Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of humoral immunity.

The abilities of concanavalin A (Con A) and phytohemagglutinin P (PHA) to selectively induce different T-cell activities affecting humoral immunity were evaluated. The mitogens were intravenously injected before, with, or after injection of sheep red blood cells (SRBC) into mice, and the 3 to 6-day plaque-forming cell (PFC) responses were assessed. Mitogenic treatment differentially influenced the resultant in vivo PFC responses to SRBC. The in vivo suppressive effects induced by Con A were shown to be temporary; only the Day 4 PFC response was inhibited. Con A given 3 hr before, with, or after the antigenic challenge enhanced the PFC response. In contrast, PHA given at all intervals inhibited both the 4- and 5-day PFC response. Neither mitogen appeared to affect the kinetics of the in vivo PFC response to SRBC. Both mitogens enhanced in vivo DNA synthesis by the splenic cells, and Con A appeared biphasic in its stimulation. Con A-induced effects on the humoral immune response were short-lived and transient, while PHA induced a longer-lasting effect on humoral immunity.     

Susceptibility of C5-deficient mice to listeriosis: modulation by Concanavalin A.

The susceptibility of normal (C5n) and C5 deficient (C5d) mice to Listeria monocytogenes infection and the effects of concanavalin A (Con A) on host resistance to Listeria were investigated. C5d mice were shown to be more susceptible to Listeria, but their morbidity was enhanced only with certain doses, 2 to 6 × 10⁴ viable Listeria. Listeria infection produced immune cells in both C5n and C5d mice as assessed in a passive transfer study, but the C5d immune cells significantly suppressed Listeria growth only when transferred into C5n mice. Con A altered the response to Listeria in both C5n and C5d mice. The growth of Listeria in the spleens of C5n and C5d mice, which were treated with Con A 1 day before infection, was clearly suppressed, and their mortality was reduced. Con A given 1 day after infection (Day +1) consistently enhanced the early (Days 1 to 3 post-infection) accumulation of Listeria in the spleens, and A/J (C5d) and CBA/J (C5n) mortality was increased but B10.D2 (C5d and C5n) mortality was reduced. Con A given 7 days before Listeria infection produced no differential effects on the early growth of Listeria in the spleens of C5n and C5d mice, but most C5n mice survived a lethal dose of Listeria and the C5d mice died. Since C5n and C5d mice differ in their resistance to Listeria, Con A (Day ?7), a selective T-cell mitogen, produced differential effects in C5n and C5d mice and Listeria is believed to induce predominantly a cell-mediated imune response, T cells and complement components independently or cooperatively appear to play a role in immunity to Listeria.     

A history of theories of acquired immunity

Alloimmune mouse spleen cells are capable of carrying out nonspecific cell-mediated cytolysis of syngeneic target cells when incubated in the presence of lectins such as Con A or PHA (lectin-dependent cell-mediated cytotoxicity). In the present study plant lectins from a variety of sources were examined for their ability to participate in alloimmune-LDCC. Reactivity was then compared to mitogenic activity and the ability to activate cytotoxic effector cells in vitro. Of the lectins tested only those reported to be T-cell mitogens were capable of participating in alloimmune-LDCC. Agglutinating but nonmitogenic lectins (e.g., WGA) or mitogens such as LPS or PWM failed to yield positive LDCC. Of the T-cell mitogens demonstrating positive reactivity in the alloimmune-LDCC assay, only a portion were able to generate cytolytic activity when incubated with normal spleen cells in vitro (Con A, GPA, lentil). Crude PHA, purified erythroagglutinin, or leukagglutinin failed to generate cytotoxic effector cells in this system even though these were mitogenic and demonstrated positive alloimmune-LDCC. The results suggest that T-cell mitogens interact with cytotoxic effector cells in a manner which specifically triggers cytolysis. The relationship of this interaction to other lymphocyte-lectin interactions is discussed.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Suppressor T cells activated by autologous B-cell derived lines inhibit blastogenic responses by peripheral lymphocytes to mitogens or autologous lymphoblastoid cell lines

EBV-transformed B-cell lines (LCL) suppressed peripheral lymphocyte responses to mitogens (PHA, PWM, and protein A). Cell separation experiments have shown that LCL cells activate autologous radiosensitive suppressor T cells that inhibit T-cell responses to mitogens (PHA and Con A) and to the autologous lymphoblastoid cell line itself. The significance of this autologous response and the way it may reflect on the effect of the suppressor T cells on the regulation of potential autoimmune responses is considered in relation to the in vivo phenomena observed in the course of acute mononucleosis.     

