Specific Antibody-forming Cells Research Articles

Primary in situ immune response in popliteal lymph nodes and spleen of mice after subcutaneous immunization with thymus-dependent or thymus-independent (type 1 and 2) antigens.

Mice were immunized subcutaneously with thymus-independent (TI)-type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH) in order to study the primary in situ immune response in popliteal lymph nodes (PLN) and spleen. The spleen responded more rapidly in developing specific antibody-forming cells (AFC) than the lymph nodes did, in spite of the fact that antigens reach the spleen only after passing several lymph node stations. This difference between lymph nodes and spleen in developing AFC was particularly significant with respect to the responses to TI (both type 1 and type 2) antigens. No differences in the distribution of specific AFC in PLN and spleen were observed after immunization with TI and TD antigens. Results are discussed with respect to the relative contributions of lymph nodes and spleen to immune responses to antigens injected subcutaneously.

The "in situ" immune response in lymph nodes: a review.

For many years data on the development of specific antibody-forming cells in lymph nodes were incomplete, fragmentary, and even contradictory. A number of recent studies have been performed, concerning 1) their overall architecture; 2) migration of B-lymphocytes; 3) localization of accessory cells and T-lymphocytes which are believed to be involved in humoral immune responses; and 4) localization patterns of specific antibody-forming cells developing during thymus dependent and thymus independent immune responses. Comparison of these new results with those of earlier studies suggests a single route of migration followed by all cells which will differentiate into antibody-forming cells. During their differentiation into antibody-forming plasma cells, antigen reactive B-cells migrate along the required accessory cells and/or T-lymphocytes.

The limitations of immunoenzyme approaches to distinguish between 'specific' and 'non-specific' antibody-forming cells, with particular respect to immunocytochemical studies on the in situ immune response.

After antigenic stimulation, specific antibody-forming cells can be detected in situ in tissue sections of lymphoid organs using an antigen specific immunoenzyme approach. Immunoglobulin-forming cells staining positively with a particular antigen-enzyme conjugate are considered to be specific antibody-forming cells (provided that the right control conjugates show a negative staining). Immunoglobulin-forming cells, staining negatively with the same antigen-enzyme conjugate, may do so for two reasons: (i) they are in fact specific antibody-forming cells, but are not detected as such because of limitations of the technique; (ii) they are real-nonspecific immunoglobulin producing cells. In this paper, the limitations of the technique are discussed.

Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections

A new general approach has been developed for the detection of one or more different specific antibody producing cells and the simultaneous determination of their Ig isotype in tissue sections, after immunization of animals. Specificity of intracellular antibodies is demonstrated after incubation of the sections with an antigen-enzyme conjugate and the isotype of the antibodies is determined using an anti-immunoglobulin (Fc chain-specific)-enzyme conjugate followed by histochemical revelation of the two different enzymes. The principles of the method, the required antigen- and antibody-enzyme conjugates and their application in single, double or triple staining studies are reviewed. The method allows the detection of specific antibody-forming cells against protein antigens as well as against haptens. By means of haptens such as trinitrophenyl (TNP), immune responses against thymus dependent, thymus independent, and particulate antigens can be studied. In a limited number of cases the method can also be used to study the localization of antigen-antibody complexes.

Influence of carriers on the development and localization of anti-trinitrophenyl antibody-forming cells in the murine spleen.

