RESUMEN
Recently, technologies have developed that allow for the culturing of antigen-presenting cells (APC), such as dendritic cells (DC). The normal function of these cells is to present antigens to T cells, which then specifically recognize and ultimately eliminate the antigen source. Over the past number of years, these cells have been used in a variety of different immunotherapeutic strategies. Paramount in the success of such endeavors is the generation of desired T cell responses through the selection of appropriate antigens. This paper will serve to discuss the development and current status of dendritic cell-based therapy focusing on antigen selection for cancer.
Asunto(s)
Células Dendríticas/inmunología , Inmunoterapia/métodos , Antígenos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Humanos , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
Dendritic cells (DC) are potent antigen-presenting cells that are integral to the initiation of T cell immunity. The ability to culture these cells in vitro has allowed DC immunotherapy to be investigated as a mechanism of enhancing immune responses against various malignancies. We examined the optimal time for generating DC and compared DC generated from normal donors for allogeneic blood stem cell transplantation, or patient's with non-Hodgkin's lymphoma or breast cancer undergoing high-dose chemotherapy and autologous stem cell transplantation. Experiments were conducted to compare DC cultured prior to and post mobilization chemotherapy. Blood was obtained from consenting patients prior to granulocyte colony-stimulating factor (G-CSF) administration with (non-Hodgkin lymphoma and breast cancer) or without (normal donors) chemotherapy. A sample of apheresis product (AP) was obtained at the time of apheresis. DC were generated from peripheral blood mononuclear cells by culturing the adherent cells in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor. Resultant DC were harvested and examined for yield, morphology, phenotype, and function. All cell populations yielded highly pure DC, as assessed by light microscopy and flow cytometry. The average cellular yield was significantly greater from AP than steady-state blood in paired and unpaired samples. Yield did not correlate with the percentage of CD14(+) cells, and it negatively correlated with CD34 counts. DC from breast cancer patients functioned significantly better than DC from lymphoma patients in a mixed lymphocyte reaction. These data suggest that the optimal timing of culturing DC is after mobilization, and that differences may exist in the functional capabilities of DC derived from different patient populations.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-4/farmacología , Adulto , Anciano , Antígenos CD34/inmunología , Neoplasias de la Mama/sangre , Recuento de Células , Células Dendríticas/inmunología , Células Dendríticas/ultraestructura , Femenino , Movilización de Célula Madre Hematopoyética , Humanos , Leucaféresis , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Receptores de Lipopolisacáridos/inmunología , Linfoma/sangre , Masculino , Microscopía Electrónica , Persona de Mediana EdadRESUMEN
In addition to eliciting antigen specific T-cell-mediated immunity, Cryptococcus neoformans possesses a mitogen (CnM) that activates naive T cells to proliferate. This mechanism of T-cell activation is accessory cell dependent and major histocompatibility complex unrestricted. CnM-induced T-cell proliferation correlates with internalization of the organism, suggesting that intracellular processing is required to liberate CnM prior to presentation to T cells. To determine whether phagocytosis and processing are required, various inhibitors of accessory cell uptake and processing were used. C. neoformans was observed within the accessory cells. Paraformaldehyde fixation of the accessory cell abrogated presentation of CnM to T cells, indicating that a dynamic accessory cell surface was required. A lysosomotropic agent abrogated the response to CnM but had no effect on a control stimulus that did not require processing. Both aspartic acid and cysteine protease inhibitors blocked effective processing of CnM, so that it was unable to stimulate T cells. Finally, an inhibitor of microfilament polymerization abrogated proliferation to CnM. These results indicate that the mitogenic activity of C. neoformans requires phagocytosis of the organism, lysosomal or endosomal processing, proteolytic activity, and microfilament polymerization and intracellular transport as a prerequisite for T-cell proliferation.
