RESUMEN
Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.
Asunto(s)
Apoptosis , Grupo Citocromo c/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Serina Endopeptidasas/metabolismo , Adenoviridae , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Vectores Genéticos , Granzimas , Humanos , Células Jurkat , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2RESUMEN
gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility complex restricted or to be correlated with target cell expression of heat-shock proteins. Based on the ability of some epithelial tumors, including colorectal, pancreatic, and renal cell cancers to effectively cold target inhibit the lysis of colorectal cancer cell lines by these Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cells are capable of recognizing cell surface Ag(s) shared by tumors of epithelial origin.
Asunto(s)
Neoplasias Colorrectales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Glandulares y Epiteliales/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/inmunología , Secuencia de Bases , Moléculas de Adhesión Celular/análisis , Línea Celular , Técnicas de Cultivo/métodos , Humanos , Interferón gamma/metabolismo , Interleucina-1/farmacología , Interleucina-7/farmacología , Neoplasias Renales/inmunología , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Neoplasias Pancreáticas/inmunología , ARN Mensajero/genética , Homología de Secuencia , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacosRESUMEN
We have recently reported that tumor-associated lymphocytes obtained from ascitic fluids of women with ovarian carcinoma (OvCA) demonstrate a marked decrease in expression of cytoplasmic CD3-zeta and surface CD3-epsilon chains, which is associated with altered function of T cell receptor (TcR). We now demonstrate that OvCAs in situ and in culture express functional Fas ligand (FasL), capable of triggering an intrinsic cell death program in Fas-expressing T cells. The possibility of a relationship between cell death and altered expression of TcR was examined. The data indicate that alterations in expression of CD3-zeta and CD3-epsilon chains in T cells coincubated with OvCA are related to tumor-induced apoptosis, as the addition of pan-caspase inhibitors, DEVD-cho or YVAD-cho, prevents both the in vitro induction of T cell death by OvCA cells and the changes in the level of expression of CD3-zeta and CD3-epsilon chains. In the presence of Fas-Fc fusion protein, but not Fc-control protein, the loss in expression of CD3-zeta and CD3-epsilon chains induced in T cells by FasL+ OvCA cells was prevented. These results suggest that the loss in expression of CD3-zeta and CD3-epsilon chains in T lymphocytes interacting with OvCA cells is associated with apoptosis mediated by FasL-expressing tumor cells.
Asunto(s)
Apoptosis , Complejo CD3/análisis , Linfocitos/fisiología , Glicoproteínas de Membrana/fisiología , Neoplasias Ováricas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Proteína Ligando Fas , Femenino , Humanos , Linfocitos/química , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , ARN Mensajero/análisis , Receptor fas/análisisRESUMEN
Lymphocytes separated from surgically resected tumor tissue, uninvolved lung tissue, and peripheral blood of lung cancer patients were investigated for cytotoxic potential and analyzed for their phenotypes at the time of surgery and after having been propagated for 4 to 5 wk in the presence of interleukin-2. Most of the tumor lymphocyte infiltrates examined were shown to have a shift in favor of T8 subsets from those found in peripheral blood. No natural killer activity and low cytotoxicity against the autologous tumor were found to characterize the tumor-derived lymphocyte population. Propagation of lymphocytes from the different tissues of the cancer patient in the presence of interleukin-2 preparation induced widespread lytic activity against K562 cells, autologous and allogeneic tumors, but not autologous normal lung or lymphoblasts. However, cytotoxic activity against autologous tumor cells exerted by cultured tumor-infiltrating lymphocytes was found to be significantly higher than the activity of cultured lymphocytes isolated from peripheral blood or uninvolved lung tissue of the same patient. The elevated lytic activity of cells derived from the tumor tissue indicates the accumulation at the tumor site of precursors of natural killer-like cells and specifically stimulated antitumor effectors. Our results suggest the coexistence of two types of anti-autotumor cytotoxic lymphocytes at the tumor site: natural killer-like and specific cytotoxic T-cells.
