Activated Suppressor Cells Research Articles

Bacterial lipopolysaccharide activates suppressor B lymphocytes.

Lipopolysaccharide (LPS) extracted from the outer cell wall of Gram-negative bacteria modulates the immune response in vivo and in vitro. Depending on the experimental conditions, it may enhance or inhibit the production of humoral antibody. The pathway by which LPS suppresses antibody production is examined in this study. C57BL/6 spleen cells incubated with LPS (greater than 10 micrograms/ml) not only fail to produce antibody to sheep erythrocytes in vitro but also, when transferred 24 hr after stimulation with LPS, inhibit antibody production in spleen cells that were not treated with LPS. This observation suggested that LPS activates suppressor cells. We have identified a suppressor B cell as mediator of LPS-induced immune suppression and determined its cell surface antigen phenotype as Ig+, Ia+, CR+, Ly-B-2+,PC1-.LPS does not induce suppressor macrophages or suppressor T cells, nor are macrophages or T cells required for the generation of suppressor B cells by LPS.

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.

Suppressor cell function in man: Evidence for altered sensitivity of responder cells with age

Concanavalin A (Con A)-activated suppressor cell function was evaluated in young and elderly adult human donors. Activated suppressor cells reduced the mitogenic response of autologous peripheral blood lymphocytes more markedly in elderly than in young adults (60.2 ± 4.8 vs 35.3 ± 8.9%, P < 0.05). As a measure of the actual amounts of suppressor factors released, we concurrently determined the effect of supernatants from the Con A-activated cells on the proliferative activity of a dividing cell line (L cells). The inhibitory effect of supernatants from activated cells was greater in young than in elderly adults (21.4 ± 3.4 vs 11.4 ± 3.1%, P < 0.05). These results suggest that cultured lymphocytes from elderly donors produce less suppressor factors than do cells from young donors but that the responder cells of the elderly are more susceptible to suppressor influence.

Suppressor cell function in multiple sclerosis: Correlation with clinical disease activity

Concanavalin A (Con A)-activated suppressor cell activity was determined in multiple sclerosis (MS) patients who had been assigned to one of three subgroups, those with active disease, those recovering from a flare-up, and those with stable disease. The level of suppression induced by the Con A-activated suppressor cells on the mitogenic response of autologous peripheral blood lymphocytes was reduced in patients with active disease (3 +/- 8%) compared with stable patients (30 +/- 8%), patients recovering from a flare-up (62 +/- 5%), and controls (40 +/- 5%). As a measure of the actual amounts of suppressor factors released, the effect of supernatants from the Con A-activated cells on the proliferative activity of a dividing cell line (L cells) was determined concurrently. The inhibitory effect of supernatants from activated cells was reduced in active and stable MS patients (7 +/- 3%) compared to controls (21 +/- 4%). Three of 4 with active MS showed mildly elevated immune complex levels as measured by the Raji cell technique; each of these patients had low suppressor activity. Levamisole (1 microgram per milliliter) failed to alter suppressor cell activity in our in vitro system.

Immunological unresponsiveness of self-recognizing B lymphocytes to the PBA property of a soluble T cell factor

Spleen cells cultured in the presence of Con A became activated to polyclonal anti-body synthesis. This effect was found to be mediated by a polyclonal factor released by the activated T cells. Such a factor, contrary to any other polyclonal B cell activator, so far tested, failed to induce resting B cells to synthetize antibodies capable of lysing autologous albumin coupled SRBC. The Unresponsiveness was found to be specific since Con A activated spleen cells were capable of lysing heterologous albumin and HGG coupled SRBC. Moreover, neither active suppressor cells, nor soluble suppressor factors seem to be involved in the lack of response to autologous antigens. It is concluded that the clones of B cells carrying the polyclonal receptor for the T factor and Ig receptors for self are eliminated or functionally inactivated. The implication of these findings for the mechanism of self-non-self discrimination are discussed in relation to the mechanism of immunocyte triggering.

