Precursor cell division during the immune response in vitro: antigen-induced and "spontaneous" antibody-forming cells

“Background” or “spontaneous” plaque-forming cells (PFC) which arise during in vitro culture of mouse spleen cells may be eliminated by a hot thymidine pulse. This does not prevent the subsequent addition of sheep red blood cells (SRBC) or trinitrophenylated (TNP) SRBC eliciting a primary immune response. Using mouse spleen cells, resently primed by carrier (SRBC), it was found that an early “hot pulse” (for the first 24 hours of culture) can eliminate the anti-SRBC response, but not the primary anti-TNP response which is dependent on carrier-specific, θ-bearing, radioresistant spleen cells.     

Antigen-induced helper and suppressor T cells in normal and agammaglobulinemic chickens

Spleen cells from chickens injected with sheep erythrocytes (SE) intravenously 2 to 14 days prior to culture were found to give faster and higher plaque-forming cell responses upon addition of antigen on day 2 rather than on day 0 of culture. Cell mixture experiments showed that this was due to the induction of suppressor T cells upon re-exposure to SE on day 0 of culture. Spleen cells taken on days 2 or 14, but not between days 4 and 7 after priming to SE were sensitive to suppression. The suppressor cells were resistant to gamma irradiation (1000 rd) and to mitomycin C, but were apparently lost after 2 days of culture in the absence of antigen. Pokeweed mitogen addition on day 0 of culture also induced suppressor cells, both in SE immune and in normal spleen. Similar suppressor cells were induced in cultures of primed spleen cells taken from agammaglobulinemic chickens. The response to Brucella abortus in vitro was not affected by induction of suppression for the anti-SE response. Suppression could also be shown after transfer of cell mixtures to irradiated recipients. Helper cell activity for the anti-SE response could readily be shown, both in vivo and in vitro, in primed spleen cells precultured for 2 days in the absence of antigen, and was also resistant to 1000 rd gamma irradiation and to mitomycin C.     

Antibody synthesis by chicken spleen cells in vitro. I. Requirements of B cells at various stages after immunization for T cells,macrophages and antigen

The requirements for T cells, macrophages and antigen during the induction of in vitro antibody responses were ascertained with chicken spleen cells obtained at various times after immunization with sheep red blood cells (SRBC). The IgM plaque-forming cell (PFC) response was T cell independent exclusively in cultures initiated 3 days after priming, but macrophage dependent at all time intervals tested. In cultures started 4 to 10 days after priming the IgG response was both T cell and macrophage independent and PFC numbers remained at a high plateau level throughout the culture period. In contrast, IgG responses initiated more than 15 days after priming showed a reversal to complete T cell and macrophage dependence and were characterized by a sharp increase in PFC numbers between days 2 and 4 of culture. Formaldehyde-fixed SRBC were immunogenic for IgG PFC 4 to 10 days after priming but failed to stimulate later IgG memory and all IgM responses. Contrasting antigen dose requirements for IgM and IgG responses were found in cultures initiated at various periods after priming. The results suggest that direct contact with fixed antigen was sufficient to maintain IgG antibody synthesizing PFC in vitro, while native antigen and cell co-operation were required for late secondary IgG and all IgM responses. These results are interpreted in terms of separate pathways of differentiation for IgM and IgG antibody-producing cells. A distinct 3rd day stage of T cell-independent but macrophage-dependent responsiveness for both classes of antibody was also defined.     

The Effects of Metallic Tin on Background Plaque-Forming Cells, Immunoglobulins and the Immune Response

Inoculation of metallic tin powder into Lewis rats resulted in marked enlargement of the draining lymph nodes. Plasma cells were the major contributors to the increase in cell mass. Certain immunologic aspects of this plasma cell lymphadenopathy were investigated. Immunoglobulin isotypes were distinguished and quantitated by fluorescence immunocytochemistry and rocket electrophoresis of lymph node extracts. Background plaque-forming cells directed against sheep red blood cells were increased. Specific activity was highest 4 days after inoculation of tin. Lymph nodes and spleen continued to increase in cell number so that total background activity attained a maximum at 14 days after inoculation of tin. Tin was a good adjuvant for plaque-forming cells in lymph nodes and for serum agglutinins (primary and secondary responses) when the rats were immunized with sheep red blood cells, even when the antigen was injected at a remote site. The plasma cell response to tin may be due to local polyclonal activation.     

