In vitro immune response of human peripheral lymphocytes. II. Properties and functions of concanavalin A-induced suppressor T cells.

Con A-stimulation of human peripheral T lymphocytes induced both suppressor and helper T cells. ConA-generated suppressor T cells inhibited PWM-induced IgG and IgM production in PBL. Lower concentrations of Con A (0.5 micrograms/ml) or shorter incubation periods (6 to 24 hr) induced mainly helper T cells, while higher concentrations of Con A (10 micrograms/ml) or longer incubation periods (at least 48 hr) induced suppressor T cells. Con A-generated suppressor T cells were sensitive to mitomycin treatment and exerted their suppressor function on the early phase of differentiation and/or proliferation of B cells but not on the final differentiation of B cells to Ig-producing cells. The identity of the MHC was not required for the expression of suppressor function. Suppressor T cells competed with helper T cells in PWM-induced Ig-production of PBL. This experimental system can be applied to estimate the regulatory function of T cells in several disease states.     

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.     

Role of the autologous rosette-forming T cells in the concanavalin A-induced suppressor cell function

It was recently reported that the human autologous rosette-forming T cells (Tar cells) are devoid of Fc receptors for IgM and IgG but that they give rise in vitro to Tμ and Tγ cells and that these cells participate actively in feedback inhibition. We now investigated whether Tar cells participate in the concanavalin A-induced suppressor cell function, using two indicator systems, namely, mitogen- or alloantigen-induced DNA synthesis and mitogen-driven polyclonal immunoglobulin synthesis. When Tar cells were removed from peripheral blood T cells (T-Tar) there was no generation of suppression determined on both DNA synthesis and immunoglobulin production. When Tar cells were readded to T-Tar cells suppressor activity was restored. When purified Tar cells were activated by concanavalin A they showed the highest degree of suppression as compared to that exerted by total T cells or T-Tar cells. Tar cells lose their capacity to generate suppression when treated with mitomycin C before their activation; however, they are no longer sensitive to mitomycin C treatment once they have completed the period of activation. These results together with our previous findings showing that Tar cells give rise in vitro to Tμ and Tγ cells after their activation by Con-A suggest that Tar cells participate in the Con-A-induced suppressor cell function as precursors of the suppressor effector T cells.     

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Summary Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.     

An in vitro system for the generation of suppressor cells and the requirement for B cells in their induction

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.     

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.     

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.     

Con A-inducible suppression of MLC: evidence for mediation by the TH2 + T cell subset in man.

Human peripheral lymphoid cells pretreated with Concanavalin A for 48 hr can markedly suppress the proliferative response of untreated autologous lymphoid cells in MLC. Isolation studies with Sephadex G-200 anti-F(ab')2 affinity chromatography, nylon adherence, and E rosetting indicate that the Con A-induced suppressor cell is a T cell. Further fractionation into TH2+ and TH2- cell subsets with an equine-anti TH2 serum show that both subsets can be activated by Con A to an equivalent degree. After activation only the TH2+ subset can suppress autologous responder cells in MLC. The TH2- subset, which comprises 80% of peripheral human T cells, although induced by Con A to proliferate, cannot itself suppress the MLC response. Nevertheless, the TH2- subset can be shown to modulate the generation of suppressor TH2+ cells at 24 hr but not at 48 hr. These studies support the notion that the Con A-induced suppressor cell is confined to a distinct T cell subset in man and that T-T interactions are important in the overall expression of the immune response.     

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.     

The role of Ia-bearing cells in T-cell proliferative response to concanavalin A

Ia-bearing cells are required for the concanavalin A (Con A)-induced T-lymphocyte proliferative response. We attempted to determine how and when the Ia-bearing cells are required in this response. As Ia-bearing cells, mitomycin C-treated nylon wool- and plastic dish-adherent cells (AACmc) were used. AACmc didn't lose their restoring capacity on treatment with anti-Thy-1, anti-Ig, but they did with anti-Ia. Using a Marbrook chamber system, it was shown that cellular contact between T cells and Ia-bearing cells is necessary for restoring the response. Results with AACmc addition after various times from Con A addition, or Ia-bearing cell elimination after various times from Con A addition indicate that the Ia-bearing cells are required for about 12 hr from the start of incubation with Con A. And it was shown that Con A-pretreated AACmc could stimulate Ia-depleted T cells, and allogenic AACmc could replace syngenic ones.     

