Amplification of suppressor inducer pathway with monoclonal antibody, anti-2H4, identifying a novel epitope of the common leukocyte antigen/T200 antigen

The 2H4 antigen, comprised of a 200/220-kDa glycoprotein of the leukocyte common antigen (LCA) family, is expressed on a suppressor inducer, but not a helper inducer subset of T4 cells. Earlier studies have demonstrated that the T4+2H4+ subset of cells maximally responded to the AMLR and this molecule has an important role in generated suppressor signals in AMLR/Con A-activated T cell systems. In the present study, we examined the effect of a series of monoclonal antibodies including anti-2H4 antibody on the initial activation of T4 cells in response to self-Ia antigens. We found that the addition of anti-2H4 antibody resulted in an augmentation of the proliferative response of T4 cells in AMLR, whereas other antibodies reactive with LCA/T200 antigens lacked this ability. Furthermore, anti-2H4 antibody enhanced both IL-2 production and IL-2R expression in this AMLR system. This enhancing effect was inhibited by anti-T3 antibody. Moreover, the suppressor inducer function of AMLR T4 cells was enhanced with anti-2H4 antibody by increasing the number of 2H4+ cells with high antigen density. Taken together, these results suggest that the 2H4 antigen may serve as an accessory structure for enhancing the activation of the T4+2H4+ suppressor inducer subset at initiation of cell triggering.     

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.     

The isolation and characterization of the human helper inducer T cell subset

Monoclonal antibody anti-4B4 was produced by fusing NS1 myeloma with spleen cells of a mouse immunized with Saguinus oedipus lymphocyte. This anti-4B4 antibody defines a 135-KD cell surface protein that is widely distributed throughout the hematopoietic system. More importantly, anti-4B4 is reactive with functionally unique human T cell subsets. Anti-4B4 antibody was reactive with approximately 41% of unfractionated T cells, 41% of T4+ inducer cells, and approximately 43% of T8+ cytotoxic/suppressor population. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+4B4+ subset proliferates relatively poorly upon stimulation with Con A and autologous cell antigens (AMLR) but well on exposure to soluble antigens, and it provides a good helper signal for PWM-induced Ig synthesis. The T4+4B4- subset, in contrast, proliferates well to Con A stimulation and autologous cell antigen (AMLR) but relatively poorly to soluble antigen stimulation, and provides little help to B cells for PWM-induced Ig synthesis. The T4+4B4- subset is largely 2H4+ and functions as the inducer of the T8+ suppressor cells. Thus, the present results suggest that one can divide the human T4 population into two major subsets that are phenotypically and functionally distinct, the human helper inducer subset (T4+4B4+/H.I.) and its reciprocal population defined by anti-2H4, the suppressor inducer subset (T4+2H4+/S.I.).     

The isolation and characterization of the human suppressor inducer T cell subset

Immunization of mice with lower primate lymphoid cells has provided a useful strategy for raising monoclonal antibodies against functionally important surface determinants on human T lymphocytes. We have developed a monoclonal antibody, anti-2H4, which defines functionally unique human T cell subsets. This anti-2H4 antibody was reactive with approximately 42% of unfractionated T cells, 41% of T4+ inducer cells, and was reactive with approximately 54% of T8+ cytotoxic/suppressor population. Anti-2H4 was not reactive with human thymocytes, but reacted with subsets of peripheral blood B cells and null cells. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+2H4+ subset proliferate well to concanavalin A (Con A) stimulation, but poorly to soluble antigen stimulation, and provides poor help to B cells for PWM-induced Ig synthesis. The T4+2H4- subset, in contrast, proliferates poorly upon stimulation with Con A, but well on exposure to soluble antigen, and provides a good helper signal for PWM-induced Ig synthesis. What is, perhaps, most important, the T4+2H4+ subset functions as the inducer of the T8+ suppressor cells. Previous attempts to define the latter subset of cells has relied heavily on the use of specific autoantibodies present in the sera of patients with juvenile rheumatoid arthritis (JRA) and systemic lupus erythematosus (SLE). The present results suggest that anti-2H4 antibody defines the human suppressor induced subset of lymphocyte previously described as T4+JRA+. Last, the results reemphasize the previously documented remarkable structural conservation of certain T cell-specific determinants on lymphocytes of phylogenetically distant primates.     

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.     