Inhibition of a Primary DNA-Synthesis Response by Trinitrobenzosulfonate (TNBS)-Activated Suppressor Cells; Characterization of the Inhibitory Cells

Repeated intravenous injections of high doses of trinitrobenzosulfonate (TNBS) or dinitrobenzosulfonate (DNBS) activate suppressor cells which inhibit the in vivo activation of a primary DNA synthesis response against trinitroehlorobenzene (TNCB) and dinitrochlorobenzene (DNCB), respectively, almost completely and the delayed type hypersensitivity (DH) response only partially.When tested on the DNA-synthesis response, the suppressor cells show excellent specificity with little cross reactivity of TNBS (or DNBS) induced suppressor cells for DNCB- (or TNCB-) specific responses. TNBS- and DNBS-specific suppressor activity is found in spleen cells, mesenteric lymph nodes and peripheral lymph nodes. The activation of suppressor cells is resistant to the early effects of adult thymectomy (ATx), but sensitive to pretreatment with cyclophosphamide (CyP).The suppressor cells are not simply haptenated cells. They need several days for their activation and are inactivated by incubation for 30 minutes at 56°C or by 2,000 R irradiation.Attempts to obtain soluble suppressor factors by in vitro incubation or extraction of these suppressor cells failed.

Persistent Activity of Suppressor Cells in T Cell-Mediated Immune Response

Tolerance to dinitrochlorobenzene contact sensitivity induced i.v. injection of dinitrobenzenesulfonic acid in guinea pigs is a long-lasting phenomenon (up to 1 year). The tolerogen, however, was traceable in the circulation only up to 3 months after its application. In spite of that, tolerance was adoptively transferred by parabiosis 6 months after being induced. Moreover, active suppressor cells eliminated by cyclophosphamide treatment are able to regenerate in those adoptively tolerized animals. These results indicate that the tolerogenic injection stimulates precursors of suppressor cells to generate active suppressor cells and memory cells of suppression. The further formation of active suppressor cells from memory cells seems to be tolerogen independent, but the existence of specific stimulator cells for suppression may be considered. These cells may bind undetectable small amounts of tolerogen. The recovery of suppression might, however, be also due to recovery of suppressor cells which were temporarily inactivated but not destroyed by cyclophosphamide treatment.

Lack of functional immunoregulatory cells in a patient with mycosis fungoides and circulating Sézary cells.

Peripheral blood lymphocytes obtained at various intervals from normal individuals and from a patient with mycosis fungoides (MF) were cultured with phytohemagglutinin (PHA) for 3 days to activate suppressor cells. After being cultured, the PHA-treated cells were irradiated, washed, and then transferred to fresh medium with PHA. The PHA responsiveness of the cells from normal individuals was suppressed approximately 90% by autologous or normal allogeneic lymphocytes activated for 3 days with PHA, whereas the cells activated for 3 days with PHA from the patient with MF lacked the capacity to inhibit the mitogenic response of autologous or allogeneic lymphocytes. These data suggest that this patient lacked suppressor T-cells that have a specificity for helper T-cells.

Further characterization of the suppressor cells, activated by goat anti-Th-B antibody reagent and responsible for enhanced growth of sarcoma 180 in AKR mice

In our previous study, thymus cells were shown to be responsible for enhancing the growth of the allogeneic sarcoma 180 (S180) in AKR mice that had been injected with goat anti-Th-B antibody reagent (antiserum raised in goats against Balb/c myeloma MOPC 104E cells and purified). We suggested that the cells producing enhancement are suppressor T cells. We now show that the cells responsible for tumor enhancement are indeed T cells, since they carry the Thy-1 antigen on their surface. Treatment of the cells in vitro with anti-Thy-1 plus complement completely eliminates their ability to enhance tumor growth. The thymocytes responsible for tumor enhancement do not carry the Th-B determinant. Treating thymocytes in vitro with goat anti-Th-B antibody reagent plus complement does not abrogate their tumor-enhancing activity. This suggests that the suppressor T cells involved in tumor enhancement are generated by the interaction of anti-Th-B antibodies with precursor suppressor cells which do carry Th-B. Once generated, the active suppressor cells lose the Th-B antigen. This suggestion is supported by our finding that the thymic precursors of Con A-inducible suppressor cells bear Th-B, since they are killed by anti-Th-B plus complement, whereas active suppressor cells induced by Con A do not carry Th-B, since they are not killed by anti-Th-B plus complement. Neither splenic precursors of Con A-inducible suppressor cells nor the active suppressor cells thus induced carry Th-B since neither is killed by anti-Th-B plus complement. We have also found that there are apparently nonthymic suppressor cell precursors which can also be activated by anti-Th-B, since spleen cells from thymectomized mice bearing S180 and treated with anti-Th-B can transfer the tumor-enhancing effect. We conclude that precursors of suppressor cells carry the Th-B determinant. These precursors differentiate to active suppressor cells when stimulated by anti-Th-B antibodies. This process can take place either outside the thymus or in the thymus. Once differentiated, the mature suppressor cells no longer bear the Th-B marker and migrate from their sites of induction. Such cells can suppress immune mechanisms responsible for allogeneic tumor graft rejection and thus cause tumor enhancement.