Asunto(s)
Citotoxicidad Inmunológica , Linfocitos/inmunología , Neoplasias/inmunología , Anciano , Separación Celular , Células Cultivadas , Femenino , Humanos , Interleucina-2 , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
We recently reported an association between loss in T-cell receptor (TcR) zeta-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that zeta-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the zeta-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of zeta-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of zeta-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated zeta-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-AspGlu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated zeta-chain was observed. These results suggest that the loss of TcR zeta-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the zeta-chain by activated caspases. This is the first report of involvement of caspases in degradation of the zeta protein.
Asunto(s)
Caspasas/fisiología , Proteínas de la Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Apoptosis , Humanos , Células Jurkat , Datos de Secuencia Molecular , Fosforilación , Proteínas Recombinantes/farmacología , Receptor fas/fisiologíaRESUMEN
Recent reports have variously described expression of Fas ligand (FasL) or its absence in human tumors. The importance of the Fas-FasL mechanism for the immune evasion by tumors provided a strong rationale for the examination of FasL expression and function in squamous cell carcinoma of the head and neck (SCCHN), which is one of the most immunosuppressive human cancers. Using immunostaining or immunoblotting, SCCHN cell lines and tumor biopsies were examined for the presence of the components of the Fas-FasL pathway and found to express Fas, as well as both the full-length and cleaved forms of FasL. By reverse transcription-PCR, mRNA for FasL and Fas were detected in all SCCHN tested, and cross-hybridization to radioactive Fas and FasL cDNA probes confirmed the specificity of amplification. To demonstrate that FasL expressed on cell surface of SCCHN cells was biologically active, various SCCHN lines were coincubated with the Fas-sensitive Jurkat T-cell lines or activated peripheral blood mononuclear cells. Tumor-induced apoptosis of T cells was dependent on the ratio of tumor cells: lymphocytes. It was significantly but only partially inhibited by neutralizing antibodies to FasL and antagonistic antibodies to FasR. Tumor-induced apoptosis was enhanced by the pretreatment of tumor cells with metalloproteinase inhibitors, which increased expression of FasL on tumor cells. Supernatants of tumor cells transduced with FasL also induced apoptosis of Jurkat cells. Thus, coincubation of SCCHN with Fas-sensitive lymphocytes can induce apoptosis of these lymphocytes, and the Fas/FasL pathway appears to be responsible, at least in part, for tumor-induced lymphocyte death. The data suggest that the Fas/FasL pathway is potentially immunosuppressive and may be involved in the escape of human carcinoma cells from immune destruction.
Asunto(s)
Apoptosis , Carcinoma de Células Escamosas/química , Neoplasias de Cabeza y Cuello/química , Linfocitos Infiltrantes de Tumor/fisiología , Glicoproteínas de Membrana/análisis , Proteína Ligando Fas , Humanos , Immunoblotting , Células Jurkat , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , ARN Mensajero/análisis , Tiofenos/farmacología , Células Tumorales Cultivadas , Receptor fas/análisisRESUMEN
We have recently reported that apoptosis of T cells induced by squamous cell carcinoma of the head and neck (SCCHN) is partly Fas dependent. This tumor-induced T-cell death is mediated by the activities of caspase-8 and caspase-3 and is partially inhibited by antibodies to either Fas or Fas ligand. We report here that in contrast to apoptosis induced by agonistic anti-Fas antibody (Ab), the tumor-induced apoptotic cascade in Jurkat cells is significantly amplified by a mitochondrial loop. The involvement of mitochondria in tumor-induced apoptosis of T cells was demonstrated by changes in mitochondrial permeability transition as assessed by 3,3'-dihexiloxadicarbocyanine staining, by cleavage of cytosolic BID and its translocation to the mitochondria, by release of cytochrome c to the cytosol, and by the presence of active subunits of caspase-9 in Jurkat T cells cocultured with tumor cells. To further elucidate the significance of mitochondria in tumor-induced T-cell death, we investigated the effects of various inhibitors of the mitochondrial pathway. Specific antioxidants, as well as two inhibitors of mitochondria permeability transition, bongkrekic acid and cyclosporin A, significantly blocked the DNA degradation induced in Jurkat T cells by SCCHN cells. However, these inhibitors had no effect on cells triggered by anti-Fas Ab. Furthermore, a cell-permeable inhibitor of caspase-9, Ac-LEHD.CHO, which did not inhibit T-cell apoptosis induced by anti-Fas Ab, markedly inhibited apoptosis induced by etoposide or by coculture of Jurkat with SCCHN cells. These findings demonstrate that apoptotic cascades induced in Jurkat T lymphocytes by anti-Fas Ab or tumor cells are differentially susceptible to a panel of inhibitors of mitochondrial apoptotic events. It appears that besides the Fas-mediated pathway, additional mitochondria-dependent cascades are involved in apoptosis of tumor-associated lymphocytes. Inhibition of mitochondria-dependent cascades of caspase activation should be considered to enhance the success of immunotherapy or vaccination protocols in cancer.