Further characterization of mitogen-induced autorosette-forming cells: correlation with T-cell subsets and with lymphocyte proliferative responses to mitogens

Spontaneous autologous rosette-forming cells (ARFC), which form rosettes with autologous erythrocytes, have been of interest as a subset of thymus-derived lymphocytes (T cells). An association of these cells with concanavalin A (Con A)-induced ARFC has been suggested. Furthermore, the Con A-induced ARFC have been shown to be a suppressor T-cell subset in the Con A-generated suppressor system. We have previously reported the induction of ARFC from T cells by several T-cell mitogens such as phytohemagglutinin-P (PHA) and allogeneic non-T cells other than Con A. In the present report, we further characterized the mitogen-induced ARFC and have extended the study to patients with systemic lupus erythematosus (SLE). We have found that ARFC are also inducible from peripheral blood T cells by pokeweed mitogen (PWM). Studies of T-cell surface markers on the ARFC using OKT monoclonal antibodies confirmed the induction of ARFC from both OKT4- and OKT8-reactive T cells by either Con A, PHA, or PWM stimulation. However, OKT4-reactive T cells were the major cellular source of the ARFC induced by all of the mitogens. In studies of SLE patients, proportions of both Con A- and PWM-induced ARFC were found to be significantly low in PBL of SLE patients treated with moderate or large doses of prednisone, with or without concomitant immunosuppressants, but not in SLE patients without such treatment. Proportional analysis of the T cells and their subsets suggested association of these alterations in the mitogen-induced ARFC with the OKT4-reactive T cells, since a significant decrease in the OKT4-reactive T-cell subset was demonstrated in the PBL of these patients. Proportions of PHA-induced ARFC, however, were not significantly different between SLE patients and healthy adults. Moreover, positive correlations of the mitogen-induced ARFC with lymphocyte proliferative responses to each mitogen were established in both SLE patients and healthy adults. These results further support our previous observation that suggest the receptors for autologous erythrocytes are enhanced or reexpressed on those T cells which are highly activated by mitogens.     

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.     

The proliferative response of rat T cells to calcium ionophores increases with age

Splenocytes and T cells from both old and young rats proliferate to A23187 and ionomycin, and this response increases 3- to 10-fold in aged animals. Augmented responsiveness to ionomycin occurs in the absence of any defect in Con A-induced proliferation of T lymphocytes of aged rats and is dependent upon the addition of thiol compounds to the tissue culture medium. Augmented proliferative responses to ionomycin precede the significant but much smaller decline (30 to 40%) in Con A-induced proliferative responsiveness of splenocytes, which is evident only when rats reach 24 months of age. Heightened proliferation to calcium ionophores is not caused by a greater ability of T lymphocytes from aged rats to increase [Ca2+]i in response to ionomycin. The increased proliferative response of lymphocytes from aged rats to ionomycin occurs in the absence of detectable amounts of secreted IL 2 or IL 4. The ionophore response is a much more sensitive biomarker of age than the decline in Con A-induced proliferative responses of lymphocytes and identifies an activity of T lymphocytes that increases rather than decreases during the aging process.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

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.     

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.     

Murine anti-swine T4 and T8 monoclonal antibodies: distribution and effects on proliferative and cytotoxic T cells

Two murine monoclonal antibodies reactive with porcine nylon wool-nonadherent T cells were examined by flow microfluorometry analysis for cellular distribution, and by functional analysis for their effect on MLR and CTL assays. The two determinants recognized, called PT4 and PT8, were shown to be expressed on almost mutually exclusive cell populations, with only 6 to 15% of T cells expressing both antigens. Among the 15% of nylon wool-nonadherent cells that did not express either PT4 or PT8, 5% were surface Ig+. In functional assays, cells that proliferated to PHA, hen egg lysozyme, and alloantigens were blocked by PT4 and were eliminated by PT4 and complement. PT4 and complement had no effect on CTL effectors or precursors. Reciprocally, PT8 blocked and PT8 and complement eliminated CTL effectors and precursors, but had no effect on proliferative responses. These results indicate that PT4 and PT8 define the porcine helper and cytotoxic T cell subsets, respectively.     

