Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro: III. DNA synthesis of lymphocytes in clusters

The lymphocytes which engage in DNA synthesis during the in vitro immune response to PPD (purified protein derivative of tuberculin) were studied by scintillation counting and in autoradiographs prepared from cultures of macrophages and immune T-lymphocyte-enriched lymphocytes. The lymphocytes in these cultures were located in three compartments: lymphocytes in macrophage-lymphocyte clusters, lymphocytes attached to macrophages but not involved in clusters, and not macrophage-attached lymphocytes. One of the cluster cells, the central lymphocyte, which is attached directly to the macrophage, was identified as the only DNA-synthesizing lymphocyte in the cluster early in the culture period. In cultures extended beyond 20 hr the increase in percentage of DNA-synthesizing lymphocytes in the cluster and macrophage-attached compartments exceeded the increase in the compartment of not macrophage-attached lymphocytes. The total amount of radiolabeled thymidine incorporated into lymphocytes in a blast transformation assay was directly proportional to the number of macrophage-lymphocyte clusters produced by the same lymphocytes in a cluster assay.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. IV. Synergy of lymphocytes in the formation of clusters.

T-lymphocyte-enriched lymph node lymphocytes from guinea pigs immunized with Mycobacterium tuberculosis produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD)-pulsed peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. VI. Roles of T and B lymphocytes in cluster formation.

The role of immune T and B lymphocytes in the in vitro production of antigen-specific clusters with macrophages pulsed with soluble protein antigen was studied by assaying the cluster-producing capability of lymphocyte populations deprived of either T or B lymphocytes. Populations deprived of B lymphocytes were able to produce clusters to the same extent as unfractionated lymphocyte populations, whereas populations deprived of T lymphocytes produced very few clusters. Staining of the cluster lymphocytes for membrane Ig using the immunoperoxidase technique showed that Ig-bearing cells were to a large extent excluded from the clusters. We suggest that each cluster is initiated by an antigen-committed T lymphocyte and that the assay for clusters may be used to enumerate the number of antigen-committed T lymphocytes in a given population.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. V. Inhibition of macromolecular synthesis and of microfilament and microtubule function.

T lymphocytes from immune guinea pigs produce clusters in vitro with macrophages exposed to soluble protein antigens. The formation of clusters is antigen specific. Cultures containing macrophage-lymphocyte clusters produced in response to purified protein derivative of tuberculin were treated with inhibitors of macromolecular synthesis and of microfilament and microtubule systems. Cytosine arabinoside, puromycin, actinomycin D, cycloheximide, cytochalasin B, and colcemide were used. The formation of clusters is independent of DNA synthesis. Microfilaments and microtubules are needed in the formation but not in the maintenance of clusters. Protein or RNA synthesis or both are needed in the formation of clusters. Finally, continuous protein synthesis is necessary for the maintenance of clusters.     

Macrophage-lymphocyte clusters in lymph nodes: a possible substrate for cellular interactions in the immune response.

The lumens of the lymphatic sinuses in lymph nodes are traversed by fibrocellular trabeculae. Joined to these trabeculae were macrophages, which formed cell clusters with lymphocytes. It is proposed, based on structural similarities, that these cell clusters are the equivalent in vivo to those seen during primary and secondary immune responses in vitro. These intraluminal macrophages were located in the path of lymph-borne antigen, as well as in the path of newly formed and recirculating lymphocytes in sinuses. This would facilitate the possible interaction between macrophage-associated antigen and antigen-reactive lymphoid cells. The attachment of numerous lymphocytes to the surfaces of macrophages and the resulting cell clusters also afford increased opportunities for lymphocyte-lymphocyte contact.     

Role of macrophages in the immune response to soluble protein antigen and in staphylococcal infection]

In experiments on rabbits immunized with soluble protein antigen immune reactions were found to be accompanied by the production of lipofuscin in macrophages. This process was the morphological manifestation of the digestion of antigen by macrophages which thus acquired the ability to migrate in the organ and to form lymphoid follicules in the medullary zone of lymph nodes. The newly formed follicules seem to be the basis of pronounced specific immune response. In staphylococcal bacteriemia the phagocytic activity of macrophages was delayed, thus causing disturbances in lipofuscin production; as a result, the subsequent phases of immune response also lagged somewhat behind in time.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