Asunto(s)
Apoptosis , Mitocondrias/patología , Linfocitos T/patología , Antibacterianos/farmacología , Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Western Blotting , Ácido Bongcréquico/farmacología , Carbocianinas/farmacología , Carcinoma de Células Escamosas/patología , Proteínas Portadoras/metabolismo , Caspasa 9 , Caspasas/metabolismo , Técnicas de Cocultivo , Ciclosporina/farmacología , Grupo Citocromo c/metabolismo , Grupo Citocromo c/fisiología , Citosol/metabolismo , ADN/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/farmacología , Neoplasias de Cabeza y Cuello/patología , Humanos , Células Jurkat , Linfocitos/patología , Microscopía Fluorescente , Oligopéptidos/farmacología , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Células Tumorales Cultivadas , Receptor fas/inmunologíaRESUMEN
Several endogenous or viral inhibitors of apoptosis, including Bcl-2, Bcl-xL, FLIP, p35, and CrmA, have been shown to be cleaved by caspases during apoptosis. In this study, we demonstrate that the endogenous inhibitor of apoptosis, hILP/XIAP, is also cleaved in apoptotic T lymphocytes, generating at least one prominent fragment of 29 kDa. This p29 cleaved fragment was detected in Jurkat cells induced to apoptose by anti-Fas antibody, staurosporin, or VP-16. The cleavage of hILP appears to be caspase mediated because the production of the p29 protein was inhibited by the pan-caspase peptide inhibitor, Z-VAD.FMK. In Jurkat cells engineered to overexpress CrmA, cleavage of hILP in response to anti-Fas antibody or staurosporin was inhibited, whereas overexpression of Bcl-2 abrogated the cleavage in response to VP-16. Cleavage of hILP was also observed in cell-free reactions using in vitro translated hILP and recombinant caspase-3 or -7. Moreover, we found that the p29 hILP fragment retained the ability to bind caspase-3 and -7, as shown previously for full-length or BIR-2 hILP. The p29 cleavage product was also detected during T-cell receptor-mediated apoptosis in peripheral blood lymphocytes from normal donors. Furthermore, tumor-associated T lymphocytes purified from ascites of patients with ovarian cancer expressed fragmented hILP, which was not detected in control T cells purified from peripheral blood of normal donors. Our results suggest that the cleavage of hILP represents an important event in apoptosis of T lymphocytes in both normal and pathological in vivo settings.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas/metabolismo , Linfocitos T/citología , Linfocitos T/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores Enzimáticos/metabolismo , Etopósido/farmacología , Humanos , Células Jurkat , Estaurosporina/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X , Receptor fas/fisiologíaRESUMEN
Interleukin-2 (IL-2) is recognized as a T cell growth factor. We have previously reported that human carcinoma cell lines are inhibited in growth by exogenous IL-2, which binds to the IL-2 receptor beta (IL-2Rbeta) chain ubiquitously expressed on the surface of tumor cells. A possibility was considered that IL-2Rbeta on carcinomas responsible for negative signaling was different from that expressed on hematopoietic cells. To investigate this possibility, mRNA for the IL-2Rbeta chain was amplified and compared in carcinoma and lymphoid cells. Using RT-PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the IL-2Rbeta chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells as controls. The identity of the amplicons was confirmed by Southern analysis with the 32P-labeled cDNA probe coding for the entire span of the IL-2Rbeta chain. In addition, genomic DNA obtained from the tumor cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular IL-2Rbeta chain domain. No mutations or deletions were detected. The message for all three domains of the beta chain was identical in tumor cells and in normal lymphoid cells used as controls. Also, by Western blot and northern analyses no differences between IL-2Rbeta chain in tumors vs that expressed in lymphoid cells were demonstrable. The IL-2Rgamma chain, which participates in IL-2/IL-2R signaling pathway, was expressed in tumor cells. Expression of JAK1 transcripts in these cells was comparable to that in lymphocytes. However, RT-PCR analysis identified differences in expression of JAK3 splice variants (B and M) in tumor cells. These differences may be responsible for altered downstream signaling by IL-2. Overall, our data indicate that the same IL-2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells.
