Antigenic properties of subsets of splenic T lymphocytes responding to lectins.

The aim of the present study was to characterize the antigenic properties of spleen cells responding in vitro to various mitogenic stimuli. Mouse spleen cells were treated with alloantibodies and C and then cultured with various concentrations of lectins. The exposure of spleen cells to various dilutions of either anti-theta serum and C or anti-Ly serum and C inhibited the response to optimal concentrations of PHA to a greater extent than the response to optimal concentrations of Con A. With the exception of the optimal lectin concentration, theta-antiserum and C had a stronger inhibitory effect on the response to Con A than to PHA. The exposure of spleen cells to H-2 antiserum and C inhibited their response to Con A to a greater extent than to PHA. The inhibitory activity of H-2 antiserum and C was inversely correlated with the dose of PHA used for stimulation. The lower the concentration of PHA used for stimulation the more effective was the inhibitory effect of the antiserum. The inhibitory effect of H-2 antiserum and C on the response to Con A was highest at optimal concentrations of the mitogen, and somewhat less pronounced above or below this concentration. The present study suggests that subsets of splenic T-cells that react to various concentrations of mitogens, differ in their theta- and H-2 alloantigenicity.     

Immunocompetence of Murine Placental Lymphocytes. In-vitro Response to Mitogens and to Allogeneic Cells1

Murine lymphoid cells from late (16-20 day) gestational placentae were studied for their ability to respond to mitogens and adult allogeneic cells in the mixed lymphocyte reaction (MLR) and the cell-mediated lympholysis test. Placental lymphocytes from Balb/c mice expressed a weak to moderate proliferative response to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen when compared to the mitogenic responses of adult spleen cells. The stimulatory effects of Con A and PHA were abrogated after depleting the T cells from the placental lymphocyte preparation. Lipopolysaccharide-induced reactivity was similar for both cell populations. In MLR Balb/c placental lymphocytes did not undergo significant DNA synthesis after in-vitro exposure to irradiated C57BL/6 spleen cells, nor were cytotoxic lymphocytes generated against H-2^b alloantigens. Lack of an MLR response was not associated with the presence of a suppressor lymphoid-cell population in the placenta. In marked contrast, placental trophoblastic cells were refractory in their responsiveness when cultured with either mitogens or allogeneic cells. These data show that although there is a selective deficiency in the placenta of cells reactive toward alloantigens, limited differentiation of lymphoid cells does occur which may offer immunologic protection to the fetal allograft during embryogenesis.     

Effects of hydrocortisone treatment and whole body irradiation on mouse lymphocyte stimulation in vitro

Whole body irradiation was found to reduce the number of nucleated cells in the thymus and the spleen of C57B1 mice to the same extent as hydrocortisone treatment. Equal numbers of thymus and spleen cells from either hydrocortisone-treated or irradiated mice and matched controls were cultured and stimulated with various mitogens. An increased stimulation of DNA synthesis by PHA, Con A and PWM was found in thymus and spleen cells after hydrocortisone treatment as well as after whole body irradiation. In contrast, the response to LPS, known as a typical B-cell mitogen, was not affected by any of the procedures.     

Tumour-induced suppressor macrophages in rats: differences in their suppressive effects on the Con A and PHA responses

Spleen cells obtained from ACI rats bearing a syngeneic hepatoma (9098) (TBR spleen cells) showed a strongly depressed mitogen responses to concanavalin A (Con A) and to phytohaemagglutinin-P (PHA) at various concentrations of the tested mitogens. The activity of suppressor cells in TBR spleens was demonstrated in mixtures with normal spleen cells where a marked depression of the mitogen response was observed. The properties of tumour-induced suppressor cells were adherent to plastic or nylon wool, phagocytic, and radioresistant (maybe macrophages). The Con A response of TBR spleen cells was more completely restored than was the PHA response after the removal of adherent or phagocytic cells. The suppression when TBR spleen cells (2,000 rad) were added to normal spleen cells at 0, 24, and 45 h after culture initiation was greater in the PHA response than in the Con A response. The PHA assay appeared to be more sensitive method than the Con A assay for the detection of suppressor cell activity in tumour bearing rats.     

