In vitro activation of human T and B lymphocytes by pokeweed mitogen.

In previous in vitro studies the DNA synthesis in human blood lymphocytes induced by low concentration of PWM correlated with the percentage of bone marrow-derived (B) lymphocytes in cell suspensions. No correlation with lymphocyte subpopulations was noted when lymphocytes were activated by high concentrations of PWM. In this paper the hypothesis that low concentration of PWM mainly activates B lymphocytes was tested by measuring the [14C]thymidine incorporation into purified T or B lymphocytes from healthy donors. B lymphocytes were purified to 90--95% by buoyant density centrifugation of T lymphocytes rosetted with sheep red blood cells. T lymphocytes were enriched by passage of lymphocytes through an IgG-anti-IgG-coated column. Low concentration of PWM-stimulated B lymphocytes but not T lymphocytes, while high concentrations of the stimulant activated both cell types. It was also noted that the B- but not the T-lymphocyte fraction contained cells which synthesized DNA in the absence of PWM.     

Plaque-forming cell response of pokeweed mitogen stimulated frozen human lymphocytes

The plaque-forming cell (PFC) response of pokeweed mitogen (PWM) activated fresh or frozen human peripheral blood lymphocytes (PBL) was monitored by the protein A hemolytic plaque assay. Fresh PBL and PBL stored in nitrogen for 7 days to 8 years were tested. On the basis of our preliminary results we conclude that cryopreserved cells secrete the same amounts of immunoglobulin (Ig) as freshly prepared cells.     

Plaque-forming cell response of frozen human lymphocytes stimulated with pokeweed mitogen. II. Results obtained in cultures of irradiated T and untreated B lymphocytes

The PFC capability of cryopreserved lymphocytes was tested after T/B cell separation and irradiation of the T cells. It is concluded that there is no selective loss of function of the involved subpopulations. Thus nitrogen-stored lymphocytes can safely be used in PFC assays after conventional separation and irradiation procedures.     

Rosette formation with mouse erythrocytes defines a population of human B lymphocytes unresponsive to pokeweed mitogen.

Our studies indicate that rosette formation with mouse erythrocytes defines a subpopulation of human B lymphocytes positive for both sIgM, sIgD and C3 receptors but largely negative for IgG Fc receptors. These cells respond very poorly to PWM stimulation, even when reconstituted in culture with optimal numbers of autologous T cells and adherent cells.     

Induction of protein disulphide-isomerase and immunoglobulins by pokeweed mitogen in human lymphocytes.

The Protein disulphide-isomerase (PDI, EC 5.3.4.1, Thiol-proteindisulphide oxidoreductase, EC 1.8.4.2) is thought to regulate the sulfhydryl status of cells and to catalyze thiol/disulphide exchange reactions involved in the post-translational processing of disulphide containing secretory proteins. The aim of the present investigations was to study the possible function of this enzyme in differentiation of B lymphocytes and immunoglobulin synthesis. Non-adherent human mononuclear cells or purified T cells were cultured in presence and absence of Pokeweed mitogen over 3, 5 and 7 days. Monoclonal antibodies and a rabbit polyclonal antiserum specific for human liver PDI were produced to determine the concentration of PDI by an ELISA technique and cytoplasmic immunofluorescence. After PWM stimulation, both, the cellular content of PDI as well as that of immunoglobulin, particularly IgM, have been found to be induced in a time dependent manner with a 2-3 fold increase in comparison to unstimulated cells. The specific induction of PDI in human B lymphocytes was also confirmed in Western blotting. Our findings suggest that PDI plays a critical role in the final stages of B cell differentiation and immunoglobulin synthesis by activated B cells and plasma cells, respectively.     

