Late apoptotic effects of taxanes on K562 erythroleukemia cells: apoptosis is delayed upstream of caspase-3 activation.

The efficacy of taxanes on human leukemia cells is the object of intensive in vitro investigation concerning the influence of cell-type-specific characteristics on cytotoxic response to drugs. The present study dissects the response to taxanes of HL60 acute myelomonocytic leukemia and of K562 chronic myelogenous leukemia, in parallel over a 72-hr time-span. The kinetics of cytotoxicity following pulsed and continuous exposure to either taxol or taxotere showed a delayed response of K562 cells independently of dose and type of exposure. In K562 cells, apoptosis became evident at 48 hr and prominent at 72 hr of treatment. These events were mirrored by delayed kinetics of caspase-3 activation. Comparable microtubule targeting was demonstrated in HL60 and in K562 cell lines, as bcl-2 and raf-1 were phosphorylated following treatment with taxanes. These observations indicate that early activation processes were responsible for apoptosis, but that the delay was determined by other factors. In addition, cell-free-system experiments excluded the presence of excess nuclear and/or cytoplasmic inhibitory factors and demonstrated that K562 cells possess a fully competent caspase system which can be readily activated. Processing of caspase-3 pro-enzyme was in fact increased by addition of cytochrome c. These results extend to taxol and taxotere the notion that drug-induced apoptosis is delayed upstream of caspase-3 activation in K562 cells, that such kinetics is independent of drug concentration and exposure time, and that it is linked to intrinsic cellular characteristics mapping between bcl-2 phosphorylation and cytochrome c release.

Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation.

Tissue transglutaminase (tTG) is a Ca2+-dependent cross-linking enzyme that participates in the apoptotic machinery by irreversibly assembling a protein scaffold that prevents the leakage of intracellular components. In the present study a single-chain antibody fragment (scFv) detecting tTG is described. We demonstrate that TG/F8 scFv, selected from a phase display library of human V-gene segments by binding to guinea-pig liver tTG, can react with human tTG both in Western blot and in immunohistochemistry. The specific detection of tTG by TG/F8 in human thymocytes is verified by mass spectrometric analysis of the purified protein. Furthermore, we demonstrate that in lymphoid cells tTG is cleaved by caspase 3 during the late phase of apoptotic death, concomitant to DNA fragmentation, and that such cleavage causes loss of cross-linking function. We propose tTG cleavage as a valuable biochemical marker of caspase 3 activation during the late execution phase of apoptosis.

8B4/20, a private CD43 epitope on developing human thymocytes, is involved in thymocyte maturation.

The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3-/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We therefore used specific Ab to mimic putative 8B4/20 interactions with natural ligand and examined the effect on isolated thymocytes. Treatment with 8B4/20 had no effect on in vitro apoptosis of isolated thymocytes. In contrast, 8B4/20 ligation enhanced the conversion of isolated thymocytes to differentiated phenotypes. Increased numbers were found in 8B4/20-treated cultures of CD3high and single-positive thymocytes and decreased numbers of CD3-/low and double-positive thymocytes, strongly suggesting that engagement of 8B4/20 delivers a positive signal that favors completion of the thymocyte maturation program. The ability of 8B4/20 mAb to drive thymocyte maturation in vitro suggests that CD43 molecules bearing the 8B4/20 epitope participate in early events of thymic selection.

A single protein immunologically identified as CD38 displays NAD+ glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes.

The three ectoenzyme activities, NAD+ glycohydrolase, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase were purified to homogeneity from solubilized human erythrocyte membranes. The purification procedure involved three sequential chromatography steps on hydroxylapatite, immobilized Cu++ and immobilized anti-CD38 monoclonal antibody resins. The final step yielded a single 46 kDa protein displaying all three enzymatic activities. Since the protein bound specifically to the anti-CD38 resin, it was immunologically identified as CD38, a 46 kDa surface antigen involved in activation and proliferation of lymphocyte populations.

CD69 molecule in human neutrophils: its expression and role in signal-transducing mechanisms.