Immunosuppressive effect of a trichothecene mycotoxin, Fusarenon-X in mice.

The in vitro treatment with Fusarenon-X, a mycotoxin produced by Fusarium nivale Fn 2B, depressed the mitogenic responses of mouse lymphocytes to the T-cell mitogens, phytohaemagglutinin (PHA) and concanavalin A (Con A), but to a lesser extent to B-cell mitogen, a bacterial lypopolysaccharide (LPS). The in vitro treatment of mice with Fusarenon-X also decreased the responsiveness of splenic lymphocytes to the T-cell mitogen, Con A. Administration of Fusarenon-X to BALB/c mice before immunization significantly reduced anti-DNP IgE and IgG1 responses, while the treatment of mice after immunization was less effective. The number of spleen cells of the treated mice was increased with a reduction of T lymphocytes and an increase of granulocytic elements. In vitro antibody responses of spleen cells from Fusarenon-X treated mice by pokeweed mitogen (PWM) or LPS were also suppressed. Reconstruction experiments using sIg positive and negative spleen cells from normal and Fusarenon-X treated mice showed that this inhibitory effect resided in the sIg negative cell population.     

Leopard frog (Rana pipiens) spleen lymphocyte responses to plant lectins. Kinetics and carbohydrate inhibition

Mitogenic effects of the plant lectins phytohemagglutinin (PHA) and concanavalin A (Con A) on leopard frog (Rana pipiens) spleen lymphocytes in vitro were examined. At 22 ± 1°C, maximum stimulation in response to PHA and Con A occurs on day 4 with 1 pg PHA and 5–10 μg Con A. Lymphocyte stimulation can be inhibited by adding N-acetyl-D-galactosamine (NADG) to PHA cultures or α-methyl-D-mannopyranoside (αMM) to cultures containing Con A at time zero. Saccharide inhibition is concentration dependent: maximum inhibition is obtained with 75 mM NADG or αMM. Similarities between the kinetics of amphibian and mammalian mitogen stimulated lymphocytes and carbohydrate inhibition of lymphocyte activation suggest that certain biochemical characteristics of mitogen stimulation and cell surface receptors for mitogens have been phylogenetically conserved since amphibians first evolved during the Devonian period approximately 350 million years ago.     

During frog ontogeny, PHA and Con A responsiveness of splenocytes precedes that of thymocytes

The in-vitro proliferation of splenocytes and thymocytes from Xenopus laevis-gilli (hybrid clone LG-15) to the T cell mitogens, concanavalin A (Con A) and phytohaemagglutinin-P (PHA), were examined at specific stages of larval development (stages 51-66 of Nieuwkoop & Faber, 1967) and at 2 months post-metamorphosis. The responses of splenic lymphocytes to each mitogen were significant at all stages with stimulation indices ranging from 1.9 to 50.5 and 2.6 to 45.5 for PHA and Con A, respectively. Stage-related differences in responses of splenocytes to both mitogens suggest two waves of emergence of proliferative activity during development, divided by periods of diminished responsiveness during the metamorphic crisis. In contrast to the responses observed with splenocytes, proliferation of thymocytes cultured with either mitogen was barely detectable, with stimulation indices ranging from 1.2 to 6.9 and 1.4 to 2.9 for PHA and Con A, respectively. These minimal responses were observed only when thymocytes were cultured at relatively high cell density (5 X 10(5) cells/ml); they were not improved by increased or decreased concentrations of mitogen or by increased concentrations of fetal calf serum (5 or 10%) in the medium. Co-culture of larval thymocytes with autologous splenocytes and each mitogen did not consistently increase thymocyte responses suggesting that the defect in thymocyte responsiveness is not due to lack of accessory cells. These findings suggest that if PHA- and Con A-reactive cells are present in the thymus, they are present in relatively low numbers at all stages of larval development. The pattern of early mitogen responsiveness in the spleen at a time when the thymus is unresponsive contrasts with that observed in mammalian development in which thymocytes become responsive to mitogens in fetal stages and mitogen responsiveness appears in the spleen only around the time of birth. The apparent inactivity of larval thymocytes may reflect a population of cells that can become tolerant to those neo-self-antigens that arise during and after metamorphosis. If so, the larval amphibian thymus may provide a model to study the early events of thymocyte 'education' and differentiation in a broader time framework than is possible with fetal mammals.     

