The Sheep Erythrocyte Receptor and Both α and β Chains of the Human T-Lymphocyte Antigen Receptor Bind the Mitogenic Lectin (Phytohaemagglutinin) from Phaseolus vulgaris

We have studied the interaction of mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A) with both surface molecules which, by the use of monoclonal antibodies, are known to trigger T-cell mitogenesis. Monoclonal antibodies recognizing the T-lymphocyte receptor for antigen (Ti) and/or its associated structure, CD3, activate T cells. More recently, a second pathway of activation has been described which involves the sheep erythrocyte binding glycoprotein CD2, a surface molecule distinct from Ti-CD3. Lysates from surface-iodinated T-leukaemia cell lines were treated with lectin and affinity purified anti-lectin antibodies coupled to protein A-Sepharose. We have shown that eluates from Con A/anti-Con A or PHA/anti-PHA immunoprecipitates contained Ti, since a rabbit anti-T alpha serum, which recognizes the native and denatured forms of the constant region of the alpha chain, immunoprecipitated Ti from these eluates. Furthermore, Ti immunoprecipitated by anti-T alpha serum from lysates of surface iodinated E+ lymphocytes was binding to PHA after elution from the immunoprecipitate. When the purified Ti molecule was reduced and alkylated, allowing the permanent dissociation of its alpha and beta subunits, PHA interacted with both chains, whereas anti-T alpha serum immunoprecipitated the alpha chain only. Altogether, these results demonstrate that PHA interacts with both chains of the T cell receptor for antigen on human peripheral T lymphocytes. With the HPB-ALL tumour line, a similar approach showed that both alpha and beta chains of Ti bind to Con A and Ulex europaeus 1 but not Helix pomatia. Affinity chromatography on immobilized lectins and immunoprecipitation with lectin/anti-lectin antibodies were employed to test whether CD2 binds to PHA and Con A. The results show that CD2 from human peripheral T lymphocytes binds both lectins but with a lower affinity for PHA than Con A.     

Human tonsillar T lymphocytes: an immature or activated T-lymphocyte population

In this study we compare the phenotype and the enzyme content of T lymphocytes purified from tonsils and from circulating blood before and different times after in vitro activation with PHA. The phenotype was studied with the following panel of monoclonal antibodies for the presence of activation antigens: OKT9, OKT10, anti-Dr, and Tac antigen. The enzyme content was studied by estimation of the LDH isoenzyme distribution and the enzymes of the purine metabolism ADA and PNP. In addition, the in vitro responsiveness of the T lymphocytes from tonsils and blood to human recombinant interleukin 2, to the lectins PHA, Con A and PWM was also studied. The LDH isoenzyme pattern expressed as B:A ratio decreases progressively after in vitro activation of T lymphocytes. The B:A ratio is already significantly different as early as 12 hr after activation, at a time that OKT9 and Tac are already expressed, but before the expression of OKT10, HLA-Dr, and the onset of the DNA synthesis. Tonsillar T lymphocytes exhibit also a significantly lower B:A ratio, but the activation antigens are not detected on their surface, except for the Tac antigens by means of a sensitive protein A rosette assay. Tonsillar T lymphocytes have a better responsiveness to lectins and recombinant II-2 than the corresponding circulating T lymphocytes. Therefore we are in favor of the hypothesis that tonsillar T lymphocytes contain activated T lymphocytes.     

Heterogeneity of concanavalin A-induced suppressor T cells in man defined with monoclonal antibodies.

