Chicken thymocyte-specific antigen identified by monoclonal antibodies: ontogeny,tissue distribution and biochemical characterization

Two monoclonal antibodies, CT-1 (IgG₁,χ) and CT-1a (IgG₃,χ), were prepared against chicken thymocytes. The antigen identified by these antibodies was found to be a glycoprotein with a major polypeptide component having an apparent molecular weight of 63000 and a minor polypeptide component of approximately 103000. Immunoprecipitation and blocking experiments revealed that the two antibodies react with different antigenic determinants of the molecule. Ontogenic studies employing immunofluorescence failed to reveal the antigenic determinants on cells from the embryo or embryonic yolk sac on days 3 and 6 of incubation. The number of embryonic thymocytes bearing the molecules detected by these antibodies increased from 6% on day 12 to 54% on day 13; the frequency of thymocytes expressing this glycoprotein reached adult levels (> 90%) by the 15th day of embryonic age. In contrast, the CT-1 and CT-1a antibodies reacted with only 2-5% of blood and splenic cells and less than 0.05% of cells from bursa or bone marrow of young adult chickens. In the quail CT-1 reacted with cortical, but not medullary, thymocytes, while the CT-1a antibody was unreactive with quail thymocytes. The surface glycoproteins detected by these discriminating monoclonal antibodies may provide an important discriminating marker for thymic lymphocytes in the chicken and the quail.     

Chicken thymocyte-specific antigens identified by monoclonal antibodies: characterization and distribution in normal tissues and in tumoral tissues from Marek's disease chicken

Monoclonal antibodies (MAbs) were obtained against purified thymocyte membrane extracts. Five MAbs TA3, TB1, TB6 (IgG1), TC4, and TA1 (IgG2a), were tested by immunofluorescence and by immunoperoxidase tests against normal cells from different organs, Marek's disease (MD) cell lines, and MD tumoral cells from chickens. Three of them, TA3, TB1, and TB6, reacted exclusively with lymphoid cells in both cortical and medullary areas of the thymus and with less than 8% bursa cells. They identified a protein of apparently 40 kD. The other two revealed antigenic determinants on most medullar thymocytes and some cortical thymocytes, and on some splenic and peripheral blood lymphocytes. They were positive with MD cell lines and cells deriving from MD tumors. TC4 and TA1 detected molecular masses of about 110 kD and 16 kD, respectively. No MAbs reacted with erythrocytes, bone marrow, liver, brain, and skin cells. Not all of the tested cells were stained after contact with an anti-chicken immunoglobulin serum. In this paper, we determine a specific antigen restricted to T cells from thymus and different markers belonging to the mature T cells. The latter are also present on MD cell lines and MD tumoral cells.     

Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistological and immunochemical studies.

Human thymus and T cell antigens were identified by using four distinct monoclonal antibodies (MoAb), designated 2D5, 5B3, 7A5 and 9D4. 2D5 antibody reacted with most human thymocytes and a few peripheral lymphocytes as well as with a subpopulation (20%) of bone marrow cells, and precipitated a 45K molecular weight (mol. wt.) component from 125I-labelled thymus cell lysate. 7A5 antibody also reacted with the majority (80%) of thymocytes but neither with peripheral lymphocytes nor with bone marrow cells. The antigen detected by 7A5 was a glycoprotein consisting of 48K and 12K mol. wt. components, which were non-covalently associated with each other. 5B3 reacted with virtually all of human thymus and T cells but not with the majority of B cells and bone marrow cells. This reagent precipitated a 72K mol. wt. glycoprotein from thymus and T cells. An additional 65K mol. wt. glycoprotein was precipitated by 5B3 together with the 72K mol. wt. component, but with poor reproducibility. 9D4 antibody, on the other hand, reacted with a 200K mol. wt. component from thymus and T cells as well as 220K and 210K components from the non-T cell fraction of tonsil lymphocytes. Whereas antigens detected by 2D5 and 7A5 appeared to be highly expressed on cortical thymocytes, the antigen defined by 5B3 occurred much more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Based on the data described above, it is suggested that 7A5, 5B3 and 9D4 MoAb recognize human homologues of mouse TL, Ly-1 and Ly-5 antigens, respectively, whereas 2D5 antibody seems to resemble OKT10, as described by others.     

Chicken thymocyte antigen which participates in cell proliferations of thymocytes and tumour-derived lymphoid cell lines

A novel antigen on chicken thymocytes was defined by CETHB1 and CETH46 monoclonal antibodies (mAbs) that were prepared against chick embryonic thymocytes. CETHB1 and CETH46 mAbs recognized different epitopes on the same membrane antigen (molecular weight: 76.2 kDa). These mAbs reacted with >80% of thymocytes of 14-day-old embryos to 8-week-old chickens. Almost all splenocytes, peripheral blood lymphocytes and bursa cells were negative, and only 7.7% of bone marrow cells were positive for both antibodies. In two-colour analysis with mAbs reacting to T cell markers (CD4 or CD8), most CETHB1 positive cells were CD4 (- )CD8 (-) or CD4 (+) CD8 (+) . However, a proportion of CD4 (+) CD8 (-) and CD4 (- )CD8 (+) cells were negative for CETHB1 mAb. The proportion of thymocytes reacting with CETHB1 in chickens immunosuppressed by cyclophosphamide treatment increased gradually in parallel with the restoration of the thymus. An increase of CETHB1-positive cells was observed in thymocytes stimulated with Con A. Hence, it seems that the CETHB1 antigen expression on thymocytes is influenced by the thymic micro-environment and that the antigen may take part in thymic differentiation. Interestingly, CETHB1 antigen was expressed not only on T cell tumour-derived lymphoid cell lines, but also B-lymphoma-derived cell lines. The antigen expressions on these cell Unes were observed only in the prohierative phase of the cells. Hence, the molecule which reacted with CETHB1 may be an antigen commonly expressed on lymphoma cells and may be involved in cell proliferation.     

Guinea pig T lymphocyte development analyzed by enzyme histocytochemistry, monoclonal antibodies, and flow cytometry

Studies of T (thymus-derived) lymphocyte ontogeny in the guinea pig have been hampered by the lack of suitable antigenic or other markers for various T cell subpopulations in this species. Monoclonal antibodies that recognize three distinct surface proteins of guinea pig T cells and react with all peripheral T cells have been used in combination with membrane alkaline phosphatase (AP) to characterize stages of guinea pig T cell development and to determine anatomical localization of different T cell subpopulations. Flow cytofluorographic analysis of thymus, spleen, and lymph node lymphocytes was used to characterize monoclonal antibody specificity. Cortical thymocytes in tissue sections expressed membrane AP activity and contained nuclear terminal deoxynucleotidyl transferase; medullary thymocytes reacted strongly with one of the monoclonal antibodies (8BE6), minimally with a second (5CD2), and not at all with a third (11AE3). In contrast, polyclonal rabbit antiguinea pig T cell antiserum reacted with both cortical and medullary thymocytes. Staining of tissue sections of lymph node and spleen revealed AP+ lymphocytes to be present peripheral to the mantle region of lymph node follicles and to be randomly scattered throughout the splenic red pulp. T cells reactive with monoclonal antibodies were located primarily in paracortical regions of lymph node and the central region around the periarteriolar regions of the spleen. Dual staining of frozen sections and cell suspension of guinea pig lymphoid tissues for AP activity and surface proteins unique to T cells showed that AP+ cells lacked T cell markers. Dual staining for AP activity and surface immunoglobulins or esterase activity showed that AP+ cells are not likely to be derived from either B cell or monocyte-macrophage lineages. AP+ cells in guinea pig secondary lymphoid tissue may represent a unique subset of lymphocytes of unknown function.     

Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.     

抗人淋巴细胞T8抗原样McAb的研制和初步鉴定

用杂交瘤技术,将经人外周血E花环阳性细胞免疫小鼠脾细胞与NS-1骨髓瘤细胞融合后,产生一分泌IgG_1亚型McAb杂交瘤株。其所分泌抗体经微量放射免疫测定及间接免疫荧光法分析,表明它只能与T细胞系、76％的胸腺细胞及22％的外周血T淋巴细胞反应,不与其它各种不同细胞反应。将此抗体所识别入外周血T淋巴细胞亚群与抗Leu-2a识别T8~+细胞、抗Leu-3a识别T4~+细胞比较,发现此抗体与抗Leu-2a识别同一群细胞。此抗体能从T细胞表面沉淀出30KD(还原条件)或78KD(非还原条件)分子,并完全阻断FITC标记抗Leu-2a与T细胞的结合,说明此抗体是识别T8抗原样的McAb。     

A monoclonal antibody recognizing human Thy-1: distribution on human and non-human primate haematopoietic cells

A monoclonal antibody designated 'antibody 390' (Ab 390) with anti-human Thy-1 reactivity was prepared by the hybridoma technique from the splenocytes of BALB/c mice immunized with human fetal brain. This antibody was shown to have anti-human Thy-1 reactivity because (1) it precipitated a molecule with a molecular weight of about 24,000 daltons, (2) it had a pattern of reactivity similar to that of previously described anti-human Thy-1 antibodies and (3) purified human Thy-1 antigen specifically inhibited binding of Ab 390 to a known antigen-positive cell line. It was the intent of this study to investigate the distribution of Thy-1 on normal and malignant haematopoietic cells in humans and non-human primates. We show here that Ab 390 did not react with human peripheral blood leucocytes, bone marrow cells or splenocytes by immunofluorescence but did react with subcapsular and cortical fetal thymocytes by peroxidase-antiperoxidase immunohistology. A section of fetal spleen demonstrated staining of connective tissue and blood vessels and rare reactive lymphocytes. Adult spleen contained Thy-1-positive cells surrounding the white pulp and in the marginal zone, but single-cell suspensions of splenocytes did not react with Ab 390. Ab 390 was tested against a variety of fresh human leukaemia cells and human cell lines and was shown to react with only the acute lymphoblastic leukaemia T cell lines RPMI 8402 and HPB-MLT. Non-human primate studies revealed reactivity with a number of T cell lines from New World primates (cotton-topped and red-bellied marmosets) and peripheral blood granulocytes (owl monkey). Our studies support previous findings that suggest that human Thy-1 may be a marker for early T lymphocytes in man, and its distribution on non-human primate T cell lines suggests the same for certain species of non-human primates. Not consistent with the distribution on human cells was the demonstration of Ab 390 reactivity with owl monkey granulocytes.     

Quantitative analysis of tissue distribution of Ly10-like murine alloantigen(s) with antisera and monoclonal antibodies

Tissue distribution of non-Lyt1.1 ("Ly10-like") antigen or antigens encoded by short chromosomal segment differentiating B6-Ly-1a congenic strain from B6 strain of mice was studied by quantitative absorption of (BALB/c X B6)F1 anti B6-Ly-1a antiserum and by direct cytotoxicity of Ly-10-132-12-26 monoclonal antibody on lymphoid cell populations. Identical strain but not tissue distribution pattern does not allow to conclude whether antiserum and monoclonal antibody detect the same or closely linked antigens. Absorption experiments revealed the highest antigen content in the brain tissue, lower in testis and kidney, still lower in lymphoid organs and the lowest in liver and lung. Among lymphoid cells, bone marrow cells had highest absorbing capacity, followed by thymus, spleen and lymph nodes. Monoclonal antibody lysed almost 100% of thymocytes, 30% bone marrow cells and 10-20% of spleen and lymph node cells (both T-cell and B-cell enriched populations contained the same proportion of positive cells). Cortisone resistant thymocytes showed the same sensitivity as cortical thymocytes to Ly-10-132-12-26 antibody which is distinguishable characteristics of medullary thymocytes from peripheral T cells. Mitogen activated lymphocytes exhibited significantly higher expression of Ly10-like antigen than resting peripheral lymphocytes.     

Two distinct antigenic markers for rat thymus and T cells defined by monoclonal antibodies.

Rat thymus and T-cell antigens were defined by using two distinct monoclonal antibodies (R-1-3B3 and R-1-12C5). R-1-3B3 antibody, when tested for its reactivity with rat lymphoid cells by immunofluorescence, labelled virtually all thymus and T cells but not B cells and bone marrow cells. The antigen defined by this R-1-3B3 antibody occurred more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Immunochemical data showed that R-1-3B3 antibody recognized a single glycoprotein with a molecular weight of 67,000, which were able to interact with Lens culinaris haemagglutinin. R-1-12C5 antibody, on the other hand, reacted with all of thymus and T cells as well as with a subpopulation (approximately 20%) of bone marrow cells. In contrast to the antigen defined by R-1-3B3, that detected by R-1-12C5 antibody existed largely on cortical thymocytes and to a much lesser extent on medullary thymocytes and peripheral T cells. R-1-12C5 antibody detected a single glycoprotein with a 95,000 molecular weight, which could also interact with Lens culinaris haemagglutinin. Based on these data described above and since both antigens defined by R-1-3B3 and R-1-12C5 antibodies were absent from rat brain tissue, we concluded that they were distinct from brain-associated thymic antigens in rats including Thy-1 and W3/13 antigen systems.     

Recognition of a human T-lymphocyte differentiation antigen by an IgM monoclonal antibody.

A monoclonal antibody directed at a determinant on human T cells was produced and characterized. This IgM antibody, MBG6, bound to human peripheral blood T lymphocytes and to medullary thymocytes. It was unreactive with normal B cells, B-cell lines and granulocytes. Apart from T lymphocytes, bone marrow cells (including cells positive for the terminal transferase marker, myeloid colony-forming cells, myeloblasts, and differentiating myeloid and erythroid cells) were negative. Peripheral blood cells that were treated with MBG6 and rabbit complement were no longer capable of proliferating in response to phytohaemagglutinin or concanavalin A; MBG6 did not have any direct mitogenic action on T lymphocytes. Double immunofluorescence studies using IgM MBG6 and OKT3, and IgG2a monoclonal antibody that recognizes all peripheral T cells, showed that these two antibodies identified exactly the same cell populations. Competitive binding studies, however, indicated that MBG6 and OKT3 recognized different epitopes. The antibody may have clinical applications in bone marrow transplantation.     

Lymphocyte subsets in normal human lymphoid tissues

A series of B, T, natural killer (NK) cell, and monocyte-specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulations in frozen tissue sections of human lymph node, spleen, tonsil, and thymus by means of an immunohistochemical technic. In thymus, most cortical thymocytes reacted with Leu 1, Leu 2a, Leu 3a, Leu 4, Lyt 3, OKT3, and OKT6 antibodies. Except for OKT6, Leu 2a, and Leu 3a, these antibodies also reacted with medullary thymocytes. The majority (70-80%) of medullary thymocytes reacted with Leu 3a and a smaller fraction (20-30%) with Leu 2a antibody. The staining pattern of thymic medulla approximates the staining pattern of peripheral T cells. In peripheral lymphoid tissues, the majority of cells in the paracortical region of lymph node and in the periarteriolar sheath of spleen stained with Leu 1, Leu 4, OKT3, and Lyt 3 antibodies. Staining with Leu 3a and Leu 2a identified 60-80% and 20-40% of total T cells, respectively, as defined by Lyt 3 positivity. In addition, a substantial number of Leu 3a+ and Leu 7+ cells were found in the germinal centers of secondary follicles. This finding supports the importance of these subsets of lymphocytes in regulation of the human immune response. Leu 2a+ cells were rare in tissues with prominent follicular hyperplasia, but appeared in considerable number in the red pulp of spleen. In the mantle zone of lymphoid follicles, the majority of lymphocytes were positive for IgM, IgD, and B1. Approximately 60-70% of these cells bore kappa chain and 30-40% lambda chain immunoglobulin. The extracellular substance in germinal centers was positive for B1, IgG, IgM, kappa, and lambda. The majority of germinal center cells appeared to contain no surface or cytoplasmic immunoglobulins. Small mononuclear cells bearing OKM1 marker were abundant in the marginal zone of white pulp and in the red pulp of spleen but, rarely were observed in other portions of lymphoid tissues. OKM1 also reacted with granulocytes. Leu 7+ (NK) cells were rare in the thymus, but frequent in the GC of secondary follicles. The distribution of Leu 7+ cells did not correspond to staining with Lyt 3 and Leu 2a.     

The in vivo and in vitro use of monoclonal antibody for the deletion of phagocytic cells in guinea pigs

A monoclonal antibody (MAb) was raised against guinea pig macrophages and its reactivity to bone marrow derived cells was tested. The Ab selectively reacted to phagocytic cells such as polymorphonuclear leukocytes (PMNL) and macrophages, but not to erythrocytes, lymphocytes, hepatocytes and platelets, as determined by the binding and complement-dependent cytolytic activity. In vivo effect of the Ab on peripheral leukocytes was tested. More than 90% of PMNL were deleted 3 hours after the intravenous administration of the antibody. The cells started to reappear in peripheral blood on the 3rd day and returned to the level before the administration on the 6-7th day. Erythrocytes in peripheral blood were not affected. Phytohemagglutinin (PHA) and muramyl dipeptide(MDP) induced proliferation of thymocytes were tested after removal of macrophages by the selective binding of the MAb reactive cells to the plastic dish. Proliferation of the macrophage-deleted thymocytes was significantly suppressed, however, it was restored by addition of a small number of peritoneal macrophages. Those results strongly indicate that the MAb presented here is directed selectively to phagocytic cells of guinea pigs and should prove useful to study both in vivo and in vitro functions of the cells.     

Lym 7.2: monoclonal antibody defining an alloantigen similar or identical to Ly 7.2

Immunization of B10.D2 Ign mice against a (BALB/c X NZB)F1 murine B lymphoma cell line (WEHI-5) and subsequent fusion of immune spleen cells with a drug sensitive myeloma (P3 X 63-Ag8) has resulted in the generation of a series of hybridoma cell lines. One of these clones, BD5-334.5, secretes an antibody which reacts with a determinant, designated Lym 7.2, which demonstrates an identical tissue and strain distribution as the conventionally defined Ly 7.2 antigen. Furthermore, anti-Ly 7.2 alloantiserum significantly blocks reaction of the anti-Lym 7.2 monoclonal antibody with BALB/c splenocytes. Lym 7.2 is present on a majority of splenocytes, peripheral blood leukocytes, and lymph node cells. However, B cells express considerably higher cell surface density of this antigen than peripheral T lymphocytes. This antigen was not detected on erythrocytes, kidney, liver, or brain. Moreover, Lym 7.2 is present on approximately 15% of normal thymocytes, and 15% of bone marrow cells, and is expressed on cortisone resistant thymocytes. Quantitative flow cytometry analysis demonstrated that the antigen is present at approximately half the cell surface density on spleen cells from F1 mice between Lym 7.2 positive and Lym 7.2 negative mice, but on the same percentage of cells as Lym 7.2 positive inbred strains. Data from backcross analysis suggest that a single locus is encoding this antigen. Mitogen blasts, induced with PHA, ConA, and LPS, all express the Lym 7.2 marker.     

Monoclonal antibody against a "Pan-T-cell" antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias

A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.     

Antiserum specific for reticulin of the bursa of fabricius.

Antiserum produced by immunizing rabbits with embryonic chicken spleen cells showed, after appropriate absorption with cell suspensions, specificity for fetal spleen cells in membrane immunofluorescence tests. However, when the original antiserum was absorbed with tissue homogenates and tested on cryostat tissue sections, it reacted specifically with the reticular framework which separates the cortical and medullary lymphocytes in the follicles of the adult bursa of Fabricius. It is suggested that the reticulin may influence bursal lymphoid differentiation.     

PERSISTENT and ENHANCED EXPRESSION OF c-fos GENE PRODUCTS IN VARIOUS KINDS OF HUMAN HEMATOPOIETIC CELL LINES. Immunofluorescence Study Using Prepared Monoclonal Antibodies

Two kinds of monoclonal antibodies recognizing fos proto-oncogene (c-fos) products were prepared using a synthetic oligopeptide corresponding to amino acids 127-152 of the fos oncogene products. These monoclonal antibodies (FO-120 & FO-145) detected fos gene products induced in a human monocyte cell line (U-937) by phorbol acetate (TPA) and induced in both human and mouse fibroblast cell lines (284, BALB/c 3T3) by serum-stimulation. One of the monoclonal antibodies (FO-120) reacted with 50-kDa and 42-kDa proteins and the other antibody (FO-145) reacted with a 30-33-kDa protein. The expression of the fos gene in various human hematopoietic cell lines was investigated using these prepared monoclonal antibodies. While almost all hematopoietic cell lines tested reacted with these monoclonal antibodies to various degrees, the majority of normal peripheral blood lymphocytes cultured with lectin (PHA) and interleukin 2 (IL-2) did not, suggesting that cells of some permanent hematopoietic cell lines, irrespective of their lineage specificity and growth factor dependency, continuously express the fos oncogene. These monoclonal antibodies may be useful for detecting early neoplastic changes in hematopoietic cells. ACTA PATHOL JPN 38: 1523-1536, 1988.     

Persistent and enhanced expression of c-fos gene products in various kinds of human hematopoietic cell lines. Immunofluorescence study using prepared monoclonal antibodies

Two kinds of monoclonal antibodies recognizing fos proto-oncogene (c-fos) products were prepared using a synthetic oligopeptide corresponding to amino acids 127-152 of the fos oncogene products. These monoclonal antibodies (FO-120 & FO-145) detected fos gene products induced in a human monocyte cell line (U-937) by phorbol acetate (TPA) and induced in both human and mouse fibroblast cell lines (284, BALB/c 3T3) by serum-stimulation. One of the monoclonal antibodies (FO-120) reacted with 50-kDa and 42-kDa proteins and the other antibody (FO-145) reacted with a 30-33-kDa protein. The expression of the fos gene in various human hematopoietic cell lines was investigated using these prepared monoclonal antibodies. While almost all hematopoietic cell lines tested reacted with these monoclonal antibodies to various degrees, the majority of normal peripheral blood lymphocytes cultured with lectin (PHA) and interleukin 2 (IL-2) did not, suggesting that cells of some permanent hematopoietic cell lines, irrespective of their lineage specificity and growth factor dependency, continuously express the fos oncogene. These monoclonal antibodies may be useful for detecting early neoplastic changes in hematopoietic cells.     

Monoclonal antibodies to rabbit lymphoid cells: Preparation and characterization of a T-cell-specific antibody

Spleen cells of mice immunized with rabbit spleen and mesenteric lymph node (MLN) cells were fused with mutant mouse myeloma cells. Twenty-six clones which react with rabbit lymphoid cells were obtained. By membrane immunofluorescence, as analysed visually and by the fluorescence-activated cell sorter, one of these clones, 9AE10, produced an antibody that reacted with nearly all thymocytes (> 90%) and with from 46 to 78% of spleen, MLN and peripheral blood lymphocytes. Double-membrane immunofluorescence with the 9AE10 monoclonal antibody (MAb) and anti-Ig showed that 9AE10⁺ and Ig⁺ cells of spleen, MLN and peripheral blood were distinct and non-overlapping populations. Thus, the 9AE10 MAb is a T-cell-specific antibody.The 9AE10 MAb also reacted with most brain cells and with approximately 30% of bone marrow cells. Biochemical analysis of the antigen recognized by the 9AE10 MAb indicated that the antigen is a glycoprotein with an apparent molecular weight of 25,000. These data indicate that the 9AE10 MAb may be directed against a Thy-1-like antigen.