Demonstration of significant differences in the proliferative capacity of lymphocytes from normal human subjects

Lymphocytes were obtained from six normal human subjects on three to five occasions. Lymphocyte response to pokeweed mitogen (PWM), phytohemagglutinin (PHA), and a pool of allogeneic lymphocytes was measured by incorporation of [³H]thymidine in cultures established in flat- and round-bottom multiwell plates. Unstimulated and mitogen-stimulated thymidine incorporation were not correlated. Therefore, incremental counts rather than stimulation indices were used to express data. Individual lymphocyte responses were more reproducible in round-bottom well plates. Using these data, correlation was found between the responses to PWM and PHA. Neither of these responses was correlated with the mixed lymphocyte reaction. There were no significant differences in the responses of these subjects to allogeneic lymphocytes. This response may, therefore, allow clear distinction between normal and abnormal lymphocyte reactivity. Among these normal subjects significant differences were found in their responses to both PWM and PHA. The biological import of these differences is not known.     

Lipid synthesis: an indicator of antigen-induced signal transduction in antigen-binding cells.

A biochemical parameter of lymphocyte activation, lipid synthesis, has been measured in a purified specific antigen-binding cell population (ABC). ABC isolated form immune and nonimmune animals by sequential centrifugation on buoyant density and sedimentation velocity gradients have a 2- to 7-fold higher rate of 14-C choline incorporation into phospholipid than either unfractionated spleen cells or cells depleted of ABC. Aslo ABC from immune animals were shown to have a 4- to 7-fold higher rate of 14C-acetate incorporation into their neutral lipids than nonbinding controls. The elevated lipid synthesis seen in both nonimmune SRBC-ABC and TNP-SRBC ABC indicates that antigenic contact via the B cell immunoglobulin receptor results in signal transduction and activation of the specific receptor-bearing lymphocyte population. Binding of the same particle (SRBC) to B cells via their Fc receptors did not regularly result in activation of lipid synthesis. The magnitude of the increased lipid synthesis in ABC populations approached that seen in LPS-stimulated spleen cells. We propose that the measurement of early activation events in purified ABC may be a more appropriate criterion for antigen-induced signals that later events such as thymidine incorporation or antibody secretion.     

The role of the thymus in maturational development of phytohemagglutinin and pokeweed mitogen responsiveness

A sensitive culture system for measuring lymphocyte transformation under physiological conditions by thymidine incorporation into DNA has been developed to study mouse and chick cell responses to mitogens. Both phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulated thymus and spleen lymphocytes. Reduced but definite responses were obtained with lymph nodes, but negligible response with bone marrow cells.Thymocytes of newborn mice did not respond to PHA, but responded well to PWM. PHA responsiveness of thymocytes increased with aging until 12 weeks of postnatal life and then decreased in older animals. The level of background thymidine incorporation increased with advancing age. Spleen cells of 2-week-old mice were transformed by PHA and PWM, but in contrast to mouse thymus there was no decrease in older animals.Neonatal thymectomy of mice reduced the response of spleen cells to both PHA and PWM, especially in younger animals. The reduction was almost complete in the case of the PHA response, but only partial with the PWM response. Spleen cells from bursectomised chickens, checked for absence of B cell function, still responded well to both PWM and PHA.The results suggest PHA is a marker for T-lymphocytes in a certain “mature” stage of differentiation. PWM appears to stimulate a wider spectrum of cells.     

A nonimmunomodulatory effect of chronic cadmium exposure on lymphocyte transformation response in rhesus monkeys (Macaca mulatta)

The immunomodulatory effects of cadmium, an environmental pollutant, were assessed in male rhesus monkeys orally exposed to 5 mg cadmium/kg body weight for a period of 4 months. A lymphocyte transformation test was carried out in peripheral blood lymphocytes using different concentrations of phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). Cadmium exposure of monkeys resulted in a statistically nonsignificant increase in [3H]thymidine incorporation in PHA- and Con-A-stimulated lymphocytes. However, there was a nonsignificant decrease in [3H]thymidine incorporation in PWM-stimulated lymphocytes. These results suggest that cadmium may not have immunosuppressive effects in primates which are phylogenetically close to man.     