Human peripheral blood T lymphocytes were enriched for OKT4+ or OKT8+ subpopulations using complement mediated lysis with OKT8 or OKT4 monoclonal antibodies. These subpopulations and unfractionated T cells were separately stimulated with concanavalin A (Con A) for a period of 48 hr and were then examined for their suppressive influence on proliferative response of autologous T cells to phytohaemagglutinin (PHA) or allogeneic non-T cells. Con A-activated unfractionated T cells, OKT4+ and OKT8+ T cell subsets markedly suppressed both these responses. Both OKT4+ and OKT8+ T cell subsets when enriched following Con A-activation of unfractionated T cells also caused significant suppression of proliferative responses of autologous T cells to PHA and allogeneic non-T cells in mixed lymphocyte cultures. The suppressive influence of Con A-activated T subsets was abolished by irradiation (2,000 rad) of activated cells. These studies indicate that Con A-induced suppressor T cells are heterogeneous. Precursors of Con A-induced suppressor T cells appear to reside in both OKT4+ and OKT8+ T cell populations.     

Mitogen-induced membrane changes and cell proliferation in T lymphocyte subpopulations.

Rabbit thymus-dependent lymphocytes were exposed to phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) or anti-immunoglobulin at various stages of maturation. Proliferation (induction of DNA synthesis) and early membrane events (turnover of membrane phospholipids) were measured in neonatal thymocytes, normal adult thymocytes, prednisolone-resistant thymocytes and lymph node lymphocytes. In immature thymocytes PHA induced only a marginal increase in DNA synthesis. The mitotic response increased with maturation, but only peripheral T lymphocytes exhibited maximum stimulation. Con A and PWM were able to induce DNA synthesis in immature thymocytes and the degree of stimulation was shown to increase with maturation. In contrast to the different degree of proliferation of thymocytes induced by PHA or Con A the incorporation of [14C]oleate, [14C]choline or [14C]acetate into phospholipids was stimulated to the same degree by these lectins. Reactivity of T lymphocytes, as measured by early membrane changes at different stages of maturation, to different T cell mitogens appears to be identical. Differences in degree of cell proliferation therefore may be secondary phenomena due, in part, to tissue culture conditions. Reactivity to mitogens as measured by phospholipid turnover appears to be an early acquired function in the maturation of lymphocytes of the T cell line.     

Inhibitory effect of somatostatin on human T lymphocytes proliferation

Somatostatin (SOM) was originally described as a growth hormone release inhibiting factor, but SOM and its specific receptors (SOM-r) have been shown to be expressed on both normal and activated T and B lymphocytes and other immunocompetent cells. In the present study we have demonstrated that SOM strongly inhibits the proliferation of human T lymphocytes when stimulated by PHA, Con A or alloantigens. However, SOM was most effective when the T cells were stimulated by an alloantigen rather than a polyclonal activator such as PHA and ConA. Moreover, SOM strongly inhibited the expression of activation markers such as CD69 and CD25 that are expressed on T lymphocytes during alloantigen stimulation. SOM also inhibited both CD28 and CD2 mediated T cell proliferation. Whereas proliferation of T cells induced by the engagement of CD3 antigen using specific mAbs was only marginally affected. Our results would support the concept that in humans SOM plays a key role in the modulation of T cell activation by interfering with the antigen-independent pathways CD2 and CD28.     

The Interaction of Nonmitogenic and Mitogenic Lectins with T Lymphocytes: Association of Cellular Receptor Sites

The relationship between the surface receptors on neuraminidase-treated human blood lymphocytes for the mitogenic lectins Phaseolus vulgaris leukoagglutnin (La), concanavalin A (Con A), and soy bean agglutinin (SBA) and the nonmitogenic lectin Helix Pomatia A hemagglutinin (HP) was investigated. Two different techniques, co-capping with different fluorochrome-labeled lectins and cell binding-inhibition experiments with 125 I-labled lectins, were used. The results demonstrated that the nonmitogenic lectin HP and the mitogenic lectins SBA, La, and Con A bind either to the same macromolecule(s) or to different but physically linked macromolecules on the surface of human T lymphocytes. In contrast, only part of β2-microglobulin (β2-m), or β2-m-bearing complexes, appear to be physically linked to the lectin receptor complex(es). On the lectin-binding substance(s) at least two saccharide structures were recognized, one of which binds both HP and SBA and another which hinds SBA and La (and probably also Con A) but not HP.     