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes and it is constitutively expressed on thymocytes and platelets. Here we report its presence on neutrophils and on bone marrow-derived myeloid precursors. Indeed, promyelocytic cells are CD69+ on the cell membrane, while in resting neutrophils this molecule is located inside the cell. However, intracellular CD69 molecules are rapidly mobilized to the cell surface upon activation by PMA or fMLP. This translocation is independent on a new protein synthesis, as it is not inhibited by cycloheximide; furthermore, CD69 molecules are likely stored in a trans-Golgi structure since their expression is not affected by brefeldin A, a drug that blocks molecular trafficking from ER to Golgi vesicles. Immunoprecipitation of CD69 molecules either from activated neutrophils or from bone marrow cells showed that this protein has the same molecular size (28-34 kDa) as observed in platelets, T and B lymphocytes, and thymocytes. This similarity is reflected also in the functional role played by this molecule: in neutrophils as well as in lymphocytes and platelets, CD69 stimulation induced Ca2+ influx through cellular membrane; furthermore, the perturbation of the CD69 antigen on PMA-activated neutrophils enhances the lysozyme release, suggesting a role of this molecule in the regulation of granule exocytosis, probably through a Ca(2+)-dependent mechanism.

CD69 in resting and activated T lymphocytes. Its association with a GTP binding protein and biochemical requirements for its expression.

CD69 is a phosphorylated disulfide-linked homodimer that appears on the surface of human T, B cells and thymocytes in the early steps of activation; its molecular mass is 28 to 34 kDa under reducing conditions. This molecule is able to mediate positive signals to the lymphocytes as the anti-CD69 mAb (MLR3, AIM, Leu 23) in synergism with phorbol esters induce IL-2 production and proliferation of lymphocytes. Here we show that this molecule is associated to a GTP binding protein that is a substrate for Bordetella pertussis toxin. The relevance of CD69 in the activation process is also suggested by the broad range of signals able to modulate CD69 on T cells. In fact, not only the mitogens or the CD3-promoted activation, but also the alternative pathways mediated by CD2 or CD28 are accompanied by CD69 expression; moreover a very rapid and transient appearance of CD69 on the cell surface is observed also in response to a stimulus not specifically involved in T cell activation such as heat shock. Finally we demonstrate that CD69 is present in the cytoplasm of nonactivated T cells; accordingly its surface expression at the onset of activation is independent on a new RNA or protein synthesis.

MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors.

MLR3 molecule is a membrane glycoprotein (mol. mass range 28-34 kDa) present on activated, but not resting human peripheral T cells, B cells and thymocytes. Its kinetics of appearance on the cell surface (3 h after the addition of the inductive signal to the cells) suggests that it is an early activation antigen. The proliferative response of cultured T and B lymphocytes and thymocytes to different activation signals is inhibited by the addition of MLR3 monoclonal antibody. Moreover the antibody in combination with non-mitogenic doses of phorbol myristate acetate leads to proliferation of thymocytes and resting B and T lymphocytes. In the latter, synthesis of interleukin 2 is also induced. Biochemical analysis of MLR3 antigen indicates that it is a phosphorylated protein with N-linked sugar moieties. Together these data suggest a role for MLR3 antigen in the signal transduction process during activation, both for mature lymphocytes and for T cell precursors.

Functional and biochemical characterization of a human T-cell antigen related to the T3-Ti activation pathway.

In this report we studied the antigen identified by the 5/9 monoclonal antibody. This antigen is expressed on approximately 15% of resting T lymphocytes with helper activity and increases following T-cell activation both in vivo and in vitro. The 5/9 monoclonal antibody triggered T-cell proliferation in the presence of suboptimal doses of phorbol12-myrisate 13-acetate (PMA) but this effect was strongly inhibited by antibody-induced modulation of the CD3 T-cell-receptor complex. The observation that a number of T-cell lines were brightly stained by the 5/9 monoclonal antibody after being activated with phytohemagglutinin (PHA) and PMA allowed the molecular characterization of the 5/9 antigen as well as the analysis of the biochemical mechanisms occurring after cell stimulation with 5/9 monoclonal antibody (Mab). An activated Jurkat T-cell line was labeled with 125I on the membrane: the monoclonal antibody immunoprecipitated a molecule displaying an apparent molecular weight of 34 kDa. In addition, 5/9 molecules, purified by immunoprecipitation from Jurkat cells, were found associated to a Ca2+ phospholipid-dependent protein kinase activity.