Protein profile and stimulation potential of partially purified lectins from two varieties of Phaseolus vulgaris L

Phytohaemagglutinin obtained from P. vulgaris is a commonly used mitogen in lymphocyte cultures. The mitogenic potential of different cultivars of P. vulgaris vary. The present investigation encompasses the characterization of partially purified lectins from two varieties of P. vulgaris on the basis of their protein profile, lymphocyte transformation test by light and scanning electron microscopy and incorporation of radioactive thymidine.     

Immunophenotype and sister chromatid differentiation: a combined methodology for analyzing cell proliferation in unfractionated lymphocyte cultures

Combination of the MAC (morphology, antibody, chromosomes) and harlequin staining procedures offers a method for direct analysis of cell kinetics in cultures of unfractionated hematopoietic cells. In the present study unfractionated human mononuclear leukocyte cultures were stimulated with PHA or PWM mitogens and exposed to bromodeoxyuridine for various periods. For MAC, cytospin preparations were made and cells were classified with monoclonal B and T antibodies by the immunoperoxidase technique. After differentiation of the different lymphocyte subsets, the cells were stained by a fluorescence-plus-Giemsa method to distinguish sister chromatids and to determine the proportions of first, second, third, or subsequent mitoses among the previously identified subsets. The results showed (1) that the relative proportions of mitotic T and B cells are the same regardless of the mitogen used; (2) that T and B lymphocytes proliferate faster in cultures stimulated by PWM than in those stimulated by PHA; and (3) that T cells enter mitosis earlier than B cells when PHA or PWM are used as mitogens.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

A suppressor cell which interferes with antiallotype stimulated DNA synthesis of rabbit B cells

Responsiveness of rabbit spleen cells to anti-allotype antibody was measured in terms of increased thymidine incorporation. Incorporation was enhanced after removal of cells which had ingested or had adhered to magnetic particles. B lymphocytes, prepared from spleen cells by the removal of adherent cells and of RTLA bearing T cells, were more responsive to anti-allotype antibody than were the original spleen suspensions. This increase could not be explained by enrichment in B cells. It was concluded that an adherent cell suppressed B cell transformation. The addition of 2-mercaptoethanol to the cell cultures stimulated with mitogen augmented the incorporation of thymidine. Adherent cells interfered with 2-mercaptoethanol potentiation in the response to anti-allotype antibody but not in the response to Con A. Fractionation of spleen cells, over glass bead columns, yielded nonadherent and adherent cell populations. The responsiveness of nonadherent cells to anti-allotype induced thymidine incorporation was two to six times that of unfractionated cells. The responsiveness of nonadherent cells to stimulation by anti-allotype antibody was reduced after addition of adherent cells. Findings were discussed in terms of the inhibitory role played by adherent cells on anti-allotype antibody induced responsiveness of rabbit B cells and of the possible participation of a third cell type which functions as a promotor of mitogenic T cell stimulation.     

Stimulation of human lymphocytes by galactose-specific abrus and ricinus lectins.

Human lymphocyte cultures were incubated with the nontoxic abrus agglutinin and with ricin B chain, and the incorporation of 3H thymidine was measured. Abrus agglutinin stimulated strongly the thymidine incorporation whereas ricin B chain had a much lesser effect. When galactose or lactose was added to the cultures together with the lectins, the abrus agglutinin and ricin B chain induced thymidine incorporation was strongly reduced. There was a linear relationship between the concentration of lectin and the concentration of lactose required for inhibition of lymphocyte stimulation. N-acetyl-galactosamine had a much lesser inhibiting effect and alpha-methyl-mannoside did not cause any inhibition. The abrus agglutinin induced thymidine incorporation was not demonstrable before 36 to 40 hr and reached its maximum after 2 to 5 days. If lactose was added within the first 4 hr of incubation with abrus agglutinin no stimulation was observed.     

