RESUMEN
We have used a fractionation procedure involving bovine serum albumin gradient floatation, adherence to glass, and rosetting with antibody-coated sheep erythrocytes to purify an accessory cell fraction from Lewis rat spleens. The fraction which is of light buoyant density, nonadherent, and FcR- is markedly Ia+, lacks phagocytic ability, is nonspecific esterase negative and under scanning electron microscopy has a heterogeneous morphology with a variety of protuberences described for rat dendritic cells. This putative dendritic cell preparation is very effective at stimulating proliferative responses with concanavalin A and allogeneic cells. When used to reconstitute the reactivity of peritoneal exudate cells of rats immunized with azobenzenearsonate-N-acetyl-L-tyrosine (ABA-Tyr) and subsequently depleted of Ia+ cells, it was shown to be highly effective with ABA conjugates of tyrosine, Ficoll, and polystyrene beads. Thus, despite the apparent absence of phagocytic or degradative abilities, this cell was very efficient at presenting large soluble and insoluble antigens. It is suggested that processing may occur at the cell surface without requiring internalization.
Asunto(s)
Compuestos Azo/inmunología , Haptenos/inmunología , Linfocitos T/inmunología , Tirosina/análogos & derivados , p-Azobencenoarsonato/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Separación Celular , Epítopos , Femenino , Tejido Linfoide/citología , Ratas , Ratas Endogámicas Lew , Tirosina/inmunología , p-Azobencenoarsonato/análogos & derivadosRESUMEN
A series of antigens was synthesized in which peptide spacers were inserted between the ABA group and TNP-Ficoll. When these antigens were used to assess helper activity in Lewis rats immunized with ABA-tyr, it was found that an increase in the ABA-epitope density and an increase in the peptide spacer size both increased the efficacy of the antigen in eliciting ABA-specific help, manifest by an enhancement of anti-TNP PFC. Substitution of D- for L-amino acids progressively decreased the ability of these conjugates to elicit help until D-tyr-D-ala-D-ala, which when used for coupling ABA to the TNP-Ficoll resulted in a nonimmunogenic molecule. When these same Ficoll conjugates were used to study ABA-specific in vitro proliferative responses, it was found that introduction of even a single D-amino acid into the spacer greatly reduced reactivity. By contrast the ABA-peptides, free of the Ficoll backbone, were all equivalent on a molar basis in their ability to elicit ABA-specific in vitro proliferation, regardless of their content of D-amino acids. These results suggest some form of digestion is required in the processing of these antigens before they can elicit helper activity. This processing can occur only if one or more L-amino acid residues are present. If the ABA-peptides are free of the Ficoll backbone, they are all capable of stimulating T cell proliferation without apparent further processing.
Asunto(s)
Compuestos Azo/inmunología , Interleucina-1/biosíntesis , Oligopéptidos/inmunología , Linfocitos T/inmunología , Tirosina/análogos & derivados , p-Azobencenoarsonato/inmunología , Animales , Fenómenos Químicos , Química Física , Epítopos , Femenino , Ficoll/inmunología , Haptenos/inmunología , Isomerismo , Activación de Linfocitos , Ratas , Ratas Endogámicas Lew , Trinitrobencenos/inmunología , Tirosina/inmunología , p-Azobencenoarsonato/análogos & derivadosRESUMEN
In an effort to study T cell functions in Lewis rats immunized with ABA-N-acetyl-L-tyrosine (ABA-tyr), we developed an antigen that provides a sensitive assay of ABA-specific helper function that is read as an increase in TNP-specific plaque-forming cells (PFC). This antigen has ABA coupled to AECM-Ficoll by virtue of a tripeptide (tyr-ala-ala) spacer and TNP coupled to the AECM side chains. At subimmunogenic doses, this antigen induced 400 anti-TNP PFC/10(6) spleen cells in ABA-tyr-immunized rats. As many as 8000 PFC/10(6) spleen cells were induced with larger doses of antigen (200 micrograms). By contrast, only 490 PFC/10(6) spleen cells could be induced with 1 mg of the conventional doubly haptenated protein carriers such as ABA-BSA-TNP. Both direct and indirect PFC were induced by this antigen in primed rats. The use of this antigen and passive transfer techniques to study ABA-specific helper activity revealed some differences from ABA-specific delayed-type hypersensitivity (DTH) and in vitro proliferation, which were studied previously. Cells responsible for helper activity appeared sooner after immunization and were found most prominently in peritoneal exudates but also significantly in spleen where the cells responsible for DTH or in vitro proliferative responses were never found. By contrast, helper cells were not seen in lymph nodes, where some proliferative activity could be found. Of these three ABA-specific T cell functions, helper activity was least easily suppressed by the previously used regimens of ABA-tyr in incomplete freunds adjuvant (IFA). Moreover, helper activity appears after injection of ABA-tyr in IFA, a method that has never in our hands yielded detectable DTH or in vitro proliferative responses. Despite these differences, phenotyping with monoclonal antibodies indicated that cells responsible for helper and proliferative activities were both W3/25+ and OX8-.