Asunto(s)
Cryptococcus neoformans/inmunología , Activación de Linfocitos , Mitógenos/metabolismo , Fagocitosis , Linfocitos T/inmunología , Citoesqueleto de Actina/fisiología , Células Presentadoras de Antígenos/fisiología , Cryptococcus neoformans/metabolismo , Citocalasina B/farmacología , Endopeptidasas/fisiología , HumanosRESUMEN
Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrated by numerous animal studies and the prevalence of the infection in AIDS patients. Previous studies have established that the polysaccharide capsule contributes to the virulence of C. neoformans by suppressing T-lymphocyte proliferation, which reflects the clonal expansion of T lymphocytes that is a hallmark of cell-mediated immunity. The present studies were performed to identify the major mechanism by which polysaccharide impairs lymphocyte proliferation, since capsular polysaccharide has the potential to affect the development of T-lymphocyte responses by stimulating production of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing shedding of cell surface receptors. We demonstrate that polysaccharide inhibits lymphocyte proliferation predominantly by blocking uptake of C. neoformans, which is crucial for subsequent lymphocyte proliferation. In addition, we show that polysaccharide did not suppress lymphocyte proliferation via an IL-10-dependent mechanism, nor did it affect critical surface receptor interactions on the T cell or antigen-presenting cell. Having established that polysaccharide impairs phagocytosis, we performed studies to determine whether opsonization with human serum or with anticapsular antibody could reverse this effect. Impaired uptake and lymphocyte proliferation that were induced by polysaccharide can be enhanced through opsonization with monoclonal antibodies or human serum, suggesting that antipolysaccharide antibodies might enhance the host defense by restoring uptake of the organism and subsequent presentation to T lymphocytes. These studies support the therapeutic potential of stimulating cell-mediated immunity to C. neoformans with anticapsular antibody.
Asunto(s)
Anticuerpos Antifúngicos/inmunología , Antígenos Fúngicos/inmunología , Cryptococcus neoformans/inmunología , Fagocitosis/inmunología , Polisacáridos/inmunología , Linfocitos T/inmunología , Adulto , Células Presentadoras de Antígenos/inmunología , División Celular , Humanos , Interleucina-10/inmunología , Linfocitos T/citologíaRESUMEN
The mechanism of human T-lymphocyte activation by the pathogenic yeast Cryptococcus neoformans has not been established. Previous investigations have suggested that C. neoformans contains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response to C. neoformans, the present studies were performed to determine whether C. neoformans contains a mitogen for T lymphocytes. C. neoformans stimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA+ cells from adults, indicating that it stimulates naive T cells. The T-cell response to C. neoformans was dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigen Staphylococcus enterotoxin B. Precursor frequency analysis established that 1 in 7,750 +/- 2, 270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect of C. neoformans should be considered when interpreting the immune response to C. neoformans, since even weak mitogens can have profound effects on host defense.
Asunto(s)
Cryptococcus neoformans/inmunología , Mitógenos/inmunología , Linfocitos T/inmunología , Adulto , Ciclo Celular , División Celular , Membrana Celular/inmunología , Pared Celular/inmunología , Células Madre Hematopoyéticas/inmunología , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Linfocitos T/citologíaRESUMEN
Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared. One of the P. aeruginosa virulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients. It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF. The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role in P. aeruginosa pathogenesis. The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated. The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens. To determine the mechanism of activation, the requirement for accessory cells was investigated. The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody. Exoenzyme S activated both CD4(+) and CD8(+) T cells, but CD4(+) T cells were preferentially activated. The Vbeta repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen. Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen. Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Activación de Linfocitos/efectos de los fármacos , Mitógenos/farmacología , Poli(ADP-Ribosa) Polimerasas/farmacología , Pseudomonas aeruginosa/patogenicidad , Superantígenos/farmacología , Linfocitos T/inmunología , Adulto , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Fibrosis Quística/inmunología , Fibrosis Quística/terapia , Humanos , Memoria Inmunológica , Complejo Mayor de Histocompatibilidad/fisiologíaRESUMEN
The current studies were performed to determine the contribution of T-cell subsets to lymphocyte proliferation in response to Cryptococcus neoformans, the most common invasive mycosis in acquired immune deficiency syndrome. We demonstrate for the first time that both human CD4 and CD8 cells are activated in response to C. neoformans. Both CD4 and CD8 cells express interleukin-2 receptor alpha (IL-2R alpha) and transferrin receptor and proliferate in response to C, neoformans, however proliferation of CD8 cells was dependent upon CD4 cells. The requirement for CD4 cells was complex, since CD8 enriched cells failed to express mRNA for IL-2, suggesting that CD4-dependent IL-2 production was required for CD8-cell proliferation. However, IL-2 was not sufficient to restore CD8-cell proliferation. These studies provide experimental evidence in humans to support the clinical impression that CD4 cells are important in cryptococcosis, and suggest that the appropriate CD4-derived signals could allow CD8 cells to assist in host defence.