Secondary Antibody Response Elicited in vitro with Chicken γ Globulin

The plaque-forming cell (PFC) response of spleen cells from mice primed 30-90 days previously with chicken y globulin (CGG) plus adjuvant was studied after restimulation in vitro with 1 pg to 100 μg CGG per culture. The peak response (100 to 500 PFC per 10⁶ cells), elicited with 0.1 μg CGG, was detected five days after restimulation. The 100 μg level of CGG suppressed the PFC response to CGG, but did not decrease the primary PFC response to sheep red blood cells (SRBC) of other, control cultures. Plaques detected with CGG coupled immunologically to SRBC were clearer but not more numerous than those detected with CGG coupled chemically to SRBC. The system described permits the investigation of cellular mechanisms involved in the antibody response to a soluble protein antigen and is, therefore, ideally suited to elucidation of factors which enhance or suppress antibody biosynthesis and immunologic memory.     

Enhancement by the adjuvant, endotoxin, of an immune response induced in vitro

Effects of endotoxin on the immune response to sheep red cells added in vitro to spleen cell suspensions from unimmunized mice were studied. The optimal dose of endotoxin (1 μg/ml) gave a six-fold increase in the number of plaque-forming cells per 10⁶ viable cells, after 6 days of culture, if it was present from the 12th to the 24th hour after antigen. There was little or no enhancement when endotoxin was given at other times during the 6 days of culture. The response was depressed when the suspension was incubated with endotoxin before the addition of red cells. The periods of RNA and DNA synthesis were determined by adding tritiated uridine and tritiated thymidine, respectively, for 24-hour periods during the 6 days of culture. Synthesis of RNA occurred throughout the culture period whereas DNA synthesis was not detected before 24 hours of culture. We found that the adjuvant action of the endotoxin which was maximum during the 1st day of culture, was associated with an initial phase of RNA synthesis, before DNA synthesis could be detected.     

Opposite effects of the synthetic adjuvant N-acetyl-muramyl-L-alanyl-D-isoglutamine on the immune response in mice depending on experimental conditions

The influence of N-acetyl-muramyl-L -alanyl-D -isoglutamine (muramyl dipeptide, MDP) on the plaque-forming cell (PFC) response of mice to sheep red blood cells (SRBC) was studied. The effect of MDP depends on the relative doses of antigen and adjuvant and on the timing of adjuvant addition. For a given concentration of antigen, when SRBC and MDP were simultaneously injected in the i.v. route, MDP led to an enhancement of the response; in contrast, when antigen was injected i.p., a depression of the PFC response was observed. In vitro, the same duality of effect was obtained, for the same spleen cell concentration; addition of MDP led to a stimulation of the response when cultures were set in petri dishes and to an important decrease in the response when cultures were set up in plastic tubes. This inhibitory effect of MDP did not appear when it was added one night after the initiation of the cultures. These contrasting results confirm the adjuvant properties of MDP, but they show that under certain conditions, MDP can stimulate suppressive processes.     

The primary antibody response of individual spleen cells in diffusion chambers

Spleen cells from non-immunized mice were mixed with sheep red blood cells and incubated in diffusion chambers implanted in the mouse peritoneal cavity.

At 6 and 7 days an increase was noted in the proportion of plaque-forming cells in the chambers and this was accompanied by the appearance of haemagglutinin and haemolysin in the chamber fluid. Excess antigen was not observed to depress this proportion of plaque-forming cells.