Mice were i.v. immunized with various 2,4,6-trinitrophenyl (TNP)-carrier conjugates, and the development and localization of specific (i.e. anti-TNP) antibody-forming cells (AFC) in the spleen was studied. The nature of the carrier used, thymus dependent (TD) or thymus independent type 1 or type 2 (TI-1, TI-2), had marked effects on the number and time of appearance of AFC. However, no influence of the different carriers on the localization of specific AFC in the spleen was observed. By using a double immunocytochemical technique we could simultaneously determine specificity (anti-TNP) and isotype (class and subclass) of the AFC. The TNP-carrier conjugates evoked (intracellular) immunoglobulins with carrier-characteristic isotype distribution. No carrier-dependent localization pattern, in the lymphoid compartments of the spleen, of these AFC was found. These results show for the first time the actual in situ effects of various TI and TD carriers on the development of specific AFC. Based on the present findings, no direct evidence for a special role of the marginal zone in the immune response against TI-2 antigens could be demonstrated. We suggest that this new method for in vivo investigation, in combination with existing standard enzyme- and immunohistochemical techniques, can provide more insight into the precise role of lymphoid and nonlymphoid cells in TD and TI immune responses.

Specific antibody-forming cells in bronchus-associated lymphoid tissue (BALT) and lung of the rat after intratracheal challenge with horseradish peroxidase.

After intratracheal or subcutaneous priming with horseradish peroxidase (HRP), no anti-HRP-forming cells were present in the bronchus-associated lymphoid tissue (BALT) or the lung of the rat. After intratracheal priming and intratracheal boosting with HRP no specific antibody-forming cells were observed either in the BALT or the lung. A few blast cells containing anti-HRP antibody were found in paratracheal lymph nodes, which is possibly the source of anti-HRP-forming cells. After subcutaneous priming in the hind footpad and intratracheal boosting specific antibody-forming cells were present in both the BALT and in the lung. In the BALT these cells were found peripherally, and in the lung perivascularly and peribronchiolarly. The simultaneous appearance of these anti-HRP-forming cells at both sites, their localization and their morphology strongly indicate that they are recruited from the circulation and not formed in situ; the probable source is the popliteal lymph node.

The influence of the route of antigen administration on the development of specific antibody-producing cells in the follicles of the popliteal lymph nodes of rabbits.

Rabbits received an injection of liposome-associated human serum albumin (HSA) and six weeks later a booster injection of HSA free in solution. Anti-HSA antibody-forming cells were demonstrated in the popliteal lymph nodes by use of a HSA-horseradish peroxidase (HRP) conjugate for their detection. After a single subcutaneous injection into the footpads, specific antibody-forming cells were found, but after a single intravenous injection no anti-HSA-forming cells were observed in the lymph nodes. During the secondary immune response specific anti-HSA antibody-producing cells were found in the lymph nodes of all animals. The localization pattern of anti-HSA antibody-forming cells within the lymph nodes depended on the route of antigen administration used for the primary and booster injection. Anti-HSA-forming cells were mainly found in the cords of the medulla and in the outer cortex. During the secondary immune response, however, they were also detected in the follicles but only when at least one of the two antigen injections was given subcutaneously.

Lymph node involvement in a humoral immune response.

Mice were primed subcutaneously in the hind footpads with horseradish peroxidase (HRP) and boosted intravenously 10 weeks later. The appearance of cells with cytoplasmic anti-HRP antibody was studied in several lymph nodes. It appeared that following intravenous boosting antibody-forming cells appeared in significant numbers in the popliteal, the lumbar, and (in some animals) the sciatic lymph nodes exclusively. In the other lymph nodes examined specific antibody-forming cells were observed only occasionally. By subcutaneous injection of Evans blue in the hind footpads it was shown that the subcutis of the hind footpads of these animals is drained by the popliteal lymph nodes, the lumbar lymph nodes, and (to a lesser degree) the sciatic lymph nodes. The presence of trapped immune complexes within lymph node germinal centers was confined to these three nodes. Based on these findings, it is concluded that specific antibody-forming cells during the secondary response in these mice are induced exclusively in the lymph nodes draining the site of primary immunization.