5-bromodeoxyuridine-light inactivation of human lymphocytes stimulated mitogens and allogeneic cells: evidence for distinct T-lymphocyte subsets.

The study of human peripheral blood currently permits enumeration of circulating B and T lymphocytes as well as the analysis of functional responses in vitro following stimulation with mitogens, antigens or allogeneic cells. In the present experiments, subsets of these major lymphocyte populations were analyzed by dissecting in vitro responses using an ablative technique. After an initial culture period of lymphocytes with a mitogen, the proliferating cells were inactivated with 5-bromodeoxyuridine and light, then the capacity of the remaining lymphocytes to respond to the same or a different mitogenic influence was tested. Responsiveness to a different stimulant in the presence of no further response to the first stimulant was taken as evidence for a different responding cell population. A large subset of peripheral blood lymphocytes was responsive to both phytohemagglutinin (PHA) and concanavalin A (Con A); ablation of the cells responsive to one left little or no cells responsive to the other. Pokeweed mitogen (PWM) stimulated a portion of the PHA-Con A-responsive subset and an approximately equal subset unresponsive to PHA or Con A. Other evidence indicates that with each of these mitogens (especially with PHA and Con A in a soluble form), most of the proliferative response of peripheral blood B lymphocytes is indirectly triggered and is dependent on T cell stimulation. The population of PHA-Con A-responsive cells is, therefore, interpreted to represent a major T cell subset plus recruited cells; the PWM-responsive population would include a T cell subset having also PHA and Con A responsiveness, and another subset of T (or perhaps B) cells. The mitogen-sensitive population showed no overlap with cells responsive to allogeneic stimulation in mixed leukocyte culture. Ablation of the mitogen-responsive cells potentiated the mixed leukocyte reaction, suggesting that a suppressive influence was removed with the inactivation of the mitogen-responsive cells. It appears, therefore, that distinct subsets of T lymphocytes differentially responsive to PHA-Con A, to PWM and to allogeneic stimulation are present in the human peripheral blood.     

Carrageenan-induced suppression of chicken lymphocyte proliferation

Mitogenic responsiveness of peripheral blood lymphocytes (PBL) of chickens was suppressed by either pretreatment with or addition to the culture medium of various concentrations of carrageenan (CGN). Pretreatment for 1 hr significantly suppressed response to Concanavalin A (Con A) and Pokeweed mitogen (PWM) but did not affect Phytohemagglutinin (PHA) induced stimulation. Extension of the pretreatment period to 4 hrs suppressed response induced by all three mitogens. On the other hand, addition of carrageenan to the culture medium caused a dose-dependent suppression of PHA and Con A mediated response, but the effect on stimulation due to PWM was equivocal. In addition, low concentrations of CGN were weakly mitogenic to PBL and splenic lymphocytes.     

Immunoregulation in experimental filariasis. IV. Induction of non-specific suppression following in vitro exposure of spleen cells from infected jirds to Brugia pahangi antigen.

