RESUMO
By using bifunctional T cell populations, we have shown in this report that elicitation of helper versus cytolytic function depends on the stimulatory signal at the membrane. Interestingly enough, the transduction of these signals is likely to be achieved via different metabolic pathways. Thus, helper function is associated with intracellular Ca2+ mobilization and PLC activation, while cytolysis can occur even in the absence of detectable levels of these second messengers. These results indicate that selective activation through the same membrane-transducing molecule may orientate T cell function through qualitatively or quantitatively different second messengers. This would be an important part of immune regulation.
Assuntos
Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD2 , Cálcio/metabolismo , Células Clonais , Humanos , Interleucinas/biossíntese , Fosfatidilinositóis/metabolismo , RNA Mensageiro/biossíntese , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Imunológicos/imunologia , Formação de Roseta , Transdução de SinaisRESUMO
Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglobulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibodies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents.
Assuntos
Anticorpos Monoclonais/imunologia , Diversidade de Anticorpos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Genes , Hibridomas/imunologia , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Camundongos , Mutação , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Preclinical models and clinical studies have shown that anti-CD20-based treatment has multifaceted consequences on T-cell immunity. We have performed a prospective study of peripheral T-cell compartment in FL patients, all exhibiting high tumor burden and receiving rituximab-chemotherapy-based regimen (R-CHOP). Before treatment, FL patients harbor low amounts of peripheral naive T cells, but high levels of CD4+ TEM, CD4+ Treg and CD8+ TEMRA subsets and significant amounts of CD38+ HLA-DR+ activated T cells. A portion of these activated/differentiated T cells also expressed PD-1 and/or TIGIT immune checkpoints. Hierarchical clustering of phenotyping data revealed that 5/8 patients with only a partial response to R-CHOP induction therapy or with disease progression segregate into a group exhibiting a highly activated/differentiated T cell profile and a markedly low proportion of naive T cells before treatment. Rituximab-based therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype and of CD8+ T cells to a naive phenotype. In parallel, a decrease in the number of peripheral T cells expressing both PD-1 and TIGIT was detected. These observations suggest that the standard rituximab-based therapy partially reverts the profound alterations observed in T-cell subsets in FL patients, and that blood T-cell phenotyping could provide a better understanding of the mechanisms of rituximab-based treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imunidade Celular , Memória Imunológica/imunologia , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/imunologia , Subpopulações de Linfócitos T/imunologia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores , Ciclofosfamida , Doxorrubicina , Feminino , Humanos , Memória Imunológica/efeitos dos fármacos , Imunofenotipagem , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Linfoma Folicular/diagnóstico , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prednisona , Rituximab/administração & dosagem , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , VincristinaRESUMO
The purpose of these studies was to investigate whether the cell-differentiating effect of anthracyclines can trigger an over-production and secretion of molecules that may interfere with the tumor-host relationship. We exposed mouse hybridoma B-cells, which are devoted to immunoglobulin production, to doxorubicin (10-40 ng/ml). We found that most doxorubicin-treated cells secreted 3- to 5-fold higher amounts of immunoglobulin than untreated cells, along with an accumulation of 50% of them in the G2+M phase of the cell cycle. The antigenic specificity of the immunoglobulin and its size pattern as determined by polyacrylamide gel electrophoresis were similar whether or not cells were treated with doxorubicin. The enhancement of immunoglobulin secretion by doxorubicin was associated with an increase of the intracellular pool of heavy and light chains of the immunoglobulin. Furthermore, an elevated synthesis of immunoglobulin was observed. The synthesis of other proteins also appeared to be modified in these circumstances. These data suggest that doxorubicin can potentiate the biological functions of target cells when used at low concentrations, elevating the production and secretion of effector molecules that interfere with the tumor-host relationship.
