Con A-induced Suppressor Cells Research Articles

Interference of the induction of suppressor cells by a streptococcal preparation, OK-432 through the blocking of suppressor inducer T-cells

The effect of OK-432 on suppressor inducer T cells in the generation of suppressor cells was investigated to determine its mechanism of action as an immunopotentiating agent. Suppressor cell activities induced by sera from patients with advanced cancer (stage III, IV or recurrence) were found to be as high as those induced by Con-A. Suppressor activity induced by Con-A or serum from cancer patients resided in CD8 + T cells, although CD4 + T-cells were required for the induction of suppressor cells. Significant increases in the CD4 +2H4 + T cell population after stimulation with either Con-A or sera from the advanced cancer patients were observed when compared with stimulation by normal serum. Stimulation with Con-A induced suppressor cells as well as a significant increase of CD4 +2H4 + T-cells. The presence of OK-432 during the generation of suppressor cells, however, significantly reduced the suppressor activity and apparently blocked the increase of CD4 +2H4 + T-cells. Thus, it is suggested that OK-432 may interfere with the induction of suppressor cells through the blocking of CD4 +2H4 + suppressor inducer T-cells.

Human Alveolar Macrophage Suppression of Lymphocyte Proliferation: Accessory Characteristics for the Generation and Functional Expression of Con A-induced Suppressor Cells

Human alveolar macrophages (AM) provide less accessory support for mitogen- and antigen-stimulated lymphocyte proliferation than do autologous blood-derived macrophages (BM). This is at least in part due to suppression mediated by AM, but the mechanism of such suppression is not understood. To determine whether AM-mediated suppression is related to AM interaction with suppressor lymphocytes (Ts), we examined the accessory cell function of both AM and BM for the generation and functional expression of Ts induced by concanavalin A (Con A). The data indicate that human AM are equivalent to BM for the generation of Con A-induced Ts, but AM mediate less suppression of Con A-induced Ts, once generated, than do BM. Addition of indomethacin did not increase lymphocyte proliferation when AM served as accessory cells. Supernatant fluids of Con A-exposed AM promoted greater proliferation of human T-lymphocytes and mouse thymocytes than did supernatant fluids from Con A-exposed BM. Interleukin-1 inhibitor activity was not detected in the supernatant fluids. These observations make it unlikely that soluble factors alone account for AM-mediated suppression. Thus, at least for the Con A-induced Ts system, AM do not mediate suppression either via better generation or greater functional expression of suppressor cells relative to BM. The paradoxically greater proliferation of lymphocytes stimulated by the supernatant fluids of Con A-exposed AM raises the possibility that suppression of Con A-stimulated lymphocyte proliferation observed when AM serve as the accessory cells in primary culture may be related to AM secretion of a molecule with IL-1-like activity.

Effect of B-activin on human T suppressor cells

The authors studied the influence of B-activin on the effect of human concanavalin A (con A)-induced T suppressor cells and also on the process of induction of T suppressor cells by con A and stimulation of proliferative activity of lymphocytes by phytohemagglutinin (PHA). Con A-induced suppression and the effect of B-activin on it were studied in a system in which the test cell culture and the culture for induction of suppressors were prepared simultaneously. Peripheral blood was obtained from blood donors for the experiments and during the preparation of the experiments, /sup 3/H-thymidine was added. The results of investigation of the influence of B-activin on the effect of con A-induced suppressors and also on the process of their induction are given. It is concluded that B-activin blocks the effect of con A-induced human suppressor cells but does not affect their induction, and B-activin does not affect proliferative activity of lymphocytes induced by PHA.

