Binding of purified lectins to guinea pig lymphocytes. Studies of the number, binding constant, and distribution of lens culinaris lectin A and Agaricus bisporus lectin molecules on lymphocyte surfaces

The surface membrane of guinea pig lymphocytes contains discrete receptors for several different homogeneous lectins. Two very similar lectins from the lentil, LcL-A and LcL-B, bind to the same receptor. Three other lectins, AbL, WGA, and Con A, do not bind detectably to the LcL receptor. AbL binds to another discrete receptor to which none of the other lectin molecules bind. The LcL and AbL receptors are contained on different units, each of which is capable of independent movement on the cell surface. The normal distribution of LcL molecules on the surface is different than the distribution of AbL molecules. However, when lymphocytes are simultaneously incubated with LcL and AbL, the distribution of bound LcL molecules on the cell surface parallels the normal distribution of AbL molecules, which indicates that the movement of AbL molecules and AbL receptors on the surface can strongly influence the movement of LcL and LcL receptors. When lymphocytes are held in close contact by LcL or AbL, the lectin molecules and receptors appear to concentrate at the area of contact between two cells. A model is presented which suggests that the migration of lectins and lectin receptors can occur by a multicellular process.     

Effect of orally and intraperitoneally administered plant lectins on food consumption of rats

A panel of orally administered lectins (100 mg/kg b.w.) of different binding specificities was tested for suppression of voluntary food consumption in prefasted rats. PHA isolectins (Phaseolus vulgaris) and RPA-I (Robinia pseudoacacia) were found to exert a marked and significant effect, but two other gut-binding lectins, i.e. SBA (Glycine max) and WGA (Triticum vulgar) and several non-binding lectins were ineffective. In cannulated rats PHA infused into the duodenum induced food suppression, i.e. binding of the lectin to the mouth or stomach was unnecessary. Suppression of food consumption lasted through the whole nocturnal feeding period, control (BSA) and experimental (PHA) curves of cumulative food consumption showed a V-like divergence. Suppression by PHA or RPA-I showed very similar time courses, but a long-lasting inhibition of gastric emptying was only observed in the RPA-treated animals. Intraperitoneally administered lectins suppressed food consumption much more effectively than the oral ones, whereas Galanthus nivalis agglutinin (ONA) had little or no effect. It is concluded that lectins can be used as effective tools for the modulation of food consumption and gastric emptying in experimental animals.     

Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

Isolation and characterization of rat skeletal myoblast mutants resistant to lectins.

Mutants resistant to the lethal action of the lectins phytohemagglutinin A (PHA) and wheat germ agglutinin (WGA) have been made in a line of differentiating rat skeletal myoblasts. The WGA mutants are of two types, WGArII, resistant to low concentrations of the lectin, and WGArI, resistant to high concentrations of the lectin. WGArII and PHAr mutants are unable to differentiate, whereas WGArI mutants differentiate normally. WGArII mutants are not impaired in the binding of wheat germ agglutinin, but WGArI mutants bind the lectin only to the extent of about 50% of the wild-type values. All of the mutants are cross-resistant to lectins other than those used in their selection.     

Lectin-induced actin polymerization in human lymphocytes: A possible signal for mitogenesis

Employing the DNase I inhibition assay, a decrease in G-actin is demonstrated in human mononuclear cells following stimulation with mitogenic lectins concanavalin A (Con A) and phytohemagglutinin (PHA), as well as a nonmitogenic lectin, wheat germ agglutinin (WGA). The decrease in G-actin can be prevented by pretreatment of cells with cytochalasin E, indicating that the decrease is likely due to conversion to F-actin. Thus, the receptor-mediated actin polymerization is common to both the mitogenic as well as the nonmitogenic lectins. The maximal decrease in G-actin with Con A and PHA occurs at the same concentrations of the lectins that give optimal mitogenic responses. It is a distinct possibility that actin polymerization could be one of the signals necessary for the initiation of mitogenesis. The difference between a mitogenic and a nonmitogenic lectin may lie in the fact that a second signal (or signals), derived from macrophages, may not be generated by a nonmitogenic lectin such as WGA.     

Fractionation of lymphocytes using affinity chromatography with 9 lectins

Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.     

