Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide. II. Ability of suppressor T cells to act on both B cells and amplified T cells to limit the magnitude of the antibody response.

When administered 2 days after immunization with 0.5 microgram Type III pneumococcal polysaccharide (SSS-III), the T lymphocyte mitogen concanavalin A (Con A) stimulates a 2.6-to 7-fold enhancement of the plaque-forming cells (PFC) response to SSS-III in vivo. This enhancement requires the presence of amplified T cells, which act by driving PFC or their precursors to extra rounds of proliferation. The extra proliferation that can be stimulated by Con A is not seen in the normal primary response to SSS-III; but treatment with anti-lymphocyte serum (ALS) to remove suppressor T cells will permit the additional proliferation to occur. This indicates that in the primary response to SSS-III, suppressor T cells act on amplifier T cells to limit the magnitude of the antibody response. Only suppression of B cells can account for the further suppression induced by Con A given at the time of immunization or by low-dose paralysis of the SSS-III response. The relatively late development of amplified activity compared to suppressor activity appears to account for the absence of amplifier activity after primary immunization with SSS-III. It is apparent that one can explain the regulatory effects observed during the development of an immune response to SSS-III only by considering both T cell- B cell and T cell- T cell interactions, together with the temporal relationships involved in those interactions.     

Generation of low-dose paralysis in the absence of the ability to secrete antibody.

(CBA/N female x BALB/c male)F1 male mice carry an X-linked defect, originating from CBA/N mice, which renders them unable to generate an antibody response to SSS-III. Histocompatible (BALB/c female x CBA/N male) reciprocal F1 male hybrids do not carry the X-linked defect and therefore generate a readily detectable PFC response to SSS-III, which can be adoptively transferred into nonresponding reciprocal F1 male mice. In the present work, we show that this adoptive response could be inhibited in recipient (CBA/N female x BALB/c male)F1 male nonresponding mice in which low dose paralysis had been induced. Evidence is presented which indicates that such suppression is of host rather than donor cell origin. The capacity to develop low-dose paralysis, a phenomenon that is antigen specific and has been attributed to the action of suppressor T cells, indicates that nonresponding (CBA/N female x BALB/c male) F1 males (and presumably the CBA/N progenitor strain) have the ability to recognize this antigen. Furthermore, since these animals fail to make a serum antibody response to SSS-III, the signal that activates suppressor T cells cannot be circulating antibody or antigen-antibody complexes. These findings are most consistent with the view that low-dose paralysis of the response to SSS-III is not dependent on antibody-mediated feedback inhibition; rather, it is an active process mediated by suppressor T cells.     

The in vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A.

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to Br-MRBC. Mitogenic concentrations of Con A prevented the development of both the PFC and TDTH responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and TDTH response against BrMRBC.     

Activation of human B lymphocytes. III. Concanavalin A-induced generation of suppressor cells of the plaque-forming cell response of normal human B lymphocytes.

Con A-induced suppression of the direct PFC response to polyclonal stimulation in human B cells has been described. Two types of experiments are presented. First, Con A was added directly to PWM-stimulated PB or tonsil cells resulting in a dose-dependent suppression of the PFC response, with maximal suppression occurring at a Con A concentration of 10 mug/ml. This suppression is completely removed by the simultaneous addition of alphaMM to the cultures. Secondly, Con A stimulation of tonsil or PB lymphocytes generated a population of cells which when added to autologous lymphocyte cultures induced a marked and reproducible suppression of the PFC response. The generation of suppressor cells is dependent on cell division and is blocked by alpha MM. Once generated the process of suppression is indpendent of the presence of Con A itself and is mediated by an activated lymphocyte population. These studies demonstrate a simple and reproducible model for the generation of a population of suppressor cells capable of inhibiting the direct PFC response to PWM-induced polyclonal activation of normal human B lymphocytes.     

Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of humoral immunity.