Inhibition of suppressor cell generation by mouse serum in culture

Normal C57BL/6 mouse spleen cells cultured for five days in the presence of fetal calf serum (FCS-induced suppressor generation culture) were shown, in mixing experiments, to suppress the primary humoral response of freshly explanted C57BL/6 spleen cells against sheep erythrocytes (SRBC). This suppressor cell generation was largely dependent on the FCS concentration in the suppressor generation culture. Ten or 5% FCS effectively supported the generation of suppressor cells, but 1% FCS only marginally supported it. When mouse serum (MS) from normal C57BL/6 mice was added to the suppressor generation culture, it inhibited the generation of the suppressor cells. Sera from allogenic mice and athymic nude mice were also effective. The effect of MS was resistant to heat treatment (56°C, 30 min). The inhibitory activity of MS was not dialyzable, and concentrated into the fraction which was not precipitated by 50% saturation of ammonium sulfate and which was eluted at a concentration of about 0.2 M NaCl from a DEAE-Sephadex A-50 column. The active fraction of MS also effectively inhibited the growth of Ehrlich tumor cells in culture. Further, MS also inhibited the generation of Con A-induced suppressor cells. The inhibition by MS of FCS-induced suppressor generation was eliminated by an interleukin 2-containing preparation.

ConA induced suppressor cells in scrapie-infected mice

Lymphocyte suspensions prepared from the spleens of mice (LACG/COM) clinically affected with scrapie (ME7) were cultured for 48 hrs in the presence of Concanavalin (ConA) in order to induce suppressor cells. These cells behaved exactly as similarly prepared cells from normal age-matched mice in suppressing the response of fresh normal splenic lymphocytes to phytohaemagglutinin (PHA).

α-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 α- 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 α- 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 α- 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.

Activity of concanavalin A-induced suppressor cells in human MLR: differential effects on primary MLR, secondary MLR, and memory cell precursors.

The relative sensitivity of the MLR responses of freshly isolated human lymphocytes to Con A-induced suppressor cells (SC) was compared to that of lymphocytes that had been primed previously in vitro. Fresh and primed cells were suppressed 72 and 17%, respectively, when cultured under similar conditions (p less than 0.0005). Titration of SC indicated that equivalent suppression in the two populations would require greater than a 50-fold excess of SC in the primed cells. Neither preincubation of the primed cells for 24 or 72 hr with the SC before restimulation, addition of fresh autologous cells to the primed cells, nor preincubation of the SC with fresh cells before addition to the primed cells increased suppression of primed cells. SC added at the beginning of a primary MLC were, however, very suppressive for both the primary response (67% suppression) and the subsequent secondary response (81% suppression). These data indicate that although the human primary MLR and the precursor of the memory cell are both sensitive to the suppressors induced by Con A, the memory cell itself possess an intrinsic resistance to such suppressors that is not related to simple kinetic phenomena nor to the loss in vitro of an intermediate regulatory cell. Cell depletion experiments suggest that resistance to nonspecific suppression may occur at the level of the helper cell.

Abnormal generation of concanavalin A-induced suppressor cell function in human autoimmune hemolytic anemia

Lymphocyte reactivity to the mitogens Con A, PHA, and PWM and the generation of Con A-induced suppressor cells were assessed in 12 patients with autoimmune hemolytic anemia (AIHA) and in 20 normal controls. With suboptimal doses of Con A and PHA most patients (9 and 8 out of 12, respectively) gave a response above normal control values. However, no significant difference was observed when optimal doses of mitogen were used. Mononuclear cells from most patients (8 out of 10) also showed a decreased capacity to generate Con A-induced suppressor cell activity. These results support the hypothesis that in AIHA exists an immunoregulatory defect which might have a pathogenic role in the development of this disease.

Effect of Irradiation on Concanavalin A-Induced Suppression of Mitogen Responses in Human Lymphocyte Cultures

Concanavalin a (Con A)-induced and Con A-inducible human suppressor lymphocytes were examined for their radiosensitivity (16 Gy). It was observed that X-ray exposure of Con A-induced suppressor cells abolished some of the suppressive activity. Exposure of lymphocyte preparations did not prevent the subsequent induction by Con A of suppressor cells capable of inhibiting mitogen responses of lymphocytes. The relative size of the Con A-inducible suppressor population in the blood was not changed in breast cancer patients after local radiation therapy.

Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10 −3 to 10 −9 M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.