     

Suppressor cells in rabbit peripheral blood

Rabbit peripheral blood leukocytes (PBL) added to cultures of autologous spleen cells, primed in vivo to sheep red cells, are able to suppress with high efficacy the secondary in vitro response of the spleen cells to that antigen. Removal of nylon wool adherent cells from PBL abolishes the suppressive effect. When the PBL are fractionated by velocity sedimentation the suppressor cells can be separated from the responding cells. The circulating lymphocytes, freed from the inhibiting effect, either by nylon wool absorption or by velocity sedimentation fractionation, are able to give a strong secondary in vitro anti-SRC response, in which the long latency period, usually observed when PBL are stimulated with antigen in culture, is abolished or at least reduced. The suppressor effect present in the PBL is not due to granulocytes, platelets or erythrocytes.     

Stimulation of humoral immunity by peptidoglycan monomer from Brevibacterium divaricatum.

Peptidoglycan monomer (PGM), a water soluble and nontoxic disaccharide pentapeptide unit obtained from Brevibacterium divaricatum, was administered intravenously into mice, and the humoral immune response to sheep erythrocytes was assayed by means of Jerne's technique for plaque-forming cells (PFC) in the spleen. The PFC response was evidently stimulated. The counts were increased to practically the same extent over a great range of doses of PGM (from 25 to 1600 microgram per animal), and the effect was present in the mice immunised with optimal, as well as in those immunised with suboptimal, doses of antigen. The magnitude of the immunostimulation depend only on the timing of PGM administration: it was maximal if PGM was injected 1 or 2 days after the antigen. In vitro, in a 4-day culture of spleen cells, PGM did not stimulate PFC formation. We conclude that stimulation of the humoral immune response to sheep red blood cell antigens by PGM probably occurs without cell multiplication and probably involves more than simply a contact of immunocompetent cells with PGM.     

Separation of T and B lymphocytes by preparative cell electrophoresis

Free flow electrophoresis can separate mouse spleen and lymph node lymphocytes into two fractions: a high mobility (more negatively charged) and a low mobility (less negatively charged) fraction. The high mobility lymphocytes are sensitive to anti-Θ serum and complement but resistant to an anti-serum against mouse specific B lymphocyte antigen (MBLA). They produce rosettes with sheep red cells and induce a graft-vs-host reaction when injected into F₁-hybrid recipients. The high mobility lymphocytes also respond to phytohemagglutinin in vitro and are cytotoxic to allogeneic target cells after appropriate immunization. The low mobility lymphocytes are sensitive to anti-MBLA serum and complement but resistant to anti-Θ serum, form rosettes and hemolytic plaques against sheep red cells after immunization, but neither respond to phytohemagglutinin in vitro nor induce a graft-vs-host reaction. In CBA mice the percentages of high and low mobility lymphocytes in the spleen and lymph nodes correspond to the known distributions of T and B cells in these organs. The separated cells are viable since they are able to perform biological functions. Thus free flow cell electrophoresis seems to produce viable populations of mouse T and B lymphocytes.     

Analysis of immunological memory in terms of increased PFC responsiveness and increased focus-forming capacity

The increase in splenic plaque-forming cell (PFC) responsiveness which occurs in mice following the injection of a primary dose of 10⁸ sheep red blood cells was measured using the spleen cell transfer system. As for grafts of normal spleen cells, the peak direct (19S) and indirect (7S) responses obtained with grafts of primed spleen cells were found to be related to the number of cells transferred in a near-parabolic fashion. Splenic direct PFC responsiveness as measured by the regression defining the corresponding graft-response relationship was increased about five times 4 weeks after priming; focus-forming capacity was also increased by a similar factor. At the same time indirect PFC responsiveness was increased almost twenty times. These results are discussed in terms of antigen-induced changes in the populations of PFC precursors and antigen-reactive auxiliary cells in the spleen.     