Isotype-specific human B lymphocytes that produce immunoglobulin E in vitro when stimulated by pokeweed mitogen

Normal human peripheral blood lymphocytes can be stimulated by PWM and T cells to produce IgE in vitro. Between 4% and 10% of circulating B lymphocytes were found to bear noncytophilic membrane IgE. In contrast, limiting dilution analysis of reactive B cell precursor frequency revealed that only about 1 in 50,000 B cells was responsible for the synthesis of IgE in vitro. Variation in the precursor frequency was primarily responsible for differences in the amount of IgE produced in vitro by different individuals while the amount of IgE produced per precursor cell was relatively constant. B lymphocytes with surface IgM, IgG, or IgE were depleted. Depletion of IgE-bearing cells decreased IgE production 70% to 83% while IgM-bearing-cell removal lowered IgE synthesis by 59% to 72%. Dual removal of IgM- and IgE-bearing cells did not give an additive effect. IgE- and IgM-positive B cells showed enhanced IgE synthesis of 327% and 182%, respectively. The consequences of crosslinking different surface isotypes on subsequent IgE production were also assessed. Anti-? treatment inhibited IgE production up to 68% while anti-μ, treatment decreased it by up to 38%. Thus only a very limited subset of B lymphocytes is responsible for PWM and T cell-dependent IgE synthesis in vitro and these cells probably bear both surface IgE and IgM.     

Defective immunoglobulin secretion in response to pokeweed mitogen in sarcoidosis.

We studied in vitro immunoregulation of immunoglobulin (Ig) secretion in 21 patients with sarcoidosis. While peripheral blood mononuclear cells from normal individuals responded to pokeweed mitogen with a 10-fold or greater increment in Ig-secreting cells, cells from sarcoid patients failed to respond to pokeweed mitogen at any concentration employed (P less than 0.001, Student's t-test, two-tailed). More monocytes were found in sarcoid mononuclear cell preparations (44.8 +/- 2.0% vs 30.4 +/- 1.4% in normal donors, P less than 0.001), but removal of monocytes improved the response to pokeweed mitogen in only four patients. Mononuclear cells from seven of 19 patients suppressed Ig secretion in co-cultures with normal donor cells. Patients exhibiting excessive suppressor cell function were older, with longer standing and less clinically active disease than non-suppressing patients. Monocyte removal reversed the suppression in only four of the suppressor patients, but excessive suppressor monocyte function was later demonstrated in two sarcoid patients whose cells initially did not suppress Ig secretion when cultured with normal cells. While the immunological defects in sarcoidosis may be complex, heterogenous, and dynamic, these data suggest that suppressor monocytes, when present in sarcoidosis, may have developed secondarily.     

Endothelial cells enhance the human pokeweed mitogen lymphocyte response

The effect of endothelial cells (EC) on lymphocyte mitogen responses was examined. Irradiated or mitomycin C treated EC were co-cultured with allogeneic peripheral blood mononuclear cells (PBM), and proliferative responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were assessed by 3H-thymidine incorporation. Compared to lymphocyte responses in the absence of EC, EC co-culture enhanced PWM responses at 72 hours by 55 +/- 28%, 103 +/- 24%, and 96 +/- 9% at EC:PBM ratios of 1:30, 1:10, and 1:3, respectively. The EC co-culture also resulted in significant lymphocyte responses to otherwise submitogenic doses of PWM (10(-4) micrograms/ml) as well as an accelerated kinetics of response. There was no effect of EC on PHA responses. The EC effect appeared not to require cell contact for its expression; however, supernates of EC cultures were not capable of reproducing the effect. On a cell-for-cell basis, EC were more potent in enhancing responses of adherent-cell-depleted lymphocytes than either allogeneic or syngeneic monocytes. Fibroblasts could not substitute for EC in enhancing PWM response, suggesting that the effect was not a nonspecific feeder phenomenon. The EC may play a role in modulating some immune responses in vivo, especially those occurring in areas of inflammation, neovascularization, and endothelial cell proliferation.     