Assuntos
Linfócitos B/imunologia , Doxorrubicina/farmacologia , Hibridomas/imunologia , Imunoglobulinas/biossíntese , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Hibridomas/citologia , Hibridomas/efeitos dos fármacos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Cinética , Camundongos , Biossíntese de ProteínasRESUMO
BALB/c 3T3 cells transformed in vitro with polyoma virus were cloned and passaged once in syngeneic mice. Resulting tumors from each clone were explanted and recultured. Expression of receptor for Fc of IgG (Fc gamma RII) in the original in vitro maintained clones and in cells derived from tumors elicited by the respective cells was measured at the protein level as well as at the mRNA level. Clones were assayed in pairs. The ancestor in vitro maintained clones [designated cultured cells (C)] were compared with cells derived from the same clones after a single passage in vivo followed by explantation and reculturing [designated cultured-tumor-cultured cells (CTC)]. C cells of any of the tested clones did not express Fc gamma RII. On the other hand, certain CTC cells were positive. The Fc gamma RII-positive cells were derived from tumors appearing after a long precancer latency period (greater than 140 days). CTC cells derived from tumors that appeared after shorter latency periods (less than 80 days) were Fc gamma RII negative. These results were obtained both by using radioimmunoassay and monoclonal antibodies against mouse Fc gamma RII as well as by Northern blot analysis using the Fc gamma RII complementary DNA probe. The involvement of macrophages as the Fc gamma RII-expressing cells in CTC cells was excluded. Fc gamma RII expression was down-regulated in CTC cells as a function of time following their explantation into culture. Fc gamma RII expression could be up-regulated in these cells and induced on C cells by maintaining the cultured cells in the presence of normal mouse serum or recombinant interferon. We also tested the expression of Fc gamma RII on CTC cells following their inoculation into syngeneic mice for a second time (CTCx2 cells). The results showed a positive correlation between Fc gamma RII expression in the inoculated ancestor CTC cells and on the CTCx2 cell progeny.
Assuntos
Antígenos de Diferenciação/genética , Transformação Celular Neoplásica , Neoplasias Experimentais/genética , Polyomavirus/genética , Receptores Fc/genética , Animais , Antígenos de Diferenciação/análise , Biomarcadores Tumorais/análise , Linhagem Celular , Membrana Celular/imunologia , Células Clonais , Imunoglobulina G/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , RNA Neoplásico/genética , Receptores Fc/análise , Receptores de IgGRESUMO
Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.
Assuntos
Fragmentos de Imunoglobulinas/administração & dosagem , Neoplasias Experimentais/terapia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose , Genes p53 , Genes ras , Humanos , Imunoterapia , Camundongos , Camundongos Nus , Microinjeções , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Xenopus laevisRESUMO
We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.
Assuntos
Fragmentos de Imunoglobulinas/genética , Transcrição Gênica/imunologia , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Vetores Genéticos , Humanos , Fragmentos de Imunoglobulinas/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.
Assuntos
Clonagem Molecular/métodos , Fragmentos de Imunoglobulinas/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Camundongos , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Células Tumorais CultivadasRESUMO
Alteration of the p53 gene is the most frequent genetic alteration in human cancer and leads to the accumulation of mutant p53 in the nucleus of tumor cells. In addition, it has been shown that patients with various types of neoplasia have p53 antibodies in their sera which could be used as an indirect diagnostic procedure for p53 alteration. Using a new ELISA, we have analyzed the sera from more than 1000 patients with various types of cancer and from healthy blood donors. We demonstrate that p53 antibodies are detected mainly in cancer patients and are strictly proportional to the occurrence of p53 mutations. Using various immunological approaches, these antibodies were unambiguously demonstrated to be directed toward the human p53 protein. Isotyping analysis of these antibodies strongly suggested that they correspond to a humoral response to the p53 protein which accumulates in the tumor cell. This finding suggests that serological analysis, combined with histochemistry, is suitable for assessing the integrity of the p53 gene in cancer patients.
Assuntos
Anticorpos Antineoplásicos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Genes p53/imunologia , Neoplasias/imunologia , Proteína Supressora de Tumor p53/imunologia , Especificidade de Anticorpos , Feminino , Genes p53/genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Neoplasias/sangue , Neoplasias/genéticaRESUMO
Soluble Fc gamma receptors have been identified in biological fluids of mice and humans. They are produced either by alternative splicing of the exon encoding the transmembrane region of the receptor (Fc gamma RII) or by proteolytic cleavage at the cell membrane (Fc gamma RII and Fc gamma RIII). They inhibit B cell proliferation and immunoglobulin production. Their concentrations in plasma seem to be modified during the development of certain diseases, as for instance in multiple myeloma, where plasma concentrations of soluble Fc gamma RIII are correlated with the stage of the disease.