Action of Con A-induced suppressor cells on a B hybridoma line

It has been shown that myeloma immunoglobulin production may be regulated by carrier-specific T H or T S cells, by idiotype reactive or alloreactive T cells, by antigen itself, or by immune complexes. However, regulation by nonspecific suppressor cells generated by CON A has not hitherto been shown in that system, possibly, it was proposed, due to the committed state of that cell line. We describe here a system in which immunoglobulin production by a hybridoma line is subject to regulation by CON A-activated spleen cells. This system allows us to show clearly that the suppression we observe here is not due to absorption of T H factor since no such factors are required for antibody production by the hybridoma line. It could still, however, be argued that the suppression observed in these experiments is mediated by macrophages activated by T-cell products. This system has the advantage of being easily dissected to allow further analysis of the mechanism of nonspecific suppression.

Spontaneous and ConA-Induced Suppressor Lymphocytes: A Comparative Study

Suppressor monocytes, Concanavalin A(ConA)-induced suppressor T cells, and short-lived suppressor lymphocytes have been described in humans. The present study was performed to evaluate spontaneous suppression in a test system similar to that employed for the demonstration of ConA-induced suppressor cells: Lymphocytes were either stimulated by ConA ( = induced suppressor cells) or immediately mitomycin-treated ( = spontaneous suppressor cells). Both preparations were tested for their capacity to suppress mitogen-induced proliferation of autologous cells. Depletion of monocytes or B lymphocytes did not affect spontaneous suppression. The active cells were short-lived in vitro. Therefore the net increase in suppressor activity generated by preculture with ConA is in part related to a loss of spontaneous inhibitory activity in the control cultures kept without mitogen. Spontaneous suppressor cell activity was comparable to that of ConA-induced suppressor cells.

T Cell Abnormalities in Atopic Dermatitis Patients: Imbalances in T Cell Subpopulations and Impaired Generation of Con A-induced Suppressor Cells

It has been hypothesized, but not yet proven, that T cell dysfunction is operative in the pathogenesis of atopic dermatitis. In order to test this hypothesis, we assessed different quantitative and functional parameters indicative of T cell reactivity in atopic dermatitis patients and, for control purposes, in age-matched normal individuals. We found that mononuclear cells from atopic dermatitis patients proliferated in response to various concentrations of the mitogens concanavalin A (Con A) and phytohemagglutinin as well as did mononuclear cells from controls. However, atopic dermatitis patients, regardless of their clinical characteristics, have a significant decrease in the relative percentages of circulating total E rosette forming cells. The reduction in total T cell percentages may be due, in part, to a highly significant reduction in the percentages of Fc-IgG receptor bearing T cells (T γ cells) that we found in atopic dermatitis patients at every level of clinical involvement. Fc-IgM receptor bearing T cell values (T μ cells) were found to be within the normal range. A defect related to the severity of the eczematous eruption was revealed when we investigated the capability of Con A-activated mononuclear cells from either atopics or normals to suppress mitogenic responses of autologous and allogeneic mononuclear cells. We found that cells from atopic dermatitis patients with wide-spread clinical involvement could not be educated to express suppressor signals as effectively as could cells from clinically inactive patients or normal healthy individuals. Based on these findings, we propose that a regulatory cell defect operating at the T cell level participates in the pathogenesis of various aspects of atopic dermatitis.

Con A-induced suppressor cells in man. I. Induction and characterization of suppressor cells

Con A-induced suppressor cells were studied in autologous co-cultures of normal human lymphocytes. Lymphocytes pretreated with 5 µg/ml, 20 µg/ml, or 50 µg/ml of Con A were cultured for 24, 48 and 72 hours. Subsequently, the whole mononuclear (MN) cell subpopulation or T-enriched, B-enriched, and monocyte-enriched fractions were added to freshly obtained lymphocytes, stimulated with 5 µg/ml of Con A and cultured for a further 96 hours. MN cells pretreated with 5 µg/ml of Con A did not significantly affect the DNA synthesis in co-cultures with unfractionated MN cells, while cells pretreated with 20 µg/ml or 50µg/ml for 48 and 72, but not for 24 hours, possessed the suppressive activity. The suppressor cells were found in T-, but not B-enriched subpopulations. Moreover, suppression was induced by T-enriched fraction from MN cells pretreated with 5 µg/ml of Con A, while unfractionated cells failed to exhibit inhibitory activity. The degree of T-cell-induced suppression was dose-dependent. Irradiation with 3000 R diminished the suppressive activity of cells derived from 48-hour cultures and abrogated the activity of cells from 72-hour cultures. The present data indirectly prove that Con A-induced suppressor cells are radiosensitive T lymphocytes. The observation that induction of suppressor cells by Con A is dose- and time-dependent provides the further insight into regulatory immunological mechanism in humans.