696 IDENTIFICATION AND CHARACTERIZATION OF HUMAN SUPPRESSOR LYMPHOCYTES

The capacity of human peripheral blood lymphocytes (PBL) to suppress in vitro immune responses was investigated.PBL were incubated with Concanavalin A (Con A) (50 ug/ml) for 48 hours, washed in Hank's Balanced Salt Solution containing α-methyl-D-mannoside (5 mg/ml), and subsequently added at a 1:1 ratio to cultures of normal untreated lymphocytes. Con A treated cells suppressed the proliferative response of untreated lymphocytes to mitogens (phytohemagglutinin, Con A), antigens (tetanus toxoid) and alloantigens (one-way mixed lymphocyte culture), as measured by 3H-thymidine incorporation into DNA. Con A treated cells also suppressed pokeweed mitogen induced immunoglobulin production in vitro by normal lymphocytes, as measured by radio-immunoassay.Con A treated cells lost their suppressor activity following treatment with mitomycin C (50 ug/ml) or X-irradiation (> 500 RADS). It was shown that the Con A activated suppressor cell was a T cell which was nylon nonadherent and which bore surface receptors for histamine. Treatment of PBL or of T cells with antisera to B cell alloantigens (Ia like antisera) either before or after activation with Con A did not interfere with the suppressor activity of these cells.Assay of suppressor activity of Con A treated PBL could be helpful in the study of immunodeficiency diseases, graft-versushost disease, autoimmune diseases and atopic states.

Activation of Human B Lymphocytes

Abstract 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 preocess of activation of human B cells toward antibody synthesis, 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 than 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) of 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.

Circulating suppressor cells in man as a function of age

Concanavalin A (Con A) was used to activate suppressor cells derived from the peripheral blood of human donors. Activated suppressor cells reduced the mitogenic response of autologous peripheral blood lymphocytes (PBLs) more markedly in elderly than in young adults (72.2 ± 5.0 vs 41.8 ± 7.2%, P < 0.02). The baseline mitogenic response of PBLs from elderly adults was significantly lower than that of PBLs from normal young adults (9497 ± 2609 vs 28,068 ± 3973 cpm, P < 0.01). A group of young multiple sclerosis patients showed poor mitogenic responsiveness (11,017 ± 2679 cpm) and low suppressor activity (36.0 ± 3.0%) suggesting that suppressor influence need not depend on the activity of responder cells. Unconcentrated supernatants from Con A-activated cells did not suppress the mitogenic response of autologous lymphocytes but did suppress a human lymphoblast cell line (Raji).

Key antigenic determinants in regulation of the immune response.

The immune response to beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23)is characterized by a wave of early help followed by a wave of suppression to a subsequent in vitro challenge with galactosidase-fluorescein. A cyanogen bromide peptide of beta-galactosidase, CB2, mimics the suppression seen with the enzyme. It is time dependent, carrier specific, and anti-theta sensitive; however, this suppression is not preceded by a wave of help. It is possible that CB2 cannot stimulate helpers, and is only able to activate suppressor cells. These data indicate that one small region of an antigen, capable of activating suppressors, can nullify the positive effect induced in helper T cells reactive with other epitopes on beta-galactosidase. Key determinants on macromolecules may in this way be influential in regulating the immune response to the entire antigen molecule.

Immune Suppression with Supraoptimal Doses of Antigen in Contact Sensitivity

Abstract Immunologic suppression was induced in a mouse model of contact sensitization to DNFB by using supraoptimal doses of antigen. In these studies, in vivo measurement of ear swelling as an indication of immunologic responsiveness correlated well with measurement of in vitro antigen-induced cell proliferation. This unresponsiveness was specific, since supraoptimal doses of DNFB did not interfere with the development of contact sensitivity to another contactant, oxazolone. The decrease in responsiveness is a form of active suppression, as lymphoid cells from supraoptimally sensitized donors transferred suppression to normal recipients. Furthermore, pretreatment with cyclophosphamide (Cy) reversed the suppression seen in supraoptimally sensitized animals but had no effect on the optimal sensitization regimen. These results indicate that supraoptimal doses of contactants can activate suppressor cells and that precursors of these cells are sensitive to Cy. Such suppressors regenerate within 7 to 14 days after Cy treatment. The ability of Cy pretreatment to affect supraoptimal sensitization without affecting optimal sensitization confirms other reports indicating that the observed results of Cy treatment depend critically upon the dose of antigen used.

