent effects of the formulated hIL-12 plasmid on body weights or on serum chemistry, haematology, coagulation or urinalysis parameters. No treatment-elated ocular abnormalities were evident. In addition, examination of the electrocardiograms from all monkeys at the pre-study, week-4, and week-8 time points did not reveal any treatment-related effects. No treatment-related gross lesions were noted at days 28 or 57. Slight histopathological changes associated with high doses of PVP vehicle were observed at both time points. These results suggested that the administration of formulated hIL-12 plasmid at a dose level up to 18 mg kg(-1) dose twice per week for four weeks to experimentally naïve Cynomolgus monkeys did not result in significant toxicity. These results support further testing of this gene therapy in clinical trials.
Asunto(s)
Interleucina-12/toxicidad , Povidona/toxicidad , Animales , Peso Corporal/efectos de los fármacos , ADN/análisis , Electrocardiografía/efectos de los fármacos , Ojo/efectos de los fármacos , Femenino , Vectores Genéticos , Humanos , Inyecciones Subcutáneas , Interleucina-12/química , Interleucina-12/farmacocinética , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos , Plásmidos , Povidona/química , Povidona/farmacocinética , Piel/química , Pruebas de ToxicidadRESUMEN
We have characterized the fusogenic activity of a plasmid expression system encoding vesicular stomatitis virus G protein (VSVG) in vitro and in vivo. Over 70% of murine colon and renal carcinoma cells (MC38 and Renca, respectively) transfected with VSVG plasmid in vitro fused and formed polykaryons upon incubation with pH 5.5 media. Using a plasmid expression system encoding VSVG and bacterial green fluorescent protein (GFP) formulated in a polyvinyl pyrrolidone (PVP) delivery system, diffusion of GFP throughout the VSVG-induced syncytia was shown in vivo in MC38 and Renca tumors. Moreover, tumor-bearing mice showed tumor growth inhibition following in vivo transfection with VSVG plasmid at an optimal dose of 48 microg. We have previously shown that direct injection of interleukin -12 (IL -12) plasmid complexed with PVP into tumors induces a strong immune response. In the current study, we assessed the ability of VSVG to elicit an antitumor response by enhancing cytokine gene delivery within the tumor mass. Tumor-bearing mice treated intratumorally with both VSVG/PVP and IL-12/PVP (48 and 24 microg, respectively) showed increase in tumor rejection when compared to IL- 12 plasmid alone (75% vs. 50%, respectively). These data suggest that VSVG gene therapy can be used in combination with other therapeutic genes to induce an antitumor response in vivo by enhancing the expression of the gene of interest.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/terapia , Neoplasias del Colon/terapia , Terapia Genética/métodos , Interleucina-12/genética , Neoplasias Renales/terapia , Glicoproteínas de Membrana , Proteínas del Envoltorio Viral/genética , Animales , Western Blotting , Sistemas de Liberación de Medicamentos , Sinergismo Farmacológico , Femenino , Células Gigantes/citología , Células Gigantes/metabolismo , Rechazo de Injerto/inmunología , Proteínas Fluorescentes Verdes , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Interleucina-12/metabolismo , Interleucina-12/farmacología , Neoplasias Renales/inmunología , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Fluorescente , Plásmidos , Povidona/química , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/farmacologíaRESUMEN
To improve the immunogenicity of melanoma self-antigens, we used a novel strategy of nonviral genetic vaccination coupled with muscle electroporation. Electroporation-enhanced immunization with plasmids encoding either human gp100 or mouse TRP-2 antigens induced only partial rejection of B16 melanoma challenge. However, immunization with a combination of these two antigens caused tumor rejection in 100% of the immunized mice. Splenocytes from combination-immunized animals killed syngeneic targets loaded with peptides derived from both gp100 and TRP-2. Immune cell depletion experiments identified CD8+ T lymphocytes as the primary effectors of antitumor immunity. Most importantly, polyimmunization led to the generation of a therapeutic immune response that significantly improved the mean survival time of mice bearing established lung metastases. These results validated the usefulness of electroporation-enhanced, nonviral genetic immunization for the active immunotherapy of cancer and indicated that using a combination of different tumor antigens may be a decisive strategy for a successful therapeutic vaccination.