In vitro immune response of human peripheral lymphocytes. VI. Distribution and characterization of precursors for PHA- and protein A-induced colony-forming B cells

B cells from peripheral blood or cord blood formed colonies by stimulation with either PHA or protein A. On the other hand, tonsillar B cells did not form protein A-induced colonies, although PHA-induced colony formation was comparable to that observed in peripheral B cells. Lack of protein A-induced colony formation in tonsillar B cells was not due to the defect of helper T cells in preculture or to the presence of suppressor cells but was due to the absence of precursors for colony formation. The result showed that PHA- and protein A-induced colony-forming cells belonged to distinct subsets of B cells. Depletion of mu-bearing cells from peripheral B cells abrogated both PHA- and protein A-induced colony formation. Depletion of delta-bearing cells did not affect PHA- and protein A-induced colony formation and the population enriched with delta-bearing cells also showed colony formation. Depletion of complement receptor (CR)-positive cells removed precursors for both PHA- and protein A-induced colony formation. These results showed that precursor cells for PHA- and protein A-induced colony formation were IgM+, IgD+ and CR+ or IgM+, IgD- and CR+.     

Immunopotentiating effects of amphotericin B. II. Enhanced in vitro proliferative responses of murine lymphocytes.

Enhanced in vitro proliferative responses to DNBSO3 were seen in lymph node cells and spleen cells after in vivo sensitization of mice with DNFB plus AmB compared with mice primed with DNFB alone. The T cell proliferation in the nylon column nonadherent fraction for both groups was highly similar, and the enhanced lymph node cell proliferation with AmB was demonstrated to be in the nylon adherent population consisting of both T and B cells. These and earlier studies of immunopotentiation by AmB are consistent with a mechanism that depends on selective interaction of the polyene with a subset of T cells and a resultant impairment of the normally induced suppressor regulation that limits the magnitude and duration of immune responses.     

Characterization of two different Ly-1+ T cell populations that mediate delayed-type hypersensitivity

This paper describes two functionally different T cell populations that mediate delayed-type hypersensitivity (DTH) reactions in contact-sensitized mice. Both of these T cells are Ly-1+, Qa-2-, and Vicia villosa lectin nonadherent. One of these T cell subpopulations is responsible for the classical 24- to 48-hr component of DTH reactions, is induced 3 to 4 days after immunization, is H-2 restricted, is sensitive to irradiation and to antigen-specific T cell-derived suppressor factors, and is found in nylon wool-nonadherent as well as nylon wool-adherent populations. In contrast, the T cell population that is responsible, via an antigen-specific T cell factor, for a recently described early component of DTH, which is an obligatory initial step for expression of DTH, is induced within 24 hr after immunization, requires much less antigen for immunization, is not H-2 restricted, is not sensitive to irradiation nor to T suppressor factors, and is found exclusively in the nylon wool-nonadherent fraction. These results support a new formulation of DTH. According to this formulation, Ly-1+ T cells produce an antigen-specific, tissue-sensitizing, mast cell-activating factor, and via this factor induce the early component of DTH, which is an obligatory first step in which local antigen challenge induces increased local vascular permeability. This required opening of gaps between endothelial cells is due to T cell factor-dependent release of the vasoactive amine serotonin from cells such as mast cells. This first step allows the second, H-2-restricted, Ly-1+ T cell population to enter the reaction site, and to then be triggered by antigen to release lymphokines that attract the subsequent influx of blood-borne, bone marrow-derived leukocytes to constitute the classical delayed-in-time component of DTH reactions.