Asunto(s)
Neoplasias/inmunología , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/genética , Carcinoma de Células Renales , Cartilla de ADN , Neoplasias de Cabeza y Cuello , Humanos , Janus Quinasa 1 , Células Jurkat , Neoplasias Renales , Linfocitos/enzimología , Linfocitos/inmunología , Sustancias Macromoleculares , Neoplasias/genética , Oligonucleótidos Antisentido , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Interleucina-2/química , Neoplasias Gástricas , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Considerable effort in the research of adhesion molecules in recent years has been directed toward elucidation of their structure, identification of their ligands, and evaluation of their role in growth, differentiation, and migration. More recent evidence suggests that integrins function not only as adhesive proteins, but also as receptors capable of transducing biochemical signals to the cell interior. In natural killer lymphocytes, integrins have been shown to have a costimulatory role in proliferation, cytotoxicity, and cytokine production. This review will focus on recent advances in the characterization of biochemical pathways activated via integrins on NK cells and elucidation of the participating second messengers.
RESUMEN
TRAIL-induced apoptosis has been considered a promising therapeutic approach for tumors that are resistant to chemotherapy, which is usually mediated via mitochondrial apoptotic cascades. Recent studies have shown that in certain cancer cells, TRAIL-mediated apoptosis is also dependent on mitochondrial involvement, suggesting that similar mechanisms of resistance to chemotherapy might be implicated in the resistance of tumor cells to TRAIL. We have used TRAIL-resistant leukemic cells that are deficient in both Bax and Bak to determine the roles of these Bcl-2 members in TRAIL-mediated apoptosis. Exposure of these cells to TRAIL did not have an impact on cell viability, although it induced the processing of caspase-3 to its active p20 subunit. The activity of the p20 caspase-3 appeared to be inhibited as no autoprocessing of this p20 subunit or cleavage of known caspase-3 substrates were detected. Also, in the absence of Bax and Bak, no release of mitochondrial apoptogenic proteins was observed following TRAIL treatment. Adenoviral transduction of the Bax, but not the Bak gene, to the Bax/Bak-deficient leukemic cells rendered them TRAIL-sensitive as assessed by enhanced apoptotic death and caspase-3 processing. These findings demonstrate preferential utilization of Bax over Bak in leukemic cell response to specific apoptotic stimulation.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia/metabolismo , Glicoproteínas de Membrana/farmacología , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adenoviridae/genética , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Caspasa 3 , Caspasas/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Leucemia/patología , Proteínas de la Membrana/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas/genética , Linfocitos T/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2RESUMEN
Tumor-associated lymphocytes (TALs) freshly isolated from patients with cancer usually manifest reduced proliferative and cytolytic functions. To determine whether alterations in signal transduction contribute to functional impairments seen in TALs, we purified populations of T and natural killer (NK) cells by negative selection from ascites of seven patients with ovarian carcinoma. The average purity was 84 +/- 5% for CD3(+) TALs and 77 +/- 10% for CD3(-)CD56(+)CD16(+) TALs. Expression of several signal transduction molecules, including the CD3-epsilon, CD3-zeta, and FcepsilonRI-gamma chains, p56(lck) protein tyrosine kinase, and phospholipase C-gamma1, was studied in these cells using Western blotting. A marked decrease in expression of zeta and FcepsilonRI-gamma associated with CD3 or FcgammaRIIIA was observed in T or NK cells obtained from TALs, as compared to T or NK cells purified from normal peripheral blood. Expression of CD3-epsilon, as assessed using flow cytometry, Western blotting, or ELISA was also reduced in purified TAL-T cells relative to that in normal peripheral blood T cells. Surface expression of CD3 on T cells and FcgammaRIIIA on NK cells obtained from TALs was significantly decreased in comparison to normal peripheral blood lymphocytes (PBLs): the mean fluorescence intensity of CD3 was 277 +/- 18 for TAL-T (n = 7) versus 349 +/- 13 for PBL-T (n = 9) and that of CD16 was 58 +/- 1 for TAL-NK (n = 7) versus 385 +/- 55 for PBL-NK (n = 23) cells. These observations suggest a defect in assembly of T cell receptor and FcgammaRIIIA multicomponent transmembrane receptors, which are zeta and gamma dependent. In addition to alterations in expression, the function of these receptors was also modified, since cross-linking of CD3 on TAL-T and CD16 