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Oxidorreductasas Intramoleculares/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Animales , Antígenos de Neoplasias/genética , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/uso terapéutico , Electroporación , Femenino , Oxidorreductasas Intramoleculares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Melanoma Experimental/prevención & control , Melanoma Experimental/secundario , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Plásmidos/administración & dosificación , Plásmidos/genética , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas , Vacunas Combinadas/genética , Vacunas Combinadas/inmunología , Vacunas Combinadas/uso terapéutico , Antígeno gp100 del MelanomaRESUMEN
The antitumor effect and mechanism of action of IL-12 gene therapy combined with IFN-alpha gene therapy were investigated in tumor-bearing mice using renal and colon carcinoma models, Renca and CT26, respectively. Tumors were treated with murine IL-12 plasmid alone or in combination with IFN-alpha plasmid formulated with a polymeric interactive noncondensing (PINC) gene delivery system. Intratumoral injection of IL-12 DNA/polyvinyl pyrrolidone (PVP) alone induced rejection of 58 and 17% of Renca and CT26 tumors, respectively, whereas 25% (Renca) and 0% (CT26) rejection was observed in mice treated with IFN-alpha plasmid/PVP. Combination gene therapy of formulated plasmids, IL-12 with IFN-alpha, synergistically increased the antitumor response against Renca (100% tumor rejection) and CT26 (50%). In vivo depletion of leukocyte subsets indicated that CD8(+) T and NK cells were the primary effectors of the antitumor response induced by the combined cytokine gene therapy. Moreover, mice that rejected the primary tumors after combined treatment with IL-12 and IFN-alpha plasmid formulation developed protective immunity against a subsequent tumor challenge. Analysis of tumor-infiltrating leukocytes from mice treated with the combined IL-12 and IFN-alpha gene therapy showed upregulation of CD40 molecules on antigen-presenting cells (Mac-1(hi) cells). Finally, levels of mRNA for the chemokines IP-10 and TCA-3 were higher in tumors treated with the combination of cytokine plasmids than in tumors treated with either cytokine gene alone. These data provide evidence that IL12 gene therapy combined with IFN-alpha gene therapy synergistically induces regression of established tumors and may represent a novel therapeutic strategy for cancer treatment.
Asunto(s)
Carcinoma de Células Renales/terapia , Neoplasias del Colon/terapia , Terapia Genética/métodos , Interferón-alfa/genética , Interleucina-12/genética , Neoplasias Renales/terapia , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/inmunología , Quimiocinas/genética , Neoplasias del Colon/inmunología , Citocinas/genética , Sinergismo Farmacológico , Femenino , Rechazo de Injerto/inmunología , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Interleucina-12/metabolismo , Interleucina-12/farmacología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/terapia , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/análisisRESUMEN
Using a novel cationic lipid delivery system consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride and cholesterol, we delivered murine interleukin-2 (IL-2) cDNA directly into an established murine renal cell carcinoma (Renca). Production of IL-2 within the tumor induced rejection of established tumors (62% on average), whereas control plasmid had little or no effect (17% on average). Surviving animals treated with IL-2:lipid were highly resistant to Renca rechallenge, but not to cross-challenge with a syngeneic mammary adenocarcinoma. Experiments on selectively immunosuppressed animals indicated a requirement for CD8+ T, natural killer, and polymorphonuclear cells. By contrast, depletion of CD4+ T cells did not disrupt the ability of IL-2:lipid to induce tumor rejection. A combination of IL-2 gene therapy with 5-fluorouracil treatment increased the antitumoral efficacy and survival of mice bearing primary and metastatic Renca tumors (42% survival with IL-2:lipid compared with 94% survival with IL-2:lipid plus 5-fluorouracil). These data indicate that rejection of primary and metastatic tumors can be achieved after intratumoral delivery of a nonviral IL-2 gene therapy, and is increased in combination with systemic delivery of a conventional chemotherapeutic agent.