T cell stimulation and expansion using anti-CD3/CD28 beads

Following appropriate stimulation, T lymphocytes will proliferate extensively in vitro. Traditionally, mitogenic lectins such as phytohemagglutinin (PHA) and concanavalin A (Con A) have been used for polyclonal T cell stimulation. A more physiologically relevant approach uses beads coated with anti-CD3 and anti-CD28 to stimulate T cells in a manner that partially mimics stimulation by antigen-presenting cells. This protocol describes the steps involved in T cell stimulation and their subsequent in vitro expansion using anti-CD3/CD28 beads.     

T-Cell Chemiluminescence

The binding of mitogenic lectins phytohaemagglutinin (PHA), concanavalin A (Con A) and/or of monoclonal antibodies to different receptors such as antigen receptor complex or CD2 on human T cells generates increases in the concentrations of inositol triphosphate (IP3) and cytoplasmic free calcium. This T lymphocyte requires the delivery of two signals; the first can be provided by specific monoclonal antibodies or by mitogenic lectins, and the second by a phorbol ester, phorbol myristate acetate (PMA). In other cells such as macrophages, the rise of intracellular calcium via the generation of IP3 and stimulation of protein kinase C can activate the phospholipase A₂, a calcium-dependent enzyme. This enzyme initiates the release of reactive oxygen intermediates and metabolites of arachidonic acid. In order to know whether this other metabolic pathway can be generated in T cells, we tested the capacity of different T-cell lines and clones to produce superoxide anion after stimulation by the above-mentioned activating agents. In this paper, we demonstrate that treatment of the Jurkat human cell line with Con A, PHA, and PMA results in a significant release of reactive oxygen metabolites. Of the various T-cell lines and clones tested, only Jurkat exhibited an oxidative burst. Moreover, none of the antibodies tested (anti-CD3, anti-CD2, and anti-CD28) and known to activate T cells, and none of the immune complexes was able to mediate such an effect. The existence of an oxidative metabolism in at least one T-cell line suggests that T-cell activation may in some instances use another metabolic pathway.     

Surface phenotype of T cells producing growth of mucosal mast cells in normal rat bone marrow culture.

We have previously shown that lymphocytes from Nippostrongylus brasiliensis infected rats, when stimulated with antigen or concanavalin A (Con A) release factors which are comparable with murine IL-3. On addition of these factors to rat bone marrow cultures, mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) proliferate and mature. Here, we use this system, along with monoclonal antibodies against rat T cells and the fluorescence-activated cell sorter (FACS), to isolate the subset of T cells responsible for the production of this MMC growth factor. Lymphocytes from N. brasiliensis infected rats were separated on the FACs into populations with and without the antigens defined by OX19, W3/25 and OX8 monoclonal antibodies; these antibodies label all T cells, T-helper cells and T-cytotoxic/suppressor cells, respectively. The resultant subsets were cultured in vitro with Con A. The supernatants were tested for the ability to induce MMC growth and differentiation in liquid cultures of normal rat bone marrow. The phenotype of the T cells producing this factor was established as being OX19+, W3/25+ and OX8-.     

beta2-Microglobulin production by highly purified human T and B lymphocytes in cell culture stimulated with various mitogens.