Con A-induced suppressor cells in man II. Role of monocytes and their interaction with soluble inhibitory factors

Monocyte dependence of Con A-induced suppression was studied in co-cultures of normal human lymphocytes. Con-A-pretreated (25 µg/ml for 48 hours) peripheral blood mononuclear (MN) cells inhibited the 3H-thymidine uptake by fresh autologous or allogeneic lymphocytes stimulated with Con A. Monocyte-depleted lymphocytes pretreated with 25 µ/ml of Con A failed to exhibit the suppressor properties. Supernatants of MN cell activated with 25 µ/ml Con A for 72 hours inhibited the mitogenic response of fresh allogeneic cells when added in proportion 1 : 1 to the culture medium. No inhibition was observed when supernatants of monocyte-depleted, Con A activated cultures were tested. The short preincubation with suppressive supernatants rendered blood adherent mononuclear cells suppressive for 3H-thymidine incorporation by autologous MN cells stimulated with Con A. These studies indicate that monocytes are essential for induction of suppressor cells in Con A stimulated cultures. The suppressor cells produce a soluble inhibitory factor(s) which has the property of binding to the surface of fresh monocytes. Such monocytes become responsible for suppression of mitogenic response of autologous lymphocytes.

Inhibition of concanavalin A-induced human lymphocyte mitogenic factor (Interleukin-2) production by suppressor T lymphocytes.

Abstract Supernatants of fresh human MNL stimulated with concanavalin A (Con A) generally contain little or no lymphocyte-derived mitogenic factor (LMF or Interleukin-2). However, preincubation of MNL in culture medium for 2 to 5 days before stimulation with Con A rendered them capable of producing increasing LMF. The production of LMF by reincubated MNL was inhibited by readdition of autologous fresh MNL or E-rosetting (T) lymphocytes. The producing cells were T lymphocytes; however, the production of LMF was monocyte dependent. LMF eluted from a Sephadex G150 column in 2 peaks at 12 to 27,000 and 50 to 70,000 daltons. Both species of LMF were mitogenic for mouse (C3H/HeJ) thymocytes and for human T and B cells. LMF production was not affected by x-irradiation (less than or equal to 2500 R) before preincubation; it was, however, abolished by the addition of 10(-7) M hydrocortisone during the preincubation period. LMF production by fresh MNL could be moderately enhanced by the addition of cimetidine, a drug that is known to interfere with histamine H2 receptors on suppressor T cells. Presumably, preincubation of MNL reduces the activity of Con A-induced suppressor cells. We conclude that human peripheral blood contains suppressor T cells that when activated by Con A regulate the production of LMF by other T cells in response to Con A.

Concanavalin A-induced suppressor cell activity in Hodgkin's disease

Peripheral blood mononuclear cells from 20 previously untreated adult patients with Hodgkin's disease were evaluated for the ability to induce suppressor cell activity following in vitro exposure to concanavalin A (Con A). Suppression of proliferative responses by autologous as well as normal allogeneic peripheral blood lymphocytes to the mitogen phytohemagglutinin (PHA) was assessed using suppressor/responder cell ratios of 2:1 and 1:1. The generation of Con A-induced suppressor cell activity (T-T suppression) was deficient in 35% of the patients ( 7 20 ) by the criteria employed. Peripheral blood leukocytes from 18% of patients ( 2 11 ) with localized disease (stages I and II), and 56% of patients ( 5 9 ) with widespread disease (stages III and IV) exhibited subnormal suppressor activity. There was no correlation between absolute lymphocyte count, absolute T-cell number, or proliferative response to PHA, and Con A-induced suppressor cell activity. This impaired ability to generate Con A-induced suppressor cells in Hodgkin's disease may reflect a functional T-cell deficit, or the suppressive influence of abnormal circulating monocytes.