The kinetics and the distribution of antigen and antibody were shown to be similar in four species of experimental animals and in two species of wild rodents immunized with the protein-polysaccharide capsular plague antigen. Serologically active antigen and antibody were detected in homologous conjugating serological tests. Soluble antigen persists at the injection site for as long as a week and adsorbed antigen for two weeks or more. Antigen persists in the blood of animals for 2–4 days. In regional popliteal lymph nodes, antigen was detected for the first days, followed by antibody in both lymph node and blood. Plasma cell response was more intensive in animals inoculated with adsorbed antigen. The gradual decrease of antigen at the injection site shows superimposed up-and-down changes, mostly parallel with the antibody in the popliteal lymph node and blood, as well as with plasma cell response in the regional lymph node. Serological cycles were related to the resistance of immunized white mice to plague infection. Cyclic kinetics of specific polysaccharide in the faeces of dysentery patients was found.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

The injected dose of antigen determines not only the duration of its persistence in the injection site but also the intensity of plasma cell response in the regional lymph node. It was found that the logarithmic sum of antigen quantity in the injection site was related to the sum of cell response values, the correlation coefficient approaching 1. The antigen-lymphoid system interrelations appear to obey Weber-Fechner’s law for afferent systems of the organism. The sum of plasma cells appeared to be in direct connection with the logarithm of the dose injected, with antigen persistence in the injection site and also with the tangent of the acute angle adjoining the ordinate. The basic components of the primary immune response of the organism to soluble antigen,viz. logarithm of the dose injected, antigen persistence in the injection place, plasma cell quantity, tangent of the acute angle, transition modulus from antigen to plasma cells, are interconnected by rather simple equations, which represent the structural elements of the mathematical model described in the text.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

The conveyer hypothesis is based on the fact that because of clone predetermination, antibody production takes place in an organism without the presence of antigen as a result of natural cell differentiation. Soluble antigen is an analogue of a specific mitogen which gives rise to reproduction mainly of cells carrying on their surface the immunoglobulin receptors to the given antigen. The mathematical model of the conveyer hypothesis takes into account the initial conditions, among them the background level of antibody-producing cells before injection of a soluble antigen, migration of precursor cells in the draining lymphoid organ, and the rate of precursor differentiation, including the rate of the change of the immunoglobulin receptor number on the cell surface. Changes of antigen concentration in blood determine the intensity of precursor proliferation. Comparison of real experiments (intraperitoneal injection of capsular antigen ofPasteurella pestis into inbred mice) with calculations done on the basis of the developed mathematical model shows a definite qualitative resemblance with the kinetics of antibody-producing cells and free antibodies as well as with the decrease of free antigen concentration in blood. In spite of some differences between model experiments and real experiments the conveyer hypothesis and its mathematical model appear suitable for describing the primary immune response of mice immunized with low doses of capsular antigen ofPasteurella pestis.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

A mathematical expression of the accumulation of the plasma cells in the spleen of CBA mice immunized intraperitoneally is presented. The dependence of the plasma cell reaction in the spleen on the kinetics of antigen concentration in the blood was confirmed. For the transition from antigen to plasma cells, index A was proposed. The mean values of index A were used for comparison of the calculated and experimental values of the plasma cell reaction and the recorded differences were not great. In a similar way, index A was used for prediction of plasma cell accumulation in the spleen of animals, immunized with a mixture of two soluble antigens — capsular antigen ofPasteurella pestis and complete antigen ofFrancisella tularensis. The calculated values of plasma cell reaction corresponded to experimental values.     

Cyclic kinetics and mathematical expression of the primary immune response of soluble antigen

An analysis of the plasma cell reaction caused by injecting simultaneously two substances with cellular activity is presented. The plasma cell accumulation in the drained lymph node of immunized white rats is in a good agreement with the proposed equations.     

Cyclic kinetics and mathematical expression of the primary immune response of soluble antigen

The cellular activity of an antigen is understood as its power to cause plasma cells to accumulate in the regional lymph node. Two plasma cell units (PCU) is the dose causing one plasma cell addition (as compared with the “background”), whereas 1 PCU causes neither increase nor decrease in plasma cell number in the regional lymph node. Injections of antigen in less than 1 PCU causes plasma cells to decrease in number. Interrelation between antigen and plasma cells changes with time in different regions of the lymphatic system.     

In vitro immune response by murine bone marrow cells stimulated against soluble immune complexes.

Synchronized nonadherent bone marrow lymphocytes were stimulated with soluble immune complexes, in antigen excess formed by C3H/HeJ antibodies and various noncross-reacting protein antigens, in a suspension culture which allowed longterm cultivation. On binding of these complexes, lymphocytes underwent blast transformation with mitosis and formation of plasma cells which secreted specific antibodies to the antigen; a cyclic sequence of lymphocytes, blasts, and plasma cells was observed until the majority of the cell population appeared to be plasma cells. The relative percentage of mature plasma cells then decreased leaving mostly small lymphoid cells among which evidence suggests the presence of memory cells. Complexes at equivalence stimulated for the first few days, whereas antibody excess caused stimulation only initially followed by inhibition of the response. Antibodies passively added to the cultures inhibited the proliferative reaction; free antigen induced a typical secondary-type response.     