This study attempts to evaluate beta2-microglobulin production by highly purified (greater than 98%) peripheral and tonsil T and B lymphocytes cultured with various mitogens. beta2-Microglobulin was measured by the radioimmunoassay method. It was found that PHA and Con A markedly stimulated beta2-microglobulin production in cultures of T but not B lymphocytes. B lymphocytes were greatly activated, on the other hand, by Staphylococcus aureau Cowan I organisms cSpA), though the level of beta2-microglobulin production was less than that observed in PHA- and Con A-stimulated T lymphocytes. PWM only slightly increased beta2-microglobulin production of T lymphocytes, although the incorporation of [3H]-thymidine was highly enhanced. The highest level of beta2-microglobulin obtained with PHA or Con A was observed when the T/B lymphocyte ratio was between 90/10 and 80/20. These results lead to the conclusion that: (1) SpA is a specific mitogen for B lymphocytes, and its mitogenicity is independent of the presence of T lymphocytes, while PHA, Con A, and PWM are ineffective as stimulants of B lymphocytes; (2) the beta2-microglobulin producing ability of B lymphocytes is less than that of T lymphocytes, even when the lymphocytes are markedly activated; (3) the beta2-microglobulin production and DNA synthesis by T lymphocytes is markedly enhanced by the helper effect of B lymphocytes; (4) the level of beta2-microglobulin production reflects lymphocyte activation, especially in T lymphocytes stimulated with PHA or Con A.     

Selective effects of vasoactive intestinal peptide on the mitogenic response of murine T cells.

Murine lymphocytes have been shown previously to possess high-affinity specific receptors for the neuropeptide vasoactive intestinal peptide (VIP). This study examines the cellular basis for modulation of concanavalin A (Con A)-induced T-cell responses by this neuropeptide. VIP was most effective as an inhibitor when added at the initiation of the mitogen response. The loss of potency when VIP was added later in the response was accompanied by a decrease in the affinity of stimulated cells for the neuropeptide. The inhibitory influence of VIP was reversible if the neuropeptide was removed from stimulated cell cultures up to 6 hr after the initiation of stimulation. In contrast, VIP-mediated inhibition was fully developed once the stimulated cells had been exposed to the neuropeptide for 24 hr. The presence of VIP led to a decreased production of interleukin-2 (IL-2) by the stimulated lymphocytes, but did not affect the expression of Con A-induced suppressor cell activity by cultured lymphocytes. Studies of the effect of selective, complement-mediated killing of cells with Thy 1, L3T4 and Lyt 2 monoclonal antibodies showed that the majority of the VIP bound by the lymphocytes was accounted for by binding to L3T4+, Lyt 2- T cells. It was concluded that VIP exerts its influence over Con A-stimulated proliferation by selective regulation of T-cell subsets.     

Immunological studies of ageing. X. Impaired T lymphocytes and normal monocyte response from elderly humans to the mitogenic antibodies OKT3 and Leu 4.

Lymphocytes from old and young humans were cultured with PHA or the monoclonal antibodies OKT3 or Leu 4. The incorporation of [3H]TdR was significantly lower in cultures from old as compared to young donors, and the response of lymphocytes stimulated with OKT3 was the best discriminator of donor age. The mitogenic response of lymphocytes to these monoclonal antibodies requires monocytes. The response of T cells containing less than 5% adherent cells was diminished and the difference between old and young donors was not seen. The age-associated response was recovered when autologous or allogeneic monocytes were added to T cells. The age-associated response of T cells was the same, whether cultured with monocytes from young or old donors. Thus, monocytes from elderly subjects are not impaired with respect to their capacity to facilitate the proliferative response of T cells stimulated with monoclonal antibodies. Although lymphocytes from elderly donors were more sensitive to the inhibitory effect of prostaglandin E2, this did not account for the age-associated defect as indomethacin did not eliminate this defect. We conclude that the proliferative response of lymphocytes to OKT3 and Leu 4 is a more sensitive discriminator of lymphocyte donor age than is response to plant lectins, and that the age-associated defect in this response appears to reside within the T-cell population and not the monocyte population.     