Genetic control of effector cell characteristics: Con A-induced suppressor cells carry H-2 I region encoded determinants

Activation of splenic lymphocytes with Con A leads to the formation of suppressor cells capable of interfering with the activity of several polyclonal B-cell-activating substances. Thus, these suppressor cells, or their products, most probably act directly on B cells. Suppressor cells could be recovered from the effluent cell population of nylon wool columns, and they were absent from the spleens of athymic nude mice. Furthermore, they were absent from the thymus of normal as well as cortison-treated mice. Cortisone treatment did not abolish the formation of Con A-induced suppressor cells in the spleen. Treatment of activated suppressor cells with antisera specific for distinct products of the H-2 I region revealed that they carried I-J cell surface antigens. We conclude that the suppressor cells in our test system, which unlike other Con A-induced suppressor cell populations have a direct effect on B cells, had antigenic characteristics similar to those previously described for I-J carrying suppressor cells.

Specificity and Suppressor Function of Human T Cells Responsive to Autologous Non-T Cells

Abstract Human T cells proliferate in response to autologous non-T cells. This reaction, called the autologous MLR has been shown to have both immunologic specificity and memory. Our present study first demonstrates that specific secondary responses can be demonstrated in the autologous MLR. A BUdR and light technique was then used to ablate cells proliferating in response to either autologous or allogeneic non-T cells. The removal of T cells responsive to autologous non-T cells left the responsiveness to allogeneic T cells relatively intact, and conversely the removal of T cells responsive to allogeneic non-T cells left the responsiveness to autologous non-T cells relatively intact. Thus, the T cells responsive to autologous non-T cells and those responsive to allogeneic non-T cells are largely separate cell populations. Since the lymphocytes from patients with SLE fail to demonstrate a normal autologous MLR and also have a defect in the development of Con A-induced suppressor cells, we investigated whether the T cell population responsive in the autologous MLR was particularly enriched in cells capable of developing suppressor capacity. A BUdR and light treatment was used to remove T cells responsive to either autologous or allogeneic non-T cells. The remaining cells were then activated by Con A, and their subsequent suppressor activity was measured. Removal of T cells proliferating in response to autologous non-T cells produced significantly greater failure of suppressor cell development than did the removal of T cells responsive to allogeneic non-T cells. Thus, T cells capable of responding to autologous non-T cells are particularly enriched in cells capable of becoming suppressor cells.

Synthesis of IgM by Cells of NZB and SWR Mice and Their Crosses

Abstract Spontaneous and LPS-induced secretion of IgM by cultured spleen cells of NZB and SWR mice were measured after a 4-hr pulse with 3H-leucine. Similar experiments were done with fetal liver cells of NZB, CBA, BALB/c, and C57BL/6 mice. In short-term (4-hr) cultures there was marked hypersecretion of IgM by NZB cells relative to SWR cells. This abnormality was attributed to two factors: an increased number of IgM-containing cells and an increased secretion of IgM per cell. These two factors segregated as independent phenotypes in the progeny of the crosses. Liver cells from 18- to 19- day NZB fetuses matured into IgM hypersecretors in vitro in the absence of T cells. This phenomenon was uninfluenced by addition of thymocytes to cultured fetal liver cells. By contrast with the foregoing, NZB spleen cells maintained in vitro for 5 days, with or without LPS, made as much IgM during a 4-hr pulse with 3H-leucine as identically treated SWR cells. Con A-induced suppressor cells from NZB spleen cells inhibited LPS-stimulated IgM synthesis normally. There appear to be two populations of B cells in NZB mice: abnormal, spontaneously activated hypersecretors of IgM and cells that respond normally to LPS. The former population seems to arise early in life under the influence of two or three different genetic systems and functions independently of T cells.