Morphology, Kinetics and Secretory Activity of Antibody-forming Cells

Specific antibody-forming cells from spleen, bone marrow and popliteal lymph nodes were studied in mice after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP). Functional antibody-secreting capacity of these cells was correlated with their morphology at the cell population level. For this purpose, cells synthesizing anti-HRP antibody from the same cell suspensions were studied simultaneously by light and electron microscopy and by a plaque assay. It appeared that the population of cells responsible for antibody synthesis as well as antibody secretion was morphologically heterogeneous: besides plasma cells, considerable numbers of antibody-forming lymphocytes, antibody-forming plasmablasts and antibody-forming immature plasma cells were observed. Immature plasma cells constituted the majority of antibody-forming as well as antibody-secreting cells. Among the immature plasma cells in the popliteal lymph nodes proliferation occurred. Evidence is presented that the light-microscopically identified mature plasma cell is not the main antibody-forming cell. It does not show 3H-Thymidine incorporation and should be considered as a non-dividing end-cell.

Migration of specific antibody-forming cells during the secondary immune response against horseradish peroxidase.

Mice were primed subcutaneously in the hind footpads with horseradish peroxidase (HRP) and boosted intravenously 10 weeks later. Removal of the popliteal lymph nodes (draining the site of primary immunization) before the booster injection markedly depressed the secondary immune response in the spleen and bone marrow. This was taken as evidence that the secondary humoral immune response against HRP in the spleen and bone marrow is largely dependent upon immigration of cells from the popliteal lymph nodes after the booster injection. In rats primed subcutaneously in the hind footpads with HRP, antibody-forming cells were demonstrated in the blood, but not in the thoracic duct lymph 3 days after an intravenous booster injection with HRP.

Specific antibody formation in mouse spleen. Histology and kinetics of the secondary immune response against HRP.

Specific antibody formation in the spleen was described in mice primed subcutaneously with horseradish peroxidase (HRP) and boosted intravenously with the same antigen. The first specific antibody-forming cells responding to the booster injection were observed after one day in the periarteriolar lymphocyte sheath (PALS). The number and staining intensity of these cells greatly increased subsequently. Between day 3 and 6 after the booster injection a shift was observed in the distribution of antibody-forming cells from the PALS to both the site of entry of the central artery in the PALS and the site where the blood passes via terminal arterioles to the red pulp. Specific antibody-forming cells became highly concentrated in these areas, which constitute the so-called marginal zone bridging channels. After day 6 the number of antibody-forming cells decreased sharply. On the basis of this distribution pattern it was suggested that after subcutaneous priming and intravenous boosting specific antibody-forming cells migrate from the popliteal lymph nodes to the spleen and gradually leave the spleen at later stages of the response. Specific antibody-forming cells did not occur in the germinal centers during any stage of the response.

The mechanism of appearance of specific antibody-forming cells in lungs of inbred mice after intratracheal immunization with sheep erythrocytes.

Immunization, ablation, and adoptive transfer studies were performed in inbred mice to define in vivo the cellular mechanisms for the appearance of specific antibody-forming cells (AFC) in pulmonary parenchyma. Mice were immunized locally or systemically with sheep erythrocytes (SRBC), and the concentrations of IgM- and IgG-producing AFC were measured in lung and extrapulmonary lymphoid tissues with a hemolytic-plaque assay. Splenectomized mice and recipients of adoptively transferred, sensitized lymphocytes were examined. We found that primary intratracheal (IT) immunization regularly failed to induce the appearance of AFC in lungs, whereas IT boosting of primed animals consistently succeeded. Immunization experiments showed that mice could be primed by any of a variety of local or systemic routes, but that the IT route of boosting was an absolute requirement for the induction of pulmonary AFC in primed mice. Recruitment of AFC into lungs by IT boosting of systemically primed and boosted animals was antigen-specific. Splenectomy performed prior to priming reduced, but did not ablate, the pulmonary AFC-response to IT boosting. Adoptive transfer of sensitized lymphocytes to naive recipient mice substituted for antigen-priming, which is required for induction of pulmonary AFC by IT challenge. Results of adoptive transfer studies demonstrate that IT challenge with specific antigen recruits systemically administered sensitized lymphocytes into the lung. We conclude that local primary immunization of mice results in the generation of AFC in extrapulmonary lymphoid tissues and that the major mechanism for the appearance of AFC in lungs is through recruitment of sensitized cells from systemic sources by intrapulmonary boosting with specific antigen.