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.     

Proliferation of PHA- and PWM-stimulated lymphocytes measured by sister chromatid differential staining.

Lymphocyte proliferation in PHA- and PWM-stimulated cultures was measured by BrdU incorporation followed by sister chromatid differential staining. Both the PHA and PWM cultures showed a steady increase with time in the number of second- and third-division metaphases, accompanied by a corresponding decrease in the number of first-division metaphases. There was no difference between the PHA and PWM cultures in the number of first-, second-, and third-division cells, but the mitotic index was lower in the PWM cultures. During the 96-hr culture period the mitotic index of the PHA cultures decreased, whereas the PWM cultures showed an increase. It is suggested that the BrdU-Giemsa technique may prove to be a useful adjunct in the study of lymphocyte proliferation in response to mitogens.     

Relationship between the capacity to be stimulated of lymphocyte subpopulations and the RAI staging in patients with chronic lymphocytic leukemia]

By means of the incorporation rate of 3H thymidine into the lymphocytes of patients with chronic lymphatic leukaemia the possibility of stimulating them by using different mitogens was checked and compared with normal persons. The examination covered 11 patients treated with extracorporeal irradiation of the blood (ECIB), 5 patients treated with a chlorambucil therapy, and 10 untreated patients who were classified according to the staging system proposed by RAI. The lymphocytes of the peripheral blood were stimulated as mixed and isolated T and B-lymphocytes in the microculture by using the mitogens PHA, PWM, ConA, and LPS. In all CLL patients there was a diminished stimulation rate of a mixed lymphocyte population. A relation existed between the seriousness of the stage and the diminution of the incorporation rate of 3H thymidine. A corresponding correlation could not be identified in untreated CLL patients. Isolated T-lymphocytes revealed better results of stimulation than the total population. As to their function B-lymphocytes showed a dependance on the kind of therapy. In the mixed lymphocyte culture of normal persons the best findings could be observed after stimulation with PHA, that is also valid for CLL patients. PHA, PWA, ConA, and LPS were suitable as substances stimulating B-lymphocytes with different efficacy in normal persons and CLL patients. Both collectives showed the best results in the T-lymphocyte culture after stimulation with LPS.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Modulation of myelopoiesis by different bacterial cell-wall components: induction of colony-stimulating activity (by pure preparations, low-molecular-weight degradation products, and a synthetic low-molecular analog of bacterial cell-wall components) in vitro.

Chemically pure preparations of three structurally unrelated components of the cell wall of gram-negative bacteria (BCWC), lipid A, outer-membrane lipoprotein, and murein, were tested for lymphocyte mitogenicity and the ability to induce colony-stimulating activity (CSA) in various serum-free tissue-culture systems. All three components were B-cell mitogens and induced CSA in spleen-cell cultures. However, in lymphnode-cell cultures the concentrations of these agents required for either mitogenicity or CSA induction differed markedly. Moreover, in contrast to thymidine incorporation, CSA induction was not influenced by pre-irradiation of the cells. Conversely, after removal of phagocytic cells with the iron-magnet technique, CSA was no longer inducible by BCWC, while lymphocyte proliferation was barely impaired. All three BCWC readily induced CSA release in cultures of adherent peritoneal cells without influencing the release of a cytoplasmic enzyme. BCWC-dependent CSA release from adherent peritoneal cells was not influenced by pretratment of the cultures with anti-immunoglobulin, but completely suppressed by preincubation with anti-macrophage-1.2 alloantiserum and complement. CSA induction in macrophage cultures was also achieved with a low-molecular-weight synthetic muramyldipeptide and degradation products of lipoprotein. The results suggest that the induction of CSA is not directly related to the mitogenic, immunogenic, or antigenic properties of the BCWC, but that BCWC-mediated CSA production is caused by a direct “hormone-like” interaction of the agents with mature macrophages.     

