Appearance Of Antibody-forming Cells Research Articles

Editorial: A Living History of Immunology.

This Research Topic was conceived to provide a venue for investigators to document their critical contributions to our understanding of the cellular and molecular mechanisms that provide our remarkable immune system the capacity to protect us from environmental insults while simultaneously remaining unreactive to our internal molecular milieu. Although the scientific literature gives one a history of what happened, when it happened, and who were responsible, it fails to capture precisely how things came together, and why some investigators were successful, while others working contemporaneously failed. Thus, seminal contributors were asked to recount the various aspects of their experiments and people who were instrumental in making the progress that moved our understanding forward. Looking back over the brief history of immunology, a discipline that arose after the pioneering approaches of Edward Jenner introduced the smallpox vaccine in 1798 (1) and Louis Pasteur catapulted immunology into universal awareness 100 years later (2–4), the science has only become mature within the past 50 years. For almost 100 years after Pasteur, experimentalists focused on observations of the reactions of whole experimental animals or humans to the administration of putative antigenic substances. For the first time, around 1960, it was appreciated that lymphocytes are the cells that mediate the immune reaction (5–7), and experimentation moved for the first time from in vivo to in vitro, which allowed one to manipulate and investigate an immune reaction of cell populations “outside of the black box.” During the 1960s, various techniques were improved so that it was possible to discern that several different types of cells cooperated to ultimately generate a measurable immune response, usually monitored by the appearance of antibody-forming cells (AFCs) (8, 9). As detailed in this Research Topic, by the 1970s, experiments culminated in the demonstration that two distinct types of lymphocytes existed, termed thymic-derived cells (T cells) and bone marrow-derived cells (B cells), which generate AFCs. Furthermore, a third type of cell derived from myeloid cells, termed an antigen-presenting cell (APC), also played a role. Evidence was also presented that there are at least two distinct subsets of T cells. Moreover, investigators detected activities in culture supernatants of activated lymphocyte populations that seemed to enhance or suppress the generation of AFCs as well as the proliferation of various lymphocytes. However, the molecular basis of these activities remained enigmatic and essentially unapproachable, given the experimental biochemical methods then available. Four new and novel experimental methods were introduced in the 1970s that revolutionized all of biological sciences, which enabled a further reduction from cells to molecules, and led to the discipline that now can be recognized as molecular immunology. In 1972, the flow cytometer introduced a new method to identify and isolate individual cells present in cell populations (10). Also, genetic engineering approaches enabled investigators to identify and isolate complimentary DNA molecules encoding gene products, which then allowed the ready determination of their primary structures, and provided a means to generate essentially unlimited quantities of critical proteins (11). Third, the advent in 1975 of the capacity to isolate and clone individual AFCs, which could produce unlimited quantities of monoclonal antibody molecules (12), could then be used to identify and isolate both individual cells using the flow cytometer, but also new reagents that could be used to isolate and purify individual protein molecules from complex mixtures. The fourth critical technical advance, which like monoclonal antibodies was also special to immunology, was the creation in 1979 of the methods to select, clone, and grow the functional progeny of individual T cells (13). This advance, for the first time, allowed one to circumvent the tremendous heterogeneity of individual cells within T cell populations so that the molecules responsible for antigen recognition, histocompatibility restriction, and the molecular mechanisms underlying T cell function, including T cell help of antibody production and T cell mediated cytolysis, could be uncovered. The contributions comprising this compilation of the stories about how the transition from experiments on whole living organisms to cell populations to individual cells and finally to homogeneous molecules represent a unique aspect of scientific communication, in that they tell the behind the scenes dramas that are usually left out of the scientific literature. This Research Topic tells how science gets done, and what the people are like who actually do it. I hope that you enjoy the stories.

Synthesis and immunomodulating properties of poly(N-vinylformamide)

A series of poly(N-vinylformamide) (PNVF) samples with molecular masses in the range (8.7 – 280) × 103 were synthesized. The effect of the polymer molecular mass on the PNVF-stimulated antibody response was investigated using BALB/C mice (Rappolovo nursery). The immunostimulating activity of the polymer was estimated by its influence on the appearance of antibody-forming cells to sheep red blood cells by the local hemolysis method in gel. A strong dependence of the antibody response on the polymer molecular mass was observed. The maximum activity was manifested for PNVF with molecular masses in the range (55 – 85) × 103.

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5−, Lyb5+) and CBA/N (Lyb5−) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.

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.

Cell interaction (CI) and anti-ci molecules: Experiments and concepts on cell/cell interactions in the immune system

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5−, Lyb5+) and CBA/N (Lyb5−) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.

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.