Asunto(s)
Antígenos Fúngicos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Cryptococcus neoformans/inmunología , Activación de Linfocitos , Adulto , Técnicas de Cultivo de Célula , División Celular/inmunología , Humanos , Inmunofenotipificación , Interleucina-2/biosíntesis , Interleucina-2/genética , ARN Mensajero/genéticaRESUMEN
Cryptococcus neoformans is an encapsulated yeast that infects patients who have defective cell-mediated immunity, including AIDS, but rarely infects individuals who have intact cell-mediated immunity. Studies of the immune response to C. neoformans have been hampered by a paucity of defined T-lymphocyte antigens, and hence, the understanding of the T-cell response is incomplete. The goal of this study was to separate C. neoformans into its component parts, determine whether those components stimulate lymphocyte proliferation, perform preliminary characterization of the proteins, and establish the potential mechanism of lymphocyte proliferation. The lymphocyte response to fungal culture medium, whole organisms, disrupted organisms, and the yeast intracellular fraction or cell wall and membrane was studied by determining thymidine incorporation and by determining the number of lymphocytes at various times after stimulation. The cell wall and membrane of C. neoformans stimulated lymphocyte proliferation, while the intracellular fraction and culture filtrate did not. The optimal response occurred on day 7 of incubation, with 4 x 10(5) peripheral blood mononuclear cells per well and with 13 microg of cryptococcal protein per ml. The number of lymphocytes increased with time in culture, indicating that thymidine incorporation was accompanied by proliferation. Proteinase K treatment of the cell wall and membrane abrogated lymphocyte proliferation, indicating that the molecule was a protein. [35S]methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were performed to analyze the proteins contained in the cell wall and membrane, intracellular fraction, and culture filtrate. At least 18 discrete bands were resolved from the cell wall and membrane. Since a large percentage of healthy adults responded to the cryptococcal cell wall and membrane, a mitogenic effect was investigated by testing proliferation of fetal cord blood lymphocytes. The percentage of fetal samples that proliferated in response to the cell wall and membrane was similar to the percentage of fetal samples that proliferated in response to Staphylococcus enterotoxin B, a microbial mitogen. Thus, a protein in the cell wall and membrane of C. neoformans is a potent stimulant of lymphocyte proliferation and has mitogenic properties, which may have important implications for cell-mediated immunity to C. neoformans.