Asunto(s)
Carcinoma de Células Renales/terapia , Fluorouracilo/uso terapéutico , Terapia Genética , Interleucina-2/genética , Neoplasias Renales/terapia , Animales , Terapia Combinada , ADN Complementario/administración & dosificación , Femenino , Interleucina-2/biosíntesis , Interleucina-2/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Compuestos de Amonio Cuaternario/administración & dosificaciónRESUMEN
Receptors used by natural killer (NK) cells to mediate natural cytotoxicity are poorly defined, although it is now clear that a number of adhesion molecules can serve this function. CD38 transduces signals on T- and B-cell lines, and we asked whether it could trigger lytic and secretory responses in human NK cells. By using an anti-CD38 monoclonal antibody in reverse antibody-dependent cellular cytotoxicity experiments, it is shown that CD38 engagement triggers cytotoxic responses by activated NK cells, but not by cytotoxic T lymphocytes or fresh NK cells. Cross-linking with anti-CD38 F(ab')(2) caused activated NK cells to release granzymes and cytokines, but did not trigger an increase in intracellular Ca(2+). Fresh NK cells acquired CD38-dependent lytic function during activation with interleukin-2 (IL-2), and inhibitor studies suggested that IL-2 stimulated the de novo expression of proteins that act between CD38 and the lytic machinery in NK cells. The induction of proteins that link commonly expressed adhesion molecules to effector mechanisms could provide a paradigm for pathogen recognition by the innate immune system.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación/inmunología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , NAD+ Nucleosidasa/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Humanos , Interleucina-2/inmunología , Glicoproteínas de Membrana , Linfocitos T Citotóxicos/inmunologíaRESUMEN
We have utilized a nonviral, polymeric interactive non-condensing (PINC) gene delivery system to deliver IL-12 to two different types of murine tumors, an immunogenic renal cell carcinoma, Renca, and a non-immunogenic colon cell carcinoma, CT26. The delivery of IL-12/polyvinyl pyrrolidone (PVP) complexes into Renca led to the expression of IL-12 (146 +/- 89 pg/mg) and IFN-gamma (160 +/- 82 pg/mg) from explanted tumors in culture supernatants. Treated tumors showed increased infiltration of NK, CD4+ and CD8+ T cells and up-regulation of MHC class I molecules on leukocytes in both tumors and lymph nodes. Fifty per cent of tumor-bearing mice rejected Renca or CT26 tumors following IL-12/PVP treatments given at optimal doses of 24 and 48 micrograms, respectively. While polymorphonuclear cells (PMNs) were partially involved in the development of the antitumor immune response elicited by IL-12/PVP treatment, CD8+ T cells were found to be the primary effectors. In contrast, CD4+ T cells did not appear to play a significant role in the development of tumor specific immunity. Finally, mice that rejected the tumors following IL-12/PVP treatment were protected against a second challenge with the same tumor. These data provide evidence that a nonviral IL-12 gene delivery system is well tolerated and generates a potent immune response against established tumors.