Interaction of pokeweed mitogen with monocytes in the activation of human lymphocytes

The present study examines the role of monocytes in the in-vitro activation of human T cells and B cells by pokeweed mitogen (PWM). The T cell-dependent PWM-induced B-cell activation process was found to be monocyte dependent. Fluorescence-activated cell sorter (FACS) analysis revealed that upon addition to peripheral blood mononuclear cells, fluoresceinated PWM, at concentrations that provided optimal B-cell and T-cell activation, bound predominantly to human monocytes. The binding of PWM to monocytes was reversible and could be displaced within the first few hours of binding by oligomers of N-acetylglucosamine (GlcNAc). As a functional correlate of the binding studies, it was shown that PWM-pulsed monocytes could induce B lymphocytes to become plaque-forming cells (PFC) and T lymphocytes to undergo proliferation. In contrast, markedly reduced PFC and blastogenic responses were observed when monocyte-depleted B lymphocytes and T lymphocytes were respectively pulsed with PWM and washed, followed by the addition of non-PWM-pulsed monocytes to the cultures. Thus, the initial event in the PWM-induced activation of human lymphocytes, for both in-vitro T-lymphocyte blastogenic responses and B-lymphocyte Ig secretion, appears to be binding of the mitogen to sugar residues on the surface membrane of the monocyte, followed by subsequent interaction with the appropriate lymphocytes. The process of PWM binding to monocytes did not appear to affect the baseline production of interleukin-1 (IL-1) by human monocytes, nor could soluble factors from PWM-pulsed monocytes substitute for intact cells in the initiation of the lymphocyte-activation process.     

Co-operation between T and B lymphocytes from human tonsils in the response to mitogens and antigens.

Purified B lymphocytes obtained from human tonsil cell populations by removing E rosette-forming cells by density sedimentation did not proliferate at three days in response to PHA and Con A, but showed a significant 3H-labelled thymidine incorporation when the PHA response was assessed at day 6 of culture. The 6th-day responses, which was completely abolished by the reduction of T-cell contamination to less than 0-1% by re-rosetting and a second separation, was due in part to a direct activation by PHA of contaminating T cells and in part to a T cell-mediated B-cell response. When purified B cells were stimulated for 3 days by PHA in the presence of an equal number of autologous or homologous mitomycin-treated T lymphocytes a highly significant uptake of 3H-labelled thymidine was demonstrated. The majority of blast cells obtained at day 4 in these cultures were unable to form E rosettes and showed surface immunoglobulin by immunofluorescence stain. This response was markedly decreased by previous treatment of B cells with mitomycin C and it was abolished when B cells were killed by heating at 56degrees C for 1 hr. Purified B lymphocytes from human tonsils did not respond in vitro when cultured for 6 days in the presence of soluble antigens (PPD and Candida). However, a highly significant response to the same antigens could be demonstrated when B cells were cultured in the presence of autologous mitomycin-treated T cells. These models of T-B co-operation could provide an interesting tool for studying the differentiation and antibody production in vitro of human B lymphocytes.     

Functional behaviour and immunological phenotype of circulating B lymphocytes in multiple myeloma. Studies with pokeweed mitogen.

Peripheral blood B lymphocytes, depleted of adherent cells, from 10 patients with multiple myeloma were cultured in the presence of PWM with autologous or donor T lymphocytes. The results show that: (1) co-cultures with allogeneic T lymphocytes produced more plasma cells than those with autologous ones; (2) the kappa/lambda ratio overlapped the values obtained in normal controls, irrespective of the light chain produced by the neoplastic plasma cells and (3) the immunological phenotype of plasma cells obtained from PWM stimulated peripheral B cells (RFA2+, RFA3+, A10+) was clearly different from that one of myelomatous plasma cells (RFA2-, RFA3-, A10+). These data confirm the T cell imbalance already seen in myeloma patients; moreover they show that PWM responsive B cell are functionally normal and phenotypically different from bone marrow myeloma cells. These results support the view that most of the peripheral B lymphocytes, previously identified as monoclonal are in fact normal cells bearing adherent monoclonal Ig molecules.     

