A series of monoclonal antibodies was used to define three discrete stages of human intrathymic T-cell differentiation. The earliest stage was confined to <10% of thymocytes, which were.reactive with both OKT9 and OKT1O. Subsequently, approximately 70% of human thymocytes acquired a thymocyte-restricted antigen, OKT6, lost OKT9 antigen, and expressed reactivity with OKT4 and OKT5. These last two monoclonal antibodies were previously shown to define inducer (helper) and cytotoxic/suppressor populations, respectively, in peripheral blood. The OKT4+, OKT5+, OKT6+ "common" thymocyte population represents the majority of thymocytes and accounts for more than 70% of thymocytes. With further maturation, thymocytes lose OKT6 reactivity, segregate into OKT4+ and OKT5+ subsets, and acquire reactivity with OKT3 (and OKT1). This latter stage corresponds to the more functionally mature subset. The possible relationship of acute lymphoblastic leukemia of T-cell lineage to these proposed stages of intrathymic differentiation was determined. Analysis of25 tumor populations showed that 21 could be related to one or another differentiative stage. The majority (15/21) were derived from an early thymocyte or prothymocyte subpopulation, 5/25 were derived from a common thymocyte subpopulation, and 1/25 was derived from a mature (OKT3+) subpopulation. These data suggest that is it now possible to define stages of T-cell differentiation that can be related to T-cell malignancies in humans.The importance of a thymic microenvironment in the differentiation and functional maturation of T cells has been demonstrated in several species. Moreover, profound changes in cell-surface antigens mark the various stages of T-cell ontogeny (1-7). For (12). These thymcytes were the most functionally mature and probably ready for peripheral exportation. Human peripheral T cells also consist of functionally distinct subsets (13)(14)(15)(16)(17)(18)(19).In the present study, we used a series of monoclonal antibodies reactive selectively with subpopulations of human thymocytes and inducer and suppressor T-cell subsets in order to further define stages of thymic differentiation (18, 19). We show that three major stages of intrathymic differentiation exist and that the majority of tumor populations from patients with acute lymphoblastic leukemia of T-cell lineage (T-ALL) can be shown to
Three novel nonoclonal antibodies (designed OKT1, OKT3, and OKT4) were generated against surface determinants of human peripheral T cells. Both OKT1 and OKT3 reacted with all human peripheral T cells and 5 to 10 percent of thymocytes but differed in their reactivities with T cel- lines. By contrast, OKT4 reacted with 55 percent of human peripheral T cells and 80 percent of thymocyted in that they did not react with normal B cells, null cells, monocytes, or granulocytes.
A polypeptide of 8500 molecular weight is described that induces the differentiation of T (thymusderived) cell and B (bone-marrow-derived) cell immunocytes in vitro, apparently via fl-adrenergic receptors and adenylate cyclase activation. This polypeptide shows a high degree of evolutionary conservation, exhibiting close structural, functional, and immunological similarity when isolated from such diverse origins as cells of mammals and higher plants. This polypeptide was detected in animal cells, yeast, bacteria, and higher plants, and so may well be a universal constituent of living cells.The isolation from bovine thymus of the polypeptide hormones thymopoietin I and II has been described previously (1 2). [We are now using the term thymopoietin (3) because valid objections (4) were raised to our previous term "thymin," which is easily confused with the pyrimidine base thymine. ] Pure thymopoietin I and II specifically induce prothymocytes to differentiate into thymocytes (5-7), but these polypeptides were recognized originally not by this evidently physiological faculty of T (thymus-derived) cell differentiation but by a presumably incidental property discovered in the course of studies related to the human disease myasthenia gravis, in which a characteristic neuromuscular disturbance is consistently associated with thymic pathology. When reliable assays of T-cell differentiation in vitro became available, based on the manifestation of T-cell antigens on precursor cells (5,8) it was realized that, although certain pure thymic products (thymopoietins) could induce this step in T-cell differentiation specifically, extracts of tissues other than thymus were also active (10).We describe here the isolation and purification from bovine thymus of a polypeptide subsequently detected in many other tissues, which like thymopoietin induces the differentiation of pro-thymocytes, but which unlike thymopoietin induces differentiation of B (bone-marrow-derived) cells, and is thus distinguished in its mode of action from the physiological inducer thymopoietin. This The isolation of UBIP from bovine thymus is summarized in Fig. 1. Bovine thymus was obtained fresh on wet ice, dissected clean and stored at -20°. Batches were homogenized 25% wet weight/volume in 0.1 M ammonium bicarbonate with a Waring blender. The extract was heated to 700 for 30 min and then centrifuged at 500 X g for 30 min. The supernatant was filtered through gauze and cotton and 0.1% thimerosal was added as a bacteriostatic agent, since the subsequent steps were carried out at room temperature. The extract was processed through a Diaflo XM1OOA membrane (Amicon) in a TCF10 apparatus. The dialysate was then concentrated over a Diaflo UM2 membrane at 55 lb./in.2, using a 402 stirred cell and a reservoir; 3 liters were concentrated to 15 ml. This retentate was fractionated on a 2.5 X 100-cm column of medium Sephadex G-50 (Pharmacia) in 0.1 M ammonium bicarbonate. The fractions shown in Fig. 1 were lyophilized and rerun on the same column. The lyophili...