Requirement for adherent cells in the primary and secondary immune response in vitro

Treatment of spleen cells with iron powder, followed by removal of the iron with a magnet, was found to be a simple and reliable method for obtaining a pópulation of non-adherent cells, which does not give an antibody response against heterologous red cells in vitro. It was possible to reconstitute the antibody response to 40 - 90% of that obtained with untreated cells by addition of irradiated or non-irradiated adherent spleen cells. The secondary response was also depressed by treatment with iron and it could be completely reconstituted with adherent cells from normal mice. Removal of adherent cells also caused a depression of spontaneous cell proliferation, which could be reconstituted by addition of adherent cells. Comparative kinetic studies on cell proliferation and antibody response indicated that the adherent cells are necessary for a normal cell proliferation and that they do not act by breaking down the antigen. This conclusion was further supported by the finding that an immune response against a soluble sheep red cell antigen was also dependent on adherent cells.     

Carrier inhibition in the in vitor response to hapten-erythrocyte conjugate

The in vitro induction of 4-hydroxy-5-iodo-3-nitrophenacetyl (NIP)-specific plaque-forming cells by NIP-SRC (sheep red blood cells) can be specifically inhibited by the presence of free SRC in the cultures, but only when low doses of hapten-erythrocyte conjugate and of free SRC are used. At high concentrations of free SRC, the NIP response is specifically enhanced. Thus, only at low antigen concentrations can the in vitro system be seen as a model of physiological T-B interactions.     

Haemolytic activity of mouse peritoneal exudate cells in vitro

Mouse peritoneal exudate cells, and to a lesser degree spleen cells, can cause haemolysis of both syngeneic and allogeneic mouse red blood cells in vitro whereas lymph node and tumour cells are ineffective. The reaction was clearly detectable after 0.5–1 hour, and is usually complete after 5–6 hours at 37°. An analogous reaction was found also with sheep red blood cell targets, but haemolysis was weaker and a significant effect was not obtained until 24 hours later. The degree of haemolysis did not increase after the addition of complement. Hemolysis was not potentiated when specifically sensitized cells were employed or when PHA was present. Viability and active metabolism of the PE cell was essential for haemolysis. Only red blood cells were affected by the PE cells, nucleated target tumour cells being resistant. It was concluded that macrophages were responsible for haemolysis, which was not mediated by antibody and complement, but represented an active macrophage reaction unrelated to previously known mechanisms of haemolysis in vitro.     

Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharides

Lipopolysaccharides (LPS) from E. coli bacteria were found to be mitogenic for bone marrowderived (B) lymphocytes, but had no effect on thymusderived (T) lymphocytes. When added to normal spleen cells in culture, LPS selectively stimulated the secretion of 19 S proteins, whereas there was no demonstrable increase of 7 S protein synthesis. Spleen cell cultures treated with various doses of LPS exhibited atypical dose response curve with regard to induction of DNA synthesis, 10 μg/ml being optimal, higher and lower concentrations giving lower responses. When direct antibody producing cells to horse and sheep red cells were studied in normal spleen cell cultures exposed to different concentrations of LPS in vitro, it was found that their number increased in parallel with stimulation of DNA synthesis. In contrast, the number of antibody producing cells to LPS itself did not parallel activation of DNA synthesis. Spleen cells from LPS tolerant animals responded with increased numbers of antibody producing cells to heterologous red cells after treatment with LPS in vitro to the same extent as normal spleen cells. Thus, a B cell mitogen, such as LPS, could activate division in B cells, resulting in an increased number of cells producing antibodies to non-cross-reacting antigens, mimicing the effect of specific antigen.     

Oral administration of SSG, a beta-glucan obtained from Sclerotinia sclerotiorum, affects the function of Peyer's patch cells.

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of a fungus, Sclerotinia sclerotiorum IFO 9395, on the function of Peyer's patch (PP) cells was investigated in comparison with that on spleen cells in mice. Oral administration of SSG enhanced the proliferative response of PP cells to a T-cell mitogen, concanavalin A (Con A), and a B-cell mitogen, lipopolysaccharide (LPS), although the response of spleen cells was not affected. Peyer's patch cells taken from mice which had received oral administration of SSG two days before, showed enhanced plaque-forming cell (PFC) response to sheep red blood cells (SRBC) after antigen (SRBC) stimulation for 5 days in vitro. These results suggest that oral administration of SSG can modulate the mucosal immune response.     