Thyroid autoantibody synthesis by cultures of thyroid and peripheral blood lymphocytes. I. Lymphocyte markers and response to pokeweed mitogen

Thyroid lymphocytes from Graves' and Hashimoto patients have been investigated and compared with lymphocytes from the peripheral blood. Considerably more lymphocytes (20-30 X 10(6)/g) could be isolated from Hashimoto thyroids than from Graves' tissue (1-5 X 10(6)/g) but the cell suspensions extracted from Hashimoto and Graves' glands were similar in terms of cell surface markers and the ability to synthesize immunoglobulin. Thyroid lymphocytes contained a lower proportion of T cells (OKT3+ cells) and in some cases more B cells than the peripheral blood but the ratio of helper to suppressor T cells (OKT4+:OKT8+ cells) was similar to the values obtained for blood lymphocytes. Further, thyroid lymphocytes (unlike blood lymphocytes) synthesized relatively large amounts of microsomal and/or thyroglobulin antibody when cultured in medium only and these levels were significantly decreased by the addition of pokeweed mitogen. The results of this study provide further evidence for the role of the thyroid as a major site of thyroid autoantibody synthesis and emphasize the importance of characterizing the cells infiltrating the gland in autoimmune thyroid disease.     

Suppression of pokeweed mitogen-induced differentiation of human B cells by in vitro levamisole-treated T lymphocytes

The generation of immunoglobulin-producing cells from human peripheral blood lymphocytes in pokeweed mitogen-simulated cultures was significantly suppressed by levamisole. Greatest suppression was observed when cells were incubated with levamisole at a concentration of 10(-6) M for 20 min or more prior to culture. Such suppression occurred when untreated as well as levamisole-treated B cells were co-cultured only with levamisole-treated T cells. Adding of levamisole-treated T cells to a combination of untreated autologous B and T cells resulted in the suppression of B cell differentiation. The results suggest that levamisole treatment of lymphocytes exerts suppression on pokeweed mitogen-induced B cell differentiation through its effect on T cell regulatory properties.     

Differentiation of human lymphocytes by pokeweed mitogen in vitro: Studies in normal and in immunodeficient subjects

Summary Immunoglobulin (Ig) synthesis and secretion by peripheral blood lymphocytes activated by pokeweed mitogen (PWM), and by phytohemagglutinin (PHA) was measured in normal individuals and in subjects with primary immunodeficiency. Unstimulated cultures demonstrated stable, low Ig synthesis of all major Ig classes; PWM showed a peak of Ig synthesis at day 5; PHA cultures demonstrated a late peak of Ig production at day 9. Three cases of common variable immunodeficiency showed different patterns of data when the percentage of B cells in blood and Ig production in vitro were used as parameters. Two persons with immunodeficiency and thymoma showed decreased Ig production in vitro, and had suppressor cells capable of blocking Ig production by normal lymphocytes in co-cultivation experiments.

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Abbreviations used PWM pokeweed mitogen - PHA phytohemagglutinin - Ig immunoglobulin - IDD immunodeficiency disease - GARIg goat-anti-rabbit immunoglobulin serum - PBS phosphate buffered saline Supported by: United States Public Health Service Grants AI 09239, AM 20122, AM 11796     

Cell numbers in human lymphocyte cultures stimulated with pokeweed mitogen.

As determined by electronic cell counting, the cell numbers in pokeweed mitogen (PWM) stimulated cultures of normal lymphocytes decreased by about 13% during the first day and then remained almost constant up to day 8. In contrast, a progressive decrease of the cell count was observed in cultures of chronic lymphocytic leukaemia (CLL) lymphocytes reaching about 40% of the initial number on day 8. In PWM cultures of normal lymphocytes the transformed cells increased to about 20% of the cells present on day 4, whereas in cultures of CLL lymphocytes these cells reached only 11% on day 7.     

Reactivity of ovine lymphocytes to phytohaemagglutinin and pokeweed mitogen during pregnancy and in the immediate post-parturient period.