Asunto(s)
Cryptococcus neoformans/inmunología , Proteínas Fúngicas/inmunología , Activación de Linfocitos , Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Adulto , Pared Celular/inmunología , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Enterotoxinas/inmunología , Sangre Fetal , Humanos , Cinética , Linfocitos/citología , Glicoproteínas de Membrana/inmunología , Timidina/metabolismoRESUMEN
Pseudomonas aeruginosa is a gram-negative bacterium that is responsible for devastating acute and chronic infections, which include bronchiectasis in cystic fibrosis, nosocomial pneumonia, and infection of burn wounds. Previous studies have demonstrated that these patients have impaired host responses, including cell-mediated immune responses, which are important in anti-Pseudomonas host defense. The P. aeruginosa exoproduct, exoenzyme S, has a number of characteristics which suggest that it might be important in cell-mediated immunity. To determine whether exoenzyme S activates lymphocytes to proliferate, peripheral blood mononuclear cells (PBMC) from normal volunteers were stimulated with purified exoenzyme S, and the lymphocyte response was assessed by measuring [3H]thymidine uptake and by counting the number of cells after various times in culture. Ninety-five percent of healthy adult donors had a lymphocyte response to exoenzyme S. The optimal lymphocyte response occurred on day 7, with 4 x 10(5) PBMC per microtiter well when cells were stimulated with 10 micrograms exoenzyme S per ml. [3H]thymidine uptake correlated with an increase in the number of mononuclear cells, indicating that proliferation occurred. In unseparated PBMC, T cells, and to a lesser extent B cells, proliferated. Purified T cells proliferated, while purified B cells proliferated only after the addition of irradiated T cells. Thus, T lymphocytes are necessary and sufficient for the proliferative response to exoenzyme S. We speculate that exoenzyme S from P. aeruginosa is important in T-lymphocyte-mediated host defense to P. aeruginosa. In strategies to enhance impaired cell-mediated immunity, exoenzyme S should be considered as a potential stimulant.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Inmunidad Celular/inmunología , Activación de Linfocitos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/farmacología , Pseudomonas aeruginosa/inmunología , Linfocitos T/inmunología , Antígenos CD/inmunología , Antígenos CD19 , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/inmunología , Complejo CD3/inmunología , Humanos , Subgrupos Linfocitarios/inmunología , Pseudomonas aeruginosa/enzimologíaRESUMEN
Cryptococcus neoformans is a pathogenic encapsulated yeast that infects patients that have defective cell-mediated immunity, including AIDS patients. Whole cryptococcal organisms that are killed by heating stimulate normal human lymphocytes to proliferate. However, strains of C. neoformans vary widely in virulence and therefore in their ability to cause disease in humans. To determine the effect of virulence factors such as the cryptococcal capsule, serotype, and the state of the organisms on the lymphocyte response to C. neoformans, human peripheral blood mononuclear cells were stimulated with C. neoformans in vitro and lymphocyte proliferation was determined. The major determinant of the lymphocyte response to C. neoformans was the amount of polysaccharide present. The response was greater after stimulation by minimally encapsulated strains (strains C3D, 68, and 613) than by heavily encapsulated strains (strains 6 and 145). A heavily encapsulated strain (strain 6) did not suppress the response to an acapsular mutant (strain 67). However, the response to an acapsular strain was suppressed by the addition of purified polysaccharide. Human lymphocytes responded to both serotypes of C. neoformans var. neoformans. The antigen responsible for lymphocyte stimulation was preserved despite various techniques of inactivation, including heat, paraformaldehyde fixation, irradiation, and mechanical disruption. Finally, lymphocytes responded equally to live and killed organisms. These results suggest that capsular polysaccharide, a known virulence factor, may suppress the human lymphocyte response to C. neoformans during an infection. Lymphocytes could respond to C. neoformans regardless of the viability of the organism, and they could also respond to disrupted organisms. We speculate that lymphocyte proliferation in vitro could be related to the protective immune response in host defense to C. neoformans and that it is suppressed by virulence factors of C. neoformans.
Asunto(s)
Cryptococcus neoformans/inmunología , Activación de Linfocitos , Polisacáridos/fisiología , Células Cultivadas , Cryptococcus neoformans/patogenicidad , Humanos , Mutación , VirulenciaRESUMEN
In-vitro fertilization of hamster oocytes with human spermatozoa has been used to study heterozygotes for structural chromosome abnormalities. To date only four men heterozygous for Robertsonian translocations have been examined. In this study, 150 sperm chromosome complements from a 21;22 translocation were investigated. There was no evidence of an interchromosomal effect since the frequency of abnormalities unrelated to the translocation was within the range of normal donors. The frequency of unbalanced complements was 3.4%, which is similar to other Robertsonian translocations. As expected, an equivalent number of normal (n = 74) and balanced (n = 70) karyotypes was observed.