The abilities of concanavalin A (Con A) and phytohemagglutinin P (PHA) to selectively induce different T-cell activities affecting humoral immunity were evaluated. The mitogens were intravenously injected before, with, or after injection of sheep red blood cells (SRBC) into mice, and the 3 to 6-day plaque-forming cell (PFC) responses were assessed. Mitogenic treatment differentially influenced the resultant in vivo PFC responses to SRBC. The in vivo suppressive effects induced by Con A were shown to be temporary; only the Day 4 PFC response was inhibited. Con A given 3 hr before, with, or after the antigenic challenge enhanced the PFC response. In contrast, PHA given at all intervals inhibited both the 4- and 5-day PFC response. Neither mitogen appeared to affect the kinetics of the in vivo PFC response to SRBC. Both mitogens enhanced in vivo DNA synthesis by the splenic cells, and Con A appeared biphasic in its stimulation. Con A-induced effects on the humoral immune response were short-lived and transient, while PHA induced a longer-lasting effect on humoral immunity.     

Two defects in old New Zealand Black mice are involved in the loss of low-dose paralysis to type III pneumococcal polysaccharide

Treatment of normal mice with a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) results in the development of an antigen-specific state of unresponsiveness termed low-dose paralysis. This unresponsiveness is mediated by T suppressor cells and can be transferred by Lyt-2+ T cells, but not by L3T4+ T cells, obtained 18 hr after priming. As autoimmune New Zealand Black (NZB) mice age, there is a progressive decrease in low-dose paralysis to SSS-III. The defect in older NZB mice resulting in decreased suppressive activity was investigated by transferring primed Lyt-2+ T cells from young into old mice, and vice versa. Enlarged Lyt-2+ T cells from old NZB mice could not suppress the SSS-III response of young recipients. However, Lyt-2+ T cells of normal cell size were efficient in inhibiting the antibody response upon transfer. Primed Lyt-2+ T cells from young NZB mice did not affect the response of old recipients, but effectively suppressed the response of young mice. These results suggest that there are two defects involved in the decline of low-dose paralysis to SSS-III in aging NZB mice: Enlarged Lyt-2+ T cells may lose their ability to function as mediators of suppression; and B cells may become resistant to T cell-mediated suppression.     

Suppression of the in vitro secondary antibody response of rabbit lymphoid cells by Concanavalin A.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 mug/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 mug/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 mug/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 mug/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance(s).     

Kinetics of the antibody response to type III pneumococcal polysaccharide (SSS-III). I. Use of 125I-labeled SSS-III to study serum antibody levels, as well as the distribution and excretion of antigen after immunization.

A simple method was described for the preparation of 125I-labeled type III neumococcal polysaccharide (SSS-III) with a high specific radioactivity which retained the physical and immunologic properties of native SSS-III. SSS-III was used to study the serum and tissue levels of antigen, as well as its excretion, after i.p. injection. When an optimally immunogenic dose (0.5 mug) of antigen was given, greater than 90% of the injected antigen was excreted during the first 3 days after injection; however, after day 3, the SSS-III which remained in each mouse was firmly bound to various tissues, and less than 5 ng SSS-III was released into the circulation daily. SSS-III was also used in a Farr test to measure serum antibody levels; the kinetics for the appearance of PFC/spleen and serum antibody levels were measured at 24-hr intervals after immunization with 0.5 mug of antigen. Maximum PFC/spleen were observed on day 4 after immunization whereas the peak serum antibody level was seen on day 5. The decay of serum antibody level from its maximum value was much slower than that of the PFC/spleen. The data describing the distribution of SSS-III in vivo and the measurement of serum antibody levels indicated that treadmill neutralization was not a factor in determining the serum antibody levels after immunization with an optimally immunogenic dose of SSS-III.     