A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and macrophages. The OKT4-T cell population contained the previously described TH2+ subset that has been shown to contain cytotoxic/suppressor cells. With cell-sorter separation of OKT4+ and OKT4-cells, it was shown that these T cell subsets were functionally discrete. Both-gave proliferative responses with concanavalin A, alloantigens, and phytohemagglutinin, although OKT4+ cells were much more responsive to the latter. OKT4f cells alone responded to soluble antigens whereas OKT4-cells alone were cytotoxic after alloantigenic sensitization of unfractionated T cells. However, both OKT4+ aid OKT4-cells were required during sensitization for optimal development of cytotoxicity. These data suggest that the OKT4+ subset represents a helper population and that the OKT4-subset contains the cytotoxic effector population. OKT4 could be a valuable reagent for determining alterations of these functional subsets in human diseases. T cell heterogeneity has been shown to exist in several species including man (1-6). Prior studies utilizing differential Fc receptor binding, autoantibodies, and heteroantisera have facilitated the dissection of human T cell populations into distinct subsets with unique functional properties (4-6). By the use of a heteroantiserum termed anti-TH2, approximately 20% of peripheral blood T cells were shown to be TH2+ (4). These TH2+ cells are phenotypically stable after activation and contain both the mature suppressor and cytotoxic effector cells (3, 4). The T cell population unreactive with anti-TH2 (TH2-) was heterogeneous with respect to binding with an autoantibody found in serum of juvenile rheumatoid arthritis patients (anti-JRA) (7). The TH2-subset was therefore divided into JRA+ and JRA-subsets. In addition, the TH2-subset of cells was shown to contain a helper population and could also modulate the generation of concanavalin A (Con A)-inducible suppressor cells (4).The recent development of monoclonal antibodies to cell surface antigens has provided an additional tool for the orderly dissection of subsets of T cells bearing distinct antigens (8-13). Recently, we described a hybridoma antibody that defined the entire human peripheral T cell population but was unreactive with lymphocytes of non-T lineage (14). In the present study, we have produced a hybridoma secreting monoclonal antibody reactive with a subset of human T cells. In the studies to be described, it will be shown that the hybridoma antibody, termed OKT4, reacts selectively with 55-60% of human peripheral blood T lymphocytes distinct from the TH2+ subset. (ii) Selection and growth of hybridoma. After cell fusion, cells were cultured in hypoxanthine/aminopterin/thymidine medium at 37°C with 5% CO2 in a humid atmosphere. Several weeks later, 40-100 MI of supe...
We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM1O, all directed against the C3bi receptor of human monocytes and macrophages (M4). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on MO adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from MO lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM1O binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from '251-surface-labeled M4. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human MO is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.The third component of complement, C3, binds covalently to cell surfaces (1) yielding a species, C3b, that is recognized by receptors on leukocytes (2). Surface-bound C3b is rapidly cleaved by the serum enzyme, I, to yield an altered form, C3bi, that is also recognized by receptors on leukocytes (3, 4). There are separate receptors for C3b and C3bi on human monocytes, and it has been shown that each type of receptor can independently mediate phagocytosis of C3b-or C3bi-coated particles (5). We are particularly interested in the receptors for C3 because their ability to promote phagocytosis is regulated: human macrophages (MO) bind but do not ingest C3b-or C3bi-coated erythrocytes, but MO readily ingest both C3b-and C3bi-coated erythrocytes after a brief treatment with the tumor-priomoting compound phorbol myristate acetate (5).Fearon (6) (8). Monoclonal antibodies against the human C3b receptor (antiC3bR) have been described (9), as have monoclonal antibodies, OKM1, OKM9, OKM10, and OKM5 (10). Fab fragments of 1B4 were prepared by papain digestion (11).Cells. Human blood monocytes were purified and cultured for 5-8 days in Teflon beakers as described (5). Cultured monocytes (MO) were surface labeled with 1"I by the lactoperoxidase-glucose oxidase procedure (12).Sheep erythrocytes bearing C3b (EC3b) or C3bi (EC3bi) were prepared as described (5). The attachment of EC3b or EC3bi to monolayers of phagocytes was scored visually in duplicate wells (5). T...
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