Studies with inbred jirds chronically infected (greater than 5 months) with Brugia pahangi have demonstrated splenic suppressor cells which modulate in vitro responsiveness to mitogens and parasite antigens. The stimuli which induce suppression were characterized by analysing the effect of activated cells from inbred normal or B. pahangi-infected jirds on the PHA and PWM responsiveness of cultures on normal cells. Regulatory cells were stimulated in vitro with concanavalin A (Con A; 5 micrograms/ml) or an extract of adult B. pahangi (20 micrograms/ml) for 72 hr and irradiated (1500 rads) prior to cocultivation with normal cells. Addition of Con A-activated normal spleen cells to normal cells produced moderate suppression of PHA and enhancement of PWM responsiveness. However, Con A-stimulated spleen cells from infected animals consistently suppressed both the PHA and PWM responsiveness of normal cells by 80-90%. Spleen cells from chronically infected jirds were also induced by B. pahangi antigen to suppress both the PHA and PWM responsiveness of normal lymphocytes. In contrast, spleen cells from animals 3-15 weeks after infection and lymph node cells from all time points were capable of suppressing only PWM responses when stimulated by antigen. Normal spleen cells were not induced by B. pahangi antigens to exhibit immunoregulatory activity. The suppression mediated by antigen-induced spleen cells from chronically infected jirds was partially or totally alleviated by removal of non-specific suppressor cells which are plastic adherent and cyclophosphamide-sensitive, or by removal of antigen-specific suppressor cells which bear receptors for histamine. the results suggest the involvement of regulatory cell circuits in experimental filarial infections.     

Stimulation of pig lymphocytes with anti-immunoglobulin serum and mitogens.

Mitogenic stimulation of pig lymphocytes by anti-immunoglobulin serum and by Con A, PHA, PWM and LPS has been measured by 3H-thymidine incorporation. Lymphocytes from blood, spleen and lymph node were readily stimulated by antiglobulin. Specific anti-micron serum was mitogenic for blood lymphocytes, indicating that those B cells with surface IgM can be stimulated by complexing of this surface component with antibody. An initial 2-hour incubation with anti-Ig was as effective as continuous incubation. Differentiation of stimulated cells into high rate synthesising plasmablasts was not observed. Pig blood lymphocytes were reactive to Con A, PHA, PWM and LPS. LPS was also tested against spleen and lymph node cells, which were responsive to this mitogen.     

Lymphocyte stimulation and plasma inhibition in patients with malignant neoplasms. A comparative study with three mitogens.

The blastogenic response of lymphocytes from patients with malignant neoplasms was evaluated by stimulation with three phytomitogens (PHA, PWM, and Con A). The response of patient lymphocytes to all three mitogens was significantly lower than that of control lymphocytes, and most patients with abnormal PHA responses also responded abnormally to PWM and Con A. However, a few patients with normal PHA responses were abnormal to Con A, suggesting the suppression of a Con A-sensitive population. The observation that PWM responses were abnormal in patients with lowered PHA lymphocyte stimulation indicates that both T and B lymphocyte mitogen responses were suppressed in these patients. Plasma from patients was capable of either inhibiting or enhancing lymphocyte mitogen stimulation. However, inhibitory plasmas were generally from patients with abnormal mitogen responses.     

Mycoplasma neurolyticum: a potent mitogen for rat B lymphocytes.

A mitogen prepared from Mycoplasma neurolyticum has been demonstrated to induce extensive transformation of in vitro cultured rat B lymphocytes. The data summarized in this report show that rat thymus cells as well as hydrocortisone-resistant thymocytes were not activated by this mitogenic agent. On the other hand, spleen cells obtained from thymectomized, lethally irradiated and bone marrow-reconstituted rats were extensively activated by M. neurolyticum. Furthermore, M. neurolyticum was shown to induce the development of antibody-producing cells, as attested by the appearance of direct plaque-forming cells against sheep red blood cells and trinitrophenylated sheep red blood cells in spleen cell cultures exposed to this mitogen. It was also demonstrated that stimulation of rat lymphocytes by this mitogen was inhibited by anti-rat immunoglobulin antibodies. In view of these data, it was suggested that M. neurolyticum, which activates mouse B lymphocytes, is a potent mitogen for rat B lymphocytes as well. This mitogen is a significantly more powerful mitogen for rat B lymphocytes then any other known mitogens. The availability of such mitogenic material in the rat system will enable studies on control mechanisms of action and differentiation of rat B lymphocytes.     

Radiation-induced augmentation of the response of A/J mice to SaI tumor cells.