Asunto(s)
Carcinoma de Células Renales/terapia , Neoplasias del Colon/terapia , Terapia Genética/métodos , Inmunoterapia Activa/métodos , Interleucina-12/genética , Neoplasias Renales/terapia , Animales , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/inmunología , Neoplasias del Colon/inmunología , Femenino , Expresión Génica , Vectores Genéticos , Inyecciones Intralesiones , Interferón gamma/genética , Neoplasias Renales/inmunología , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Células Tumorales CultivadasRESUMEN
Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase required for T cell development and activated by cytokines that utilize the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)). Genetic inactivation of JAK3 is manifested as severe combined immunodeficiency disease (SCID) in humans and mice. These findings have suggested that JAK3 represents a pharmacological target to control certain lymphoid-derived diseases. Here we provide novel evidence that AG-490 potently inhibits the autokinase activity of JAK3 and tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription 5a and 5b (STAT5a/b). Similar inhibitory effects were observed with other cytokines that use gamma(c). AG-490 also inhibited IL-2-mediated proliferative growth in human T cells with an IC50) = 25 microM that was partially recoverable. Moreover, we demonstrate that this inhibitor prevented tetanus toxoid antigen-specific T cell proliferation and expansion but failed to block activation of Zap70 or p56Lck after anti-CD3 stimulation of human T cells. Taken together, these findings suggest that AG-490 inhibits the JAK3-mediated Type II signaling pathway but not the T cell receptor-derived Type I pathway and possesses therapeutic potential for T cell-derived pathologies such as graft-versus-host disease, allergy, and autoimmune disorders.
Asunto(s)
Citocinas/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas/fisiología , Linfocitos T/inmunología , Factores de Transcripción/fisiología , Tirfostinos/farmacología , Animales , Antígenos/fisiología , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Humanos , Interleucina-2/antagonistas & inhibidores , Janus Quinasa 3 , Activación de Linfocitos/inmunología , Ratones , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Linfocitos T/citologíaRESUMEN
Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pulmonary diseases represents a new strategy of gene therapy. In this study we present evidence that intratracheal administration of a plasmid encoding murine IL-12 complexed with N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride:cholesterol inhibits the growth of lung metastases, using a renal cell carcinoma model. Instillation of pIL-12/lipid complexes resulted in expression of biologically active IL-12 (170-240 pg/ml) and IFN-gamma (100-190 pg/ml) in the bronchoalveolar lavage fluid. A significantly reduced number of lung metastases (26+/-24) was observed in mice instilled with IL-12/lipid complexes 24 hr after tumor challenge, whereas more than 250 metastatic foci were counted in lungs of untreated mice. Moreover, IL-12/lipid inhibited the growth of 3-day-old established metastases when compared with empty plasmid/lipid or IL-12 plasmid in saline. Mice receiving IL-12 gene therapy survived significantly longer (median survival of 43 days) than untreated mice (median survival of 31 days) or mice treated with control plasmid/lipid complexes (median survival of 35 days). These data demonstrate that a nonviral IL-12 gene therapy employing synthetic cationic lipids as a delivery system can be used to inhibit the development of lung metastases. Thus, this method provides support for the use of IL-12/lipid complexes to control the growth of pulmonary metastases and represents a potentially safer alternative to IL-12 protein immunotherapy.