Rabbit leukocyte surface antigens defined by monoclonal antibodies

Several monoclonal antibodies (mAb) against rabbit leukocytes were characterized in binding and functional studies. mAb 1.24 stains thymocytes, bone marrow cells, peripheral T and B cells and blood monocytes. T cells express more 1.24 antigen than B cells. In the absence of added complement (C), mAb 1.24 inhibits alloantigen-, concanavalin A (Con A)-, and phytohemagglutinin (PHA)-, but not pokeweed mitogen (PWM)- or anti-immunoglobulin (Ig)-induced cell proliferation. It also strongly blocks anti-sheep erythrocyte plaque-forming cell responses. A second mAb, designated 4.B9, binds to 20% of thymocytes and to most, if not all, peripheral T cells and in vitro-activated T cell blasts. A third one, 10.B3, is reactive with the nearly entire thymocyte and a major peripheral T cell population. Two-color membrane immunofluorescence reveals the presence of a small population of peripheral blood leukocytes which bear surface Ig and are weakly stained by mAb 4.B9 and 10.B3. Without C, both 4.B9 and 10.B3 inhibit Con A- and PHA-induced mitogenesis, but have no effect on PWM-, antigen-, or alloantigen-induced cell proliferation. Depletion of 4.B9+ cells by panning or complement lysis completely abrogates proliferative responsiveness to antigen and alloantigen, significantly reduces responsiveness to the T cell mitogens Con A and PHA, but enhances that to the B cell mitogen anti-Ig. A fourth mAb, 12.C7, binds to 60% of thymocytes and to 10-30% of peripheral T lymphocytes at high-level fluorescence. T cell blasts obtained in mixed leukocyte reactions are partially stained by mAb 12.C7, while those obtained after Con A or PHA activation are not. In addition, mAb 12.C7 is completely unreactive with B cells or monocytes. Without complement, it does not seem to interfere with any of the in vitro functions tested. All antigens studied here do not appear to be expressed in nonleukon tissues, as they do not bind to erythrocytes and are absent from brain, heart, liver and kidney as shown by quantitative absorption analysis.     

Role of the Ly 1 antigen in interleukin 1-induced thymocyte activation

Interleukin 1 (IL 1), but not IL 2-induced thymocyte proliferation was augmented by monoclonal antibodies (mAb) against the Ly 1 antigen. The anti-Ly 1 mAb could also co-stimulate thymocytes with phytohemagglutinin (PHA) alone, mimicking the effect of IL 1. Moreover, overnight culture with PHA of thymocytes, but not of spleen T lymphocytes led to selectively enhanced expression of the Ly 1 antigen on the cell surface. Similar data were generated at the clonal level. Thus, a thymocyte hybridoma (22A6) could be stimulated to release IL 2 in response to PHA plus IL 1, but also to PHA plus anti-Ly 1 mAb. In addition, 22A6 exhibited enhanced cell surface expression of the Ly 1 antigen after overnight culture with PHA. These data suggest a critical role for the Ly 1 antigen in thymocyte proliferation, perhaps by serving as an IL 1-receptor.     

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.     

Different pathways of human T-cell activation revealed by PHA-P and PHA-M.

Antigen-specific T-cell activation is mediated via the CD3-Ti (antigen receptor) complex, and monoclonal antibodies to both CD3 and Ti cause a rapid rise in intracellular Ca2+. This calcium mobilization is not inhibited by monoclonal antibodies to CD2. The rise in calcium mobilization induced by purified PHA (PHA-P) does not occur in a cell line which lacks CD2 expression, and can be blocked in other T cells by anti-CD2 antibodies. A combination of monoclonal antibodies to different epitopes of CD2 causes calcium mobilization and mitogenesis. Reagent grade PHA (PHA-M) induces calcium moblization in cells that lack CD2, and its effects in other T cells cannot be blocked by anti-CD2 antibodies. The effects of PHA-P and PHA-M are thus mediated predominantly through different activation pathways.     

Determination of the T3-3A1 antigen in PHA-induced human T-cells by standardized cell-ELISA

The treatment of mononuclear leukocytes (MNL) with lectins induces marked changes in the cell's morphology, physiology and the composition of the cell surface. We used an immunoassay to monitor the PHA-induced expression of the T-lymphocyte-specific antigen T3-3A1 in fixed MNL with a monoclonal antibody (MoAb) specific for this antigen. This assay permits the detection and quantitation of the T3-3A1 antigen in a few thousand cells without the use of a FACS. The test was calibrated with isolated plasma membranes and, combined with a total protein determination, the relative content of T3-3A1 antigen in each sample could be calculated. Maximal T3-3A1 synthesis required a 10-fold lower concentration of PHA than was necessary for optimal DNA synthesis. The test may be used to screen for PHA stimulation.     