Antibody Formation in Mouse Bone Marrow During Secondary Type Responses to Various Thymus-Independent Antigens

The data presented in this paper show that different thymus-independent (TI) antigens have a differential capacity of inducing antibody formation in mouse bone marrow, both after primary and secondary intravenous immunization. Primary immunization with certain TI antigens (e.g., lipopolysaccharide [LPS], TNP-LPS, DNP-Ficoll) induces the appearance of antibody-forming cells not only in the spleen, but also in the bone marrow. A single injection of certain other TI antigens (e.g., pneumococci [Pn], TNP-conjugated detoxified LPS [TNP-dLPS], TNP-conjugated Brucella abortus bacteria [TNP-BA]), on the other hand, induces antibody formation in the spleen only. After secondary immunization with these TI antigens only TNP-BA induces a PFC response in the bone marrow. Pn, TNP-dLPS and TNP-BA, but also DNP-Ficoll, are unable to induce bone marrow antibody formation after secondary injection of the antigen, in spite of the clear-cut secondary type character of the splenic response. Thus, the absence of a bone marrow PFC response after secondary immunization with these antigens is not due to a failure to induce memory B cells. This data implies that either two subpopulations of memory B cells exist, one giving rise to antibody formation in the spleen and the other accounting for the bone marrow response, or that antibody can selectively inhibit the secondary bone marrow antibody response to certain TI antigens.

Immunosuppressive properties of leukotriene D 4 and E 4[formula omitted][formula omitted

Leukotrienes D 4 and E 4 whose physiological function has been associated with smooth muscle contraction, are demonstrated to be potent suppressors of immunocompetent cell function. In concentrations as low as 10 −12 M both leukotriene D 4 and E 4 can inhibit mitogen-induced lymphocyte transformation. Higher concentrations of leukotrienes E 4 and D 4 will inhibit appearance of antibody-forming cells in tissue culture. These data suggest another role for the leukotrienes in addition to their function as slow-reacting substances.

Effects of histamine receptor antagonists metiamide and cimetidine on antibody formation in vitro by murine cells.

AbstractThe effects of the H2 receptor antagonists cimetidine and metiamide on antibody responsiveness by murine splenocytes immunized in vitro with sheep erythrocytes were examined. These agents had a dose-related effect on antibody formation. Incubation of normal mouse spleen cells with 10 μg metiamide or cimetidine resulted in a marked stimulation of the antibody response, but increasing the dose to 1000 μg or more depressed the response. The higher doses also suppressed viability of the cells, but to a lesser extent than suppression of antibody formation. There was no effect on the cytokinetics of appearance of antibody-forming cells, since the peak day of the response was similar regardless of presence or absence of the drugs. Optimum enhancement occurred when the drugs were added to cultures on Day 0 or plus one day, indicating that immunomodulation was associated with early events of the immune response.

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.

Pulmonary immune responses of inbred mice. Appearance of antibody-forming cells in C57BL/6 mice after intrapulmonary or systemic immunization with sheep erythrocytes.

Pulmonary immune responses of inbred mice. Appearance of antibody-forming cells in C57BL/6 mice after intrapulmonary or systemic immunization with sheep erythrocytes.

Appearance of antibody-forming cells in lymphocytes from the lower respiratory tract of the dog after intrapulmonary or intravenous immunization with sheep erythrocytes.

To investigate the mechanisms of expression of humoral immunity in the lower respiratory tract, dogs were immunized either locally (intrapulmonary) or systemically (intravenous) with low doses of sheep erythrocytes. Bronchoalveolar cells obtained by pulmonary lavage and hilar lymph nodes were assayed during the primary immune response for their concentrations of antibody-forming cells with the Jerne plaque technique. Antibody-forming cells consistently appeared among bronchoalveolar cells after intravenous immunization but failed to accumulate in this cell population after local administration of the identical dose of sheep erythrocytes. By contrast, after local immunization, antibody-forming cells appeared in the hilar lymph nodes in generally greater concentrations than after intravenous immunization. The results demonstrated that the anatomic site of presentation of this particulate antigen to the lung (intravascular versus intra-alveolar) as well as the dose of antigen are important determinants of the distribution of antibody-forming cells in these 2 pulmonary lymphoid tissues. The data suggested that bronchoalveolar lymphocytes and hilar lymph node cells function independently in the expression of humoral immunity in the lower respiratory tract.

The clonal origin of cells contributing to successive phases of a cyclical immune response.

A single injection of mice with a low dose of sheep red blood cells results in a cyclic appearance of antibody-forming cells of the IgG classes. The peaks of the responses occur at the same time in the spleen and the thoracic lymph nodes. We have investigated the phenomenon with respect to IgG2a response by using the isoelectric focusing overlay assay and have assumed that in the great majority of cases, after immunization with limiting amounts of antigen, an individual spectrotype represents a "clonal" product. Enumeration of spectrotypes which arise only in the first peak and those which arise only in the second, suggests that the second peak of plaque-forming cells can be accounted for on the basis of the stimulation of a second population of precursor cells. Several possible mechanisms whereby new antigen-sensitive precursor cells can be turned on late in a primary response are discussed.