Asunto(s)
Interleucina-12/administración & dosificación , Interleucina-12/genética , Lípidos/administración & dosificación , Neoplasias Pulmonares/prevención & control , Animales , Femenino , Proteínas Fluorescentes Verdes , Interferón gamma/uso terapéutico , Liposomas/uso terapéutico , Proteínas Luminiscentes/metabolismo , Pulmón/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Plásmidos , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
Following an infection or immunization, a primary CD8+ T cell response generally rises then falls rapidly before giving rise to a "memory" response. When we immunized mice with recombinant viral immunogens optimized to enhance the lytic capability of CD8+ T cells, we measured a profound depression in Ag-specific effector function after early restimulation. Indeed, a "mirror image" cytolytic capability was observed: the most powerful immunogens, as measured by cytolytic capacity 6 days after immunization, elicited the weakest secondary immune response when evaluated following an additional 6 days after restimulation. To understand the mechanism of this suppression, we examined the fate of splenocytes immunized with a vaccinia virus encoding Ag and IL-2 then restimulated ex vivo. We found that these splenocytes underwent an apoptotic cell death, upon early restimulation, that was not dependent on the engagement of the FasR (CD95). Unlike previously described mechanisms of "propriocidal cell death" and "clonal exhaustion," the cell death we observed was not an inherent property of the CD8+ T cells but rather was due to a population of splenocytes that stained positive for both the Mac-1 and Gr-1 surface markers. Deletion of these cells in vitro or in vivo completely abrogated the observed suppression of cytolytic reactivity of Ag-specific CD8+ T cells. These observations could account for the apparent absence of Ag-specific immune responses after some current vaccination regimens employing powerful immunogens. Finally, our results may shed new light on a mechanism for the suppression of CD8+ T cell responses and its effect on vaccine efficacy and on immune memory.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos , Antígeno de Macrófago-1/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD8-positivos/patología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Comunicación Celular/inmunología , Membrana Celular/inmunología , Citotoxicidad Inmunológica , Femenino , Células HeLa , Humanos , Tolerancia Inmunológica , Inmunización , Inmunización Secundaria , Interleucina-2/genética , Recuento de Linfocitos , Depleción Linfocítica , Antígeno de Macrófago-1/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Virus Vaccinia/genética , Virus Vaccinia/inmunología , beta-Galactosidasa/genéticaRESUMEN
A plasmid expression system encoding murine IFN-alpha4 and complexed with a protective interactive noncondensing polymeric (PINC) delivery system was used for in vivo immunotherapy treatment of an immunogenic murine renal cell carcinoma, Renca, and a nonimmunogenic mammary adenocarcinoma, TS/A. Mice bearing established tumors were treated with IFN-alpha/polyvinylpyrrolidone (PVP) expression complexes via direct intratumoral injection. Up to 100% inhibition of tumor growth was observed in the treated mice. By using an optimal dose of 96 and 48 microg of formulated IFN-alpha plasmid for the treatment of Renca and TS/A, respectively, 30% (Renca) and 10% (TS/A) of the treated animals remained tumor free. Inhibition of tumor growth was dependent on activation of the immune system. The antitumor activity elicited by IFN-alpha gene therapy was abrogated when mice were selectively depleted of CD8+ T cells. By contrast, depletion of CD4+ T cells resulted in enhanced tumor rejection following IFN-alpha/PVP treatments. Finally, mice that remained tumor free following IFN-alpha gene therapy displayed immune resistance to a subsequent tumor challenge. These data provide evidence that IFN-alpha gene therapy can be used to induce an efficient antitumor response in vivo.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Terapia Genética/métodos , Interferón-alfa/genética , Neoplasias Experimentales/terapia , Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Animales , Western Blotting , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/terapia , Femenino , Interferón-alfa/inmunología , Neoplasias Renales/inmunología , Neoplasias Renales/terapia , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/inmunología , Plásmidos/genética , Povidona , Células Tumorales CultivadasRESUMEN
HIV-1 infection is accompanied by qualitative and quantitative defects in CD4+ T lymphocytes. Loss of immune function in HIV patients is usually associated with a profound dysregulation of cytokine production. To investigate whether cytokine signaling defects occur during HIV infection, PHA blasts from healthy human donors were infected with two strains of HIV-1 and screened for the expression of STAT proteins used in cytokine signaling. A selective decrease in STAT5B was seen 8 days after infection with the BZ167 dual-tropic HIV isolate, but not with the Ba-L, M-tropic strain. Based on these findings, purified T cells from HIV-infected patients in different stages of disease were also tested for STAT expression; decreases in STAT5A, STAT5B, and STAT1alpha were observed in all patients. The reduction in STATs seen in vivo and in vitro after HIV infection may contribute to the loss of T cell function in HIV disease.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Infecciones por VIH/metabolismo , Proteínas de la Leche , Linfocitos T/virología , Transactivadores/metabolismo , Células Cultivadas , Regulación hacia Abajo , Humanos , Pruebas de Precipitina , Factor de Transcripción STAT5 , Linfocitos T/metabolismo , Proteínas Supresoras de TumorRESUMEN
