In vitro response of lymphocytes to phytohemagglutinin (PHA) as studied with antiserum to PHA : II. Cell cycle analyses

The cell cycle and phase times of human lymphocytes responding to PHA have been analysed with the percent labelled metaphases (PLM) technique. The range of generation times (13–18 h) and DNA synthesis times (6.5–10.5 h) reported here compare well with previous measurements in the literature. Cycle analyses of the early responding cells of the initial response, selected with partial anti-PHA serum inhibition, and of restimulated cells yield relatively well-defined PLM curves. The short cycle times measured from these curves may reflect the early cycles after stimulation or a subpopulation of responding cells. Analyses at two times during both the initial and restimulation responses suggest that cycles lengthen with time after stimulation. The poor PLM curves of the initial response and the restimulation response of cells released from anti-PHA inhibition indicate considerable intercellular variation in cycle times. Cells in the initial long G 1 phase contribute to this variation. PHA dose does not appear to affect the cycle time.     

Endocytosis and exocytosis of phytohemagglutinin (PHA) cell surface receptors of human lymphocytes during blast transformation

The ultrastructural distribution of T lymphocyte surface membrane receptors for phytohemag-glutinin (PHA) during blast transformation is examined using PHA covalently coupled to ferritin (PHA-Fe). Human peripheral blood lymphocytes from normal donors were enriched for T cells by nylon wool elution and cultured in vitro with PHA-Fe at a concentration known to cause maximal stimulation of DNA synthesis as measured by [³H]thymidine incorporation. Over the course of a 72 h incubation period, cell samples were harvested at regular intervals and examined by transmission electron microscopy. Within several minutes of culture at 37 °C the majority of the ferritin (Fe)-labeled PHA surface receptors on almost all cells undergo rapid endocytosis; some Fe label remains at the cell surface. After several hours, endocytotic vesicles containing Fe-labeled receptors coalesce and undergo condensation. Within 36–48 h, most endocytotic vesicles transform into multivesicular bodies (MVBs). After 48–72 h, 70–80% of the cells had the ultrastructural appearance of blast transformation as characterized by increased size, euchromatic nuclei, nucleolonema and polyribosomes. In 40 % of the blast cells the Fe-labeled MVBs are exocytosed to the cell surface; cytoplasmic MVBs in the remaining portion of the blasts and non-blast lymphocytes do not appear to undergo exocytosis. Although endocytosis and exocytosis of lymphocyte surface receptors during mitogen-induced blast transformation are observed, the role and significance of receptor redistribution to cell activation remains unclear.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Activation of leopard frog (Rana pipiens) spleen lymphocytes by concanavalin A and phytohemagglutinin.

Activation times for concanavalin A (Con A)- and phytohemagglutinin (PHA)-induced lymphocyte mitogenesis in leopard frogs were determined by competitive inhibition analysis using α-methyl-d-mannopyranoside and N-acetyl-d-galactosamine. Con A activation required approximately 18–20 hr whereas PHA was required for at least the first 24 hr. Temporal events for mammalian lymphocytes are similar suggesting that activation parameters of lymphocyte biology have remained unchanged since the first vertebrate tetrapods evolved.     

Mixed lymphocyte reaction in mice genetically selected for high (Hi/PHA) or low (Lo/PHA) responsiveness to phytohemagglutinin.

Lymph node cells from Hi/PHA and Lo/PHA mice were evaluated for proliferative response after stimulation by allogeneic lymphocytes (MLR) originating from four inbred strains of different H-2 haplotype (C57B1/6, DBA/2, CBA, A). Reactivity to MLR and PHA were compared in these two lines and in the four inbred strains. The high and low responder status of Hi/PHA and Lo/PHA, as determined by T mitogens lymphocyte responsiveness, was also observed when one measured T responsiveness after MLR. Values obtained with the four inbred strains are included in the range of those measured in Hi/PHA and Lo/PHA cells when stimulated by PHA as well as by allogeneic cells. In contrast, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. These experiments support the hypothesis of a common genetic control of proliferative response following PHA or MLR stimulation. The genes implicated would be different from those coding for I region associated antigens.     

Morphological characterization of the pathway of endocytosis and intracellular processing of transferrin and alpha-fetoprotein in human T lymphocytes stimulated with phytohemagglutinin (PHA)

Covalent conjugates of transferrin (Tf) and alpha-fetoprotein (AFP) with horseradish peroxidase (HRP) have been used to follow, at the ultrastructural level, the uptake and the intracellular pathway of these proteins in peripheral blood human lymphocytes stimulated by phytohemagglutinin (PHA) to blast formation. Both proteins enter specifically the cells via vesicles (60-70 nm in diameter) and endosomes. They are then observed in multivesicular bodies and tubular vesicular elements in the Golgi region. AFP is thus found in the same subcellular compartments as Tf and is probably also recycled, as most of the 125I-labeled protein leaves the cells undegraded. Unstimulated lymphocytes do not internalize significantly AFP-HRP. The uptake of a noncovalent conjugate of AFP-HRP and [3H]-arachidonic acid [3H-(20:4)] is usually poor, at 37 degrees C, in unstimulated lymphocytes as well as, at 4 degrees C, in lymphocytes stimulated for 72 h. Stimulated lymphocytes incubated at 37 degrees C with the radioactive conjugate show a heavy labeling of cell organelles and more particularly of lipid droplets. AFP could regulate the intracellular delivery of fatty acid molecules.     

The effects of pokeweed mitogen (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation and embryo development in vitro

The effects of two commonly used cell culture mitogens, pokeweed (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation in vitro (IVM) and preimplantation embryo development in vitro were evaluated by randomized complete block experimental design with three treatments. Effects were measured by quantifying subsequent embryo development. Oocyte maturation was adversely affected by PWM-containing medium as indicated by a decrease in cleavage rate and subsequent embryo development to morula and blastocyst stages. Embryo developmental competence was also adversely affected by PWM. Development in PHA-containing medium was significantly better (P<0.05) than in the PWM treatment, although there was no difference (P>0.05) when compared to Control. We conclude that there are no beneficial effects in adding mitogenic agents to culture medium to enhance in vitro embryo production and development.