A monoclonal antibody to a novel differentiation antigen on human macrophages associated with the down-regulatory phase of the inflammatory process

A monoclonal antibody (RM3/1), raised by immunizing mice with human monocytes, is described which detects a surface antigen on about 20% of freshly isolated peripheral blood monocytes and is increasingly expressed upon cultivation, reaching a maximum between day 2 and 3. By incubation of monocytes with interferon-gamma, 12-O-tetradecanoylphorbol-13-acetate and lipopolysaccharide, antigen expression is decreased but strongly enhanced after incubation with dexamethasone. In cryostat sections of normal tissue, the antibody detects histiocytes in the skin, Kupffer cells in the liver, few alveolar macrophages in the lung, macrophages in the red pulp of the spleen and in the cortex of the thymus, and many macrophages in the placenta. In acute inflammatory tissue, e.g. gingivitis, the antigen is preferentially expressed by macrophages appearing late in the inflammatory process. In chronic inflammation, e.g. BCG granulomas and rheumatoid arthritis, RM3/1-positive macrophages are seen to varying degrees. Double-staining experiments with the antigen 25F9, specific for resting mature macrophages, revealed that RM3/1 and 25F9 are expressed by distinct populations in normal and acute inflammatory tissues. From this it is concluded that the antibody RM3/1 specifically detects a macrophage phenotype which seems to be associated with the healing phase of the inflammatory process.     

A monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at Mr 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-gamma (IFN-gamma), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) but not with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphocytes, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG granulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr. From this it is concluded that the antibody 27E10 detects an antigen expressed by a subset of peripheral blood monocytes. In situ the antigen is found only in inflammatory tissues and is absent from normal resident mononuclear phagocytes.     

Production and characterization of a monoclonal antibody (A1-3) that binds selectively to activated monocytes and inhibits monocyte procoagulant activity

We describe the generation and characterization of a new monoclonal antibody, A1-3, which possesses two unique properties. First, A1-3 binds selectively to stimulated human monocytes. Secondly, A1-3 inhibits the procoagulant activity expressed by stimulated monocytes and by human brain tissue factor. Unstimulated human peripheral blood cells (granulocytes, lymphocytes, monocytes, red blood cells, and platelets), prepared in the absence of detectable endotoxin, express no procoagulant activity and fail to bind A1-3. Stimulation of peripheral blood monocytes. alveolar macrophages, or the monocyte-like cell line U937, however, results in the expression of procoagulant activity and the binding of A1-3. The surface antigen recognized by A1-3 was recovered from endotoxin-stimulated human monocyte vesicles by immune precipitation and demonstrated an apparent m.w. of approximately 52,000. It is proposed that the monoclonal antibody A1-3 detects a differentiation antigen on human monocytes that is expressed in response to stimuli for monocyte activation.     

Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus

In this study we report the identification of an antibody in the sera of some patients with autoimmune disease that reacted with a cytoplasmic antigen localized within the Golgi apparatus. The antibody reacted with all tissues investigated, which included pancreas, kidney, testis, liver, thymus, and spleen. In addition, it reacted with some human peripheral circulating lymphocytes, murine peritoneal macrophages, and a variety of tissue culture cell lines, which included HEp-2 cells (human epithelial carcinoma), baby hamster kidney cells, a canine thymus cell line, a primary kidney cell line, Ehrlich ascites cells, Wil-2 cells, and Raji cells. The antigen is located in the same region stained by the histochemical reaction for thiamine pyrophosphatase, thus indicating that the antigen is located within the Golgi apparatus. The antigen was not demonstrated by immunodiffusion of saline extracts of rabbit thymus, pancreas, or liver. The antigen in HEp-2 cells was resistant to RNase A, DNase I, micrococcal nuclease, and to extraction with 0.1 N HC1, but was sensitive to trypsin and Proteinase K. Eight patients with anti-Golgi antibodies have been identified. Six of the eight had systemic lupus erythematosus. Autoantibodies to a Golgi apparatus antigen might serve as a useful biologic marker to study the functional relationship of the Golgi apparatus to lymphocytes and macrophages.     