Impaired immune responses are frequently observed in tumor-bearing hosts during progression of tumor growth, but the molecular basis of these functional defects remains unclear. To investigate tumor-induced immunosuppression, we first established that lymphocytes from mice bearing s.c. mammary adenocarcinoma (TS/A) tumors were severely impaired in their ability to generate cellular and humoral Ag-specific responses. Lymphocytes from these mice were then screened for abnormalities in the expression of signal transducing proteins known to be involved in the regulation of cellular immunity. Interestingly, purified T and B cells isolated from immunocompromised tumor-bearing mice displayed a marked decrease in the transcription activators STAT5a and -b at the protein level and to a lesser extent at the mRNA level. By contrast, no change in the expression of STAT1, -3, and -6 or of the TCR itself were detected. The correlation in the loss of T and B cell function with the selective decrease in STAT5a/b expression suggests that regulation of the STAT5 signaling pathway may provide a molecular mechanism for modulating the immune system.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/biosíntesis , Huésped Inmunocomprometido , Proteínas de la Leche , Neoplasias Experimentales/inmunología , Linfocitos T/metabolismo , Transactivadores/biosíntesis , Animales , Linfocitos B/inmunología , Proteínas de Unión al ADN/inmunología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Factor de Transcripción STAT5 , Linfocitos T/inmunología , Transactivadores/inmunologíaRESUMEN
In this study we provide evidence that a human breast carcinoma cell line, MDA-MB-231 (MDA), can be made immunogenic following B7 transfection and that full T cell activation is obtained through cooperation of T-B lymphocytes via CD40-CD40L interactions. Tumor cells transfected with either B7 gene (MDAB7), neomycin-resistant gene only (MDAneo), or untransfected (MDA) were used in an allogeneic mixed lymphocyte tumor culture (MLTC) to investigate their ability to stimulate T cell proliferation and generate cytotoxic T lymphocytes (CTL). MDAB7 induced moderate T cell proliferation while MDAneo or MDA did not. Substantial T cell proliferation and de novo generation of cytolytic T cells was obtained only in response to MDAB7 when B cells were present during the MLTC. CD8+-purified T + B cells proliferated to a greater extent than whole T cell populations + B or CD4+ + B in response to MDAB7. Addition of alpha-B7-1 or alpha-CD40 in the MLTC inhibited T cell proliferation by 65 and 40%, respectively, whereas T cell proliferation and generation of CTL was completely abrogated when MLTC was performed in the presence of both antibodies. These data suggest that the engagement of CD40L on T cells with CD40 on B cells provides a costimulatory signal which, in synergism with TCR-dependent MDAB7-T cell recognition (signal 1) and B7/CD28 interactions (signal 2), leads to full T cell activation.
Asunto(s)
Antígeno B7-1/farmacología , Neoplasias de la Mama/inmunología , Antígenos CD40/farmacología , Activación de Linfocitos , Glicoproteínas de Membrana/farmacología , Linfocitos T/inmunología , Linfocitos B/inmunología , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Neoplasias de la Mama/metabolismo , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Ligando de CD40 , Linfocitos T CD8-positivos/inmunología , Humanos , Ligandos , Cooperación Linfocítica , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Transfección/inmunología , Células Tumorales CultivadasRESUMEN
In this study, we present evidence that CD44 is a cytotoxic triggering molecule on freshly isolated polymorphonuclear cells (PMN). PMN constitutively express high levels of CD44 as determined by FACS analysis, and immunoprecipitation studies using PMN lysates and an anti-CD44 mAb show a band of 80 to 90 kDa that migrates slightly faster than CD44 from PBL. A bispecific Ab consisting of anti-CD44 Fab cross-linked to anti-DNP Fab (anti-CD44(Fab) x anti-DNP(Fab)) induces PMN to lyse DNP-coated tumor cells in an 18-h assay, and this lysis is specifically inhibited by a polyclonal anti-CD44 F(ab')2. A second bispecific Ab, anti-CD16(Fab) x anti-DNP(Fab), that binds to Fc(gamma)RIIIb on PMN does not induce lysis, indicating that the bridging of target cells to PMN per se is not sufficient for killing. Moreover, CD44-directed killing by PMN results in the lysis of bystander cells, suggesting that the mechanisms of tumor cytolysis by CD44-targeted PMN does not require cell-cell contact. Lastly, PMN lyse target cells coated with hyaluronic acid (HA), the principal ligand for CD44, and this cytolytic activity is specifically blocked by the polyclonal anti-CD44 F(ab')2 and by an anti-CD44 mAb. We suggest that the interaction of HA with CD44 on neutrophils might initiate cytotoxic or inflammatory responses in vivo when neutrophils encounter high amounts of HA, for example on tumor cells, or in the extracellular matrix.