on TAL-NK cells with the respective monoclonal antibodies resulted in a pattern of protein phosphorylation that was distinct from that observed in normal PBLs. Expression of tyrosine kinase p56(lck) and its kinase activity were also depressed, while expression of phospholipase C-gamma1 appeared to be normal in most preparations of the TALs tested. In vitro proliferation of TAL-T in response to anti-CD3 monoclonal antibody and TAL-NK cells to interleukin 2 were significantly depressed as was the ability to produce IFN-gamma. In contrast, TAL-T cells were able to produce interleukin 10 at levels similar to those secreted by normal PBLs. Thus, in TALs obtained from patients with advanced ovarian cancer, alterations in expression and activity of signaling molecules were associated with reduced cellular functions such as proliferation and production of certain cytokines.
Asunto(s)
Células Asesinas Naturales/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Ováricas/inmunología , Transducción de Señal , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Membrana/análisis , Persona de Mediana Edad , Fosforilación , Receptores de Antígenos de Linfocitos T/análisis , Receptores de IgE/análisis , Receptores de IgG/análisis , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismoRESUMEN
Recent studies have demonstrated altered expression and function of signaling molecules in T and natural killer cells in patients with cancer. The impairment of immune cell functions in advanced cancer may result from defects in signal transduction. We studied purified T cells obtained from peripheral blood or tumor-involved lymph nodes (LNs) of 45 patients with advanced metastatic melanoma for the presence of abnormalities in expression or activity of various signaling molecules. Western blot analyses demonstrated reduced expression of CD3-zeta in 10 of 11 preparations of T cells obtained from tumor-involved LNs. Similar reduction in expression of CD3-zeta was demonstrated by immunostaining performed in situ on frozen sections of melanoma tissues. Expression of p56(lck) and Zap-70, but not phospholipase C-gamma1, was reduced in these patients' T cells relative to those obtained from normal individuals. In 50% of the patients, reduced expression of CD3-zeta and p56(lck) was observed in T lymphocytes obtained both from tumor-involved LNs and from peripheral blood. To determine whether deficient expression of these signaling molecules is reversible, T cells from melanoma-involved LNs were incubated in the presence of interleukin 2 (IL-2) for 48 h, and lysates from fresh or cultured lymphocytes were compared for changes in expression of signaling molecules. Cells cultured in the presence of IL-2 demonstrated increased expression of CD3-zeta and p56(lck), which approached the levels detected in normal T cells. However, the level of p56(lck) kinase activity did not normalize in any of the LN-derived lymphocytes cultured in the presence of IL-2. Decreased expression of CD3-zeta or p56(lck) observed in the patients' T cells was not reversed by immunotherapy with IL-2 at low or high dose in those patients with metastatic melanoma who failed to respond to therapy. However, in three patients who achieved clinical responses, the initially reduced expression of zeta in peripheral blood T cells normalized following IL-2 therapy.
Asunto(s)
Interleucina-2/uso terapéutico , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Melanoma/inmunología , Melanoma/secundario , Proteínas de la Membrana/análisis , Receptores de Antígenos de Linfocitos T/análisis , Linfocitos T/química , Humanos , Ganglios Linfáticos/química , Melanoma/terapia , Fosfolipasas de Tipo C/análisisRESUMEN
Melanoma escapes host defenses through a variety of means, including the elimination of immune effector cells within the tumor microenvironment. We have reported recently that murine and human tumors including melanoma induce premature apoptosis of dendritic cells both in vitro and in vivo. In this study, we have demonstrated that overexpression of the Bcl-2 protein family member Bcl-xL rescued murine dendritic cells (DCs) from melanoma-induced death in vitro. Another successful protection approach was tumor necrosis factor (TNF)-alpha-promoted sustained expression of the antiapoptotic protein Bcl-2 within dendritic cells. This effect of TNF-alpha was mediated by inhibition of mitochondrial cytochrome c release. Thus, both Bcl-xL and Bcl-2 enhance survival of dendritic cells within the tumor microenvironment. In addition, mature DCs were more resistant to melanoma-induced apoptosis than immature dendritic cells. This finding suggests a stage-dependent sensitivity of DCs to tumor-induced cell death. We conclude that: (a) mature DCs might be more suitable for the use of cancer vaccination; and (b) Bcl-2 protein family members such as Bcl-xL and Bcl-2 rescue DCs from tumor-induced premature apoptosis.