Cell surface antigens and other characteristics of T cells regulating the antibody response to type III pneumococcal polysaccharide

The administration of a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) produces an antigen-specific T cell-dependent phenomenon termed low-dose paralysis (immunologic unresponsiveness). This form of unresponsiveness can be transferred by spleen cells obtained 5 to 24 hr after priming, and the suppressive activity of the transferred cells is abolished by prior treatment with monoclonal anti-Lyt-2 and anti-I-J antibody in the presence of complement, indicating that suppression is mediated by a distinct subset of T cells (suppressor T cells). If primed spleen cells are transferred 24 to 72 hr after immunization with SSS-III, however, the resulting antibody response of immunized recipients is enhanced. Greater enhancement is noted when transferred cells, pretreated with monoclonal anti-Lyt-2 antibody plus complement to remove suppressor T cells, are used; such enhancement is attributed to amplifier T cells. These findings indicate suppressor T cells regulate the antibody response to SSS-III by influencing the expansion of SSS-III-specific clones of B cells as well as the expression of amplifier T cell activity; the latter causes B cells to proliferate further in response to SSS-III.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

Cytokine modulation of immune activation associated suppression of macrophage cyclooxygenase activity in vivo.

Intraperitoneal infection with Listeria monocytogenes (LM) results in activation of the peritoneal macrophage population which displays increased surface expression of major histocompatibility (MHC) Class II (Ia) antigen and markedly suppressed prostaglandin (PG) synthesis. We demonstrate here that this decrease in PG production is also seen after treatment by mitogen (Con A) and endotoxin (LPS), and can be explained by reduced cyclooxygenase activity in these cell populations. We show that, whereas Ia expression was augmented at all doses of LM and Con A tested, it displayed a biphasic response to LPS in vivo: increase at the lowest dose and inhibition at higher doses. In order to identify possible endogenous mediators of these responses, we used highly purified preparations of recombinant murine (rMu) cytokines and neutralizing cytokine specific monoclonal antibodies (MAbs) to examine whether interferon-gamma (IFN-gamma) and/or tumor necrosis factor (TNF) down-regulate macrophage cyclooxygenase activity in vivo. We found that IFN-gamma induced Ia expression but had no effect on PG secretion. In contrast, TNF-alpha suppressed PG synthesis and inhibited Ia surface expression. Similarly, in our model of Con A-induced peritoneal macrophage activation, pretreatment of animals with a neutralizing MAb to rMuIFN-gamma completely blocked the induction of Ia positive macrophages by Con A but did not affect Con A-dependent suppression of PG synthesis. Pretreatment with MAb to TNF had no effect on Con A-induced Ia levels, but significantly inhibited suppressed PG synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A reverse effect of in vitro and in vivo concanavalin A-induced spleen cells on anti-sheep red blood cells response detected with a new method based on flow cytometry

A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes, but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.     

Effects of atomic bomb radiation on the differentiation of B lymphocytes and on the function of concanavalin A-induced suppressor T lymphocytes

The differentiation of peripheral blood B lymphocytes into immunoglobulin-producing cells (Ig-PC) by pokeweed mitogen (PWM) and the function of concanavalin A (Con A)-induced suppressor T lymphocytes were examined to elucidate the late effects of atomic bomb radiation. A total of 140 individuals, 70 with an exposure dose of 100 rad or more and an equal number with an exposure dose of 0 rad matched by sex and age, were selected from the Nagasaki Adult Health Study (AHS) sample. Both the differentiation of peripheral blood B lymphocytes into Ig-PC by PWM and the function of Con A-induced suppressor T lymphocytes tended to be more depressed in the exposed group than in the control group, but a statistically significant difference could not be observed between the two groups. The function of Con A-induced suppressor T lymphocytes tended to decrease with age, but a statistical significance was detected only for percentage suppression against IgM-PC.     

