Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.     

Lack of generation of killer cells in the mixed lymphocyte reaction between mitogen-stimulated and unstimulated autologous lymphocytes.

The present study has demonstrated that the Con A-activated cell-mediated autologous mixed lymphocyte reaction (MLR) is not associated with the generation of cytotoxic effector cells that kill autologous targets. Thus, the suppression of antibody production of PWM stimulated lymphocytes by autologous Con A-activated suppressor cells cannot be explained by detectable cytotoxicity. We have further demonstrated that the stimulator cell in this system is a nonadherent non-T cell.     

Biological expressions of lymphocyte activation. IV. Concanavalin A-activated suppressor cells in mouse mixed lymphocyte reactions.

Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.     

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.     

Cellular and genetic restrictions in the immunoregulatory activity of alpha-fetoprotein. III. Role of the MLC-stimulating cell population in alpha-fetoprotein-induced suppression of T cell-mediated cytotoxicity

Alpha-fetoprotein (AFP), a normal component of fetal and newborn sera, exerts selective suppressive effects on various functions of thymus-derived (T) lymphocytes, including the T cell-mediated cytotoxic reaction. In the present study, AFP-induced suppression of the T cell-mediated allogeneic response was examined to define more precisely the mechanism by which AFP acts. Data presented indicate that AFP elicits its effect within the first 25 to 36 hr of culture. However, AFP apparently has no direct effect on T lymphocytes. T cells incubated in the presence of AFP retain full capacity to respond in MLC and CML. In contrast, AFP induces major changes in the functional status of monocyte-enriched, MLC-stimulating cell population. This monocyte-enriched population, after pretreatment with AFP, possesses strong stimulating capacity for the T suppressor cell limb of the immune response, while at the same time, suppresses the activity leading to the normal generation of cytotoxic T lymphocytes. These data correlate with the immune activity present in the newborn mouse.     

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.     

Mitomycin C-treated or irradiated concanavalin A-activated T cells augment the activation of cytotoxic T cells in vivo

The activation of cytotoxic T lymphocytes (CTL) in vivo after immunization of normal or cyclophosphamide-treated mice with allogeneic cells was strongly augmented by the administration of mitomycin C-treated or irradiated concanavalin A-activated spleen cells (Con A-spl). This effect of the Con A-spl was abrogated by treatment with Anti-Thy 1 antibody plus complement, and was therefore presumably mediated by activated "helper" T cells. (The term "helper" cell is only operationally defined in this context and indicates that the augmenting irradiation resistant T cells are obviously not CTL precursor cells). These observations indicated (i) that even the cytotoxic response against allogeneic stimulator cells suffers in vivo from insufficient "helper" T cell activity, and (ii) that the injection of Con A-spl may serve as a simple procedure to apply this "helper" activity in vivo. This procedure was at least as effective as the repeated injection of interleukin 2 (IL-2)-containing cell supernatants with up to four 30-unit doses of IL-2 per mouse. IL-2-containing cell supernatants were found to mediate similar effects only if injected into the footpads but not intravenously. This was in line with the reported observation that IL-2 has an extremely short half-life in vivo. The injection of Con A-spl was also found to augment the proliferative response in the regional lymph nodes.     

Characteristics and mechanisms of action of the concanavalin A-activated suppressor cell in man.

Human peripheral blood lymphocytes treated for 24 to 48 hr with optimally mitogenic doses of concanavalin A suppressed the proliferative response of autologous T cells to mitogens and antigens. Con A-treated cells also suppressed the proliferative response and the immunoglobulin synthetic response of autologous B cells stimulated in vitro by T cell helper factor. The human Con A suppressor cell was sensitive to treatment with mitomycin C and to exposure to radiation doses exceeding 1000 rads. The Con A suppressor cell was shown to reside in the nylon wool-nonadherent, sheep red cell rosette-forming, histamine receptor-bearing population of lymphocytes and to lack surface DRW antigens. One mechanism of action of Con A suppressor cells was shown to be the inactivation of nonspecific T cell helper factor.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells

The molecular basis for the suppression generated in a concanavalin A (Con A)-activated T cell culture remains unknown. In this study, we have attempted to determine whether the 2H4 and 4B4 molecules on Con A-activated T cells play some role in the generation of suppression by such cells. We have shown that Con A-activated suppressor cells belong to the 2H4+ subset of T cells but not the 4B4+ (2H4-) subset. Con A-activated T cells exerted their optimal suppressor function on day 2 in culture, a time at which the expression of 2H4 on such cells was maximal and 4B4 was minimal. Furthermore, the stimulation of T cells with the higher concentration of Con A generated the stronger suppressor function. At the same time, both 2H4 expression and density were increased and 4B4 expression and density were decreased on such Con A-activated T cells. More importantly, the treatment of Con A-activated T cells with anti-2H4 antibody but not with anti-4B4, anti-TQ1, or anti-T4 antibodies can block the suppressor function of such cells. Taken together, the above results strongly suggest that the 2H4 molecule itself may be involved in the generation of suppressor function in Con A-activated T cells. The 2H4 antigen on such cells was shown to be comprised of 220,000 and 200,000 m.w. glycoproteins. Thus this study indicates that the 220,000 and 200,000 m.w. structure of the 2H4 molecule may itself play a crucial role in the generation of suppressor signals of Con A-activated cells.     

Simultaneous suppression of allogeneic cytolytic activity and stimulation of lectin-dependent cytolytic activity by con A.

It is well known that when mouse lymphocytes are cultured with irradiated allogeneic stimulator lymphocytes, T cells capable of mediating specific cytolysis are generated. We noted that when the stimulator cells were pretreated with concanavalin A (Con A), the generation of T cell-mediated specific lysis (TSL) is largely abolished, but large amounts of T cell-mediated lectin-dependent lytic (LDL) activity are nevertheless produced. Data are presented indicating that generation of TSL is inhibited by suppressor cells which are generated by the Con A in the culture.One possible source of the LDL would be a specific clonal response to Con A-altered H-2 molecules. Evidence concerning this was equivocal since the amount of lectin required tor expression of the LDL is suffcient to cause non-specific T cell-mediated lysis of any target cell type, making inoperative the conventional tests for H-2 restricted recognition.Use of dead cell fragments for stimulation in MLC has previously been reported to produce LDL without TSL, but Con A-induced premature death of the stimulator cells was ruled out as a source of the LDL in our cultures.Another possible source of LDL would be suppressor-induced blockade of killer cell differentiation at a hypothetical stage expressing LDL but not TSL. However, delayed addition of Con A induced suppressor cells to M LC's never allowed generation of LDL when TSL was suppressed. Therefore, such a blocked differentiation mechanism did not contribute significantly to the LDL produced.The LDL activity results largely from Con A-induced polyclonal activation of cytolytic progenitor cells. It is shown that generation of LDL by Con A is able to be suppressed by Con A-induced suppressor cells added at the initiation of culture. However, in cultures in which Con A is simultaneously generating LDL and suppressor activities, the LDL is generated rapidly (in 2 days) and apparently passes the suppressable stage before the suppressor cells become active.     

Immunologic functions of isolated human lymphocyte subpopulations. IV. Stimulation of MLC and CML by human T cells.

Surface immunoglobulin positive and immunoglobulin negative human lymphocyte populations were obtained by immunoabsorbent column chromatography. Both cell populations were effective as stimulating and target cells in allogeneic MLC and CML reactions. The immunoglobulin negative population was further depleted of both EAC rosette forming cells and nylon wool adherent cells. The resulting highly purified T cell population was also able to stimulate allogeneic cells in MLC, and induce the generation of specifically cytotoxic killer cells in CML.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

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.     