Analysis with monoclonal antibodies of human lymphoid cells forming rosettes with rabbit red blood cells.

Rabbit red blood cells have previously been shown to rosette with a subpopulation of thymocytes and with mitogen activated peripheral lymphocytes but not with unstimulated lymphocytes. Using monoclonal antibodies and double marker assays we studied the phenotype of these cells. In thymus, over 90% of rosetting cells express antigens of immature thymocytes (HTA1, OKT6). A proportion of the rosetting cells shows in addition antigens of mature thymocytes (OKT3, UCHT1). These cells probably correspond to a stage of intrathymic maturation between common and mature thymocytes. Virtually all rosetting cells are T cells and express an antigen related to T cell activation (TAC) when lymphocytes are activated by mitogens like PHA or Con A. Few rosetting cells are Ia positive. Two other antigens (OKT9, OKT10) known to be associated with proliferating and immature cells, are found in variable proportions on rosetting cells. After stimulation with allogeneic lymphocytes, fewer rosettes are detected than after stimulation by mitogens. Cells activated by a soluble antigen (PPD) and forming rosettes with rabbit red blood cells have a helper phenotype (Leu3a positive). Screening of leukaemia cell samples revealed that only cells from patients with T-ALL form rosettes with rabbit red blood cells. Rosette formation is almost totally inhibited by a polyclonal anti-thymocyte serum and two monoclonal antibodies (OKT11A,Lyt3) which have been shown to block rosettes with sheep erythrocytes.     

A Monoclonal Antibody, H2, Defines a New Surface Antigen Expressed on Human Lymphocytes

We have previously described a monoclonal antibody (MoAb), H2, which recognized a tumour-unique antigen on a human T-cell chronic lymphatic leukaemia (T-CLL, CD3,4+). However, further characterization of H2 has revealed a reactivity with the majority of T lymphocytes and a minority of B lymphocytes, some malignant T cells and a few cell lines of leukaemia or of hematopoietic tumour origin. The molecular weight of the antigen (80,000) precipitated by the MoAb H2 from the cell lines NALM-6 and Reh corresponded to that previously found. When PBL were stimulated with PHA, IL-2, or Con A a reduced reactivity of H2 could be seen. The MoAb H2 was submitted to the Fourth International Conference on Human Leucocyte Differentiation Antigens, Vienna, 1989. H2 did not cluster in any of the 78 clusters of differentiation (CD 1-78) discussed at the conference, indicating its unique reactivity. This suggests that we have defined a new antigen on lymphocytes with a possible role along the resting-proliferating axis.     

刀豆凝集素单克隆抗体特性分析和初步应用

建立了一株能分泌抗刀豆凝集素(ConA)单克隆抗体(McAb)的细胞株-CA2。ELISA证实所得 CA2 McAb仅与 ConA有反应,而与菜豆凝集素(PHA)、美洲商陆(PWM)、花生凝集素(PNA)、扁豆凝集素(LcA)以及牛血清白蛋白(BSA)都无交叉反应。免疫印迹试验进一步证实该McAb 只能与 ConA 形成免疫沉淀线。该McAb 能抑制 ConA对RLc-802细胞的凝集作用,竞争抑制试验显示α-甲基甘露糖、果糖不能抑制 CA2 McAb与 ConA 的结合,提示它可能是针对 ConA蛋白结合点部位抗原决定簇的 McAb。应用该McAb证实了急性T淋巴细胞白血病细胞株Molt_4、CEM和B淋巴母细胞CCRF-sb以及转化的扁桃腺淋巴细胞表面膜存在有分子量大小不同的 ConA受体,提示同是ConA受体,可能具有不同的生物学功能。