Functional studies of T4+ lymphocyte subsets distinguished by the monoclonal antibody 5/9: suppressor-effector T4+ lymphocytes derive from the 5/9+ subset.

Human T lymphocytes bearing the cell surface antigen T4 are functionally heterogeneous, exerting helper/inducer, suppressor-inducer, suppressor-effector, and cytotoxic activities. Other cell surface antigens with a more restricted expression may help separate T4+ lymphocytes into functionally distinct subsets. This report describes the regulatory functions of T4+ lymphocytes fractionated by the monoclonal antibody 5/9, which detects a cell surface antigen present on 50-60% of T4+ lymphocytes. The results indicate that both 5/9+ and 5/9- T4 subsets contain helper/inducer and suppressor-inducer cells. Suppressor-effector activity, however, is found predominantly within the 5/9+ T4 subset. The 5/9 antibody thus identifies the suppressor-effector subset of T4+ lymphocytes, although it does not distinguish between T4+ cells with or without helper/inducer and suppressor-inducer functions.

Functional characterization of an antigen involved in an early step of T-cell activation.

An activation antigen, identified by the monoclonal antibody MLR3, is described that is present on activated T lymphocytes and thymocytes but not on resting T lymphocytes. Immunoprecipitation of radiolabeled membranes from an activated T-cell line showed that the MLR3-binding molecule has a molecular size of 28-34 kDa. Immunofluorescence analysis showed that the appearance of the MLR3 antigen is an early event and precedes that of the interleukin 2 receptor both in T lymphocytes and thymocytes. The proliferative response of resting T cells to OKT3-Sepharose and interleukin 1 or accessory cells, but not the interleukin 2-dependent proliferation, was inhibited by the addition of MLR3 monoclonal antibodies. Similar results wer also obtained in an interleukin 1-dependent human thymocyte proliferation assay. In addition when MLR3-positive cells were cultured with purified interleukin 1, MLR3 surface antigen expression was not observed. Thus MLR3 monoclonal antibody appears to recognize an antigen involved in an early step of T-cell activation related to interleukin 1-dependent functions and on both T lymphocytes and thymocytes.

Functional characterization of a regulatory human T-cell subpopulation increasing during autologous MLR.

The present study was undertaken to investigate the heterogeneity of helper T cells in humans using two different monoclonal antibodies: 5/9 and MLR4. The former identifies 15-20% of resting T lymphocytes from peripheral blood and corresponds to an anti-helper/inducer T cell. The second antibody, MLR4, recognizes 5% of total T lymphocytes and partially overlaps with the 5/9+ T cells. In order to investigate functional differences within the 5/9+ cells, we separated two different subsets (5/9+ MLR+ and 5/9+ MLR4-) by a rosetting technique. Although both subsets provide help for Ig synthesis in a PWM-stimulated culture, only the 5/9+ MLR4- fraction gave a proliferative response in both autologous and allogeneic MLR and to soluble protein antigens. The effect of radiation on the ability of the two subsets to provide help for Ig synthesis showed that the 5/9+ MLR4+ subset is highly radiation-sensitive, while 5/9+ MLR- is relatively radiation-resistant. In a further series of experiments, 5/9+ MLR4+ cells isolated after activation in an autologous MLR but not by Con A, were no longer able to induce T-cell differentiation but now showed a strong suppressor effect. The 5/9+ MLR4- subset separated from the same cultures did not display any suppressor function. These data demonstrate in fresh PBL the existence of a radiation-sensitive regulatory subset exerting a helper activity, and which acquires suppressor activity after activation in autologous MLR.

The identification of monoclonal class switch variants by sib selection and an ELISA assay.