Production of soluble suppressor factor by spleen cells from mice immunized with type III pneumococcal polysaccharide

Supernatant fluid (SF) derived from spleen cell cultures, obtained from mice 16 hr after immunization with 0.5 microgram of Type III pneumococcal polysaccharide (SSS-III), suppressed the antibody response when SF was given (i.v.) 3 hr before immunization with SSS-III. Such suppression was antigen specific and could be reproduced by SF derived from cultures of T cells from mice immunized with SSS-III (0.5 microgram) or by SF derived from cultures of spleen cells from mice primed with a subimmunogenic dose of SSS-III (0.005 microgram). Adsorption of SF with SSS-III covalently bound to a Sepharose 4B column did not alter the ability of SF to suppress the SSS-III-specific antibody response. However, adsorption of SF with Ig+ (B) cells from mice immunized with 0.5 microgram SSS-III completely removed the suppressive activity. Significant (p less than 0.05) suppression of the antibody response was observed only when SF was administered (i.v.) 24 hr before to 24 hr after immunization with 0.5 microgram of SSS-III. These results suggest that suppressor T cells generated in response to SSS-III function by releasing a soluble factor(s) that binds to determinants on B cells rather than antigen; this soluble factor(s) acts directly on antigen-stimulated B cells or inhibits the induction of amplifier T cells.     

Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Regulation of the antibody response to type III pneumococcal polysaccharide. V. Ontogeny of factors influencing the magnitude of the plaque-forming cell response.

Mice of different ages were evaluated with respect to their ability to give a plaque-forming cell (PFC) response to Type III pneumococcal polysaccharide (SSSIII), as well as the degree of amplifier and suppressor thymus-derived(T) cell activity present. Although the magnitude of the PFC response to an optimally immunogenic dose of SSS-III for 2-and 3-week old mice was only 7% and 14%, respectively, of that produced by adult (8-week old) mice, values comparable to those of adult animals were attained by 4 weeks of age; no significant changes in the ability to respond to SSS-III occurred thereafter. Amplifier T cell activity, which was minimal at 2 to 4 weeks of age, matured slowly and did not reach a maximum until 8 to 10 weeks of age. By contrast, suppressor T cell activity appeared to be fully developed at least as early as 2 weeks of age; here, the inhibitory effects produced could by abrogated by depletion of T cells, indicating that the unresponsiveneness induced by such cells does not result in the depletion ot irreversible inactivation of B cells capable of responding to SSS-III. These findings suggest that the inhibitory effects of suppressor T cells are predominant in young mice and that such cells may play an important role in determining the ease with which unresponsiveness is induced in neonates, and in the prevention of autoimmune disease. Also, studies conducted with adult-thymectomized mice showed that both amplifier and suppressor T cells, once seeded to the periphery, are stable and do not depend upon the presence of intact thymus for the expression or renewal of their activity.     

Separation of immunomodulatory effects of mannan from Candida albicans into stimulatory and suppressive components

Mannan extracted from Candida albicans was studied for its immunomodulatory activity on in vivo antibody responses to type III pneumococcal polysaccharide (SSS-III), a helper-T-cell-independent antigen, and to sheep erythrocytes (SRBC), a helper-T-cell-dependent antigen. In some studies, the antibody response to SSS-III was converted to a helper-T-cell-dependent response by attaching it to a carrier (horse erythrocytes, HRBC); this complex then was used to immunize mice primed with a subimmunogenic dose of HRBC. Mannan enhanced the antibody response to both SSS-III and SRBC when administered at the same time or 1 or 2 days after immunogen. However, when both mannan and SSS-III were coated onto HRBC for immunization, either enhancement or suppression was noted; the effect depended upon the amount of mannan used. Larger amounts stimulated, whereas smaller amounts suppressed, the antibody response to SSS-III. The enhancing and suppressive components of mannan could be separated by molecular size or charge by chromatography on Sepharose 4B or on DEAE-Sephadex A-50 columns, indicating that mannan extracts contain individual components having opposing immunomodulatory properties. These components can be separated on the basis of molecular size and charge.