Co-expression of an epitope on human free kappa-light chains and on a cytoplasmic component in activated T cells

K-1-21 is a murine monoclonal antibody that reacts with human kappa-light chains in free form but not when they are associated with immunoglobulin heavy chains. K-1-21 was unexpectedly shown to bind to a determinant, STA (Sezary T cell antigen), detected by immunofluorescence in the cytoplasm but not on the surface of Sezary T cells isolated from peripheral blood (4/4 cases) and in Sezary T cells from lymph node and bone marrow (one patient). STA was detected in F2/F7, CCRF-CEM, Molt-4, and CCRF-HSB (four human T ALL cell lines), in JURKAT (a human T cell leukemia line), and in MLA144 (a Gibbon T cell lymphoma line). It also occurred in Leu-3a+ antigen-specific T cell clones (6/6 tested). Moreover, although STA was absent from freshly isolated normal T cells, its expression could be evoked in E+ cells from peripheral blood by in vitro culture with phytohemagglutinin. Thus, STA appears to be a cytoplasmic marker for activated T cells. Cytoplasmic inhibition immunofluorescence studies indicated that K-1-21 binding to STA in Sezary cells or T cell lines was inhibited by preincubation of the K-1-21 antibody with purified kappa-Bence Jones protein. STA from radiolabeled MLA144 cell lysates was immunoprecipitated by K-1-21 and was identified on polyacrylamide gel electrophoresis under reducing conditions as a protein of m.w. 57,000. Additional experiments are underway to define the molecular basis of the interesting cross-reactivity between a determinant in T cells and the K-1-21 reactive epitope on free kappa-light chains.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

A novel monoclonal antibody induces the differentiation of monocyte leukemic cells

A cell surface antigen (possibly a receptor) on human myeloid cell lines, that may play a crucial role in the differentiation of promonocytic cells, was detected by a murine monoclonal antibody (MAb 710F). When the myeloid cell lines, HL-60, THP-1 and U937, were cultured with the MAb 710F following pretreatment with phorbol 12-myristate 13-acetate (PMA), they displayed both cellular spreading and a strong adherence to the culture dish substratum. On transforming from their original round shape, the induced cells displayed the well-developed microvilli, spindles, or raffles that are characteristic of macrophages or dendrocytes. Similar morphological changes were not induced by treatment with either PMA or MAb 710F alone. By contrast, the lymphoid cell lines did not respond to these reagents. These results suggested that a signal for cellular adhesion and cytoskeletal reconstitution was triggered by the binding of MAb 710F to an antigen on the PMA-primed cells. Thus, the antigen 710F, which is preferentially expressed on peripheral blood monocytes, may represent a cell surface receptor closely associated with differentiation. The antigen/receptor 710F was shown to be a N-glycosylated protein with Mr. of 35-70k. This MAb may prove useful as a tool for both studies on the differentiation of myeloid lineage cells as well as on monocyte/macrophage adhesion function.     

Significance of antigen, drug, and tumor cell targets in the preclinical evaluation of doxorubicin, daunorubicin, methotrexate, and mitomycin-C monoclonal antibody immunoconjugates

We tested drug monoclonal antibody immunoconjugates in vitro in 72 h 3H-thymidine assays and in vivo in athymic mice bearing human tumor xenografts of the same target cells. Experimental arms included control, monoclonal antibody, drug, drug + antibody, the test immunoconjugate, and a negative control immunoconjugate with an equivalent molar amount of drug for in vitro experiments, and the amount of drug conjugated to 500 micrograms of antibody in the animal experiments. Monoclonal antibodies included T101, an IgG2a that reacts with a rapidly modulating antigen, 9.2.27, an IgG2a that reacts with a slowly modulating antigen, and ME7, an IgG1 that reacts with a slowly modulating antigen. Cells used in testing included MOLT-4 (T lymphoma), 8392 (B lymphoma), and M21 (melanoma). Drugs tested were doxorubicin, daunorubicin, methotrexate, and mitomycin-C. M21 cells were resistant to daunorubicin in vitro but were inhibited by the 9.2.27 daunorubicin immunoconjugate. T101, 9.2.27, and ME7 cis-aconitate anthracycline immunoconjugates and mitomycin-C-glutarate immunoconjugates were specifically cytotoxic only for antigen positive cells in vitro and were superior to free drug in vivo. These results confirm that antigen specific-cytotoxic drug immunoconjugates can be produced that are superior to the same dose of free drug. However, each monoclonal antibody drug target system is unique and must be well-characterized for appropriate interpretation of data.     