Asunto(s)
Apoptosis , Grupo Citocromo c/antagonistas & inhibidores , Células Dendríticas/metabolismo , Melanoma/patología , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Western Blotting , Grupo Citocromo c/metabolismo , Fragmentación del ADN , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Transfección , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co-culture of murine or human DC with different tumor cell lines for 4-48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase-3-like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time- and dose-dependent induction of apoptosis in DC by tumor-derived factors. This finding is both effector and target specific. The mechanism of tumor-induced DC apoptosis involved regulation of Bcl-2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumor-associated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC-based immunotherapies for cancer and other immune diseases.
Asunto(s)
Apoptosis/inmunología , Células Dendríticas/inmunología , Neoplasias/inmunología , Animales , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Fragmentación del ADN , Electroforesis en Gel de Agar , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transglutaminasas/metabolismo , Tritio , Células Tumorales CultivadasRESUMEN
Adherent lymphokine-activated killer (A-LAK) cells, selected from peripheral blood lymphocytes (PBL) of normal human donors by adherence to plastic, and cultured in the presence of interleukin 2 (IL-2), are highly enriched in CD3-CD56+ natural killer (NK) cells. These IL-2-activated NK cells proliferate extensively upon further culture in conditioned medium containing IL-2. In contrast, we previously found that with PBL of some patients with advanced cancer, the same procedure often failed to yield high enrichment of NK cells or substantial expansion in the numbers of these effector cells. To obtain sufficient numbers of A-LAK cells for adoptive immunotherapy in cancer patients, an improved method for generation of human A-LAK cells with irradiated mitogen-stimulated allogeneic PBL- or Epstein-Barr virus-transformed lymphoblastoid cell lines was introduced. In paired experiments, A-LAK cultures with feeder cells showed significantly enhanced IL-2-driven proliferation of A-LAK cells obtained from normal donors or patients with metastatic melanoma, renal cell carcinoma, and other types of solid cancers. The growth-promoting effect of feeders for A-LAK cells resulted in significantly improved expansion of CD3-CD56+ (NK) effector cells in A-LAK cultures established from normal donors. Cells in these cultures also had significantly higher levels of antitumor cytotoxicity against K562 and Daudi targets than did A-LAK cells grown in the absence of feeder cells. Enrichment in CD3-CD56+ cells and antitumor activity also occurred in patient A-LAK cultures supplemented with mitogen-stimulated feeder cells, but was not statistically significant. Overall, despite improved proliferation and CD3-CD56+ cell content of A-LAK cultures established in the presence of mitogen-activated feeder cells, only 39% (21/54) of patients tested generated A-LAK cells that would be judged acceptable for large-scale therapeutic use by criteria based on fold expansion and purity of A-LAK cells. These results suggest that in comparison to normal individuals, NK cells of many patients with advanced solid tumors are defective in their ability to respond by proliferation to IL-2 even in the presence of exogenously supplied growth factors.
Asunto(s)
Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Células Asesinas Activadas por Linfocinas/inmunología , Melanoma/inmunología , Adhesión Celular/inmunología , Línea Celular Transformada , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Humanos , InmunofenotipificaciónRESUMEN
Peripheral blood mononuclear cells (PBMC) from aged humans were found impaired in their ability to mount T mitogen induced proliferative responses and NK cell activity. The production of IL-2 in response to PHA has also been found decreased in the aged subjects. When the data from IL-2 production and cellular responsiveness of aged and young groups were pooled for regression analysis, a significant correlation was found both for mitogenic responses and NK activity. The reduced proliferative responses and NK cell activity can be enhanced by exposing them to exogenous IL-2. However, they were not fully reconstituted to the levels achieved in PBMC of the young subjects supplemented in vitro with IL-2. This would indicate that the reduced cellular responsiveness in aging may be related both to a defect of IL-2 receptor expression on T cells of aged subjects and to an impairment in the endogenous IL-2 synthesis in the aged individuals.