Monoclonal antibodies can now be generated against a wide variety of antigens. However, a potentially useful monoclonal antibody may be of the wrong class or subclass for a particular task. Antibodies of the desired class can be obtained by identifying rare subclones in which the variable region has been rearranged to a new constant region. We describe here the use of sib selection and an enzyme-linked immunoassay (ELISA) for isolating such class and subclass switch variants from 2 IgM and 1 IgG3 producing hybridoma. The relative simplicity of ELISA assays makes it feasible to apply this approach to many different hybridomas. Since commercial affinity purified class and subclass specific antibodies are now available, the method can be used by any laboratory. Furthermore, the technique does not require that the switch variants express surface immunoglobulin and enriches for hybridomas secreting higher amounts of antibody.

Thyroglobulin-induced T-cell in vitro proliferation in Hashimoto's thyroiditis: identification of the responsive subset and effect of monoclonal antibodies directed to Ia antigens.

Recently it was reported that the peripheral blood and thyroid gland of patients with Hashimoto's thyroiditis contain activated (Ia+ and/or MLR4+) T cells and high levels of 5/9+ ("helper") T lymphocytes. In normal individuals the 5/9 monoclonal antibody recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5/9+ and 5/9- T lymphocytes were isolated from patients with Hashimoto's disease, and the proliferative response induced by human thyroglobulin was investigated. The results show that the total thyroglobulin-induced lymphocyte DNA synthesis is confined to the 5/9+ T-cell fraction. Further subfractionation of 5/9+ into MLR4+ and MLR4- cells clearly indicates that no substantial differences exist in their proliferative capacities. Whether 5/9, MLR4, and Ia antigens, all expressed on the thyroglobulin-responsive T-cell subset, are involved in thyroglobulin-induced cell proliferation, was also analyzed. Although both 5/9 and MLR4 monoclonal antibodies had no effect, complete inhibition of antigen-induced blastogenesis was observed upon addition of monoclonal antibodies (D1/12 and BT2/9) directed to common determinants of Ia antigens. This inhibitory effect was also observed when T or non-T fractions were separately incubated with the monoclonal antibodies before culture. These results indicate that in humans, as in animals, the major histocompatibility complex may play a role in autoimmune thyroiditis. The data show that (a) the thyroglobulin-induced proliferative response is confined to a subset (5/9+) of T lymphocytes and (b) Ia antigens are involved in thyroglobulin-induced lymphocyte DNA synthesis in Hashimoto's disease.

Pulmonary sarcoidosis: excess of helper T lymphocytes and T cell subset imbalance at sites of disease activity.

Different lymphocyte subpopulations have been evaluated in bronchoalveolar fluid and blood obtained from six patients with active and six with inactive pulmonary sarcoidosis and from six normal subjects by means of two recently described monoclonal antibodies, 5/9 and MLR4. The percentages of OKT4 positive (helper) and OKT8 positive (suppressor) T cells were also determined. Patients with active sarcoidosis had significantly higher proportions of 5/9 positive T cells in the bronchoalveolar fluid than patients with inactive disease (p less than 0.01) or normal subjects (p less than 0.001). In contrast, the proportions of 5/9 positive blood T cells were similar in the three groups studied. Patients with active sarcoidosis had also a greater proportion proportion of MLR4 positive T lymphocytes in bronchoalveolar fluid than patients with inactive disease or normal subjects (p less than 0.01 for each comparison), but similar proportions of MLR4 positive blood T cells were found in each group. The ratio of 5/9 positive to MLR4 positive T cells was higher in the bronchoalveolar fluid (but not in the blood) in patients with either active or inactive sarcoidosis than in normal subjects. These observations suggest that the MLR4 negative fraction rather than the MLR4 positive fraction of the 5/9 positive T cells is preferentially expanded in the lungs of patients with pulmonary sarcoidosis and may indicate a secondary role for the MLR4 positive T cells in producing lung injury in this disorder. Comparisons of the OKT4 positive and 5/9 positive T cells showed that in patients with active disease most of the lung T lymphocytes expressed both the OKT4 and the 5/9 surface antigens, so the 5/9 monoclonal antibody may be considered a good marker of activity in this disorder. Pulmonary sarcoidosis may be characterised by the preferential expansion of helper T cell subsets at sites of disease activity.