Activated suppressor cell dysfunction in progressive multiple sclerosis

Concanavalin A (Con A)-induced suppressor activity has previously been shown to be reduced in multiple sclerosis (MS) patients with active clinical disease. In this study, we demonstrate that OKT3, as well as Con A induced suppressor activity mediated by unfractionated peripheral blood mononuclear cells is reduced in patients with the progressive form of MS. By performing reconstitution experiments involving E+, T4+, or T8+ cells derived from either MS patients or controls, and normal allogeneic macrophages or E- cells, we sought to define the cellular basis for this suppressor defect. In both MS and control groups, E+ cells were required to obtain measurable levels of suppression. Suppressor levels induced by Con A-activated cultures containing E+ cells from MS patients were lower than those induced by those containing control donor E+ cells. Suppression mediated by T8+ cells from MS patients was also lower than for controls. In the control group, suppression mediated by T8+ cells exceeded that mediated by T4+ cells; such differences were not apparent in the MS group. These results suggest that although Con A-induced suppression can be mediated by a number of T and non-T cell subsets, the functional suppressor defect measured in the MS population does involve the T8+ cell subset.     

Abnormal activation and loss of suppressor T cells in the spontaneously hypertensive rat.

Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.     

Role of prostaglandin E2 in the induction of nonspecific T lymphocyte suppressor activity

Activated human monocytes and concanavalin A (Con A)-activated T lymphocytes are known to suppress T and B lymphocyte proliferation and B cell maturation into immunoglobulin-producing cells. We have now shown that monocyte suppressive activity is predominantly mediated through release of prostaglandin E2 (PGE2), which is active only in the presence of a "short-lived," radiosensitive T lymphocyte subset. PGE2, at high concentration, can activate T suppressor lymphocytes (TS), which display the same characteristics as Con A-activated TS lymphocytes. Moreover, Con A activation of TS lymphocytes was obtained only in the presence of PGE2, as specific anti-PGE2 antiserum or indomethacin prevented TS activation; this suggested a double signal as a prerequisite for activation of the nonspecific TS cell subset. We propose that TS lymphocytes modified by Con A become sensitive to small amounts of PGE2 produced by monocytes that must be present during the Con A-stimulated activation phase of suppressive cells.     

The 2H4 molecule but not the T3-receptor complex is involved in suppressor inducer signals in the AMLR system

It is suggested that autologous mixed lymphocyte reaction (AMLR) may play an important role in generating suppressor inducer signals and in down-regulating the immune response following self-major histocompatibility recognition. In the present study, monoclonal antibodies directed at cell surface structures on T4+ cells activated in AMLR were used to define the molecules important in the generation of the suppressor inducer signal. The density of a 200/220-kDa structure, termed 2H4, increased on T4 cells during activation in AMLR and furthermore a strong correlation was observed between the generated suppressor inducer activity of such cells and the density of the 2H4 antigen. More importantly, we showed that treatment of AMLR activated T4 cells with anti-2H4 but not anti-T3 or T4 antibody abolished the suppressor inducer function of these cells. These results suggest that the 2H4 molecule but not the T3-receptor complex plays an important role in generating suppressor inducer signals in the AMLR system.     

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.     

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.     

Deficiencies in suppressor T cell activity seen in patients with active systemic lupus erythematosus are due to the dilution of normally functioning suppressor T cells by nonsuppressor T cells

Concanavalin A (Con A)-activated T lymphocytes from patients with active, but not inactive, systemic lupus erythematosus (SLE) failed to express normal suppressor activity, regardless of the phenotype of CD4+ or CD8+. Con A-activated CD4+ or CD8+ T lymphocytes from the SLE patients and from normal controls were further separated into two populations, using the autologous erythrocyte rosette technique. One population very rich in cells capable of forming rosettes with autologous erythrocytes from the active patients showed the same degree of suppressor activity, as did that from normal controls; the CD4+ or CD8+ population poor in autorosetting cells derived from Con A-activated T lymphocytes from both the controls and patients did not express suppressor activity. Moreover, when autorosetting T cells from the active patients and nonrosetting cells from the same patients were mixed at a normal ratio (4:6), normal suppressor activity could be restored. It was notable that the frequency of autorosette-forming cells was markedly reduced in the Con A-activated T lymphocytes from the active, but not inactive, SLE patients, regardless of the phenotype of CD4+ or CD8+. These findings indicate the presence of a normally functioning suppressor T cell population in patients with active SLE. It seems that the lack of suppressor T cell function in patients with active SLE is due to the dilution of a few normal suppressor T cells by large numbers of nonsuppressor T lymphocytes.     

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.     

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.     

CD11 molecule defines two types of suppressor cells within the T8+ population

It has been shown that T8+ cells are comprised of functionally heterogeneous subpopulations such as suppressor, cytotoxic, and NK cells. In this report, we attempted to delineate the functional heterogeneity of T8 cells defined by anti-CD11 antibody (anti-Mol). Although allospecific cytotoxic activity was restricted to the T8+Mol- subset, suppression of PWM IgG synthesis could be elicited in both the T8+Mol+ and the T8+Mol- subset of cells. However, the mechanism of suppression was different in these two subsets. Suppression by the T8+Mol- subset of cells required the interaction with the T4+2H4+ suppressor inducer cells, whereas the T8+Mol+ subset of cells could suppress in the absence of the suppressor inducer cells. Moreover, recombinant interleukin-2 alone could augment this suppression by the T8+Mol+ subset, but did not induce suppression by the T8+Mol- subset. In contrast, NK and LAK activity was exclusively found in the T8+Mol+ subset of cells but not in the T8+Mol- subset of cells. These results suggest that the CD11 molecule is useful for distinguishing novel subsets of T8 cells.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

The suppressive effect of a supernate from concanavalin A-activated human lymphocytes: effects of concanavalin A-activated lymphocytes and their supernates on cytotoxic and mixed lymphocyte reactions.

The effects of Concanavalin A-treated human peripheral blood lymphocytes and their supernatants were evaluated on the MLC reaction and on the generation of cytotoxic lymphocytes assessed by cell-mediated lympholysis (CML). Experiments were performed with both allogeneic and xenogenic sensitization. It was found that Con A-activated cells suppressed the MLC and CML reactions in allogeneic and xenogeneic systems. On the other hand, the SIRS-like supernate was able to suppress the MLC reaction and blastogenesis, but had no suppressive effect on the generation of cytotoxic lymphocytes. We found no difference in the magnitude of suppression, whether or not Con A-activated lymphocytes were syngeneic to the responder cells. This finding suggests that there is no requirement for allogeneic restriction in the interaction between suppressor and suppressed cells, and demonstrates a soluble human suppressor substance capable of suppressing some cell-mediated reactions.     

Characterization of a soluble suppressor of human B cell function produced by a continuous human suppressor T cell line. II. Evidence for suppression through a direct action of CTC-SISS-B on human B cells

CTC-SISS-B is an antigen-nonspecific suppressive lymphokine elaborated by an interleukin 2-dependent suppressor T cell line that produces noncytotoxic inhibition of human B cell but not T cell function. Like SISS-B, a soluble suppressive lymphokine present in the supernatants of Con A-activated peripheral blood T cell cultures, CTC-SISS-B is of 60,000 to 90,000 m.w., and its action is blocked by the simple sugar L-rhamnose. CTC-SISS-B inhibits human B cell Ig production and proliferation through a direct interaction with human B cells rather than through indirect effects on immunoregulatory T cells or monocytes. CTC-SISS-B suppression occurs through inhibition of an early event(s) in B cell activation since proliferation and Ig production by established human B cell lines are not inhibited by this lymphokine. Despite sharing many biochemical and biologic properties, CTC-SISS-B and gamma-interferon appear to be distinct mediators.     

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.     

Generation of CD8 (T8) cytotoxic cells has a preferential requirement for CD4+2H4- inducer cells

CD8 (T8) cells are capable of both suppression and cytotoxicity. However, we have found that the activation of CD8 cytotoxic cells has a preferential requirement for a different CD4 (T4) subset from that previously reported for the activation of CD8 suppressor cells. We have recently characterized two monoclonal antibodies which subdivide CD4 cells into inducers of help for antibody production (CD4+ 4B4+) and inducers of CD8 mediated suppression (CD4+2H4+). We now report that CD4+4B4+2H4- cells also preferentially induce CD8-mediated cytotoxicity. Human peripheral blood T cells were fractionated into CD8, CD4, CD4+2H4+, and CD4+2H4- populations by both the adherence to antibody-coated plates and the fluorescence-activated cell sorter. The cells were cultured 6 days with irradiated allogeneic non-T cells and a cytotoxicity assay was then performed using cryopreserved non-T cells as targets. It was found that the combination of CD4+2H4- cells and CD8 cells resulted in greater cytotoxicity than either CD4 + CD8, or CD4+2H4+ + CD8. The combination of CD4+2H4+ cells with CD8 cells resulted in minimal cytotoxicity, which was similar to that generated by CD8 cells alone. These results were confirmed using anti-4B4 to positively select the reciprocal CD4 subset. Furthermore, the cytotoxicity induced by CD4+2H4- cells was alloantigen specific and Class I major histocompatibility complex restricted. As both CD4+2H4+ and CD4+2H4- cells proliferate equally well to alloantigen and produce similar levels of interleukin 2 (IL-2), it is likely that the generation of CD8 cytotoxic cells requires a signal in addition to IL-2.