Asunto(s)
Citotoxicidad Inmunológica , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/farmacología , Neutrófilos/inmunología , Animales , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Receptores de Hialuranos/biosíntesis , Ácido Hialurónico/farmacología , Ratones , Neutrófilos/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
We have previously established that human polymorphonuclear cells (PMN) express IL-2R beta- and gamma-chains and that addition of IL-2 maintains the viability of PMN by preventing these cells from undergoing programmed cell death. The purpose of this study was to examine whether IL-2-releasing tumor cells are capable of stimulating PMN tumoricidal activity. We therefore investigated the ability of PMN to kill IL-2-transfected tumor cells using normal human PMN directed against the murine mammary adenocarcinoma TS/A engineered to release high amounts of murine IL-2 (3,600 U, B6) compared with TS/A parental cells and TS/A tumor cells transfected with the neomycin-resistance (NEO) gene only. The potency of PMN as IL-2-induced killer cells was indicated by the low number of cells required for killing (effector cell:target cell ratio 10:1) and the degree of tumor cell lysis (68+/-10%). Evidence for the role of IL-2 as a mediator of tumor cytotoxicity by PMN was substantiated by inhibition of tumor killing with anti-IL-2 and anti-IL-2R beta monoclonal antibodies (MAbs). Furthermore, in vivo depletion of mature granulocytes using MAb RB6-8C5 resulted in B6 adenocarcinoma growth, thereby confirming a direct role for IL-2-activated PMN in tumor cytolysis. Lastly, we suggest that one possible mechanism involved in IL-2-induced PMN cytotoxicity against the B6 clone occurs via the nitric oxide pathway, which could be inhibited upon addition of the arginine analog, N(G)-monomethyl-L-arginine.
Asunto(s)
Supervivencia Celular , Interleucina-2/farmacología , Interleucina-2/fisiología , Neutrófilos/fisiología , Fagocitosis , Receptores de Interleucina-2/fisiología , Adenocarcinoma , Animales , Anticuerpos Monoclonales/farmacología , División Celular , Línea Celular , Células Cultivadas , Humanos , Interleucina-2/biosíntesis , Kanamicina Quinasa , Neoplasias Mamarias Experimentales , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
The lysis of antigen presenting cells (APCs) by cytotoxic T lymphocytes (CTLs) may be one mechanism whereby an immune response is downregulated by Staphylococcus superantigens. Disappearance of monocytes/macrophages from staphylococcal enterotoxin A (SEA)-activated peripheral blood mononuclear cell (PBMC) cultures, but not from control PBMC cultures was seen by flow cytometry. Recently, adenosine triphosphate (ATP) has been described as an effector molecule in CTL-mediated lysis of some murine tumor target cells. We have also shown that ATP caused the lysis of human macrophages, and that treatment of cells with interferon gamma (IFN gamma) rendered macrophages significantly more sensitive to ATP than untreated cells. To show that this purine nucleotide may play a role in modulating the immune system, we generated human CTLs that were stimulated with SEA, and used them as effector cells against SEA-pulsed autologous macrophages. CTLs were found to specifically lyse SEA-pulsed macrophages, while control, unpulsed, macrophages were unaffected. The addition of hexokinase, an enzyme that hydrolyzes ATP, significantly abrogated the killing of SEA-pulsed cells during the assay. In examining the mechanism of cytotoxicity, electron microscopy showed that macrophages incubated with both ATP and CTLs underwent necrosis, rather than apoptosis. From these results, it is suggested that ATP is released from CTLs during antigen presentation, and that IFN gamma-activated macrophages, which are inherently more sensitive to this mediator, are readily lysed and therefore removed from circulation, thus downregulating an immune response.