Whole-body exposure of A/J mice to low doses (5-25 rads) of ionizing radiation immediately prior to the inoculation of 10(4) Sarcoma I (SaI) cells results in smaller tumors than are observed in sham-irradiated control animals. The irradiated group also contains a greater proportion of mice that fail to develop tumors or that demonstrate tumor regression. Low-dose augmentation is 1) less pronounced in recipients that have undergone splenectomy; 2) not evident in adult thymectomized-lethally irradiated-bone-marrow-restored (ATxXBM) animals, where the opposite effect is seen (eg, low-dose enhancement of tumor growth); and 3) abolished by the administration of normal syngeneic spleen cells unless the latter have been depleted of T cells. On this basis, a very radiosensitive T cell with suppressor activity is implicated in this phenomenon. Low-dose exposure at various times prior to tumor inoculation suggests that this cell regenerates in 5-10 days after irradiation.     

In vitro evaluation of radiation-induced augmentation of the immune response.

Small doses (5--25 rads) of radiation augment the in vitro response of murine spleen cells to sheep red blood cells (SRBCs). Such augmentation appears to result from radiation-induced disruption of a homeostatic component of the response that exerts maximum effect soon after the introduction of antigen. Evidence is presented to support the concept that augmentation is due to injury of an exquisitely radiosensitive subpopulation of T cells with suppressor activity.     

Differences in the effect of indomethacin and preincubation on the lymphoproliferative response to concanavalin A of spleen cells from low responder C57BL/6 and high responder BALB/c mice

In vivo treatment with 100 micrograms of indomethacin each 48 h for 2 weeks enhanced the proliferative response to concanavalin A (Con A) of spleen cells from mice of the C57BL/6 (B6) strain, low responder to T cell mitogens, but did not modify the response of spleen cells from mice of the high responder strain BALB/c (C). The enhancing effect of in vivo indomethacin treatment was more marked in cultures of B6 splenocytes stimulated with high, moderately supraoptimal doses of Con A than in cultures stimulated with optimal mitogen doses. Addition of indomethacin to cultures of spleen cells from untreated donors induced greater increase of the lymphoproliferative response of cells from low responder B6 than from high responder C mice. The enhancing effect of indomethacin added in vitro was observed in cultures stimulated by optimal but not by supraoptimal doses of Con A. The addition of indomethacin did not enhance the response of B6 spleen lymphocytes depleted of adherent cells. Preincubation for 24 h prior to mitogen stimulation increased the response to high Con A doses of spleen cells from low responder B6 mice whereas this procedure did not enhance lymphocyte proliferation in cultures of spleen cells from high responder C mice. Supplementation with indomethacin in vitro combined with preincubation induced additive enhancing effects on DNA synthesis by B6 spleen lymphocytes, suggesting that each treatment acts through different mechanism(s). The results indicated that spleen cells from low responder B6 strain mice are more sensitive than cells from high responder C mice to the potentiating effect of indomethacin and preincubation on the proliferative response to Con A. These observations suggest that mechanisms sensitive to indomethacin and to preincubation contribute to the depression of mitogen induced DNA synthesis in low responder B6 mice.     

Effects of cyclosporin A on the metabolism of unstimulated and mitogen-activated lymphocytes.

The immunosuppressive drug cyclosporin A (CS-A) reduces the magnitude of T-lymphocyte activation by all mitogenic lectins tested. However, in all cases a proportion of the activation observed is resistant even to very high concentrations of the drug. This proportion depends on the mitogen used, the responses to concanavalin A (Con A) and soybean agglutinin (SBA) being much more strongly inhibited than the responses to phytohaemagglutinin (PHA) or pokeweed mitogen (PWM). The differential effects of CS-A on lymphocyte activation by these mitogens could not be accounted for by the magnitude of the mitogenic response, the mitogen concentration used or the dependence of the responses on the presence of accessory cells, and they were maintained when several different procedures were used to assess the degree of activation. CS-A effectively inhibited inhibited lymphocyte activation. CS-A effectively inhibited lymphocyte activation only when added prior to, or very shortly after, the mitogen. Its ability to inhibit the response to PHA was lost more rapidly than that of Con A. The rate of protein synthesis by unstimulated lymphocytes was also affected by CS-A over the concentration range required to inhibit activation by mitogens. Although this effect was smaller than the inhibition of mitogen activation, it was highly significant and reproducible, and could not be accounted for by inhibition of spontaneous activation occurring in the unstimulated cultures.     