Stimulation of specific antibody-forming cells in antigen-primed nude mice by the adoptive transfer of syngeneic anti-idiotypic T cells.

Stimulation of specific antibody-forming cells in antigen-primed nude mice by the adoptive transfer of syngeneic anti-idiotypic T cells.

60] General considerations in measurement of interferon-induced antibody suppression

This chapter discusses interferon-induced antibody suppression and the assay systems. The detailed analysis of the kinetics of immunosuppression, particularly in relation to the heterogeneity of responding cells are presented, which directly affects the level of suppression observed. Therefore, the discussion in the chapter concentrates on the means whereby an assay system can be devised to minimize the problems that result from having a complex cell system to assay possible complex effects of an immunosuppressive agent such as IF. The main approach has been to stimulate the appropriate precursor cells with antigens or mitogens in the presence of IF and to measure the number of specific antibody-forming cells (AFC) which are produced. In studying the effect of IF (or any other soluble mediator) on a complex, multicomponent system, two important points emerge which are (a) that there are a number of possible targets on which IF might act and (b) that its effect will depend not only on the time at which it is added, but also on the time at which the response is measured. The production of AFC involves a combination of two processes, which are the activation of antigen-specific precursor B cells and the clonal expansion of each activated precursor. By employing various lymphocyte fractionation techniques and clonal analyses, it is possible to gain a clearer understanding of the mechanics of IF-induced antibody suppression. Much of the investigation of IF-induced immunosuppression has been derived from work with mouse interferon and murine in vitro responses.

The effect of splenectomy on the appearance of specific antibody-forming cells in lungs of dogs after intravenous immunization with sheep erythrocytes.

Effector cells of both T-lymphocyte-mediated and B-lymphocyte-mediated immunity can be induced experimentally to appear in the lung. It is not known whether such effector lymphocytes arise from precursor cells resident in the lung or if they are recruited from systemic sources. Previous studies demonstrated that IV administration of sheep erythrocytes to dogs consistently resulted in the appearance of specific antibody-forming cells, B-cell effectors, in lavage preparations obtained from the lung. In the present study, experiments were performed to determine the mechanism of appearance of effector B-cells in the lung after IV immunization. Control, sham-operated, and splenectomized dogs were immunized IV with 10(10) sheep erythrocytes. The concentrations of antibody-forming cells appearing in lymphocytes obtained from the lung and from peripheral blood were measured during both the primary and the secondary immune responses. Control and sham-operated dogs developed high concentrations of antibody-forming cells in blood 5 days after immunization, but splenectomized dogs did not. Antibody-forming cells consistently appeared in substantial numbers in lymphocytes obtained by lavage from lungs of control and sham-operated animals, however, they failed to appear in lungs of animals which had undergone splenectomy. Similar results were obtained both after primary and after secondary IV immunization. The data indicate that in this model, namely after IV immunization, with sheep erythrocytes, pulmonary antibody-forming cells or their precursors are derived from systemic sources (spleen) via the circulating blood. The observations provide strong in vivo evidence that lung antibody-forming cells are not generated locally after IV priming and boosting. The results of the present study relate directly to the mechanism of appearance of antibody-forming cells in lungs after IV, not local immunization. The study defines and documents one mechanism by which immune effector cells can appear in lung tissue.

Determination of immunoglobulin classes of specific antibodies secreted by single cells. A cluster formation assay

A method is described to identify specific antibody-forming cells and their immunoglobulin classes by a multilayer cluster formation. Cells producing specific antibodies are mixed with erythrocytes coated with the corresponding antigen in presence of rabbit anti-human immunoglobulins. Clusters are formed only if the mixtures contain rabbit anti-serum against the immunoglobulin chains of the specific secreted antibody. Thus the immunoglobulin class of the specific antibody is assessed. For this study the cells of three EBV-transformed, antibody-producing human lymphoblastoid cell lines were used.