Asunto(s)
Anciano , Interleucina-2/biosíntesis , Linfocitos/inmunología , Monocitos/inmunología , Adulto , Envejecimiento , Línea Celular , Células Cultivadas , Citotoxicidad Inmunológica , Femenino , Humanos , Interleucina-2/aislamiento & purificación , Células Asesinas Naturales/inmunología , Leucemia Mieloide , Activación de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
Diminished immune responses in aging may be partially due to alteration of the lipid composition of membrane and a decrease in membrane fluidity. The effect of a daily diet of 10 g active lipid (AL 721) on the mitogen responsiveness of peripheral blood mononuclear cells (PBMC) from aged volunteers was studied. AL 721 is a mixture of lipids from hen egg yolk which was formulated for in vivo rectification of rigidified cell membranes in an attempt to restore proper physiological function. After 3 weeks on the diet, six aged participants displayed a significant increase in lymphocyte responsiveness to mitogens, while in four the responsiveness was unaffected, or increased insignificantly. In all six participants, a decline in lymphocyte responsiveness was observed a week after termination of the diet. Our preliminary results indicate that improvement of immune functions in the aged may be achieved by dietary manipulation of lipids. This innocuous approach seems to be of potential value and merits further studies.
Asunto(s)
Grasas de la Dieta , Glicéridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Fosfatidilcolinas/farmacología , Fosfatidiletanolaminas/farmacología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Combinación de Medicamentos/farmacología , Femenino , Humanos , Linfocitos/citología , Linfocitos/inmunología , Masculino , MitógenosRESUMEN
Fourteen-day mouse fetal liver was found to contain cells capable of giving rise to T as well as B cell functions. The experimental system consisted of congenic C3H/DiSn and (C3H/DiSn X C3H.SW)F1 lethally irradiated (900 R) mice reconstituted with C3H/DiSn fetal liver or bone marrow cells. Assays included thyroid allograft rejection as well as in vitro measurement of reactivity to phytohemagglutinin (PHA) and concanavalin A (Con A) and in a mixed lymphocyte culture (MLC) system in spleen, lymph node, and thymus cells. The fetal liver chimeras were found to become as capable as the bone marrow chimeras in responding in these various assays. The T cell responses lagged behind the responses to the B cell mitogens dextran sulfate (DXS) and lipopolysaccharide (LPS) (30 days after reconstitution, as compared with 14 days for DXS and 21 for LPS). The reacting cells were of the donor genotype, as revealed after treatment with C3H/DiSn (H-2k) anti-C3H.SW (H-2b) congenic sera. T cell responses were not manifest in thymectomized (TX) chimeras. Hence, the liver seems to contain cells capable of developing into T cell lineages in a thymus-dependent process.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Hígado/citología , Linfocitos T/citología , Animales , Linfocitos B/citología , Trasplante de Médula Ósea , Femenino , Feto , Rechazo de Injerto , Células Madre Hematopoyéticas/inmunología , Trasplante de Hígado , Ratones , Ratones Endogámicos C3H , Embarazo , Quimera por Radiación , Linfocitos T/inmunologíaRESUMEN
Incubation of human fetal liver cells (HFLC), obtained from 8-12-week-old fetuses, with mitogenic doses of Concanavalin A (Con A) resulted in generation of a population that could suppress alloantigen-induced lymphoproliferation. The supernatant media in which these cells were activated suppressed DNA synthesis, both in mitogen and alloantigen induced peripheral blood lymphocyte cultures. Suppression was achieved only when the Con A-activated cells or the supernatants were added during the early stages of sensitization. Treatment of the cells with mitomycin C after Con A activation did not alter their ability to mediate suppression. This study indicates the presence of suppressor or presuppressor cells in the liver in the early period of fetal life. If the suppressor activity of HFLC in vitro is also induceable in vivo, it may contribute to the fetal defense mechanism against maternal rejection.