Assuntos
Receptores de IgG/fisiologia , Animais , Humanos , Camundongos , Receptores de IgG/química , Receptores de IgG/genética , SolubilidadeRESUMO
Thirty years after their discovery by Milstein and Köhler, monoclonal antibodies have now come of age as therapeutics. Nineteen monoclonal antibodies are on the market and/or have got authorization to be used for the treatment of severe diseases. Many technical efforts have been devoted over the last two decades to the generation of second generation mAbs with better affinities, decreased immunogenicity and optimized effector functions. The development of molecular engineering techniques applied to antibody molecules has also made it possible to design bi-specific antibodies and fusion molecules exhibiting different modules with bi-functional activities. The use of proteomics and genomics combined with phage display allows now the rapid selection of antibodies directed against new targets at a high rate. Many efforts are currently focused on the selection of high-responder patients, the optimization of antibody delivery, schemes of infusion, antibody pharmaco-kinetics and bio-distribution, as well as on a better control of the severe side-effects generated by some antibody treatments.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoterapia/tendências , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Linhagem Celular Transformada/imunologia , Ensaios Clínicos como Assunto , Herpesvirus Humano 4 , História do Século XX , Humanos , Hibridomas/imunologia , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Imunoterapia/história , Imunoterapia/métodos , Camundongos , Biblioteca de Peptídeos , Engenharia de Proteínas , Ratos , Proteínas Recombinantes de Fusão/uso terapêutico , Especificidade da EspécieRESUMO
Immunoglobulin G-binding factors (IgG-BF) produced by mouse T cells or hybridoma T cells (T2D4) were used to manipulate in vitro mouse hybridoma B cells. Both IgG production by, and proliferation of, these cells was inhibited by IgG-BF, or during co-cultures with IgG-BF-producing T2D4 cells. Thus, treatment of tumor B cells, besides its potential therapeutic use, represents an invaluable model for studying the regulation of Ig production by IgG-BF at a molecular level. To further analyze the molecular events induced by IgG-BF in B cell hybridomas, a set of variant clones of a hybridoma cell line (UN2) was isolated and variants were characterized for their Ig production and their Fc gamma R expression.
Assuntos
Linfócitos B/imunologia , Hibridomas/imunologia , Linfocinas/farmacologia , Proteínas Secretadas pela Próstata , Animais , Antígenos de Diferenciação/análise , Linfócitos B/citologia , Northern Blotting , Western Blotting , Divisão Celular , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunoglobulina G/biossíntese , Camundongos , Receptores Fc/análise , Receptores de IgGRESUMO
Given the presence of ionic channels at the membrane of lymphocytes, we have analyzed the effect of various channels blockers on B lymphocytes activation. TEA and 4-AP, two K+ channels blockers, quinine, a blocker of Ca2(+)-activated K+ channels, nickel and verapamil, two Ca2+ channels blockers, all inhibited LPS-induced B cell proliferation. However, these drugs neither inhibited the induction of Ia and Fc gamma RII expression nor cell enlargement and early RNA synthesis, indicating that the entry of B lymphocytes into G1 phase was not affected. In contrast, both late RNA synthesis and the induction of the TfR, which occur while the cell progress through G1, were inhibited by these blockers. These data show that TEA, quinine and verapamil block B lymphocyte activation during the G1 phase, probably between G1A and G1B. To question whether these effects were due to the block of voltage-activated K+ channels, we compared the ability of TEA, quinine, verapamil, 4-AP and nickel to block proliferation and K+ channels. A striking correlation was found for all the drugs but less for 4-AP. Moreover, TMA, a TEA analog unable to block K+ currents, did not affect B cell proliferation. Taken together, our data suggests that functional voltage-gated K+ channels are required at a precise stage of the G1 phase of the B cell cycle.
Assuntos
Linfócitos B/fisiologia , Canais Iônicos/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fase G1 , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Fase de Repouso do Ciclo Celular , Fase SRESUMO
A rat single-chain Fv (Y238 scFv) was derived from the Y13-238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13-238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vkappa chain derived from the rat fusion partner and of a rat Vlambda chain. Primers designed for rat Vlambda amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (K(D)=4.58+/-0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13-259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170-1176.]. Finally, BIAcore analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13-238 epitope.