A monoclonal antibody to platelet glycoproteins IIb and IIIa complex: its use in purifying human megakaryocytes from sternal bone marrow aspirates for immunofluorescence studies of Ia-like antigens.

A monoclonal antibody (PBM 6.4) to platelet and megakaryocyte glycoproteins IIb and IIIa has been obtained and used to purify human megakaryocytes from sternal bone marrow aspirates by a simple method, consisting of a Percoll gradient centrifugation followed by affinity adherence on PBM 6.4-coated plastic surface, "panning." Megakaryocytes, 80-90% pure and morphologically well preserved, were attached to poly-L-ornithine-coated multi-well microscope slides and immunofluorescence was done using monoclonal antibodies to human Ia-like antigens (DR, DC1). A higher proportion of DR-positive megakaryocytes was found, in comparison to the values reported by others, while DC1 antigen was detected on about 20% of megakaryocytes. The method described has the unique feature of enriching cells of the human megakaryocytic lineage from simple diagnostic sternal aspirates in an amount adequate for immunofluorescent and morphological analysis.

Defect of T helper lymphocytes, as identified by the 5/9 monoclonal antibody, in patients with common variable hypogammaglobulinaemia.

Peripheral blood lymphocytes from 17 patients with common variable hypogammaglobulinaemia (CVH) were tested for reactivity with the 5/9 monoclonal antibody which reacts with about 15% of normal T-PBL in which all helper activity is found. In PBL from CVH patients, the proportions of OKT4 and OKT8 positive cells were also determined. Five patients had normal percentages of 5/9 cells and a normal OKT4/OKT8 ratio. Twelve patients had significantly decreased (or absent) 5/9 lymphocytes. Among these, five had decreased 5/9 cells and a normal OKT4/OKT8 ratio and seven had decreased 5/9 cells and an inversion of the OKT4/OKT8 ratio. The deficiency of the helper phenotype T cell subpopulation identified by the 5/9 monoclonal antibody in many patients with CVH may be relevant in the pathogenesis of this disease.

A monoclonal anti-DC1 antibody selectivity inhibits the generation of effector T cells mediating specific cytolytic activity.

The products of the DC locus have been shown to be structurally different from those of the DR locus. In this paper it is shown that, unlike anti-DR antibodies, a monoclonal antibody directed against DC1 does not affect proliferation of T cells in response to alloantigens or soluble antigens or production of Ig in a pokeweed mitogen-stimulated in vitro culture. However, the anti-DC1 inhibits the generation of effector T cells mediating specific cytolytic activity, whereas no inhibitory effect can be observed on natural killer and antibody-dependent cytotoxic cell activities.

In vitro production of IgE by human peripheral blood mononuclear cells. III. Demonstration of a circulating IgE-bearing cell involved in the spontaneous IgE biosynthesis.

The presence of surface membrane IgE (SmIgE)-bearing cells in the peripheral blood (PB) of atopic patients was investigated by the use of isotype-specific rosettes of human red blood cells coupled to immunosorbent-purified rabbit or monoclonal mouse antibodies against human IgE (R or M anti-epsilon-HRBC). After dissociation of cell bound IgE by treatment with acid buffer, 2.1 +/- 0.3% and 1.2 +/- 0.3% circulating non-T, non-phagocytic, cells from atopic patients were still capable of forming rosettes with R or M anti-epsilon-HRBC, respectively. IgE molecules detectable on cells after dissociation of cytophilic IgE were quite resistant, like surface membrane IgM (SmIgM), to treatment with proteolytic enzymes, but they were removed under capping conditions by soluble anti-IgE antisera. All SmIgE-bearing (IgE+) cells also bore DR determinants, but many of them lacked SmIgM. Depletion of IgE+ cells strongly reduced the ability of PB lymphocyte suspensions from atopic patients to produce spontaneously IgE protein in vitro. Likewise, depletion of cells bearing DR determinants (DR+ cells) resulted in a marked decrease of the spontaneous IgE biosynthesis, whereas depletion of SmIgM-bearing (IgM+) cells had no effect. These data suggest that cells mainly implicated in the spontaneous IgE production in vitro seen in atopic patients are DR+ IgE+ IgM- circulating lymphocytes.