Immune interferon induction by T-cell mitogens involves different T-cell subpopulations.

Recent studies of mitogen-induced DNA synthesis in cells from various lymphoid tissues indicate that certain mitogens may selectively activate specific T-cell subpopulations. Staphylococcal enterotoxin A (SEA) optimally stimulates thymocytes and spleen cells (presumed T1 cells) and phytohemagglutinin P (PHA) optimally stimulates lymph node and spleen cells (presumed T2 cells); concanavalin A (Con A) stimulates cells from all these sources well. To determine further the specificity of mitogens for T-cell subpopulations, immune interferon (IIF) production was studied in spleen cells from mice treated in vivo with cortisone acetate (CA), preferentially a T1-cell inactivator, and antithymocyte serum (ATS), preferentially a T2-cell inactivator. The effect of administering gallic acid (3,4,5 trihydroxy-benzoic acid) (GA) in vivo was also studied, since in vitro studies showed GA to be capable of blocking IIF production. Results indicate that SEA induces IIF primarily in T1 cells, PHA induces IIF primarily in T2 cells, and Con A induces IIF in both T1 and T2 cells. Furthermore, GA was shown to mimic the ability of CA to inactivate T1 cells selectively in vivo. The data indicate that more than one type of T-cell subpopulation is capable of producing IIF.     

Nature of T-cells resident in spleens of thymectomized,lethally irradiated,bone marrow-reconstituted mice

Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.     

The effect of a high external temperature on cellular immunity]

The study was made of spleen cells proliferative response to mitogens PHA, Con A or alloantigens in relation to hyperthermia effects. Acute hyperthermia (rectal temperature 42 degrees) enhanced lymphocyte function, proliferative responses to allo-antigens, PHA and Con A increased. Thermal shock was associated with suppression of the spleen cell response. Mice suffering from hyperthermia for 20 min (43-44 degrees) daily during 10, 20 and 30 days showed suppressed T-cell immune response. Normal splenocyte proliferation recovered 40 days after hyperthermia induction.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Opposite modulation of lymph node cell and spleen cell responses to mitogens by muramyl dipeptide and its desmethyl analog

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), the minimal structure necessary for adjuvant activity of mycobacterial cell wall preparations, was evaluated as an immunostimulant in mice. MDP treatment, which increased carbon clearance and nonspecific resistance to lethal Klebsiella challenge, induced lymph node cellular hyperplasia (4-fold). In contrast, spleen and resident peritoneal cell recovery was comparable to controls. Lymph node cells (LNC) from MDP-treated mice had enhanced [³H]thymidine uptake in unstimulated (4-fold) and lipopolysaccharide (LPS) (5-fold)-, concanavalin A (Con A) (2-fold)-, and phytohemagglutinin (PHA) (1.5-fold)-stimulated cultures. In contrast, spleen cells exhibited depressed responses when stimulated with LPS (2-fold), Con A (2- to 5-fold), and PHA (3-fold). Depressed responses of spleen cells to mitogens were demonstrated over a range of mitogen concentrations. The desmethyl analog produced similar effects, although spleen cells were not as hyporeactive. Opposing modulations of the immune system by MDP resembles that reported after BCG infection, and correlates with increased nonspecific host resistance to microbial challenge.     