Effect of ribonucleases on cell-mediated lympholysis reaction and on GM-CFC colonies in bone marrow culture

Natural dimer of bovine seminal ribonuclease (AS RNase) suppressed markedly DNA synthesis in allogeneic mixed lymphocyte culture (MLC) of normal human lymphocytes and simultaneously inhibited induction of cytotoxic effector cells within the sensitization phase of indirect cell-mediated lympholysis (CML) reaction. The last purification step of the AS RNase isolation procedure did not increase the suppressive activity of AS RNase compared to a less purified preparation (ZS RNase), thus, the later preparation was mostly used. ZS RNase (10 micrograms/ml) caused 50% inhibition of MLC reaction whereas pancreatic ribonuclease (A RNase) was 10 times less effective. The suppressive effect of RNases added in the beginning of the sensitization phase of the CML reaction correlated with that observed in the MLC reaction. The concentrations of ZS RNase (10 micrograms/ml), A RNase (100 micrograms/ml), and additionally tested cyclosporin A (0.5 microgram/ml) resulted in nearly total abrogation of cytolysis in CML. ZS RNase added after the sensitization of effector cells did not influence their cytolytic action on target cells within the destruction phase of CML. Natural killer and killer cell activities in normal peripheral lymphocytes were not inhibited by ZS RNase at the concentration of 330 micrograms/ml. ZS RNase (20 micrograms/ml), cocultivated 1 h with normal human bone marrow cells and then washed off, enhanced formation of GM-CFC colonies in semisolid agar culture up to 200%. Simultaneously tested antilymphocyte globulin increased the number of GM-CFC colonies at the average of 128%. This stimulating effect on colony formation appeared also in bone marrow culture of patients suffering with various hematological disorders. The possibility of utilizing the preparations gained from seminal plasma in clinical bone marrow transplantation is discussed.     

alpha-Fucose inhibits human mixed-lymphocyte culture reactions and subsequent suppressor cell generation

Carbohydrate moieties serve as important sites of interaction for many lymphocyte activities. The potential role of saccharides in the cellular interactions involved in mitogen-, antigen-, and alloantigen-induced proliferation was investigated. Eight different monosaccharides were tested for their inhibitory potential when added to uni- and bidirectional mixed-lymphocyte culture (MLC) reaction as well as to mitogen (Con A, PHA, PWM)-stimulated cultures. Only alpha-L-fucose blocked the MLC reaction in a dose-dependent fashion while having no effect on mitogen stimulation, although antigen-specific stimulation was also blocked by fucose. Similarly alpha-L-fucose specifically inhibited the MLC-induced generation of suppressor cells. Pretreatment of the MLC responder cells with fucose dehydrogenase abolished the MLC reaction while stimulator cell pretreatment had no effect, suggesting that the recognition site of the former contained alpha-L-fucose. The generation and the effector phase of Con A-induced suppressor cells was not affected by fucose, indicating that different receptors are involved in the latter. Apparent competitive inhibition by exogenous fucose of the cell-cell interaction required for the MLC reaction suggested that this monosaccharide is an essential constituent of allogeneic recognition sites.     

Identification of profound peripheral T lymphocyte immunodeficiencies in the spontaneously diabetic BB rat

The BB rat is presently the best available animal model for human insulin dependent diabetes (IDD). Because of the extreme susceptibility of the strain to opportunistic infections and because current studies suggest that they have an autoimmune diathesis, of which IDD is but one result, aspects of the immune system of the BB rat were studied. Severe T lymphopenia was observed in all BB rats, irrespective of sex or the presence of IDD, while numbers of B cells and serum immunoglobulin levels were normal. Both the helper T lymphocyte and cytotoxic/suppressor T lymphocyte subsets, defined by reactions with monoclonal antibodies, were depressed, and an inversion of the helper T cell subset to cytotoxic/suppressor T lymphocyte subset ratio occurred in all BB rats with increasing maturity. Concomitantly, severe impairments of T cell-mediated immune responses were noted. BB rats poorly rejected allografts across both major and minor histocompatibility barriers, and BB splenic or peripheral blood lymphocytes had markedly defective proliferative responses to mitogens and to allogeneic cells in MLC. Irradiated and nonirradiated BB spleen cells did not inhibit WF mitogenic or MLC responses, which suggests that the T cell defect in BB rats is not solely due to increased suppressor activity. Because irradiated WF cells and Con A supernatants did not restore BB proliferative responses, and BB lymphocytes were able to produce IL-2 normally, a reduced ability of BB lymphocytes to respond to helper factors such as IL-2 is suggested. In contrast to T lymphocytes from spleen or peripheral blood, BB thymocytes responded as well as did WF thymocytes to Con A or Con A supernatants. Percentages of T lymphocyte subsets and histology of BB thymuses were also normal when compared to WF thymuses. However, spleens and lymph nodes from BB rats were severely depleted of T lymphocytes, and thymocytotoxic autoantibodies were detected in many BB rat sera. The above findings indicate that BB rats have T lymphocyte immunoincompetence, which appears to be a post-thymic or peripherally acquired maturational defect.     