Positive selection of T-cell subsets. I. Proliferative responses of Lyt 2 separated thymocytes and splenic T cells.

Flow microfluorometry analysis of peanut lectin non-agglutinable (PNA-) thymocytes (ThC) reveals the existence of 30%-50% Lyt 1,2,3+ and 50%-70% Lyt 1+,2,3- subpopulations. Using positive selection on anti-immunoglobulin-coated (Mage) plates, we selected PNA- Lyt 2+ and PNA- Lyt 2- ThC as well as their peripheral counterparts in the spleen. These populations were tested in parallel for their ability to respond to concanavalin A (Con A) and phytohaemagglutinin (PHA), to respond to allogeneic stimulation in the mixed lymphocyte reaction (MLR); ThC subpopulations were also tested for their ability to provide synergy with lymph node cells (LNC) in the MLR. It was found that (a) Lyt 2- cells of both thymic and splenic origin responded to all doses of Con A or PHA; (b) PNA- Lyt 2+ ThC were unresponsive to Con A or PHA, whereas splenic Lyt 2+ T cells responded to low doses of mitogens; and (c) PNA- ThC of both Lyt phenotypes responded in a MLR and provided synergy with LNC in the MLR. These data support the notion that Lyt 2+ cells of either PNA- or PNA+ subpopulations must undergo post-thymic maturation before becoming responsive to low doses of T-cell mitogens.     

Blastogenic response of Toxoplasma-infected mouse spleen cells to T- and B-cell mitogens.

In order to differentially test the function of lymphocytes in Toxoplasma gondii-infected mice, the in vitro blastogenic response of spleen cell cultures to non-specific mitogens was studied. Phytohaemagglutinin (PHA) and concanavalin A (Con A) stimulation were used as tests of thymus-dependent lymphocyte (T cell) function and bacterial endotoxin lipopolysaccharide (LPS) was used as a probe of bursal equivalent lymphocyte (B cell) function. For the first 3 weeks following T. gondii infection, the uptake of tritiated thymidine ([3H]TdR) by spleen cells cultured with all three mitogens was markedly reduced in comparison to the uptake in spleen cells from uninfected control mice. Thereafter, the response to LPS returned to normal while stimulation by the T-cell mitogens (PHA and Con A) remained depressed. It is postulated that T. gondii infection either: (1) diluted out T cells in the spleen with unreactive cells; (2) modified T cells in such a way that they were less responsive to mitogens; (3) depleted the peripheral lymphoid tissues of T cells; (4) induced non-specific suppressor cells, which inhibited the T-cell function assays; or (5) activated macrophages which depressed T-cell function non-specifically.     

Differential effects of marijuana components on proliferation of spleen, lymph node and thymus cells in vitro

The effects of delta 9 THC and 11-OH THC on the proliferative response of murine spleen cells stimulated in vitro with the T cell mitogens Con A or PHA were compared with the effects of these drugs on the mitogen-induced proliferation of murine thymus and lymph node cells. Thymus cells were found to be suppressed at lower cannabinoid concentration than either spleen or lymph node cells. However, splenic cells were more easily suppressed than were the lymph node cells. Lymphoid cell numbers were varied from 1 X 10(6) to 8 X 10(6) cells and treated with a constant dose of either THC or 11-OH THC. When suppression was noted with spleen and lymph node cells, the smallest number of cells in the assay resulted in the greatest level of suppression of cell proliferation. No significant suppression to PHA induced proliferation was found for lymph node cells at any cell number tested. Thymus cells were always more readily suppressed than spleen or lymph node cells regardless of the number of cells in culture. Furthermore, 11-OH THC suppressed the responsiveness of the thymus cells to PHA more than to Con A under the experimental conditions used. Thus, the ability of cannabinoids to induce suppression of the proliferative response of lymphoid cells to mitogens depends on the organ source of the cells, nature of the cannabinoid (THC or 11-OH THC), dose of the cannabinoid, mitogen used (PHA or Con A), and number of cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)