Assuntos
Anticorpos Monoclonais/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Células 3T3 , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Sequência de Bases , Técnicas Biossensoriais , Clonagem Molecular , Primers do DNA , Mapeamento de Epitopos , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Hibridomas , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Proteínas Recombinantes/imunologia , Análise de SequênciaRESUMO
We analyzed several cellular and molecular properties of BALB/c 3T3 cellular clones transformed in vitro with polyoma virus and exhibiting a high or low tumorigenicity phenotype. We also analyzed the same clones after a single in vivo passage in syngeneic mice. This passage invariably induced and/or selected variants exhibiting a very high tumorigenicity phenotype. BALB/c mice bearing tumors induced by the inoculation of the above cells, regardless of their tumorigenicity phenotype, have a lower number of L3T4 positive splenocytes than appropriate controls. The response to Con-A of spleen cells from such mice was also suppressed. Concomitantly, an increase in Mac-1 positive splenocytes could be measured. In spite of the non-specific suppression of T cells, spleen cells from tumor-bearers showed a specific proliferative response to polyoma antigens. Molecular analysis of polyoma transformed cells showed no differences between the various cells with respect to integration of the polyoma viral genes or with respect to src, myc and fos proto-oncogenes. In vitro maintained cells and in vivo passaged cells seemed to differ, however, in the content of polyoma middle T. Whereas polyoma virus transformed cells maintained only in culture never expressed low affinity receptors for IgG (Fc gamma RII), certain in vivo passaged cells did. This expression could be measured both at the protein and the mRNA level. Those in vivo passaged cells which expressed F alpha RII gave tumors following a long latency period. Ongoing experiments will indicate whether or not Fc gamma RII expression is linked to long latency of tumor development.
Assuntos
Transformação Celular Neoplásica/imunologia , Transformação Celular Viral/genética , Animais , Antígenos de Diferenciação/biossíntese , Antígenos Transformantes de Poliomavirus/imunologia , Linhagem Celular Transformada , Mapeamento Cromossômico , Células Clonais , DNA Viral/genética , Variação Genética , Tolerância Imunológica , Camundongos , Fenótipo , Polyomavirus/genética , Proto-Oncogenes/genética , RNA Mensageiro/análise , Receptores Fc/biossíntese , Receptores de IgGRESUMO
In this study we report on some lines of ongoing research performed in our laboratory, in relation to the increased expression of FcR on tumor cells, as well as on cells present in the tumor-bearing host, and its possible role in tumor progression. In a previous study we have shown that a Polyoma virus (PyV)-induced anaplastic carcinoma (SEYF-a tumor) contained an FcR-expressing subpopulation of tumorigenic cells. We tested the effect of in vivo passaging of FcR-expressing and of non-FcR-expressing sub-populations of SEYF-a tumor cells on the expression of FcR, as revealed by the ability of these cells to bind the 2.4G2 monoclonal antibody, which is directed against mouse Fc gamma 2b/gamma 1R. It was found that upon in vivo passaging these two sub-populations became practically identical in their ability to bind anti-Fc gamma R antibody. On the other hand, in vitro passaging of FcR-expressing SEYF-a cells resulted in a gradual decrease in the expression of Fc gamma R. These results, indicating that the expression of Fc gamma R on tumor cells, per se, is dependent on a factor present in the in vivo environment were confirmed using 3T3 cells transformed in vitro by PyV (C) and forming tumors at first injection to mice (CTC). C cultures of various clones did not express Fc gamma R, while CTC cultures (cultures from tumors) became positive. We also detected an increase in the level of a soluble form of Fc gamma 2b/gamma 1R in the circulation of mice bearing PyV induced tumors. This increase paralleled the appearance of palpable tumors. A similar pattern of increase was observed in mice inoculated with the c-H-ras transformed tumorigenic clone 8/F/5, but not in mice inoculated with non-tumorigenic 3T3 cells. Data published by us show that metastatic breast cancer patients had significantly elevated Fc gamma R levels on their peripheral blood mononuclear cells (PBMC). Experiments presented here indicate a direct correlation between increased Fc gamma R levels on PBMC and tumor mass in colon, ovary and lung metastatic carcinoma patients. The possibility that malignantly transformed cells have the potential to cause proliferation of Fc gamma R expressing T cells was tested. It was found that extract derived from r-H-ras transformed 3T3 cells triggers the proliferation of a T cell hybridoma expressing Fc gamma R.
Assuntos
Antígenos de Diferenciação/análise , Neoplasias/imunologia , Receptores Fc/análise , Animais , Comunicação Celular , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neoplásicas/imunologia , Oncogenes , Polyomavirus , Receptores de IgG , Sarcoma Experimental/imunologia , Linfócitos T/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.