Cell-mediated lympholysis by fetal and neonatal lymphocytes in sheep and man

The occurrence of cell-mediated lympholysis (CML) was studied in the fetal lamb at 70 to 138 days of gestation and in the human fetus at 12 to 23 weeks of gestation, and also at the time of full-term birth in both species. The fetal lymphocytes were sensitized in mixed-lymphocyte culture (MLC) to prepare effector cells for CML. Concanavalin A-induced cells were used as target cells. No fetal capacity for CML was demonstrable during those periods of intrauterine life studied, despite the occurrence of clear MLC responses. At the time of full-term birth. CML by lamb lymphocytes was on average only one-seventh of the adult response. In man, neonatal lymphocytes may occasionally show a CML of the same magnitude as the adult lymphocytes, although on average the neonatal CML capacity was about half that in the adult.     

Human suppressor lymphocytes: I. Induction and characterization

Human peripheral blood lymphocytes were induced to become suppressor cells by exposure to Concanavalin A or Phytohemagglutinin. These cells were able to suppress the cytotoxic responses but not the proliferative responses of alloantigen activated human lymphocytes. Suppressor cells were non-specific in their activity, suppressing both allogeneic and syngeneic responders. They were labile in short-term tissue culture and required a proliferative phase for full induction of suppressor activity. Removal of macrophages prior to mitogen exposure resulted in a small loss of suppressor activity. Suppression was not the result of too much help since enhancement could not be demonstrated upon the addition of small numbers of suppressor cells to MLC.     

Soluble suppressor activity of concanavalin A-activated spleen cells on B-lymphocyte colony formation in vitro.

Supernates from concanavalin A (Con A)-activated mouse spleen cell cultures suppress the formation of B-lymphocyte colonies (BLC) in soft agar culture by 30 to 95%. Con A-induced BLC suppressive culture supernates can be heated at 80 °C for 1 hr without losing activity. The BLC suppressive activity is eliminated totally by trypsin treatment and partly by treatment with β-galactosidase. Activity is unaffected by treatment with DNAse, RNAse, and α-glucosidase. By ultrafiltration the BLC suppressive factor(s) was shown to have a molecular weight greater than 300,000. These data suggest that BLC suppression is mediated by a protein-carbohydrate complex. BLC suppression was obtained when normal spleen cells were preincubated in Con A-activated supernates for only 1 hr at 37 °C. BLC suppressor activity was absent in the supernatant fluid of Con A exposed anti-θ-treated spleen cells, nonadherent spleen cells, extensively washed spleen cells, and spleen cells from nude (athymic) mice suggesting that cells responsible for Con A-induced BLC suppression are adherent, fragile cells of the T lineage. Con A-activated spleen cell supernates do not suppress colony formation in soft agar by normal mouse granulocyte-macrophage precursors, by plasmacytoma cells, T-lymphoma cells, or by carcinoma cells. However, colony formation by Abelson's murine leukemia virus transformed B-lymphoma cells was suppressed by 95% suggesting a relationship between this immature B-lymphoma line and B-lymphocyte colony-forming cells. Con A-activated spleen cell supernates do not suppress lymphocyte activation in liquid culture by phytohemagglutinin, Con A, or lipopolysaccharide. Heat-treated supernates—which inhibited BLC development by 90–95%—did not suppress the plaque formation by spleen cells immunized in vivo or in vitro by sheep red blood cells.