The relationship between rubella hemagglutination inhibition antibody (HIA) and rubella induced in vitro lymphocyte tritiated thymidine incorporation

The parameters of lymphocyte, rubella virus interaction were studied by means of tritiated thymidine incorporation and compared with the rubella HIA level. Stimulation of lymphocyte DNA synthesis was found to depend on antigen concentration and to be independent of the presence of viable virus. When compared to HIA titer, lymphocyte transformation was found to be detectable in the absence of antibody. It is suggested that lymphocyte transformation is a sensitive method to detect both prior exposure and cellular immune reactivity to rubella virus.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

Effect of lipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets

The lipoxygenase products LTB4 and 15 HPETE have been reported to stimulate T suppressor cell function and also to inhibit [3H]thymidine incorporation into mitogen-stimulated T cells. This present report documents that although these compounds do indeed inhibit [3H]thymidine incorporation into unfractionated T cells, they significantly enhance [3H]thymidine incorporation into T cell preparation enriched for cells bearing the cytotoxic suppressor cell phenotype identified by the OKT8 monoclonal antibody. The mitogen response of T cells enriched for OKT4+ helper-inducer cells is inhibited in manner similar to the response of unfractionated T cells. Thus, LTB4 and 15 HPETE stimulate both the function and the proliferation of the cytotoxic-suppressor T cell subset.     

Analysis of lymphocytes reactive to histocompatibility antigens. II. Exponential alloantigen-dependent lymphocyte growth in vitro

Rat thoracic duct lymphocytes were maintained in continual blast transformation and cell division by repeated in vitro stimulation with allogeneic cells. This resulted in increases in responder cell numbers of up to 10,000-fold in 10-day periods. Growth of responder lymphocyte populations was dependent upon cell density, culture medium nutrients, and the presence of antigen in the form of allogeneic cells. A titration assay for mixed lymphocyte interactions (MLI) was used to relate absolute growth of cells in preparative cultures to [³H]thymidine incorporation in analytical MLI. Growth of lymphocyte populations derived by repeated stimulation with cells bearing a single foreign MHC haplotype was supported to lesser, variable degrees by stimulation with unrelated “third party” stimulator cells. The extent of this operational cross-reactivity was assessed by parallel line analysis of MLI titrations of responder lymphocytes enriched for specific alloreactivity.     

Assay for Epstein-Barr virus based on stimulation of DNA synthesis in mixed leukocytes from human umbilical cord blood.

Relationships between the rate of DNA synthesis in cultured human umbilical cord leukocytes and the multiplicity of added Epstein-Barr virus (EBV) were studied. At low multiplicities of approximately 0.1 transforming units/cell (approximately 10 physical particles/cell), inoculated cultures demonstrated increased rates of DNA synthesis, by comparison to uninoculated cultures, 3 days after inoculation. Stimulation of DNA synthesis was evident of progressively longer intervals after inoculations of 10-fold dilutions of virus. The rate of DNA synthesis, determined by short [-3H]thymidine pulses, reflected as small as twofold changes in multiplicity and thus can serve as a quantitative assay for the virus. Changes in the rate of DNA synthesis were evident before increases in cell number or alteration in morphology. Stimulation of DNA synthesis in umbilical cord leukocytes was inhibited by treatment of EBV with antibody and also in graded fashion, by progressive doses of UV irradiation to the virus. Induction of DNA synthesis by EBV was serum dependent. Estimates of the number of cells transformed were obtained by extrapolation from a standard curve relating known numbers of transformed cells to [-3H]thymidine incorporation and also by cloning cells after exposure to virus. At the low multiplicities of infection used in these experiments approximately 0.04 to 0.002 of the total cellular population was transformed. The high efficiency of cell transformation by EBV by comparison to other DNA tumor viruses is emphasized.