Assuntos
Células Dendríticas/citologia , Macrófagos/citologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD/biossíntese , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunofenotipagem , Interleucina-13/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fagocitose/efeitos dos fármacos , Saccharomyces cerevisiaeRESUMO
The long-lasting clinical response by lymphoma patients to anti-CD20 therapy has been attributed to the induction of an anti-tumor adaptive immunity. We previously demonstrated that a CD4-dependent mechanism is responsible for the long-term protection of CD20(+) tumor-bearing mice by anti-CD20 treatment. Here, we compare tumor immunity in tumor-bearing animals that did or did not receive anti-CD20 treatment. Splenic CD4(+)FoxP3(+) regulatory T cells (Tregs) expanded substantially in untreated mice that exhibited then a reduced survival, whereas Tregs depletion led to long-term survival of the animals, suggesting the establishment of a Treg-dependent immunosuppressive environment after tumor injection. Strikingly, anti-CD20 therapy reversed the initial expansion of Tregs, and was accompanied by a marked increase in the number of Th1 cells, with no detectable change in Th2 and Th17 cell numbers. Interleukin-12 serum level was also increased by the anti-CD20 treatment, and activated myeloid dendritic cells producing interleukin-12 could be detected in lymph nodes of treated animals, while interferon-γ blockade strongly reduced survival. Also, CD4(+) effector memory T cells were evidenced in surviving animals, and the transfer of CD4(+) T cells induced long-term protection. Thus, anti-CD20 therapy promotes strong anti-tumor adaptive immunity, opposes Treg expansion and inhibits tumor cells from maintaining an immunosuppressive environment.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos/farmacologia , Regulação Leucêmica da Expressão Gênica , Interferon gama/imunologia , Interleucina-12/imunologia , Linfoma de Células B/tratamento farmacológico , Linfócitos T Reguladores/efeitos dos fármacos , Imunidade Adaptativa/efeitos dos fármacos , Animais , Antígenos CD4/genética , Antígenos CD4/imunologia , Proliferação de Células/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Memória Imunológica , Interferon gama/genética , Interleucina-12/genética , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Linfoma de Células B/imunologia , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Rituximab , Transdução de Sinais , Análise de Sobrevida , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/patologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/patologiaRESUMO
Freshly isolated human Langerhans cells (LC) express two forms of Fc gamma RII: a membrane-associated form detected by monoclonal antibody (MoAb) anti-CD32, which recognize an extracytoplasmic epitope of the molecule, and a soluble secreted form, whose existence is suggested by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Indeed, RT-PCR performed on total LC RNA reveals the presence of two Fc gamma RIIA mRNA, one encoding the FC gamma RIIA with a transmembrane region (membranous form) and the other without this region (soluble form). Densitometry studies performed on the two PCR products reveal that the ratio between the membranous form and the soluble secreted form is about 1.5. LC maintained in culture for 24-48 h lose the major part of their membrane Fc gamma RII expression (shown by flow cytometry) and release soluble Fc gamma RII molecules (revealed by dot-blot assay), but maintain the same ratio of the two Fc gamma RIIA mRNA. The disappearance of the membrane-associated Fc gamma RII may be explained either by modification of its recycling pathway or by proteolytic cleavage of the receptor at the cell surface. Thus, soluble Fc gamma RII molecules generated during LC culture may result from proteolytic cleavage of the cell-surface receptor and/or secretion of a soluble form derived from the translation of an alternate spliced mRNA. Interestingly, addition of TNF-alpha (10 ng/ml) to the culture medium i) maintains the expression of the membranous form, which can be detected on the LC surface at the same level as on freshly isolated LC, and ii) reverses the ratio (to 0.6) of the two Fc gamma RII mRNA, the mRNA encoding the soluble form becoming predominant. Thus, TNF-alpha seems to modify the expression of the Fc gamma RII at the mRNA level, favoring the secretion of soluble Fc gamma RII molecules, and changes the fate of the membranous Fc gamma RII.
Assuntos
Células de Langerhans/metabolismo , Receptores de IgG/metabolismo , Humanos , Células de Langerhans/imunologia , Células de Langerhans/ultraestrutura , RNA Mensageiro/análise , Receptores de IgG/genética , SolubilidadeRESUMO
The growth-modulating effect on mouse hybridoma B cells of IgG-BF-producing Fc gamma RII+ mouse T cell hybridomas and of the IgG-BF isolated from the culture supernatants of these cells has been examined. Cocultures of IgG-secreting hybridoma B cells with IgG-BF-producing T hybridomas or with partially purified IgG-BF demonstrated a reproducible inhibition of the tumor B cell growth. The inhibition was due to a cytostatic and not to a cytotoxic effect. Hybridoma B cells cultured in liquid medium in the presence of soluble IgG-BF, or cocultured in semisolid agarose assays with IgG-BF-producing hybridoma T cells did not undergo immediate cytolysis but were prevented from proliferating. Thus, our data indicate that IgG-BF-producing Fc gamma RII+ T cells interfere with the proliferation of transformed B cells, possibly through soluble IgG-BF.