Con A induced suppressor cells: Suppression with I region incompatibility

Allogeneic Con A induced suppressor cells differing in the I and S region but not H-2 K or H-2 D regions were as efficient as syngeneic cells in suppressing the secondary IgM and IgG response to burro erythrocytes. Con A activated suppressor cells were not sensitive to anti-Ia serum and complement. However, if the spleen cell population was treated with anti-Ia serum and complement before stimulation with Con A, suppressor cells were not generated.

Control of mitogen-induced transformation: Characterization of a splenic suppressor cell and its mode of action

The present study demonstrates that mouse spleen cells contain a population of glass wool adherent T lymphocytes which exhibit the capacity to suppress non-glass adherent lymphocyte responses to mitogens. These suppressor cells are stimulated by both low and high doses of PHA 1 1 Abbreviations used: Act. D, actinomycin D; ATS, anti-thymocyte antisera; C′, guinea pig complement; con A, concanavalin A; Bu 2cAMP, dibutyryl cyclic 3′,5′-adenosine monophosphate; HBSS, Hank's basic salt solution; GAL, glass-adherent lymphocytes; NAL, non-glass adherent lymphocytes; PHA, phytohemagglutinin; Mit. C, mitomycin C; [ 3H] Tdr, [ 3H]thymidine; MEM, minimum essential medium; FCS, fetal calf serum. and high doses of con A. The suppressor cell effect is observed when UNA, but not RNA or protein synthesis, is studied. This glass-adherent suppressor cell population is characterized as being the primary DNA synthesizing cells during the early (0–8 hr) stages of culture. Suppression still occurs when the suppressor cells are treated with mitomycin C, actinomycin D or cycloheximide. This implies that new macromolecular synthesis may not be required for suppression to occur. Suppression is blocked by inhibiting synthesis of prostaglandin and is mimicked by Bu 2cAMP. This suggests that mitogen activated suppressor cells regulate T cell responses via production of prostaglandin which modulates the concentration of intracellular cyclic nucleotide levels.

In vivo suppression of delayed hypersensitivity: prolongation of desensitization in guinea pigs.

Administering moderate (milligram) amounts of antigen to a guinea pig immunized with thatntigen leads to a transient loss of all delayed hypersensitivity (DH) responses in that animal. In this study, we demonstrate that this "desentization" can be prolonged for 10 days by repeated injection of antigen. At this time, tolerance to the desensitizing antigen develops in both the humoral and cellular systems of the immune response and DH responsiveness to other antigens returns. Repeated cycles of sensitization and desensitization produce repeated episodes of generalized anergy. Neither cells nor serum from desensitized animals could be shown to exert a suppressor effect when transferred to immunized animals and the cells responded normally to antigen and mitogen in tissue culture. The best generalized depression of DH was seen in those animals producing the best DH before desensitization. The inability of antigen to react with tolerant cells to produce desensitization suggests that this phenomenon is an active rather than a passive one and may represent an exaggeration of a normal regulatory mechanism for DH triggered by a regimen of antigen administration that activates suppressor cells to produce a systemic effect.

Biological Expressions of Lymphocyte Activation

Abstract 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.

The Role of Suppressor Cells in Avian Allogeneic Tolerance: Implications for the Pathogenesis of Marek's Disease

Abstract Chicken embryos injected with pooled spleen cells from outbred embryos are tolerant to histocompatability antigens of the donor population. Tolerance can be shown by the failure of peripheral blood cells (PBC) from tolerant birds to produce graft versus host lesions on the chorioallantoic membrane of outbred chicken embryos. It is shown that PBC from tolerant chickens can reduce by up to 85% the GVH reactivity of PBC from normal chickens. These results are interpreted to indicate that the PBC of birds tolerant to allogeneic cells contain active suppressor cells that can reduce the activity of normal cells in their response to allogeneic stimulation. The implications of these results are discussed in relation to the pathogenesis of Marek's disease—a common herpes virus-induced lymphoid neoplasm of chickens. It is suggested that resistance to neoplasia in this disease occurs because of the establishment of a population of active suppressor cells that prevents a neoplastic lymphoid proliferation.