Interactions of lectins and monoclonal antibodies with human mononuclear cells. I. Specific inhibition of OKT4 and OKT8 binding by Ricinus communis agglutinin and wheat germ agglutinin

We used flow cytometry to examine effects of lectins on interactions between human lymphocytes and the anti-T cell monoclonal reagents OKT4 (T helper-specific) and OKT8 (T suppressor-specific). Wheat germ agglutinin (WGA) inhibited OKT8 binding to lymphocytes by a mean 77% and Ricinus communis agglutinin (RCA-I) inhibited OKT4 binding by 66%. Inhibition was abolished in each case by appropriate carbohydrate hapten inhibitors of lectin binding, indicating it was mediated by the lectin saccharide combining sites. Neither WGA nor RCA-I inhibited binding of OKT3, a pan-T cell monoclonal reagent. In addition, a group of other lectins with a variety of nominal carbohydrate specificities did not inhibit OKT4 or OKT8 binding. Preincubation experiments and gel filtration indicated that inhibition in each case was due to competition between lectin and monoclonal for binding to cell surfaces, not to direct lectin-monoclonal antibody interactions. Treatment of lymphoid cells with OKT8 and complement reduced OKT8- and WGA-binding cells concurrently, whereas treatment with OKT4 and complement did not reduce percentages of either type of cell. Similarly, specific depletion of OKT8-binding cells abolished the mitogenic response to WGA but not that to PHA. Cell populations enriched for WGA-binding cells prepared by flow cytometry and cell sorting demonstrated parallel enrichment for OKT8-binding and depletion of OKT4-binding cells. Therefore, these data demonstrate specific inhibition of OKT4 and OKT8 binding by the lectins, RCA-I and WGA, respectively. Inhibition was mediated by lectin binding to lymphoid cell surfaces, perhaps directly to the T4 or T8 antigens. The observations indicate that lectins may prove useful for investigating structural features of some immunologic cell surface markers. Furthermore, they provide the possibility that certain in vitro effects of lectins on immune function may result from their interactions with molecules such as the T4 and T8 antigens.     

Effect of orally administered plant lectins on intestinal liquor accumulation and amylase activity in rats

Short-term effects of orally administered plant lectins, with special reference to the Phaseolus vulgaris agglutinin (phytohaemagglutinin, PHA), were studied in growing rats. The orally administered PHA elicited a dose-dependent accumulation of liquor with elevated pH in the proximal small intestine. Although the concentration of alpha-amylase activity did not change, total alpha-amylase activity slightly, but significantly increased in the gut. When a panel of plant lectins with different carbohydrate binding specificities was tested at the dose of 100 mg/kg body weight, most of them stimulated the secretion of liquor, but the total alpha-amylase activity was increased only by PHA, ConA or WGA.     

Modulation of cytolytic T cell function by lectins

Human thymocytes cultured for 5 days in interleukin 2 containing supernatants (IL 2 Sup) virtually become a population of mature T cells (T3+, HTA-) that acquires strong cytotoxic activity against NK-sensitive and NK-resistant target cells. The addition of different lectins to the cultures abrogated the expression of the cytotoxic activity and enhanced thymocyte proliferation. The modulation of this cytotoxic activity by lectins has the following properties: a) inhibition of cytotoxicity is related to the concentration of lectins added, but does not correlate with their mitogenic properties, because either strong mitogens such as PHA or weak mitogens such as wheat germ agglutinin (WGA) are both able to strongly decrease cytotoxicity; b) lectin presence is not required at the onset of the culture but is required during the last 24 hr of the 5-day incubation period; c) reversion of inhibition with full expression of cytotoxic activity can be obtained after removal of the lectin and subsequent culture in lectin-free conditions for at least an 18 to 24 hr period; d) lack of cytotoxicity is observed regardless of target cell specificities and cannot be overcome in a lectin-dependent cytotoxicity assay (LDCC); and e) abrogation of cytotoxicity is not restricted to thymocyte cultures because it can also be observed in peripheral lymphocytes. These results cannot be explained by a simple steric blockade, the overgrowth of a distinctive noncytotoxic lymphocyte population, autologous killing, or a failure in the recognition phase of the lytic event. Modulation by lectins of function-associated cell surface structures implicated in cytolysis is discussed as an alternative hypothesis that might account for the observed phenomenon.     

Effect of calcium on the cyclic GMP elevation induced by thymosin fraction 5.