Whole-cell Bordetella pertussis vaccine component modulates the mouse immune response to an unrelated soluble antigen

Several factors are involved in the selective activation of Th1 or Th2 cells, such as different physical characteristics of antigens and the type of antigen-presenting cells involved in the immune response, among others. To study the influence of a particulate antigen on Th1/Th2 cell differentiation during the immune response to another antigen, we analysed the immune response to tetanus toxoid (soluble antigen) in BALB/c mice immunized with one of the three following vaccines: tetanus and diphtheria toxoids (DT), or DT associated with whole-cell Bordetella pertussis or its soluble antigens (DTPw and DTPa, respectively). Similar total antibody levels were observed for all vaccines. DT vaccine showed a higher IgG1/IgG2a ratio than the similar values observed for DTPw and DTPa vaccines. DT- and DTPa-primed spleen cells showed a Th2 (IL-5) profile while a Th1/Th2 (IFN gamma, IL-5) profile was observed for DTPw. IL-6 was only produced by DTPw-primed cells. Besides, IL-12 levels induced by DTPw were three times higher than the ones induced by both DT and DTPa. Our findings indicate that whole-cell B. pertussis priming modifies the tetanus immune response from Th2 to Th1/Th2 type probably via inflammatory mechanisms. In addition, in the light of conflicting reports regarding the mechanisms of protection induced by DTP vaccines, we studied the pertussis immune response. Only DTPw immunization generated memory T cells capable of proliferating with B. pertussis as an in vitro stimulus. Results might indicate that these cells may not play a key role in protecting against B. pertussis when the host is vaccinated with DTPa.     

Genetically restricted immune responses in guinea pigs primed in vivo with antigen-bearing macrophages.

Guinea pigs injected intradermally with antigen pulsed macrophages generate a population of immune T cells that proliferate in vitro on second exposure to antigen. T cells from F1 (2 X 13) guinea pigs immunized with DNP-OVA on one parental macrophage respond in vitro only to DNP-OVA on macrophages identical to those used for immunization and not to DNP-OVA associated with the other parental macrophages. These results demonstrate that the immunogenicity of antigen is dependent upon the macrophages used for priming in that, with this approach, strain 2 or 13 guinea pigs immunized with allogeneic macrophages pulsed with antigen do not respond to either allogeneic or syngeneic antigen-bearing macrophages. However, lysates of antigen-pulsed macrophages can still immunize either allogeneic or syngeneic recipient via their own macrophages. F1 (2 X 13) guinea pigs are immunized by insulin B chain pulsed strain 13 macrophages (responder) but not by strain 2 macrophages (nonresponder) suggesting that whether a F1 (nonresponder X responder) guinea pig recognizes antigen bound to a parental macrophage is genetically restricted before immunization to the same extent as the donor parental macrophages used for immunization.     

Stimulation of the immune response to immobilized antigen in an in vitro system with B-activin

The influence of B-activin, the preparation of immunomodulating myelopeptides, on the level of antibody formation after the primary immunization of mouse splenocyte cultures with immobilized antigens has been studied. The treatment of the cells with B-activin on the third day of their cultivation in the presence of peroxidase immobilized on polystyrene or protein M1 of influenza virus has been found to increase antigen-specific antibody formation by several times, while having practically no effect on the total level of IgG secretion. The stable level of the stimulation of antibody formation and the possibility of its quantitative evaluation in the enzyme-linked immunosorbent assay makes this immune response inducing system a convenient model for testing the biological activity of myelopeptides and other immunostimulators.     

Adherent cell function in murine T lymphocyte antigen recognition. II. Definition of genetically restricted and nonrestricted macrophage functions in T cell proliferation.

The mechanisms by which adherent cells, presumably of mononuclear phagocytic lineage, influence in vitro antigen-specific activation of murine T lymphocytes was examined. Two distinct functions for macrophages could be discerned. One macrophage function is dependent on a soluble factor produced by cultured adherent cells and is most easily studied with complex multideterminant antigens. This factor is neither antigen-specific nor MHC-restricted in its action in that PEC, regardless of haplotype, produce factor in the absence of antigen. A second function, antigen-specific T cell activation, is seen when antigens of more restricted heterogeneity are used, such as those under the control of Ir genes. This latter activity demands identity or partial identity between the antigen-presenting cell and the primed T cell, thus suggesting an additional specific, genetically restricted function for macrophages in in vitro antigen recognition. Whether these adherent cell functions are mediated by all or distinct subsets of cells was not established.