Asunto(s)
Compuestos Azo/inmunología , Ficoll/inmunología , Interleucina-1/biosíntesis , Polisacáridos/inmunología , Linfocitos T/inmunología , Tirosina/análogos & derivados , p-Azobencenoarsonato/inmunología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Animales , Células Productoras de Anticuerpos/inmunología , Femenino , Adyuvante de Freund/farmacología , Haptenos/inmunología , Técnica de Placa Hemolítica , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Oligopéptidos/inmunología , Fenotipo , Ratas , Ratas Endogámicas Lew , Trinitrobencenos/inmunología , Tirosina/inmunología , p-Azobencenoarsonato/análogos & derivadosRESUMEN
Immunization of Lewis rats with azobenzenearsonate-N-acetyl-L-tyrosine (ABA-tyr) in complete Freund's adjuvant (CFA), produces a hapten-specific helper T cell response measured by an increase in plaque forming cells (PFC) against a different hapten. The response seen is primarily direct (IgM) PFC unless B cells are primed by injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) prior to immunization with ABA-tyr. The response requires both ABA and TNP to be on the same carrier molecule which can be as diverse as bovine serum albumin (BSA), poly L-glutamine-lysine-tyrosine (L-GLT); however, a D-amino acid polypeptide does not work. The in vitro demonstration of such help was successful only with peritoneal exudate lymphocytes, not spleen or lymph node cells. Repeated pretreatment of rats by intraperitoneal injection of ABA-tyr in incomplete Freund's adjuvant (IFA) induced an unresponsiveness for helper activity to subsequent immunization with the same antigen in CFA. Passive transfer of lymphoid cells from spleens and lymph nodes from rats pretreated with ABA-tyr in IFA followed by boosting with ABA-tyr in CFA induced unresponsiveness to subsequent induction of hapten-specific help.
Asunto(s)
Compuestos Azo/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Tirosina/análogos & derivados , p-Azobencenoarsonato/inmunología , Adyuvantes Inmunológicos , Animales , Femenino , Haptenos/inmunología , Inmunidad Celular , Inmunoglobulina M/biosíntesis , Ganglios Linfáticos/citología , Linfocitos/inmunología , Ratas , Ratas Endogámicas Lew , Bazo/citología , Tirosina/inmunología , p-Azobencenoarsonato/análogos & derivadosRESUMEN
Pretreatment of Lewis rats with a single i.p. injection of ABA-N-acetyl-tyrosine in incomplete Freund's adjuvant induced an unresponsiveness for delayed-type hypersensitivity to subsequent immunization with the same antigen in complete Freund's adjuvant. Complete suppression of in vitro antigen-induced proliferative responses required repeated pretreatment. Passive transfer of lymphoid cells from spleen and lymph nodes but not sera from suppressed rats induced unresponsiveness of hapten-specific T cell functions. Nylon wool-nonadherent cells and cells panned on F(ab')2 of rabbit anti-Lewis rat Ig plates suppressed the induction of DTH and in vitro antigen-stimulated proliferation. Adult thymectomy increased DTH and failed to abolish the induction of suppression.