STIMULATION OF cAMP LEVELS AND MODULATION OF ANTIBODY FORMATION IN MICE IMMUNIZED WITH CHOLERA TOXIN*

Injection of mice with 1.0 mu g of a purified exotoxin derived from Vitro cholerae together with a challenge injection of sheep erythrocytes (SRBC)P OR E. coli LPS markedly influenced the immune response to these antigens. Simultaneous injection of the toxin with antigen resulted in a delayed appearance of antibody-forming cells during the first few days after immunization, followed by a marked enhancement of the peak numbers of antibody-forming cells. In the case of the immune response to SRBC, both 19S and 7S plaque-forming cells (PFC) were enhanced on the peak day of response after simulataneous immunization of toxin-injected mice. The secondary immune response to SRBC was also similarly affected when cholera toxin was given along with a second injection of erythrocytes: i.e. a delay in appearance of the first antibody-forming cells followed by a marked enhancement of the peak 19S and 7S PFC response. Injection of cholera toxin 103 days prior to SRBC or LPS was immunosuppressive. The effect of cholera toxin on the level of splenic cyclic AMP appeared related to the effects on antibody formation.

Synthesis of Antibody and Immunoglobulins without Detectable Antibody Function in Cells Responding to Horseradish Peroxidase

The kinetics of immunoglobulin-synthesizing cells (IFC) and antibody-forming cells (AFC) are compared in the popliteal nodes of mice challenged, in the hind footpads, with horseradish peroxidase (PO) in Freund's complete adjuvant. A rise in the number of IFC without antibody function precedes the appearance of AFC. IFC reach peak numbers 7 days before AFC. Control experiments show that the majority of IFC without antibody function are responding to PO and not to adjuvant. Kinetic and radioautographic data suggest that IFC arise by division and differentiation; however, the doubling time of AFC is too rapid to be accounted for by cell division alone. Double staining techniques revealed the presence of immunoglobulin and antibody in different compartments of the same cell in 5 to 15% of the AFC. It is postulated that some AFC are recruited, possibly from cells already synthesizing immunoglobulin determinants.

Antigenic Composition, Not the Degree of Polymerization, Determines the Requirement for Thymus-Derived Cells in Immune Responses to Salmonella Flagellar Proteins

Abstract Two flagellar proteins, prepared from either SW1338 or SL167 strains of Salmonella, have a differential requirement for thymus-derived (T) cells in producing a primary IgM response in mice. By using two methods for T cell depletion, the SW1338 flagellar protein, in both monomeric and polymeric forms, induced an IgM response that was essentially the same as that found in normal mice. In contrast, the SL167-derived antigen failed to initiate the appearance of antibody-forming cells in T cell-deprived mice. These findings add a further dimension to the consideration of mechanisms which underlie the collaboration between T and B cells, namely the role of epitope composition. In view of a failure to demonstrate differences between monomeric and polymeric forms of the antigen, it is argued that the arrangement of epitopes is not an essential requirement for T cell-independent IgM responses in vivo.

Interferon preparations as modifiers of immune responses.

SummaryWithin certain dose ranges, preparations of mouse serum interferon or tissue-culture interferon show a modest capacity to increase the number of antibody-forming spleen cells in mice as measured by the Jerne plaque technique. When low levels of polyadenylic polyuridylic acid (= poly A:U), i.e., an amount that causes only modest stimulation, were used together with amounts of the interferon preparations that also by themselves are ineffective, a good stimulation of early appearance of antibody-forming cells was produced. High concentrations of IF preparations reduced antibody formation, and the potent immunoenhancing effects of chlorite-oxidized amylose (COAM) were almost completely abolished in the presence of low concentrations of interferon preparations. These findings suggest certain similarities between the effects of interferon preparations and those of agents known to modify the cyclic AMP system. Such suspected relationships are supported by preliminary data demonstrating a capacity of IF pr...

Enhanced resistance of mice to virulent Japanese B encephalitis virus following inactivated vaccine and poly I:C.

The double-stranded complex consisting of polyriboinosinic and polyribocytidylic acids (poly I:C) has been shown to be a potent inducer of interferon [1], effective prophylactically against a number of experimental viral infections [2]. Poly I:C has also stimulated the appearance of antibody-forming cells [3] and potentiated the antibody response to influenza viral vaccine in adjuvant 65 [4]. The purpose of this study was to evaluate and attempt to explain the effect of poly I:C on the resistance of mice immunized with formalin-

Cell populations and cell proliferation in the in vitro response of normal mouse spleen to heterologous erythrocytes. Analysis by the hot pulse technique.

The role of proliferation in the development of 19S antibody-forming cells in the primary response has been investigated in an in vitro system. Spleen cell suspensions from normal, unimmunized mice were cultured in vitro in the presence of mammalian erythrocytes and the number of 19S hemolytic plaque-forming cells that arose 4 days later was measured. The hot pulse technique for the selective irradiation of those cells which synthesize DNA during a defined period of time has been described. The effect of such hot pulses administered at various times after the addition of antigen on the subsequent appearance of antibody-forming cells was determined. The results established that: (a) the onset of DNA synthesis does not start for approximately 24-32 hr after the addition of antigen, (b) essentially all the antibody-forming cells arise by cell division, and (c) different cell populations are involved in the response to two non-cross-reacting antigens.