Asunto(s)
Adenosina Trifosfato/fisiología , Células Presentadoras de Antígenos/inmunología , Citotoxicidad Inmunológica/fisiología , Tolerancia Inmunológica/fisiología , Macrófagos/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos Bacterianos/inmunología , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Muerte Celular , Células Cultivadas , Enterotoxinas/inmunología , Hexoquinasa/farmacología , Humanos , Interferón gamma/farmacología , Interleucina-2/farmacología , Receptores de Lipopolisacáridos , Factor Estimulante de Colonias de Macrófagos/farmacología , Necrosis , Proteínas Recombinantes/farmacología , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Functional interleukin-2 receptors (IL-2R) on lymphocytes contain both IL-2R beta and gamma chains. Whereas constitutive expression of IL-2R beta has been found on monocytes, the expression of IL-2R gamma on these phagocytes has not been examined. We performed reverse-transcription-polymerase chain reaction with Southern blot analysis on RNA derived from purified human monocytes and discovered that they constitutively produce IL-2R gamma mRNA. Western immunoblotting revealed 58- and 64-kDa forms of IL-2R gamma on YT-1 and human monocytes, whereas 58-, 64-, and 69-kDa bands were detected using peripheral blood mononuclear cells and non-adherent lymphocytes. These different forms resulted from variable N-linked glycosylation since culture of the cells in tunicamycin resulted in detection of a single 39-kDa band which corresponds to the molecular weight predicted from the deduced amino acid sequence. By co-immunoprecipitation, the IL-2R beta subunit associates with only the 64-kDa IL-2R gamma protein band in monocytes.
Asunto(s)
Monocitos/inmunología , Procesamiento Proteico-Postraduccional/fisiología , ARN Mensajero/biosíntesis , Receptores de Interleucina-2/biosíntesis , Southern Blotting , Western Blotting , Humanos , Técnicas In Vitro , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Receptores de Interleucina-2/metabolismoRESUMEN
A retroviral infection was used to introduce the cDNA coding for mouse IL-4 into the parental cells of a spontaneous adenocarcinoma of BALB/c mice (TS/A-pc). Four clones releasing between 5 to 40 U of IL-4 (10(5) cells) in 48 h culture were selected. The secretion of IL-4 does not affect their in vitro growth, whereas their ability to form tumor in vivo inversely correlates with the amount of IL-4 secreted. Although morphologic observation suggested that the rejection of clone D5.40 cells (releasing 40 U of IL-4) depends on eosinophil cytolysis, lymphocyte depletion experiments showed that this required CD8+ lymphocyte guidance. Mice that had rejected D5.40 cells were immune to a subsequent challenge with TS/A-pc. This memory rests on the interaction between noncytotoxic lymphocytes, eosinophils, and IgG1 and IgE anti-TS/A Abs. Comparison of these memory mechanisms with those elicited by IL-2 gene-transduced TS/A cells shows that the kind of cytokine released by the tumor cells determines the type of response. This Th2 memory seems to be more efficient in protecting against a subsequent challenge of TS/A-pc than the Th1-type memory elicited by IL-2 gene-transduced TS/A cells.