Lymphocytes from sheep in late pregnancy and at parturition showed markedly impaired proliferative responses to phytohaemagglutinin (PHA) in vitro when cultures were supplemented with foetal bovine serum (FBS), as compared to the responses of lymphocytes from non-pregnant sheep, sheep at 40 days of gestation and sheep at 80 days of gestation. Similar responses to PHA were observed when the medium was supplemented with autologous plasma (AP), although the responses were of a lower order. In both cases elevated responses to PHA were apparent at 10 days post-parturition. The response with FBS was more marked than with AP. Progressive reduction of lymphocyte responses to pokeweed mitogen (PWM) in the presence of FBS and AP were less obvious, although it was still apparent that responses to PWM were depressed at 120 days of gestation and at parturition, when compared with lymphocyte responses during early pregnancy (at 40 days and 80 days of gestation). The difference was much more apparent with AP than with FBS and responses during early pregnancy were markedly higher than those with FBS. An increase in lymphocyte responsiveness to PWM 10 days post-parturition was evident whether FBS or AP was incorporated in the cultures. The response with FBS was again more marked than with AP.     

Interaction between T and B lymphocytes and their migration in the immune response during methylcholanthrene-induced carcinogenesis

Methylcholanthrene (MC) was injected intramuscularly in a dose of 0.3 mg into (CBA×C57BL)F₁ mice and the ability of their T and B lymphocytes to cooperate during the immune response to injection of sheep's red blood cells and also migration of these cells from the thymus and bone marrow into the spleen were investigated. The results showed that the immunodepressant action of MC is connected with inhibition of processes of migration and cooperation of T and B lymphocytes in the immune response. It is concluded that the immunosuppression developing during carcinogenesis is complex in character and is realized at different stages of immunogenesis.Institute of Biophysics, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 10, pp. 464–466, October, 1977.     

Cytotoxicity of human lymphocytes induced by pokeweed mitogen or in mixed lymphocyte culture. Specificity and nature of effector cells

Purified human peripheral blood lymphocytes were activated in vitro with pokeweed mitogen (PWM) or with mitomycin-treated allogeneic lymphocytes (MLC). After incubation for several days, stimulation to DNA synthesis and cytotoxicity for phytohemagglutinin (PHA)-activated lymphoblasts or for Chang cells was tested. Lymphocytes activated with PWM for 3–6 days were cytotoxic for both autologous and allogeneic PHA-induced lymphoblasts. In addition, they destroyed human tissue culture cells (Chang cells) effectively. In contrast, lymphocytes activated in MLC were cytotoxic for lymphoblasts bearing stimulator cell antigens, but not for autologous PHA-lymphoblasts. Cytotoxicity for Chang cells was variable. Lymphocytes which proliferate and become cytotoxic after activation in MLC are of T cell origin, since their ability is not reduced by prior removal of non-T cells on columns charged with human IgG-anti-IgG complexes. On the other hand, this procedure reduced both DNA synthesis and cytotoxicity of PWM-activated lymphocytes. This suggests that cells equipped with surface immunoglobulin and/or Fc recptors were required for activation of these cultures, or alternatively, were among the cells which responded to PWM by DNA synthesis and cytotoxicity.     

Human tonsil B lymphocyte function. I. The proliferative response to Staphylococcus aureus and pokeweed mitogen in relation to surface heavy chains mu and delta

Human tonsil non-T cells were separated into surface IgD positive (sIgD+), surface IgD negative (sIgD-), sIgM+ and sIgM- fractions by rosetting and gradient centrifugation with ox red blood cells, coated with immunosorbent purified goat anti-human heavy chain antibodies. No activation or suppression was found as a result of the separation technique. The sIg patterns of the isolated B cell fractions showed the effectiveness of the separations; sIgD+ and sIgM+ fractions were to a great extent overlapping populations, but there was a definite population of s mu + delta- cells (about 20% of the sIgD- fraction). The isolated fractions were tested for proliferative responses to Staphylococcus aureus (Sta) and pokeweed mitogen (PWM). The sIgM+ fraction showed the best response to Sta, eight times better than the sIgM- fraction, while the sIgD+ and sIgD- fraction showed equal responses to Sta. The sIgM- fraction responded best to PWM but all fractions responded quite well. Our results indicate that PWM is a mitogen to a whole variety of rather mature B cells while the Sta target B cell is restricted to the relatively immature sIgM+D- and sIgM+D+ cells.