In vitro development of a primary antibody response with lymph node cells

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10 6 recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.

Splenic influence on the development of a local pulmonary immune response.

The role of the spleen in the development of specific antibody-forming cells (sAFC) in the pulmonary draining lymph nodes (pdLNC) of hamsters after local inoculation of sheep erythrocytes (SRBC) was evaluated. The role of the spleen was viewed from two vantage points. Panels of animals were either splenectomized with appropriate sham-operated controls before intratracheal inoculation of SRBC, or panels were immunized intravenously simultaneously with the local inoculation of antigen. The presence of an intact spleen was not necessary for the induction of a sAFC response to occur in the pdLNC. Similar numbers of immunoglobulin M (IgM) sAFC were recorded in the pdLNC on day 4 of both sham-operated and splenectomized animals. However, an enhancement of this local response occurred on day 7 if the animals were systemically immunized and therefore demonstrated active participation of the spleen in the specific immune response. The results support the hypothesis that although a local response may occur in the pdLFC in the absence of a spleen or a splenic response, the presence of a systemic or splenic response appears to be important for the enhancement of local IgM sAFC response. These observations suggest that the immune defenses involved in the lower respiratory tract may differ from those in upper respiratory tract and other mucosally lined organs in that the response of the spleen to the antigen affects the local response to that antigen.

T-cell lines producing antigen-specific suppressor factor

THE immune system is now recognised as a network of interacting cells with some interactions augmenting, and others suppressing immune responses. Regulatory interactions are often mediated by T cells, and do not always require cell-to-cell contact between appropriate cells, but may be mediated by soluble transmitters, commonly termed ‘factors’. Both antigen-specific1–7 and nonspecific8–10 factors have been described. Biochemical characterisation of these factors is hampered by the often minute quantities required to mediate biological effects and by the multiplicity of regulatory pathways involved in immune responses. Our knowledge of these factors is derived from functional tests, often combined with serological analysis of the factors. One approach to the analysis of monoclonal cell products is the somatic cell hybridisation technique described by Milstein et al.11,12. Using this method, specific antibody-forming cells have been hybridised with myeloma cells, and have yielded clones producing specific antibody11. This antibody is particularly useful as it is monoclonal and large quantities can be obtained. Although attempts to produce T-cell hybrids have been successful13–15, the products have not been functional. However, only failures in expression of cytotoxic activity have been described13–15. We describe here one antigen-specific suppressor factor (SF)-producing line S1-41 and its products.

Mechanisms of hemopoietic and immunological dysfunction induced by lymphocytic choriomeningitis virus.

Sublethal irradiation (500 R) of C3H mice is followed by a gradual replacement of radiosensitive cells in their spleens by surviving stem cells originating in bone marrow. This compensatory hemopoiesis was quantitated by counting the numbers of stem cell-derived colonies appearing on spleen surfaces, as well as those which grew in vitro after marrow cells, suspended in soft agar, were overlaid onto syngenic mouse embryo fibroblast feeder layers. Compensatory colony formation, both in vivo and in vitro, was severely depressed when mice were infected with lymphocytic choriomeningitis virus (LCMV) 1 day before irradiation, although the induction of virus-specific cytotoxic T cells in their spleens was unimpaired. Without irradiation, mice, acutely infected with LCMV, showed a dramatic reduction in the numbers of specific antibody-forming cells generated in their spleens after priming with sheep erythrocytes during week 1 post-infection, yet the ability of their marrow cells to form colonies in vitro remained normal. Therefore, the basis of immunodepression is distinct from that of defective hemopoiesis since the latter is apparent only when LCMV infection is accompanied by irradiation. However, as discussed, both phenomena may be related to alterations induced within the splenic environment by LCMV.