Thymosin fraction 5 induces an increase in cyclic GMP but not cAMP in murine thymocytes. Calcium (0.6 mM) is necessary for an optimal response in both phosphate buffered saline and hepes-buffered RPMI 1640 media. The calcium dependence of the cGMP response was most pronounced in a minimal salts medium (PBS) and higher concentrations (greater than 0.8 mM) caused a lessening of the cGMP elevation induced by thymosin. Basal cGMP levels of thymus and spleen lymphocytes vary with increasing concentrations of calcium (0–1 mM) and to a lesser extent, the levels of cAMP also are increased. Calcium uptake was measured both at mitogenic levels of Con A and at thymosin concentrations which were similar to those necessary for the increase in cGMP. The results suggest that calcium and cGMP play an important role in T cell differentiation under the influence of thymosin.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Effect of leukoagglutinating phytohemagglutinin on cAMP and cGMP levels in lymphocytes

In concentrations which do not affect the cellular concentrations of the cyclic nucleotides cAMP and cGMP, phytohemagglutinin (PHA) (crystalline, kidney-bean leukoagglutinin) can activate adenoidal lymphocytes. High concentrations of leukoagglutinin caused an increase in cAMP without stimulating mitosis in lymphocytes. Our results do not support the concept that cyclic nucleotides act as mediators for lymphocyte activation by lectins. Previous contradictory reports on the role of cyclic nucleotides may have been due to the use of impure bean extracts and lymphocytes.     

Effects of trifluoperazine and mitogenic lectins on calcium ATPase activity and calcium transport by human lymphocyte plasma membrane vesicles

The phenothiazine, trifluoperazine, and the mitogenic lectins, phytohemagglutinin (PHA) and Concanavalin A (Con A), were tested for their effects on human lymphocyte plasma membrane Ca-activated Mg-ATPase and ATP-dependent calcium uptake. Trifluoperazine completely inhibited Ca-uptake when present from the start of the assay at concentrations of 100 microM or more. When added during measurement of calcium uptake, trifluoperazine reduced the rate of vesicular calcium accumulation but was unlike the calcium ionophore, A23187, which caused a rapid release of accumulated calcium from the vesicles. Trifluoperazine also inhibited membrane vesicle Ca-ATPase activity, but this inhibition was non-specific since the Mg-ATPase and Na,K-ATPase activities were inhibited to similar extents at the same concentration of the phenothiazine. In contrast, concentrations of PHA and Con A, which are mitogenic for lymphocytes, did not cause any change in Ca-uptake when added to suspensions of membrane vesicles. Con A had no effect and PHA had a weak inhibitory effect on Ca-ATPase activity.     

Modulation of mouse anti-trinitrophenyl plaque-forming cell affinity by adjuvants or lectins.

The efffects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins.

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Phar) phenotype is stable in the absence of selection; its frequency in a sensitive-population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal alpha-mannose residues is dramatically reduced (less than or equal to 5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytoxocity of concanavalin A (Con A); (d) an increased ability to bind 125I-Con A; and (e) decreased surface galactose residues - all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistance closes from each of two complementary CHO auxitrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC, or LCA, and 4-5 "lectin-resistance" phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.     

Potentiation of the T lymphocyte response to mitogens. V. Inhibition of the response to concanavalin A by supraoptimal doses or by other lectins and its aversion by LAF.

The enhanced thymidine incorporation in murine lymphocytes induced by Concanavalin A (Con A) was markedly inhibited in the presence of other lectins, which are poorly mitogenic (phytohemagglutinin {PHA} or pokeweed mitogen), or non-mitogenic (soybean agglutinin {SBA}). The level of inhibition was found to be inversely proportional to the mitogenic effect of the lectins. Our results did not support the notions that the lectins inhibit the lymphocyte responses by competing with Con A, or by activating suppressor cells. Rather, the data suggest that the lectins cause cytotoxic or cytostatic effects. The effects of the inhibitory lectins were found to resemble those of supraoptimal doses of Con A. In particular, both effects were partly averted by the lymphocyte activating factor (LAF). The mitogenic effect of LAF was not inhibited by the non-mitogenic lectin, SBA, whereas the poor responses to PHA or to moderately supraoptimal doses of Con A were markedly potentiated by this factor. It is thus suggested that LAF activity counteracts the inhibitory processes provoked by the lectins.     

Wheat germ agglutinin and other selected lectins increase synthesis of decay-accelerating factor in human endothelial cells.

Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N-acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N-acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N-acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium.     

ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue

The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.