Adjuvant actions of polyclonal lymphocyte activators. II. Comparison and characterization of their actions in initiation and potentiation of immune responses to T-dependent and T-independent soluble antigens

Various polyclonal lymphocyte activators (PLA) such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide of Escherichia coli (LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and polyadenylic-polycytidylic acid (poly A:U) were compared in their effects on antibody response to T-dependent antigen (bovine gamma globulin (BGG) and dinitrophenylated (DNP)-BGG) and T-independent antigen (DNP-Ficoll) and on induction of tolerance to T-dependent antigen. All of these PLA acted more or less to trigger the initiation of the antibody-forming mechanism for deaggregated BGG (DBGG) or DNP-BGG through their actions on the carrier-specific T-cell function. All of these PLA tested also acted more or less to inhibit the induction of the carrier-specific T-cell tolerance to DBGG. Moreover, some of these PLA could act to augment antibody response to DNP-Ficoll. The adjuvant action of PLA in the response to DNP-Ficoll worked as well in athymic nu/nu mice as in nu/+ mice, whereas that in the response to DNP-BGG did not occur in athymic nu/nu mice. The order of the strength of the action of PLA to trigger the initiation of the whole immune response to DBGG, that to trigger the carrier-specific T-cell function to DNP-BGG, and that to inhibit the induction of the whole tolerance to DBGG was very similar to each other: i.e., CPS-K ? Con A > LPS, DS, poly A:U, PWM and PHA. By contrast, the order of the strength of the action to inhibit the induction of T-cell tolerance to DBGG was ≧ = LPS > Con A, PWM and poly A:U > DS and PHA, and that of the action to augment the antibody response to DNP-Ficoll was CPS-K > LPS > Con A. CPS-K was the most potent in all of these immunological activities. It was concluded that PLA act generally to stimulate the immune response at its initiation step in which T cells in the case of T-dependent antigen and B cells in the case of T-independent antigen play a predominant role, but that individual PLA share this adjuvant activity in different fashions.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

Insoluble immune complexes suppress mitogen-induced proliferation of human T lymphocytes bearing Fc gamma receptors.

Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (T_G) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β₂ microglobulin release.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

Enhancement of DNA synthesis and cAMP content of mouse thymocytes by mediator(s) derived from adherent cells.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.     

Effect of T-cell mitogens and T-lymphocyte deficiency on splenic colony formation

A study was made of the influence of T-cell mitogens (Con A and PHA) on the colony formation and differentiation of hemopoietic stem cells from normal and thymectomized mice, as well as of the relationship between the colony formation and the dose of injected thymocytes. The incubation of bone marrow cells with Con A and PHA was shown to inhibit the growth of spleen colonies. This inhibition is reduced by thymocytes within the dose intervals of 0.25-2.0 X 10(7) cells/mouse. Administration of these agents serially has led to the potentiation of inhibition effect and to the inability of thymocytes to reverse it. Con A and PHA exert no effect on the differentiation of stem cells. Incubation of the bone marrow cells from thymectomized mice with Con A is much less effective in the depression of colony formation, if compared with the treatment by intact bone marrow preparations. A reversed picture was observed using antiserum to mouse brain (RAMBS). It is proposed that regulation of stem cells is governed by different subpopulations of thymocytes.     

Thymopoietin-induced acquisition of responsiveness to T cell mitogens

A population of murine spleen cells, enriched by flotation in discontinuous bovine serum albumin gradients, was induced to differentiate in vitro by incubation with the purified thymic polypeptide hormone thymopoietin. These cells, normally unresponsive to both T and B cell mitogens, acquired the capacity to respond to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but remained unresponsive to the B cell mitogen lipopolysaccharide from Escherichia coli. The acquisition of responsiveness to mitogens was not impaired by treatment with anti-Thy-1 serum + complement before induction but was prevented by this treatment after induction; thus the cells acquiring the functional capacity to respond to T cell mitogens had also been induced to express the T cell alloantigen Thy-1. Like the expression of T cell alloantigens, the capacity to respond to Con A developed rapidly and reached its maximum within 6 hr. Responses to Con A always greatly exceeded those to PHA. Our data suggest that committed precursor cells, which we believe to be prothymocytes, are induced by thymopoietin to differentiate to cells with an antigenic phenotype and mitogen responsiveness similar to cortical thymocytes.     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.     

Bone marrow T cells. II. Thymic dependency

Mouse bone marrow contains cells capable of responding in vitro to the T cell mitogens PHA and Con A. These cellular responses are not demonstrable in the marrow of athymic nude mice (when compared with heterozygous littermates) and are depressed 47% in the marrow of neonatally thymectomized LAF₁ mice (when compared with sham-operated littermates). Therefore, the population of “bone marrow T cells” is thymus dependent.