Reconstitution of immunocompetence in B cells by addition of concanavalin A or concanavalin A-treated thymus cells

The effect of soluble Concanavalin A (Con A) on the primary antibody response in vitro against sheep red cells (SRC) by mouse spleen cells was studied. Stimulation of normal spleen cells with Con A caused a slight increase of the background number of plaque-forming cells (PFC). Cultures stimulated with SRC did not show an increased PFC response after Con A treatment, except in experiments where the PFC response against SRC was rather low in the absence of Con A. Concentrations of Con A higher than 0·5 μg/ml were inhibitory to the immune response of SRC-treated cultures also early during the culture period. In contrast, T cell depleted spleen cultures, incapable by themselves to respond to SRC, were reconstituted by addition of Con A at concentrations of 0·5 μg/ml or more. Presumably, residual T cells were activated by Con A. Con A activated T cells could reconstitute the PFC response in T cell-deficient cultures. In contrast, it was not possible to obtain more than a marginal stimulation of the antibody response in cultures of spleen cells depleted of adherent cells by addition of soluble Con A or Con A-activated thymocytes.

The results suggest that Con A may stimulate the antibody response by activation of T cells, and support the concept that activated T cells can non-specifically stimulate the antibody response of B cells. Large numbers of activated T cells were inhibitory to the immune response and it is suggested that this phenomenon is analagous to antigenic competition. Furthermore, activated T cells do not seem capable of substituting for adherent cells in the primary immune response in vitro, suggesting that adherent cells are important for the functions of B cells.

     

Ontogeny of adherent cells. I. Distribution and ontogeny of A cells participating in the response to sheep erythrocytes in vitro

An adherent (A) cell function required for the in vitro antibody-forming response to sheep erythrocyte (SRBC) antigen has been demonstrated in a culture medium enriched with nucleic acid precursors and 2-mercapto-ethanol. The essential function is fulfilled best by adult adherent cells of the spleen, to some extent by bone marrow and perhaps lymph node A cells, and poorly or not at all by peritoneal or lung A cells. Ontogenetic studies reveal the absence or inhibition of neonatal A cell synergistic function in the in vitro response to SRBC. The capacity of splenic adherent cells to collaborate with column-separated nonadherent cells appears only from the third or fourth day after birth, with ratio indices reaching levels significantly greater than nonsynergistic background by the fifth day and showing little further increase until the second week of age.     

Lipopolysaccharide can substitute for helper cells in the antibody response in vitro

The effect of lipopolysaccharide (LPS) from E. coli on the primary and the secondary antibody response against sheep red blood cells (SRC) by spleen cells in vitro was studied. LPS, which is a non-specific mitogen for B cells, often caused an increased antibody response. Stimulation was obtained on day 4 of the culture if the spleen cells gave a low response to SRC without LPS, whereas no stimulation was obtained if the antibody response was high. However, kinetic studies showed that cultures treated with LPS always exhibited increased numbers of plaque-forming cells (PFC) to SRC if the response was studied earlier than the 4th day. LPS could substitute for certain cell types otherwise necessary for the immune response. Thus, LPS reconstituted the immune response of spleen cells depleted of adherent cells as well as of thymus-derived lymphocytes. It was also possible to enhance the immune response of spleen cells from nude mice by addition of LPS. The results suggest that LPS increases the immune response by direct stimulation of bone marrow-derived precursor cells for antibody production. LPS makes these cells competent to differentiate and proliferate in the absence of helper cells, such as adherent cells and thymus-derived cells. This implies that cooperation with helper cells is not an obligatory event in the antibody response, but can be substituted by other means of stimulating the precursor cells.