Monoclonal antibody with specificity for monocytes and neurons

We have characterized a monoclonal antibody, called UC45, that reacts with both monocytes and neurons. It was derived from a fusion of the NS-1 plasmacytoma cell line with spleen cells from a mouse immunized with human acute monoblastic leukemia cells. The antibody reacts weakly with viable monocytes in suspension but has specificity for fibrous projections, which are found on monocytes that have adhered to a substrate. Other hemopoietically derived cells such as granulocytes and lymphocytes, and many tissue-culture lines, do not react with UC45 by cell-surface immunofluorescence. Similarly, UC45 reacts with the processes of both viable CNS and PNS neurons in tissue culture but with no other neural-tissue-derived cells. The monoclonal antibody has interspecies reactivity, in that it reacts with human, rat and mouse monocytes and neurons. The monocyte and neuronal antigen is present predominantly on a protein of 45 kd. Attempts to identify this protein on monocytes with conventional heteroantisera directed against fibronectin, complement components, fibrinogen, collagen, tubulin and actin have failed. A monoclonal antibody has therefore allowed identification of an antigen, unexpectedly shared by monocytes and neurons. The fact that it is found on cell processes of both cell types suggests that it may be performing some similar function for these cells, whose other activities differ substantially.     

Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. We show here that monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocytes, and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an 125I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.     

A monoclonal antibody against an antigen present on mouse macrophages and absent from monocytes

Summary A monoclonal antibody (BM8) raised in the rat against cultured mouse bone marrow-derived macrophages reacted only with macrophages and not with granulocytes, mast cells, platelets, lymphocytes, fibroblasts, and endothelial cells. BM8 did not detect blood monocytes. In cultured bone-marrow cells, expression of BM8-antigen was found on a few macrophages after one day of culture and reached its maximum level with 80% positive macrophages after 7–10 days of culture. The antibody BM8 belonged to the IgG_2a subclass, was non-cytotoxic and directed against a 125 kD membrane antigen. In cryostat sections of normal mouse tissues (spleen, lymph node, thymus, liver, skin) BM8 detected tissue-fixed macrophages and Langerhans cells in the skin. In spleen and lymph node, BM8 reacted with macrophages in the red pulp and in the medullary cords, respectively, but not with heavily phagocytosing marginal-zone macrophages, as revealed by in-vivo phagocytosis of colloidal carbon. In granulomata induced by complete Freund's adjuvant, BM8 detected inflammatory macrophages but not epithelioid cells. Thus, antibody BM8 detected a differentiation antigen expressed only on mature, tissue-fixed macrophages.     

Anti-T cell monoclonal antibodies in vivo. I. Inhibition of delayed hypersensitivity but not cutaneous basophil hypersensitivity reactions

As part of a study of the therapeutic potential of anti-T cell monoclonal antibodies, we studied the biologic effects of 8BE6, a mouse anti-guinea pig (GP) pan-T cell monoclonal antibody, on blood and tissue T cells and on the prototypic T cell-mediated reactions, classic delayed hypersensitivity (DH) and cutaneous basophil hypersensitivity (CBH). 8BE6 reacts to a 68,000 m.w. protein probably homologous with human CD5 (T1) and murine Lyt-1. A single dose of 1.8 to 3.4 mg 8BE6 caused lymphopenia and greater than 90% depletion of 8BE6+ peripheral T cells 1 to 72 hr later, and a significant but lesser decrease of lymphocytes reacting with another pan-T cell monoclonal antibody (p less than 0.02 at 24 hr). Free serum 8BE6 was detected for up to 48 hr after administration. Immunoperoxidase stains of tissue revealed that lymphocytes in lymph nodes and spleen were coated with mouse immunoglobulin 1 hr after antibody treatment and displayed in situ capping. Subsequently, there was a loss of T cells in all tissues (spleen, lymph node, liver, and kidney) except the thymus, with normal 8BE6 antigen staining returning by 72 hr. Areas of induration of DH reactions to PPD were reduced in 8BE6-treated GP, compared with pretreatment reactions in the same GP or in control-treated GP (p less than 0.001 for both). The numbers of infiltrating T cells and fibronectin-receptor-positive macrophages were also reduced. In contrast, 8BE6 had no effect on CBH reactions, as judged by erythema and basophil counts in 1-micron sections, although fewer T cells were found in reaction sites. There were no differences in IgM, fibronectin, or Ia staining between 8BE6-treated GP and controls. In vivo administration of a single dose of anti-T cell monoclonal antibody results in a transient, highly specific depletion of T cell populations in peripheral blood and tissues except the thymus. This treatment inhibits DH but not CBH reactions by systemic and local depletion of T cells.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells

We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.     

Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes

We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Modulation induction of the T3 antigen by OKT3 antibody is monocyte dependent

We investigated the influence of monocytes on the susceptibility of the T3 antigen on human T cells to modulation induction by OKT3 antibody. In the absence of monocytes, the T3 antigen was only minimally susceptible to modulation. After the addition of 20% monocytes to the culture, however, complete modulation was readily observed. Furthermore, we found that even in the absence of OKT3 antibody, monocytes were able to down-regulate the expression of the T3 antigen, although to a lesser extent. The ability of monocytes to enhance antigenic modulation proved to be a more general phenomenon. Each individual T cell antigen, however, differed in its susceptibility to modulation by antibody, monocytes, or both, thereby establishing its own characteristic pattern. In addition, after complete modulation of the T3 antigen, the addition of monocytes to the culture thereafter had a distinct inhibitory effect on the reexpression of the T3 antigen. Monocyte enhancement of T3 modulation is significantly reduced when using the OKT3 F(ab')2 fragment, as is OKT3 mitogenesis. After pulsing the monocytes with OKT3 antibody before adding them to the culture, T3 modulation became nearly complete even in the absence of added OKT3 antibody. Monocyte-induced modulation proved not to be MHC restricted, thus allowing for comparative analysis of this effect between monocytes and other cell types. A moderate, however, incomplete modulation enhancement was observed with the human monocyte cell line U937 and with Daudi cells. This finding proved to coincide with the distinct ability of these cell lines to bind OKT3 antibody by their Fc receptors, as was the case with monocytes. In contrast, neither Fc receptor binding nor T3 modulation enhancement was observed with the cell lines Cess and G7. In addition, no effective T3 modulation was observed with glutaraldehyde-fixed monocytes. The overall results seem to indicate that effective modulation of the T3 antigen by OKT3 antibody requires the active participation of Fc receptors on monocytes.     

Murine T lymphoma cells express a novel membrane-associated antigen with unique features

A novel membrane-associated antigen expressed on various murine T lymphoma cells has been detected by a rat monoclonal antibody. The antibody YE6/6 initially produced against Moloney leukemia virus-transformed T lymphoma line MBL-2, reacted with several other lymphoma lines including non-T lymphoma lines as well as thymocytes from leukemic AKR mice, but it did not show significant reactivities with resting or mitogen-activated normal lymphocytes by flow cytometric analysis. The antibody did not bind to some Abelson leukemia-transformed cells, which express Moloney virus antigens, suggesting that the antigen is unlikely to be encoded by Moloney virus genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the antigen molecules immunoprecipitated by the antibody revealed three major polypeptides. Two of the polypeptides, with approximate m.w. of 95,000 and 35,000, can be labeled by the cell surface iodination and, therefore, seem to be exposed on the cell surface. The third polypeptide, with approximate m.w. of 65,000, is not labeled by the surface iodination but it is readily detected by [35S]methionine labeling. The third polypeptide was labeled with [32P]orthophosphate indicating that it is a phosphoprotein. Western blot analysis showed that YE6/6 antibody primarily reacts with 35,000 m.w. polypeptide. Furthermore, the same 35,000 m.w. protein was also detected in concanavalin A-activated spleen cells at a low level by Western blot, but normal resting lymphocytes were negative. These results suggest that the antigen detected by YE6/6 antibody may be a cell proliferation-associated antigen and its expression is highly elevated on transformed lymphoma cells as compared to normal mitogen-activated lymphocytes.     

Identification of a glycoprotein, gp21, of adult T cell leukemia virus by monoclonal antibody

A mouse hybridoma cell line producing monoclonal antibody, F10, was established from mice hyperimmunized with cells bearing adult T cell leukemia (ATL) virus (ATLV). F10 antibody reacted with an ATLV structural polypeptide ( gp21 ) with a m.w. of 21,000 that was glycosylated on cell surfaces. The gp21 was expressed on cell surfaces of all ATL-associated antigen (ATLA)-positive human cell lines but not on ATLA-negative cell lines nor peripheral blood leukocytes stimulated with mitogens. The gp21 was also detected with anti-ATLA-positive human serum, and the binding of F10 antibody to ATLA-positive cell surfaces was significantly blocked by pretreatment with anti-ATLA-positive human sera. Double immunofluorescence staining with F10 antibody and anti-ATLA-positive human serum caused co-capping on cell surfaces, which suggests that gp21 co-exists with other ATLV antigens expressed on cell surfaces. Immunoprecipitation studies also suggested that the gp21 is a minor component of the ATLV envelope.     

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts.