Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody,HOP-26

Bone is a highly organized structure comprising a calcified connective tissue matrix formed by mature osteoblasts, which develop from the proliferation and differentiation of osteoprogenitor cells. The osteogenic cell lineage is thought to arise from a population of uncommitted multipotential stromal precursor cells (SPC) which reside close to all bone surfaces, in the bone marrow spaces and the surrounding connective tissue. These SPC also give rise to related cell lineages which form cartilage, smooth muscle, fat, and fibrous tissue. Due to the lack of well defined cell surface markers, little is known of the precise developmentally regulated changes in phenotype which occur during the differentiation and maturation of human osteoprogenitor cells into functional osteoblasts and ultimately, terminally differentiated osteocytes. In order to identify antibody reagents with greater specificity for osteoprogenitors we generated a series of antibodies following immunization with freshly isolated human bone marrow stromal fibroblasts. One such antibody, HOP-26, reacts with a cell surface antigen expressed by SPC and developing bone cells. We now demonstrate that this mAb identifies a member of the tetraspan family of cell surface glycoproteins, namely CD63. Western blot analysis of human bone marrow stromal cells (HBMSC) has revealed that like a well defined CD63 mAb 12F12, HOP-26 interacts with a heavily glycosylated cell surface protein with an apparent molecular weight of 50-60 kD.     

Tp44 molecules involved in antigen-independent T cell activation are expressed on human plasma cells

We have analyzed cells of the B lineage for expression of the Tp44 antigen, a 44,000 homodimer detected by monoclonal antibody 9.3 on approximately 80% of mature human T lymphocytes. Previous evidence has suggested that Tp44 may function as a receptor for accessory signals in T cell activation. High level Tp44 expression was observed on plasmacytomas grown in cell culture and on plasma cells from bone marrow biopsies of multiple myeloma patients. This antigen is not present on resting B cells from either peripheral blood or lymphoid organs, or on any other B cell tumor. The growth kinetics and Ig production in plasmacytomas are not affected by the binding of antibody 9.3. Moreover, the Tp44 molecule is co-expressed with PCA-1, an antigen characteristic of plasma cells, on peripheral blood B cells stimulated in vitro to differentiate toward plasma cells. Tp44 may represent a later stage of B cell differentiation than PCA-1 because unlike the PCA-1 antigen, this molecule could not be detected on any EBV-transformed cell line or Burkitt's lymphoma lines. The m.w. of the Tp44 molecule expressed on plasma cells and on T cells is identical, as determined by immunoprecipitation of radioiodinated cell surface proteins with monoclonal antibody 9.3. This antigen might be useful in studying the mechanism of growth and differentiation of human B cells, the heterogeneity within plasma cell populations, and B cell interactions with other components of the immune system.     

Distribution and modulation of a human leukemia-associated antigen (CALLA)

CALLA is a 100,000-dalton surface glycoprotein expressed by malignant cells of patients with clinically important subtypes of acute leukemia. Incubation of human leukemic cells expressing CALLA with specific monoclonal antibody (J5) at 37 degrees C causes rapid and selective internalization and degradation of this antigen (antigenic modulation). In these studies we show that CALLA-specific monoclonal antibodies also identify a cell surface glycoprotein having a m. w. of approximately 100,000 on 2 to 6% of nonmyeloid nucleated cells of normal adult bone marrow, on normal fibroblasts in tissue culture, and on cells of several nonhematopoietic human tumor cell lines. J5 antibody similarly modulates the surface expression of CALLA on nonleukemic cell populations, although the extent of modulation at a given concentration of antibody varied considerably. Modulation was almost complete for CALLA on cells of normal bone marrow, but was highly variable for cells of nonhematopoietic cell lines, possibly reflecting variability in antibody access to surface antigen. Using fluoresceinated or iodinated J5 antibody to modulate expression of CALLA on cells of leukemic cell lines, we show that antibody-antigen complexes undergo a temperature-dependent redistribution on the cell surface during modulation to form microaggregates. Antibody as well as antigen is then internalized. Studies of [35S]methionine-labeled cells indicate that synthesis of CALLA continues despite modulation of its surface expression by specific antibody, implying that the presence of CALLA on the cell surface reflects a dynamic equilibrium between the processes of surface expression of newly synthesized glycoprotein and its spontaneous and antibody-mediated clearance. The implications of these observations for immunotherapy are discussed.     

Inhibition of mixed lymphocyte response by a rat monoclonal antibody to a novel murine lymphocyte activation antigen (MALA-2)

A novel surface antigen expressed on activated and proliferating murine lymphocytes has been identified by a rat monoclonal antibody. The antigen, termed MALA-2, is also expressed on various lymphoid cell lines, but it is absent or present at very low densities on the majorities of unstimulated thymocytes and lymph node cells. Some cells in normal spleen and bone marrow seem to express the antigen at relatively high densities and they may represent proliferating cells in these tissues. MALA-2 has an apparent m.w. of 95,000 to 100,000 under both reducing and nonreducing conditions. The monoclonal antibody YN1/1.7 that reacts with this antigen partially inhibits Con A stimulation of spleen cells, but its inhibition of LPA stimulation is negligible. Furthermore, the antibody profoundly inhibits MLR. The inhibition of MLR by YN1/1.7 antibody is comparable to that caused by anti-transferrin receptor. The time course study suggests that the antibody may directly inhibit proliferating cell populations in MLR.     

Development and distribution of a human B cell subpopulation identified by the HB-4 monoclonal antibody

Monoclonal antibodies have been successfully used to identify B cell differentiation antigens, few of which mark discrete B cell subpopulations. We have produced a monoclonal antibody, HB-4, against a cell surface antigen on the human B cell line, BJAB, which has an unusual distribution on normal lymphoid cells. HB-4, an IgM antibody, was found to react with an antigen that is expressed by a subpopulation of B cells, approximately 50% of natural killer cells, and not by other types of cells in bone marrow, blood, and lymphoid tissues. In two-color immunofluorescence assays, the HB-4-reactive antigen was found on less than 5% of immature IgM+ B cells in fetal liver and bone marrow and on 25% of B cells in fetal spleen. The HB-4 antibody reacted with 40% of IgM+ cells in newborn blood and 60% of B cells in adult blood. In contrast, only 2 to 26% of IgM+ B cells in the peripheral lymphoid tissues of adults were HB-4+. HB-4+ B cells could be induced to proliferate by cross-linkage of their surface immunoglobulins but not by T cell-derived growth factors. The subpopulation of activated B cells that is responsive to T cell-derived differentiation factors was HB-4-, as were plasma cells. The HB-4 antibody was reactive with some but not all B cell malignancies and cell lines, and not with malignancies or cell lines of other lineages. The HB-4 antigen may therefore serve as a useful nonimmunoglobulin marker for the identification of a subpopulation of mature resting B cells that are present in the highest frequency in the circulation.     

A monoclonal antibody with reactivity restricted to normal and neoplastic plasma cells

A monoclonal antibody that defines a new and distinct plasma cell antigen, termed PC-1, was developed against human plasmacytoma cells. This antigen is strongly expressed on normal plasma cells isolated from bone marrow and on abnormal plasma cells isolated from myelomas, plasma cell leukemias, and plasmacytomas. The antigen is not detected on normal T or B lymphocytes, granulocytes, or monocytes, and with the exception of plasma cells, is absent on malignancies of B, T, or myeloid origin. Utilizing pokeweed mitogen to induce human B lymphocyte differentiation in vitro, PC-1 is expressed when B cell determinants are lost and the plasmacytoid morphology, intracytoplasmic immunoglobulin-staining, and surface PCA-1- and T10-staining characteristic of plasma cells appear. This antigen is useful for the study of the terminal stages of normal B cell differentiation to plasma cells, and may offer insight into the heterogeneity of the plasma cell dyscrasias.     

Cellular aspects of tolerance. II. Unresponsiveness of B cells

The responsiveness of bone marrow cells from tolerant donors was examined by reconstitution of lethally irradiated tolerogen-free recipients. In these animals, stem cells from tolerant donors gave rise to immunologically competent antigen sensitive B cells. The antibody produced by these cells could be detected by a sensitive plaque assay in liquid and by antigen elimination. The antibody was not demonstrable by an assay which only detected plaque forming antibody which was highly avid or was formed in large quantity per cell. In lethally irradiated animals, partially purified B cells from a tolerant animal could not cooperate with T cells from normal donors to reconstitute immunological responsiveness to immunogenic doses of the tolerance inducing antigen. We concluded that antigen sensitive B cells in the bone marrow become unresponsive following administration of tolerogenic forms of antigen. Responsiveness of the reconstituted recipient animals was due to the differentiation of donor stem cells and subsequent antibody production by their descendants. Earlier contradictory findings could be unified in terms of these observations and conclusions.     

Identification of an early activation antigen (Bac-1) on human B cells

We have produced a monoclonal antibody, Bac-1, that appears to identify a novel antigen on activated human B cells. The Bac-1 antigen can be detected between 8 to 16 hr, as well as transferrin receptors (T9), after activation of small resting B cells with phorbol myristic acetate, anti-IgM antibody, Staphylococcus aureus Cowan I, or Epstein-Barr virus. The expression of the Bac-1 antigen precedes that of IL 2 receptors (Tac-1). Peak expression of the Bac-1 antigen was observed on day 3 after activation, and decreased thereafter. The Bac-1 antigen was present on a minor subpopulation of relatively large B cells isolated from blood samples, and on "preactivated" B cells of heterogeneous size isolated from spleens and tonsils. It was not detected on bone marrow pre-B cells, blood small B cells, or plasma cells, nor was it expressed by resting or activated T cells or nonlymphoid cells. Certain B cell neoplasms and B lymphoblastoid cell lines were Bac-1+, but neoplastic cells of non-B lineage were Bac-1-. With immunoperoxidase staining, Bac-1+ cells were detected predominantly in the germinal centers of tonsil sections. The Bac-1 antigen on activated B cells was destroyed by protease treatment and was enhanced by neuraminidase treatment, suggesting that the Bac-1 antibody detects a cell surface molecule via an antigenic determinant which is partially obscured by neighboring sialic acid residues. The reactivity pattern of Bac-1 differs from the patterns of cellular reactivity reported for other monoclonal antibodies with specificity for activated human B cells.     

Acute lymphoblastic leukemia (ALL) antigens detected with antisera to E rosette-froming and non-E rosette-forming ALL blasts.

Based on the presence or absence of erythrocyte receptors(E) a T cell marker, acute lymphocytic leukemia (ALL), can be divided into E+ALL and E-ALL. We studied cell surface antigens on blasts from 12 children with untreated ALL: eight with E-ALL and four with E+ALL. Heterologous antisera were raised against thymus cells, E+ and E-ALL blasts, appropriately absorbed and tested by immunofluorescence and a radiolabeled antibody assay with normal and leukemic lymphoid cells. By both methods, anti-thymus and anti-E+ALL sera reacted with human thymocytes. Specific binding of anti-E+ALL serum to T antigens was indicated by the fact that a single absorption with thymocytes abolished its binding to allogenic thymocytes, and the reactivity of anti-E+ALL serum with thymus, blood and bone marrow lymphocytes was similar to that of anti-thymus serum. After exhaustive absorption with blood leukocytes, anti-E+ALL and E-ALL sera were negative against normal lymphocytes and bone marrow cells from children with ALL in remission. Anti-thymus and anti-E+ALL sera reacted with blasts from patients with E+ALL, but not with E-ALL. In contrast, anti-E+ALL serum reacted with 40 to 96% of blasts from all children with E-ALL, whereas of the four patients with E+ALL, two were negative and two had the lowest percentage of immunofluorescent cells (10 to 22%). These results were confirmed with the radiolabeled antibody assay. Patients with active E-ALL had cells bearing E-ALL antigen(s) in the peripheral blood and bone marrow, but the number of immunofluorescent cells was lower in blood. Cells reactive with anti-E-ALL serum did not react with thymus cells, blood lymphocytes, remission bone marrow cells, Raji cells, PWM and PHA-induced blasts and CLL cells bearing mIg (uk). These data suggest that the antigen detected on E-ALL blasts by anti-E-ALL serum is neither a HLA-related nor a cell differentiation antigen. Thus, by using antiserum to E+ALL blasts, we have confirmed the presence of a T cell-specific antigen(s) on E+ALL cells. This antiserum did not recognize other leukemia-associated antigens common to E+ and E-ALL. We have also demonstrated an antigen(s) which is regularly expressed on E-ALL blasts and is either not detectable or is present in a lower proportion of E+ALL blasts.     

A new human T-cell differentiation antigen: Unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with extablished leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.PJM is a Junior Faculty Clinical Fellow of the American Cancer Society.     

Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

Novel cell surface antigens expressed on mouse alveolar macrophages.

Two new cell surface antigens expressed on mouse alveolar macrophages were defined by rat monoclonal antibodies. One marker, AVM-1, was detected on mouse alveolar macrophages, but it was undetectable on resident peritoneal cells, thioglycollate medium-induced peritoneal cells, and splenic macrophages. Splenic lymphocytes, thymocytes and bone marrow cells were also AVM-1 negative. Anti-AVM-1 monoclonal antibody immunoprecipitated a single polypeptide with a molecular weight of 200,000. Of particular interest was the finding that the anti-AVM-1 antibody could inhibit the formation of EA and EAC rosette on macrophage line cells. A second antigen (AVM-2) was also present on alveolar macrophages, and its molecular weight was 38,000.     

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with established leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50 000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.     

HD39 (B3), a B lineage-restricted antigen whose cell surface expression is limited to resting and activated human B lymphocytes

The B cell-restricted antigen HD39, whose cell surface expression is limited to resting and activated human B lymphocytes, is described in this report. The monoclonal antibody HD39 detects a two-chain glycoprotein with apparent molecular weights of 130,000 and 140,000. During B cell ontogeny, HD39 is first expressed in the cytoplasm of bone marrow derived pre-B cells, then appears on the cell surface of sIgM+ B cells, and finally on the majority of sIgM+ sIgD+ resting B cells. After activation in vitro, the expression of HD39 on the cell surface first increases, and then the antigen is lost as cells begin to differentiate. HD39 is weakly expressed on very few non-T cell ALL and B cell CLL, on approximately 50% of B cell lymphomas, and not on Waldenstr?m's macroglobulinemias and myelomas. In contrast, it is strongly expressed on all hairy cell leukemias. Its limited cell surface expression in B cell ontogeny suggests that HD39 may be important in the events that regulate the activation of the human resting B lymphocytes.     

Immunoelectron microscopic detection of band 3 protein during erythroid cell differentiation by a monoclonal antibody

We created a monoclonal antibody, designated EB1 (IgM, kappa), that reacts with erythroblasts by fusion of P3-X63-Ag8.653 with splenocytes of rats immunized with erythroblastic islands isolated from mice spleens. Western blotting revealed that EB1 reacted with the band 3 protein of the erythrocytic membrane. It stained erythrocytes and erythroblasts, forming clusters in the bone marrow, splenic red pulp, and fetal liver, but did not stain other tissues in the cryostat sections. The EB1 antigen was detected during dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. Immunoelectron microscopy revealed that the EB1 antigen was expressed from the basophilic erythroblasts during normal erythroid differentiation. Preferential segregation of the EB1 antigen on the cell membrane of the nucleating erythroblasts was not observed. These results suggest that EB1 is specific for erythrocyte band 3 protein and may be useful for studying erythroid cell differentiation.     

Description of a murine B lymphoma tumor-specific antigen

By using monoclonal antibodies, a tumor-specific antigen (TSA 41.5) was detected on the cell surface of a B lymphoma CH-1 tumor variant, CH-1.1. This antigen is not expressed by normal lymphocytes (spleen cells, lymph node cells, thymocytes, bone marrow cells, or blast cells) of B10.A mice, the host strain of CH-1.1, or by the CH-1 lymphoma. Immunoprecipitation and biochemical characterization of TSA 41.5 with the use of two-dimensional gel electrophoresis showed this antigen to be a surface protein of CH-1.1 cells with an Mr of 80k and pI of 4.6. TSA 41.5 is not related to the murine transferrin receptor, and not to gp70, a viral envelope protein expressed by CH-1.1 cells, shown by comparative peptide map analysis of these three proteins. TSA 41.5 is a surface antigen unique to the CH-1.1 tumor, which is not expressed by the 19 different murine tumor lines that were tested nor by spleen cells of 15 independent mouse strains. In addition, treatment of spleen cells with bacterial lipopolysaccharide did not induce the expression of TSA 41.5. These characteristics of TSA 41.5 make it unlikely to be a product of viruses. Additional evidence against TSA 41.5 being a viral protein was obtained by the observation that antisera against viral proteins could not block the binding of the anti-TSA monoclonal antibody to its antigen. In vitro treatment of CH-1.1 cells with anti-TSA monoclonal antibody specifically inhibited the in vitro growth of the tumor cells in a dose-dependent fashion. The CH-1.1 tumor and monoclonal antibodies could be a useful murine model system for the exploration of the use of monoclonal antibodies for the in vivo treatment of cancer.     

Cell surface expression of CD154 inhibits alloantibody responses: A mechanism for the prevention of autoimmune responses against activated T cells?

Binding of CD40 by CD154 expressed on activated T cells is a pivotal event in T cell help to B cells, macrophages, and other antigen-presenting cells. Expression of CD154 by MHC mismatched cells, in contrast to expectations, strongly suppressed alloantibody responses against the cells. This was caused by a failure of priming of antibody responses by the CD154 expressing cells. We hypothesize that this lack of response against CD154 expressing cells may represent a mechanism that has evolved to prevent autoantibody responses being generated against the CD154 antigen itself, as B cells expressing antibody reactive with CD154 would probably escape deletion on binding antigen in the bone marrow due to rescue by the simultaneous ligation of CD40.     

Specificity and function of monoclonal antibodies directed against Ewing sarcoma cells

A selection of 16 monoclonal antibodies has been produced against a fresh Ewing's sarcoma (ES) tumor mixed with a permanent ES cell line. The majority of antibodies identify an 80-kDa molecule, which is not detected on healthy tissues except on certain cultured monocytes. One antibody recognize the CD2 ligand MIC2 and 2 antibodies (numbers 13 and 16) define a higher-molecular-mass antigen. Antibody 16 is also expressed on mesenchymal fibroblasts of bone marrow or fetal origin. Tumorspecific antigen expression is potentially linked to the chromosome 22 abnormality decribed in Ewing's sarcoma.     

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.